Follistatin-like 1 in development and human diseases

Follistatin-like 1 in development and human diseases Follistatin-like 1 (FSTL1) is a secreted glycoprotein displaying expression changes during development and disease, among which cardiovascular disease, cancer, and arthritis. The cardioprotective role of FSTL1 has been intensively studied over the last years, though its mechanism of action remains elusive. FSTL1 is involved in multiple signaling pathways and biological processes, including vascularization and regulation of the immune response, a feature that complicates its study. Binding to the DIP2A, TLR4 and BMP receptors have been shown, but other molecular partners probably exist. During cancer progres- sion and rheumatoid arthritis, controversial data have been reported with respect to the proliferative, apoptotic, migratory, and inflammatory effects of FSTL1. This controversy might reside in the extensive post-transcriptional regulation of FSTL1. The FSTL1 primary transcript also encodes for a microRNA (miR-198) in primates and multiple microRNA-binding sites are present in the 3′UTR. The switch between expression of the FSTL1 protein and miR-198 is an important regulator of tumour metastasis and wound healing. The glycosylation state of FSTL1 is a determinant of biological activity, in cardiomyocytes the glycosylated form promoting proliferation and the non-glycosylated working anti-apoptotic. Moreover, the glycosylation state shows die ff rences between species and tissues which might underlie the die ff rences observed in in vitro studies. Finally, regulation at the level of protein secretion has been described. Keywords Cardiovascular disease · Cancer · Immune disease · Inflammation · Fibrosis · Obesity · Pulmonary disease · Signal transduction · Glycosylation · miRNA Introduction 308 Aa) and mouse (Genbank Q62356; 306 Aa) protein sequences shows that the secretion signal (human: Aa 1–20 Follistatin-like 1 (FSTL1) is a glycoprotein of the secreted and mouse: Aa 1–18) is most species variable, whereas the protein acid and rich in cysteine (SPARC) family. In the remaining 272 Aa shows a very high degree of similarity literature, FSTL1 is referred to by many different names [ 1]. (94.4%). In the sequence of mouse Fstl1, three potential sites Two groups independently discovered FSTL1 and named for N-glycosylation and two for O-glycosylation are present it (I) transforming growth factor (TFG)-β-induced clone and in vitro studies have shown that only the three aspar- 142 173 178 36 (TSC36) isolated from mouse osteoblasts [2] and (II) tate residues Asp, Asp , and Asp are N-glycosylated. Follistatin-related protein (FRP) secreted from rat glioma Moreover, glycosylation at these sites shows cell-type speci- [3]. FSTL1 comprises a secretion signal, a Follistatin- and a ficity [ 4]. N-glycoproteome analysis on human blood plasma Kazal-like domain, two EF-hand domains, and a von Wille- identified only one glycosylated form of FSTL1 in which 175 180 brand factor type C domain (http://www.unipr ot.org/unipr two (Asp and Asp ) of the three sites are used [5]. From ot/Q12841 ). Comparison of the human (Genbank: Q12841, gastrulation onward, Fstl1 mRNA is broadly expressed throughout the entire mouse embryo and its expression becomes restricted to the mesenchymal component of most Electronic supplementary material The online version of this tissues at the end of gestation [6]. In the adult mouse, the article (https ://doi.org/10.1007/s0001 8-018-2805-0) contains highest levels of Fstl1 mRNA are found in heart, lung, and supplementary material, which is available to authorized users. subcutaneous white adipose tissue [7]. Interestingly, the * Maurice J. B. van den Hoff expression of FSTL1 changes with respect to its level and m.j.vandenhoff@amc.uva.nl pattern during various diseases, including cardiovascular disease [8–15], cancer progression [16–22], and systemic Department of Medical Biology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Vol.:(0123456789) 1 3 2340 A. Mattiotti et al. autoimmune diseases [23–26]. Analysis of the exomes of Cardiovascular system over 60,000 individuals revealed that the estimated probabil- ity of loss-of-function intolerance is 0.96 (http://exac.broad Cardiovascular disease (CVD), such as heart failure (HF) institute.or g/gene/ENSG0 00001 63430 ), r eflected in the find- and coronary artery disease, is a group of disorders that ing that homozygous loss-of-function mutations is never affect the heart and blood vessels. CVD is a leading cause observed and heterozygous ones have only been described of death in Western countries [34]. For the sake of clarity, in 35 individuals. In line with this, functional disruption of we have included a summary figure to accompany the text Fstl1 in mice was found to result in respiratory distress and below (Fig. 1). death within hours after birth. Gene knock-out mice display a phenotype that appears to suggest an important inhibitory Clinical relevance role of FSTL1 in BMP signaling [27, 28]. Multiple TGFβ superfamily receptors as well as disco-interacting protein In humans, circulating concentrations of FSTL1 increase 2 homolog A (DIP2A) have been shown to interact with during cardiac and vascular diseases, such as HF [9, 35], FSTL1 [28–30]. However, using other transgenic models, HF with preserved ejection fraction (HFpEF) [15], acute FSTL1 has been implicated in multiple signaling pathways coronary syndrome (ACS) [36], and chronic obstructive and its role during diseases remains unclear [1]. Recently, a pulmonary disease [37]. In patients with ACS, the increase study on the role of FSTL1 in cardiac regeneration showed in the serum level of FSTL1 correlates with mortality [36]. different effects on cardiomyocyte proliferation and pro- Moreover, the levels of FSTL1 in the circulating blood tection from apoptosis depending on the post-translational were found to correlate with the severity of chronic HF [35, modification of the protein [13], opening a new perspective 38]. Although FSTL1 has a negative prognostic value with on the interpretation of previously seemingly contradict- respect to HF, it was found that patients with end-stage HF ing data. Moreover, post-transcriptional regulation plays an who received a combination of a left ventricle assist device important role in the expression of the protein [31] and mul- (LVAD) and pharmacological therapy, who had high levels tiple functional miRNA-binding sites have been identified of FSTL1 at the time of LVAD implantation, showed recu- in the 3′UTR of FSTL1 mRNA [24, 32, 33]. For this reason, peration and recovery of ejection fraction [9]. This finding we present a review of the studies that have reported various suggests that FSTL1 could be a therapeutic target for drug roles of FSTL1 in development and disease while trying to development and serum concentration of FSTL1 can be used clarify the sometimes contradictory results. as a prognostic biomarker for CVD [35, 39]. Fig. 1 Follistatin-like 1 in car- diovascular disease. Schematic Fa�y acid oxidaon Glucoseoxidaon representation of the known signaling pathways interacting Cardiomyocytes Hypertrophy with FSTL1 in cardiovascular hypertrophy disease. Grey components Smooth muscle indicate unknown receptors. AMPK ACC cells proliferaon The coloured arrows denote the Pathological and migraon secreted Fstl1 (width relates remodeling amount). Coloured area defines Fibroblasts different conditions. Image MEK1/2 ERK1/2 proliferaon and adjusted from http://smart .servi migraon er.com Ischemia FSTL1 mTOR Cardiomyocytes DIP2A FOXO1/3 apoptosis PI3K AKT1 Endothelial cells eNOS survival, migraon ALK3/6 and differenaon BMP4 BMPR-2 / Inflammatory SMAD1/5/8 ACTR-IIA/B response Cardiomyocytes proliferaon 1 3 Follistatin-like 1 in development and human diseases 2341 using a recombinant adenovirus, have positive effects on sur - Cardiac development vival and regeneration leading to a significant reduction of the infarct size [8, 13]. To study the beneficial role of FSTL1, RNA-Seq data (https ://www.ncbi.nlm.nih.gov/gene/11167 ) show that FSTL1 mRNA is expressed in the adult human E. coli produced human FSTL1 was intravenously adminis- trated in a murine model either before inducing ischemia or heart, but the expression pattern of the mRNA has not been established. However, immunohistochemical data showed after reperfusion; to validate a therapeutic effect in a larger animal, a porcine model was subjected to 45 min of ischemia that FSTL1 is present in vascular endothelial cells of ves- sels located within the myocardium, in smooth muscle cells and 24 h of reperfusion and intracoronary administration of FSTL1 during the first 10 min after ischemia. In both of larger vessels and at a low, but significant levels in car - diomyocytes [9]. Furthermore, the pattern of expression models, administration of FSTL1 reduced ischemic damage and improved cardiac performance after reperfusion [11]. of FSTL1 during human cardiac development remains to be established. In mouse and chicken, Fstl1 is expressed Applying a collagen patch to the infarcted heart ameliorated recovery, which was even further improved when this patch throughout the entire heart during early development and with expression subsequently becoming largely restricted was enriched with epicardium-derived medium or bacteri- ally produced recombinant FSTL1 protein. The addition to the non-myocardial component, with low levels being expressed in the cardiomyocytes [6, 40]. Homozygous dele- of bacterially produced FSTL1 significantly decreases the infarct area and as a consequence increases survival after tion of Fstl1 in mice results in an overall enlargement of the heart of neonates [27]. Whether this enlargement is due to MI [13]. In pig, a collagen patch loaded with FSTL1 applied to the ventricle 1 week after ischemia/reperfusion was also hyperplasia or hypertrophy of the cardiomyocytes is not yet known, and is currently being evaluated. found to promote the regenerative response [13]. In cul- tured neonatal rat cardiomyocytes (NRCM), recombinant Myocardial infarction protein reduced hypoxia/reoxygenation-induced apoptosis both directly via (I) the MEK1/2 (mitogen-activated protein During a myocardial infarction (MI), blood flow to a portion kinase kinase) and ERK1/2 (extracellular signal-regulated kinase) signaling pathway, (II) the DIP2A, PI3K (phospho- of the heart muscle is interrupted causing local ischemia when prolonged results in cardiomyocyte death [41]. The inositide 3-kinase) and AKT1 (RAC-alpha serine/threo- nine–protein kinase) pathway with activation of downstream dying cardiomyocytes trigger an inflammatory response which is followed by a reparative process that results in the effectors mTOR and FOXO1/3 [8 , 44] and (III) AMPK (AMP-activated protein kinase) phosphorylation, and indi- formation of non-muscular scar tissue, thus reducing cardiac contractility and output [42]. rectly via (IV) inhibition of BMP4-induced apoptosis and (V) reduction of pro-inflammatory cytokines expression After inducing an MI in mice by permanent occlusion of the left anterior descending artery (LAD), Fstl1 is transiently [11]. Interestingly, a difference in the effect between bac- terially and eukaryotically produced FSTL1 was observed highly expressed in the heart, especially in the ischemic zone. Fstl1 reaches its highest level of mRNA expres- in the study of Wei and colleagues [13]. It should be noted that bacterially produced proteins are not glycosylated, sion 1 week after MI and normalizes in the remote zone of the heart at 1 month after MI, while low levels of expression while eukaryotically produced proteins are. The non-gly- cosylated FSTL1 increased NRCM proliferation, while the persist in the infarct zone [10, 14]. Non-myocardial cells have been shown to be the major source of Fstl1 in the heart, glycosylated protein, in accordance with previous reports [8, 11], protected NRCM from peroxidase-induced apoptosis with low levels also being detectable in the cardiomyocytes [13, 14]. Because Fstl1 is predominantly expressed in the [13]. In vitro experiments showed that glycosylated FSTL1 promotes fibroblast proliferation and migration via ERK1/2 ischemic zone, the serum levels seem to be a reflection of the infarct size. This might also underlie the observed correla- phosphorylation [14]. Deletion of Fstl1 from a part of the fibroblast population using the S100A4–Cre mouse line did tion between elevated FSTL1 levels and mortality in ACS patients [36]. However, other organs that serve as a source of not affect cardiac function compared to control littermates. Upon the induction of an MI, however, the number of mice FSTL1 cannot be excluded. During the healing process after an MI, the epicardium plays an important role as a source dying due to cardiac rupture within the acute phase, i.e., first 7 day post-MI, doubled from a 25% in wild type to 50% of signaling molecules and provides cells to the infarct zone [10, 43]. Fstl1 is transiently expressed in the epicardium in Fstl1-depleted animals. Analysis of cardiac function did not reveal any significant differences, though the number of overlaying the infarct tissue and in the derived mesenchymal cells that populate the infarct scar [10, 13]. myob fi roblasts was decreased and the synthesis and matura - tion of the extracellular matrix proteins were reduced [14], In both permanent and transient LAD ligation mouse models, it was found that locally produced and circulating indicating that the initial reparative response is abrogated. Fstl1 protein, assessed by overexpression of Fstl1 in the liver 1 3 2342 A. Mattiotti et al. Fstl1 expression was found to be induced in skeletal muscle Cardiac hypertrophy cells [48]. Adenoviral-mediated overexpression of Fstl1 in the latter mouse model was found to stimulate revasculariza- Cardiac hypertrophy is characterized by the abnormal thick- ening of the wall of the heart as a result of an increase in the tion. Interestingly, adenoviral-mediated overexpression of Fstl1 in non-ischemic muscle did not alter its vasculariza- volume of the individual cardiomyocytes [45]. Fstl1 expression is upregulated in mice after transverse tion. In this model of hind limb ischemia, glycosylated Fstl1 was found to promote endothelial cell survival, migration, aortic constriction (TAC), which results in pressure over- load-induced hypertrophy [12] or by aldosterone infusion and differentiation into a vascular network-like structure via phosphatidylinositol-3 kinase (PI3K), AKT, and endothelial which causes hypertension-induced HFpEF [15]. In both these models, cardiomyocytes are the major source of Fstl1 nitric-oxide synthase (eNOS) activation [48]. At that time, the receptor conveying the extracellular Fstl1 signal into the [12, 15]. Treatment of adult rat ventricular cardiomyocytes with recombinant glycosylated human FSTL1 produced in cell was unknown, but recently, DIP2A has been identified as a potential cell-surface receptor upstream of PI3K and Sf9 insect cells abrogates the increase in protein synthesis and Nppa (also known as: ANF or ANP) expression induced AKT1 phosphorylation [44]. Interestingly, an opposite effect of FSTL1 has been observed in smooth muscle cells (SMCs) by aldosterone stimulation, indicating that FSTL1 prevents hypertrophy in cardiomyocytes. In HFpEF mice, increased during pulmonary hypertension [37] and vascular injury [49, 50]. In the latter two models, FSTL1 prevents pathological levels of circulating Fstl1, mediated by overexpression in the liver via adenoviral delivery, significantly reduce car - vascular remodelling, as a result of a decrease in SMC pro- liferation and migration which is mediated via the induction diomyocyte hypertrophy and ameliorate cardiac functions [15]. Specific deletion of Fstl1 from cardiomyocytes using of AMPK and inhibition of ERK phosphorylation [37, 49, 50]. Studies on HUVEC cells showed that FSTL1 affects the alpha myosin heavy chain (αMHC)–Cre mouse line did not show structural or functional differences compared to vascular endothelial cell polarization, but not migration and tube formation [51]. control littermates [12, 15]. However, when these mice were challenged by TAC, cardiac hypertrophy was enhanced and Mitral valve disease ventricular performance decreased [12]. On the other hand, when these mice were challenged by uninephrectomy in Mitral valve disease is a major cause of morbidity, heart combination with 4 weeks of contiguous infusion of aldos- terone, HFpEF ensued [15]. Interestingly, in the latter model, failure, and death worldwide [52]. Deletion of Fstl1 from the endothelial/endocardial lineage using the Tie2–Cre mouse the role of Fstl1 in the development of HFpEF was inde- pendent of the changes in cardiac fibrosis but crucial in the line resulted in dysfunctional mitral valves, HFpEF and death [53]. The dysfunctional mitral valve leaflets became development of cardiac hypertrophy [15]. In vitro experi- ments showed that the inhibitory effect of Fstl1 on cardio- long and thick, suggestive of enhanced proliferation of the valve cells and formation of mesenchymal cells as a result myocyte hypertrophy is mediated by AMPK and acetyl-CoA carboxylase (ACC) phosphorylation [12]. of prolonged endocardial to mesenchymal transition, valve enlargement itself probably being due to enhanced TGF-β During cardiac disease, the energy consumption of car- diomyocytes changes from fatty acid to glucose [46]. When and BMP signaling. Whether the observed development of HFpEF is a direct consequence of deletion of Fstl1 or heart failure is induced by tachy-pacing in dogs, this meta- bolic switch is also observed. Treatment of these dogs with a secondary effect of the dysfunctional mitral valve or the enhanced TGF-β and BMP signaling, is not yet clear [53]. a single dose or long-term infusion (14 days) with glyco- sylated human FSTL1 (CHO cells) inhibited the pathologi- It should be noted at this point that FSTL1 was not found in a meta-analysis of genome-wide association studies identi- cal switch from fatty acid to glucose oxidation. This effect was transient, because after clearing FSTL1 from the blood, fying 23 loci with suggestive evidence of association with mitral valve prolapse in the human population [54]. consumption of glucose increased and fatty acid consump- tion decreased. Moreover, the infusion of AMPK inhibitor In conclusion neutralized the effect of FSTL1 [47]. FSTL1 is a secreted glycoprotein with both protective and Vascular system regenerative capacities of which the circulating concen- tration increases during CVD. Overexpression or lowered In some of the previously mentioned studies on MI and cardiac hypertrophy, mice also displayed a pro-angiogenic expression (hypomorphic or heterozygous KO) of Fstl1 is tolerated in healthy animal models, whereas during patho- effect of Fstl1, though the underlying mechanism was not investigated [12–14]. A similar pro-angiogenic effect was logical conditions, additional Fstl1 prevents extensive car- diac damage and abnormal vascular remodelling. A lack of also found in a mouse model of ischemic hind limb, in which 1 3 Follistatin-like 1 in development and human diseases 2343 Fstl1 exacerbates cardiac injury. Most importantly, Fstl1 In clear-cell renal cell carcinoma (ccRCC), high levels of affects multiple pathways in a cell-type specific manner and FSTL1 expression correlate with a favorable post-operative different effects are observed depending on the glycosylation survival (hazard ratio = 0.325; p = 0.030). Knock-down of state of the protein. These results suggest that FSTL1 may FSTL1 using retrovirus mediated short hairpin RNA in not just represent a biomarker, but could also be an interest- ccRCC cell lines resulted in anchorage independent growth ing candidate for the development of new therapies in CVD. and invasion. Further analysis showed that the tumour sup- pressor function of FSTL1 is mediated through repression of the NF-κB and HIF-2α signaling pathways [57]. Moreover, Cancer and tumours in line with these novel findings, a polymorphism in the second intron of the FSTL1 gene, associated with downregu- Cancer is characterized by an imbalance in growth regula- lation of its expression, correlates with an increased risk of tion caused by genetic changes. When cancer cells do not renal cell carcinoma and poor prognosis [21]. Interestingly, migrate into the surrounding tissues, the tumour is referred in nasopharyngeal carcinoma (NPC) cell lines and tumour to as a benign. Cancer cells become malignant when they biopsies, decreased levels of FSTL1 mRNA correlate with acquire the additional property of migration, allowing them hypermethylation of CpG islands in its proximal promoter to invade other tissues, this being referred to as metastasis. (− 166 to + 332 bp) and also with increased tumourigenic- When left untreated, tumour metastasis is the leading cause ity. Restoring of FSTL1 mRNA levels using demethylat- of death in cancer patients [1, 55, 56]. For the sake of clarity, ing agents decreased tumourigenicity. The reduced levels we have included a summary figure to accompany the text of expression of Fstl1 in the NPC tumours are reflected in below (Fig. 2). decreased FSTL1 serum levels [22]. A low level of expres- sion of FSTL1 in lung adenocarcinoma patients has a poor Clinical relevance prognostic value (hazard ratio = 2.09; p = 0.022), but no correlation was observed between FSTL1 expression and Compared to healthy tissue, the expression level of FSTL1 survival in squamous cell carcinoma [58]. was found to be reduced in biopsies of various types of On the other hand, increased expression of FSTL1 com- cancers, such as prostate [16], ovarian, endometrial [19], pared to healthy control tissue was observed in brain cancer kidney [20, 21, 57], nasopharyngeal carcinoma (NPC) [22], cells [18], in fibroblasts but not in epithelial cells in colon and lung adenocarcinoma [58]. In endometrial and ovar- tumours [59], in castration-recurrent prostate cancer [60], ian tumours, a trend between poorer prognostic character- in most cases of hepatocellular carcinomas (HCC) [61], in istics and decreasing levels of FSTL1 was observed [19]. head and neck squamous cell carcinoma (HNSCC) [62], and Fig. 2 Follistatin-like 1 in FSTL1 cancer and tumour. Schematic representation of the known signaling pathways interact- ing with FSTL1 during cancer growth and metastasis. Grey FASLG Smad3 proliferaon components indicate unknown AKT ERK1/2 FAS receptors. Helical structures GSK-3β G2/M represent gene expression. arrest BIM BIM-EL/L/S p/uBIM Dashed arrow indicates transla- BAX tion from mRNA to protein. The BCL2 Caspase3/9 Caspase3/7 dashed line separates different CDC2 processes: tumour growth and apoptosis PARP metastasis. Image adjusted from http://smart .servi er.com CCL2 CCR2 MMP9 CXCL12 CXCR4 migraon invasion ERK1/2 FSTL1 MMP2 WNT7a CX43 FSTL1 EGF 1 3 Metastasis Growth CyclinA/B1, CDK1 accumulaon 2344 A. Mattiotti et al. esophageal squamous cell carcinoma (ESCC) [63]. FSTL1 breast cancer [64] cells promotes proliferation. Phosphoryla- is higher expressed in metastatic brain tumours compared tion of Smad2/3 is one of the pathways that mediate FSTL1 to primary breast cancer [64]. FSTL1 is not expressed at a inhibition of proliferation [64]. Injection of NPC cells sta- detectable level in normal brain tissue and in diffuse-infil- bly transfected with FSTL1 in nude mice showed a similar trating astrocytes of grade II or III gliomas, but is expressed tumour incidence compared to non-transfected cells. How- at high levels in grade IV gliomas, referred to as glioblas- ever, the tumour growth rate was lower in FSTL1-transfected toma [18]. The level of FSTL1 was found to correlate with NPC cells compared to control cells [22]. Similar in vivo the malignancy of the astrocytoma and its coexpression results were observed in NSCLC; tumour growth was inhib- with p53 has a negative prognostic value [18]. In HCC, high ited when FSTL1 was overexpressed in the CL1-5 cell line levels of FSTL1 correlate with larger tumours, advanced and promoted when FSTL1 expression was downregulated tumour/node/metastasis (TNM) stages, metastasis, and poor in the CL1-0 cell line [58]. post-surgical outcomes (hazard ratio = 1.84; p = 0.015) [61]. Decreasing the level of FSTL1 in human NSCLC cell The high levels of Fstl1 correlate with lower survival per- lines (NCI-H460 and A549) using an siRNA induced the spective in HNSCC and ESCC patients [62, 63]. accumulation of cell cycle proteins, such as cyclin A, cyclin Most cell lines derived from human tumours express B1, CDK1, and phosphorylated CDC2 and caused G2/M lower levels of FSTL1 than immortalized non-tumourigenic arrest [69]. In this same study, it was also reported that apop- fibroblasts [65]. In vitro transfection of fibroblasts (mouse tosis increased. The increase in apoptosis was mediated by NIH3T3 and rat 208F) using oncogenes like ras, myc, or fos a decrease in ERK1/2 phosphorylation, an increase in both induces a tumourigenic phenotype and downregulation of cleaved (activated) Caspase-3, Caspase-9, and PARP, and Fstl1 expression, suggesting a tumour suppressor role [2, 65, an accumulation of the pro-apoptotic factor BIM-EL [69]. 66]. Comparison of human cancer cell lines with a differ - Overexpression of FSTL1 in a human hepatocellular car- ence in aggressiveness of their phenotype showed opposite cinoma (Huh7) cell line promoted its expansion, being the outcomes: the prostate cancer cell line LNCaP shows lower result of increased proliferation and inhibited apoptosis. The FSTL1 expression levels compared to the more aggressive inhibition of apoptosis was found to be the result of activated C4-2 variant [17], while less aggressive non-small cell lung AKT/GSK-3β signaling, with as consequence an increase carcinoma (NSCLC) cells express on average higher levels in the anti-apoptotic protein BCL-2 and a decrease in the of FSTL1 compared to more malignant small cell lung can- pro-apoptotic BIM and BAX proteins [61]. Similar results cer (SCLC) cells [67]. However, high variability in FSTL1 were reported in ESCC cells (KYSE-150), where inhibitory expression levels is observed in NSCL cell lines from both effect of FSTL1 on the BMP-signaling pathway and chem- adeno- or squamous carcinomas. oresistance could be demonstrated [63]. Overexpression of Taken together, the data on the expression levels of FSTL1 in mouse MC3T3 osteoblast precursor cells [65] or FSTL1 in relation with its effect in different tumours are rat 208F fibroblasts [66], on the other hand, did not affect highly variable from respect to growth inhibition or induc- cell morphology or proliferation. Although downregulation tion and from invasiveness to immobility. As a consequence, of FSTL1 in a squamous cell carcinoma (SCC12) cell line the expression level of FSTL1 should be regarded with cau- did not affect the proliferation rate in vitro, they were found tion, and not be associated with a specific phenotype. to form larger tumours when injected in nude mice [62]. Tumour growth Metastasis With respect to imbalanced tumour growth, contradictory Overexpression of FSTL1 in lung (PC-14 line and H446) results have also been reported upon in vitro manipulation [67, 68], ovarian, endometrial [19], or NPC [22] cell lines of the level of expression of FSTL1 in cell lines. not only decreased their proliferative capacity, but also their Transfection of lung NSCLC cell lines (PC-14 and H446) ability to migrate and invade flanking tissues. This reduction [58, 67, 68], ovarian, endometrial [19], NPC [22], and breast in migratory capacity was accompanied by a reduction in cancer [64] cell lines with FSTL1 reduces their growth rate metalloproteinase-2 (MMP-2) expression [19, 66]. In line via an unknown signaling pathway. Overexpression of with these results, downregulation of FSTL1 in ccRCC cells FSTL1 in ovarian or endometrial cells was also found to was shown to lead to a reduced migratory capacity and tissue increase the rate of apoptosis via a death receptor-initiated invasion [57]. pathway. In these cells, an increase in the mRNA of Fas Downregulation of FSTL1 in human melanoma cells cell-surface death receptor (FAS or First Apoptotic Signal using a siRNA results in the inhibition of the expression receptor) and its ligand (FASLG) was found to result in an of genes associated with migration, such as CCL2 and increase in cleaved (activated) PARP, Caspase-3, and Cas- CXCL12, and with the formation of bone metastasis, such pase-7 [19]. Downregulation of FSTL1 in ccRCC [57] and as CCR2 and CXCR4 [70]. In line with these observations, 1 3 Follistatin-like 1 in development and human diseases 2345 stimulation of these melanoma cells with recombinant Immune diseases glycosylated FSTL1 induced their migratory capacity and differentiation into a bone phenotype. FSTL1 is also lower The immune system is the collective of tissues, cells, and expressed in the breast cancer cell line MDA-MB-231 com- processes that provide a specific response to protect the pared to its metastatic version 231-BR [64]. In vivo studies, organism upon invasion by organisms, foreign cells, and tox- further showed that injection of an FSTL1 siRNA into a ins. Dysfunction of components of the immune system often subcutaneous tumour suppressed tumour growth and the for- results in immune disorders. Systemic autoimmune diseases mation of bone metastasis, increasing mouse survival com- (SADs) are disorders characterized by loss of function or pared to injections with a control siRNA [70]. Overexpres- destruction of normal tissues due to autoimmunity which is sion of FSTL1 in ESCC cells (KYSE-150) promoted tumour specific to each disease. The role of FSTL1 during inflam- growth and metastasis in vivo. In vitro analysis showed that matory processes has been studied in several models and inflammation (Fig.  3) and epithelial-to-mesenchymal transi- reported to be both pro- and anti-inflammatory. For the sake tion processes were strongly affected by FSTL1 [57, 63]. A of clarity, we have included a summary figure to accompany recent study on the metastatic effect of FSTL1 showed that the text below (Fig. 3). FSTL1 specifically interacted with WNT7a and antagonized its inhibitory effect on endothelial growth factor-mediated ERK phosphorylation which in turn induced the expression Clinical relevance of MMP9 [62], a prerequisite for cell migration. Overex- pression of Connexin-43 in pulmonary giant carcinoma cell High serum concentrations of FSTL1 are found in many inhibited their metastatic capacity. This inhibitory effect SADs, such as rheumatoid arthritis (RA) [23, 24], in par- could be reversed by adding antibodies against FSTL1 pro- ticular juvenile RA with systemic onset [25], osteoarthritis tein [71]. These observations need to be regarded with cau- (OA) [26], and Sjögren’s syndrome [23]. Synovial tissue of tion, as they might be pointing to two independent signaling RA patients expresses FSTL1 at high levels, which correlate pathways regulating cell invasion. positively with clinical and serological parameters of disease [72]. In serum of RA patients, antibodies directed against In conclusion FSTL1 appear more frequently (30%) than in other SADs, such as systemic sclerosis (17%), systemic lupus erythema- Due to the heterogeneity in the different cell lines and can- tosus (10%), and Sjögren’s syndrome (10%). Antibodies cers and the complexity of the multiple mechanisms under- against FSTL1 were not found in patients with OA or poly- lying tumour development, our knowledge on the role of myositis/dermatomyositis or healthy controls [73]. Besides FSTL1 during cancer development and progression is still the elevated serum levels of FSTL1 in RA, no association of fragmented and limited. The origin of the tumourigenic cell polymorphisms in Fstl1 with susceptibility to RA has been line appears to be a main determinant for the different, some- found to date [74], suggesting that overexpression of FSTL1 times opposite, effect of FSTL1. is a consequence of RA rather than a cause. Fig. 3 Follistatin-like 1 in FSTL1 immune diseases. Schematic representation of the known signaling pathways interacting with FSTL1 during inflamma- CD14 tory processes. Grey compo- DIP2A nents indicate unknown recep- TLR4 tors. Helical structures represent gene expression. Dashed arrow ACC AMPK Inflammatory factors indicates translation from FSTL1 mRNA to protein. The dashed ERK1/2 line separates the two opposite IKKβ Transcrip­on factors effects of FSTL1: pro- and anti- IKBα (ETS-2, c-FOS) inflammatory. Image adjusted JNK Proinflammatory from http://smart .servi er.com NF-Kβ cytokines Matrix metalloproteases IL-1β IL-6 (MMP-2, MMP-3, MMP-13) miR-27a IFN-γ MCP-1 miR-32-5p TNF-α Pro-inflammatory effects in chronic disease An -inflammatory effect during acute phase 1 3 2346 A. Mattiotti et al. Patients with Kawasaki disease present with inflamma- inflammatory response and Fstl1 expression. In CIA mice, tion of blood vessels throughout their body. Plasma levels of Ad-mFstl1 exacerbates severity of arthritis, while admin- FSTL1 are significantly elevated in acute Kawasaki disease istration of anti-mFstl1 IgG neutralizes endogenous Fstl1 patients compared to control subjects. Upon intravenous and reduces the severity of disease [81, 82]. A similar pro- immunoglobulin therapy, levels of FSTL1 were found to inflammatory effect, mediated via the CD14-TLR4 pathway, slowly decrease over time [75]. Interestingly, FSTL1 lev- was also found in CIA mice in which Fstl1 was delivered by els correlate with a high risk of developing coronary artery genetically modie fi d-T cells [ 83]. Moreover, it was observed aneurysm [75], which is a major cause of morbidity and that FSTL1 functioned in a species-specific manner: recom- mortality [76]. binant human but not mouse protein, both produced in Lumbar disc herniation (LDH) is a medical condition human cells (HEK293 and HT-1080), induces IL-6 expres- affecting the spine in which a rupture in the outer fibrous sion in cultured synovial cells from RA patients but not in ring of an intervertebral disc allows the soft central por- mouse NIH-3T3 cells and vice versa [83]. Interestingly, the tion to bulge out beyond the damaged outer rings causing mouse and human proteins are very similar (94.4%), and inflammation. Serum levels of FSTL1 are higher in LDH although the effect difference may lie in this small differ - patients compared to scoliosis patients and healthy controls ence, perhaps, a differing glycosylation state of the two and correlate with the amount of pain on a visual analogue proteins plays a major role in determining their observed scale [77]. biological activities. In the CAIA mouse model, an increase in Fstl1 expression Arthritis is observed during early stages of arthritis. In this model, the administration of E. coli-derived recombinant human To evaluate the role of FSTL1 in arthritis, two different ani- FSTL1 showed anti-destructive and anti-inflammatory mal models are used that result in apparently contradictory effects, reducing synovial cellular infiltration and retaining effects. In the collagen-induced arthritis (CIA) model, the cartilage proteoglycan. Treatment of synovial cells and joints mice are immunized by intradermal administration of an with FSTL1 resulted in downregulation of the transcription emulsion of complete Freund’s adjuvant and bovine type II factors c-Fos and Ets-2 and downstream matrix metallopro- collagen, and 21 days later, a booster or secondary immu- teases, such as Mmp-3 [84, 85]. In line with these obser- nization is given [78]; in this mouse model, FSTL1 was vations, upregulation of these genes was observed when found to promote inflammation. In the anti-type II collagen cells were treated with antibodies directed against FSTL1 antibody-induced arthritis (CAIA) model, the mice are intra- that were obtained from mouse or from RA patients [84, peritoneally injected with a cocktail of monoclonal antibod- 85]. Interestingly, both c-FOS and ETS-2 are expressed at ies directed against type II collagen, and 72 h later, they are higher levels in RA patients than in healthy controls [86]. injected with endotoxin (LPS) [79]; in this acute, destructive In vitro experiments showed that downregulation of c-FOS arthritis model FSTL1 was found to be anti-inflammatory. is mediated by FSTL1 binding to DIP2A receptor [84]. Curi- The different effects reported in these two models of arthri- ously, during differentiation of bone-marrow-derived mac- tis are likely due to the differences in disease development rophages into osteoclasts, Fstl1 induces the transcription of and in the evaluated therapeutic potential of FSTL1; in the c-Fos [87], suggesting a different role of Fstl1 during normal CIA model, adenovirus mediated overexpression of mouse development and in pathological conditions. In this CAIA Fstl1 (Ad-mFstl1) and in the CAIA model administration of model, like in the CIA model, the expression of IL-6 was recombinant non-glycosylated FSTL1. reduced [85], but it is important to note that in the CAIA Fstl1 is normally expressed in synovium, but in early mouse model, IL-6 is not responsible for the progression of stage of CIA, Fstl1 becomes highly overexpressed in fibro- arthritis [88]. blasts at the margin of the eroding bone and in cells of the mesenchymal lineage including osteocytes, chondrocytes, Pro‑inflammatory effect and adipocytes but not in cells of the hematopoietic line- age, like macrophages, neutrophils, or T cells [25, 80, 81]. In a mouse model for bacterially induced septic shock, endo- Ad-mFstl1 increased the secretion of pro-inflammatory toxin (LPS) administration in the rear footpads induces the cytokines, IFN-γ, tumour necrosis factor alpha (TNF-α), expression of both Fstl1 and IL-1β. In control mice and interleukin-1β (IL-1β), and 6 (IL-6), resulting in the induc- in transgenic mice, where Fstl1 was removed by tamox- tion of synovitis (inflammation of the synovial membrane) ifen administration, IL-1β remained low or undetectable with infiltration of inflammatory cells in the synovium [89]. Overexpression of FSTL1 in cultured monocytes and and surrounding tissue. Both in vitro and in vivo experi- macrophages or in the septic shock mouse model induces ments showed that IL-1β induces Fstl1 expression through expression of caspase-1 and NLRP3 (nucleotide-binding NF-κB [82], suggesting a positive feedback between the domain leucine-rich repeat containing (NLR) family, pyrin 1 3 Follistatin-like 1 in development and human diseases 2347 domain containing 3), confirming that FSTL1 mediates probably as a result of infiltrating CD8 T cells. It is of rel- pro-inflammatory events [89]. In cultured adipocytes, mac- evance to note that the infiltration of T cells was not related rophages, and nucleus pulposus cells, the pro-inflammatory to an inflammatory response due to surgery. Intravenous effect of FSTL1 is mediated via the signaling cascade IKKβ, administration of an adenovirus expressing FSTL1 leads IκBα, NF-κB, JNK, and ERK1/2 resulting in the induction to a reduction in expression of pro-inflammatory cytokines of expression of IL-1β, IL-6, TNF-α, MCP (monocyte (IL-17A, IL-6, and IFN-γ) and prolonging survival of trans- chemotactic protein)-1, COX (cyclooxygenase)-2, MMP- planted patients [93]. A similar effect is also observed dur - 13, and iNOS (inducible nitric-oxide synthase) [22, 77, ing cancer progression, in which FSTL1 plays an important 87, 90]. Moreover, stimulation of nucleus pulposus cells role in immune dysfunction regulating thymocyte matura- with TNF-α induces FSTL1 secretion [77], again suggest- tion: in vivo inhibition of Fstl1 increases the tumour-specific ing a positive feedback loop on the inflammatory response. CD8 T-cell response [70]. Treatment of bone-marrow-derived macrophages with gly- cosylated recombinant FSTL1 increases cell proliferation in In conclusion a dose-dependent manner [87]. Together. these data suggest that FSTL1 promotes inflammation by inducing not only Taken together FSTL1 was observed to have a dual function cytokine production, but also proliferation of inflammatory during inflammatory processes, acting as an anti-inflamma- cells. tory factor in the acute phase but having a pro-inflamma- tory effect in the long term and in chronic diseases. This Anti‑inflammatory effect is likely due to activation of different signaling pathways: initially, FSTL1 binds the DIP2A receptor and prevents tis- Observing lowered expression levels of Fstl1 using a hypo- sue degradation by MMPs through the downregulation of morphic mouse model revealed no effect on the levels of the transcription factors c-FOS and ETS-2; subsequently, pro-inflammatory cytokines, such as TNF-α, IL-6, and FSTL1 activates the inflammatory response via the TLR4/ IL-1β, in kidney [91]. However, in renal injury mouse CD14 pathway, the activation of the AMPK pathway, and models, circulating Fstl1 appeared to regulate the immune the inhibition of BMP-signaling pathway. However, it cannot response: in a cisplatin nephrotoxicity model Fstl1 inhib- be excluded that additional endogenous or exogenous factors its the synthesis of pro-inflammatory cytokines, like IL-1β are involved in the regulation. [91], and in a subtotal nephrectomy model Fstl1 decreases TNF-α, IL-6, and MCP-1 (monocyte chemotactic protein-1) expression [92]. In vitro experiments on human, mesangial FSTL1 in other diseases cells showed that this anti-inflammatory effect is mediated by phosphorylation of AMPK and activation of acetyl-CoA Fibrosis carboxylase (ACC) [92]. Upon cardiovascular injury, administration of recom- Fibrosis refers to the formation of excessive connective tis- binant glycosylated human FSTL1 produced in Sf9 insect sue in an organ or tissue during a reactive and/or repara- cells reduced pro-inflammatory cytokine expression. In vitro tive process. Compared to healthy tissue, FSTL1 expression studies on NRCM and macrophages showed that this ina fl m- increases in patients with idiopathic pulmonary fibrosis and matory response is mediated both (I) by inducing AMPK in mouse models such as bleomycin-induced lung injury phosphorylation and downstream ACC activation and (II) [94–96], CCl -induced liver injury and in kidney after uni- by inhibiting BMP4 signaling which otherwise increases lateral ureteral obstruction [97]. Haplodeficiency of Fstl1 or TNF-α and IL-6 expression via pSmad1/5/8 [11]. After liga- reduced expression of Fstl1 using siRNA results in a reduc- tion of the femoral artery in mice, in which Fstl1 expres- tion in collagen accumulation in both lung and liver injury sion from the muscle cells was deleted using the muscle [95, 97]. The effect on fibrosis is most probably due to dis- creatine kinase (MCK)–Cre mouse line, the inflammatory ruption of the TGF-β/BMP balance by FSTL1 because of response was enhanced. This was evidenced by an increase the ability of FSTL1 to inhibit Smad1/5/8-mediated BMP4 in the expression of the pro-inflammatory cytokines TNF-α, signaling and of the stimulation of expression of FSTL1 IL-1β, and MCP-1 and in the infiltration of monocytes and via Smad2/3 mediated TGF-β1 signaling [28, 95]. During macrophages [50]. nephrectomy, overexpression of circulating Fstl1 by adeno- In host-versus-graft disease, FSTL1 was also found to virus mediated delivery reduces kidney fibrosis formation play a role. An upstream morphogen of FSTL1, TGF-β, is and the expression of fibrosis markers, such as TGF-β1, known to play a central role in allograft tolerance [2, 93]. collagen-I, collagen-III, and connective tissue growth fac- In line with this, FSTL1 has been shown to be induced in tor [92]. It is important to note that unlike initial studies, in donor specific blood transfusions after heart transplantation which the mice where analyzed 1–2 weeks after induction of 1 3 2348 A. Mattiotti et al. fibrosis [95, 97], later studies analyzed mice 2 months after and microtubule-associated protein 1A/1B-light chain 3). surgery [92]. This opposite effect on fibrosis development FSTL1 stimulation of these cells induced the switch from is in line with studies in immune diseases, in which FSTL1 E-cadherin to N-cadherin expression, pointing endothe- also shows an opposite effect during the acute or chronic lial-to-mesenchymal transition. Interestingly, when these phases of disease. cells were also treated with an autophagy inhibitor, not only autophagy but also endothelial-to-mesenchymal tran- Lung development and asthma sition was attenuated [102]. During mouse development and in the adult mouse, Fstl1 mRNA is expressed in mesenchymal cells of the lung, vas- Obesity cular and airway smooth muscle cells, goblet cells of the airway epithelium and in the endothelial cells [6, 98]. Upon Serum levels of FSTL1 were found to correlate with the functional disruption of Fstl1, neonates die at birth due to body mass index with FSTL1 higher in overweight and respiratory distress, displaying tracheomalacia, hypoplastic, obese subjects than in controls [90]. During differentia- and absence of tracheal cartilage rings. Within the lung tis- tion of pre-adipocytes (3T3–L1) into adipocytes, Fstl1 sue, a thickening of the alveolar walls and a reduction in showed a transient short high level expression to become airspace were found. Moreover, differentiation of the airway subsequently downregulated to background levels at both epithelium seems to be impaired as seen in the reduced level the mRNA and protein level [7, 104]. Either by prevent- in mature surfactant protein [27, 28]. ing this initial peak in Fstl1 expression or by maintaining Asthma, a chronic respiratory disease, is characterized high levels of Fstl1 in the culture medium during induced by airway inflammation, remodelling, and hyper-respon- adipogenesis, the differentiation of 3T3–L1 cells was siveness. Proteomic analysis of sputum of patients with blocked [104]. Two different mechanisms have been iden- asthma revealed that FSTL1 is one of the highest expressed tified linked directly to the regulation of Fstl1 expression. proteins [99]. Histological analysis of post-mortem human Recently, it was discovered that the secretion of Fstl1 was lungs of asthma patients showed expression of FSTL1 in regulated via cilia. When genes essential for ciliogenesis, alveolar macrophages [100]. Moreover, the elevated levels BBS4 or IFT88, were knocked-down in 3T3–L1 pre- of FSTL1 were found in blood plasma and bronchoalveolar adipocytes, the levels of Fstl1 mRNA and protein were lavage fluid in asthma patients compared to healthy con- downregulated and the cells failed to undergo correct adi- trols [101]. The FSTL1 concentration negatively correlates pogenesis [104]. with lung function parameters and positively with airway The second mechanism involved the downregulation of remodelling markers. Interestingly, plasma levels of FSTL1 the pro-inflammatory cytokines, IL-6, IL-8, and MCP-1, significantly decrease and return to control levels 1 month during the differentiation of 3T3–L1 cells into adipocytes after treatment with inhaled corticosteroids and long-acting [90]. The addition of TNFα to 3T3–L1 adipocytes induced β-agonist therapy and/or oral leukotriene receptor antagonist their de-differentiation and was accompanied by a re-expres- therapy [101]. Along the same line, mice chronically, but sion of Fstl1 mRNA and protein [7] and an upregulation not acutely, challenged with an allergen showed an increase of the pro-inflammatory cytokines [90]. The changes in the in Fstl1 expression [100]. Haplodeficiency of Fstl1 [ 102] or ratio of pro- and anti-inflammatory cytokines are thought to deletion of Fstl1 from macrophages/myeloid cells using the underlie the chronic inflammation observed in obesity, which Lys–Cre mouse line [100] reduced inflammation (Fig.  3) and leads to insulin resistance and other obesity-associated dis- airway remodelling in OVA-challenged mice, while opposite eases [105]. This closely resembles the affects reported in effects are observed when mice were treated with recombi- arthritis, pointing to a role of FSTL1 in the regulation of the nant glycosylated FSTL1, though in this study, it is not clear balance of pro- and anti-inflammatory cytokines. whether human or mouse protein was used [100]. Increased Another, as yet, unexplored alternative mechanism could expression of Fstl1 in a chronic but not in an acute mouse be via micro RNA (miRNA) regulation. Like in the differ - model of asthma is in line with previously reported results entiation of adipocytes, a similar downregulation of Fstl1 is suggesting different roles of FSTL1 during acute and chronic observed during in vitro myogenesis of C2C12 myoblasts. In inflammation in arthritis [100]. myoblasts, the downregulation of Fstl1 mRNA is regulated Autophagy and epithelial-to-mesenchymal transi- by muscle-specific miR-206 [32]. Furthermore, during adi- tion are two associated biological processes [103]. pogenesis, regulation of gene expression levels by microR- Autophagosome formation was found to be increased in NAs has been reported [106], and candidate miRNA-binding human bronchial epithelial cell line 16HBE stimulated sites can be found when the 3′UTR of FSTLl1 is scanned with recombinant FSTL1 protein using electron micro- for potential binding sites of microRNAs (Supplemental scopical analysis and autophagy biomarkers (Beclin-1 Table 1). 1 3 Follistatin-like 1 in development and human diseases 2349 no FSTL1 protein expression is observed [113]. Disregula- Central nervous system tion of the ratio FSTL1 versus miR-198 is observed in head and neck squamous cell carcinoma, in which FSTL1 pro- During mouse development, Fstl1 is locally expressed in all the component of the central nervous system [107] and tein persists with the afore-mentioned consequences [62]. MiR-198 is also involved in suppressing colorectal cancer it is involved in the radial glial scaffold formation [108]. In the dorsal root ganglia, Fstl1 is involved in maintenance growth [114] and lung adenocarcinoma A549 cell prolifera- tion [115], but the relation between FSTL1 and miR-198 in of the normal threshold of somatic sensation: it is secreted from afferent axons and it activates the α1 subunit of the this cancer type has not yet been studied. + + MiR-206 is one of the most abundant miRNAs expressed Na ,K -ATPase, suppressing synaptic transmission. Neural deletion of Fstl1 using Na 1.8–Cre mice causes hypersensi- during skeletal myogenesis [116]. In chicken, the expression of FSTL1 (also referred to as Flik) is downregulated in the tivity of both wide dynamic range neurons and nociceptive neurons [109]. embryo during myotome formation suggesting a role during myogenesis [117]; however, the role of FSTL1 during this FSTL1 and microRNAs process has not been investigated. The group of Tapscott showed that skeletal muscle cell differentiation is coordi- MicroRNAs (miRNAs) are short (~ 22  nt) endogenous nated by the transcription factor MyoD, and expression of FSTL1 is reduced [118]. This process appears to be medi- noncoding RNAs that regulate messenger RNA (mRNA) degradation and/or translational repression [110]. Several ated by the induction of miR-206 that binds to the 3′UTR of FSTL1 mRNA [32]. studies have highlighted the relevance of the miRNA and miRNA-binding sites during human disease [111]. miR-198 During mycobacterial infection, miR-32-5p negatively regulates the inflammatory response [119] and promotes is encoded in the 3′UTR of human FSTL1 primary transcript [112]. The FSTL1 mRNA, therefore, not only encodes the the survival of infected macrophages. MiR-32-5p binds to the 3′UTR of FSTL1 decreasing mRNA and protein levels FSTL1 protein, but also an miRNA. Though the FSTL1 protein has been highly conserved during evolution from (Fig. 3). Re-expression of FSTL1 completely reverses the inhibitory effects of miR-32-5p on secretion of inflammatory tick to human [29], the encoded miRNA is only found in primates. Moreover, in silico analysis revealed multiple cytokines, indicating that inhibition of FSTL1 is a mediator of the anti-inflammatory effects [33]. However, the increased miRNA-binding sites in the 3′UTR of the FSTL1 mRNA of which three have been functionally analyzed (miR-206 level of Fslt1 protein seems to have no effect on the level of released cytokines. [32], miR-32-5p [33], and miR-27a [24]) (Table 1). A list of predicted miRNA-binding sites in the 3′UTR of human Significant lower levels of miR-27a and higher levels of FSTL1 are found in serum, synovial tissue, and fibroblast- FSTL1 with associated clinical relevance is provided in Sup- plemental Table 1. like synoviocytes of RA patients compared to healthy con- trols. Transfection of fibroblast-like synoviocytes with miR- In the normal healthy human epidermis, FSTL1 mRNA is expressed, but the protein is present at low to almost unde- 27a inhibits cell migration and invasion and downregulates TLR4, NF-κB, and MMPs. Further analysis showed that tectable, levels. Contrary to this, miR-198 is expressed in this tissue. During wound healing, a switch from miR-198 these effects are the result of miR-27a-mediated downregu- lation of FSTL1 expression via its target sequence in the to FSTL1 protein expression is observed without a change in the FSTL1 mRNA level. TGF-β1 indirectly regulates this 3′UTR (Fig.  3). Moreover, overexpression of FSTL1 via adenoviral vector rescues the miR-27a effects [24]. switch via KH-type splicing regulatory protein (KHSRP). In the healing skin, FSTL1 protein promotes keratinocyte migration during re-epithelization. Interestingly, in chronic non-healing ulcer wounds in patients with diabetes mellitus, Concluding remarks From a clinical point of view, it is important to consider that Table 1 MicroRNA-binding sites in FSTL1 gene during pathological conditions, such as cancer and cardio- Human microRNA Binding position in Biological relevance vascular disease, not only individual cell behaviors such as 3′UTR proliferation and migration are important but also paracrine miR-27a 1537 Inflammation [24] communication between cells, inflammation, and vasculari- miR-32-5p 142 Inflammation [33] zation. Because FSTL1 has been shown to be involved in miR-206 2101; 2375 Myogenesis [32] multiple signaling pathways and processes, results observed in specific cell types and in defined conditions should be List, position, and biological processes of the verified microRNA- regarded on their specific merits and not generalized, as yet. binding sites in the 3′UTR of FSTL1 gene 1 3 2350 A. Mattiotti et al. 5. 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Ogura Y, Ouchi N, Ohashi K, Shibata R, Kataoka Y, Kam- bara T, Kito T, Maruyama S, Yuasa D, Matsuo K, Enomoto T, the oligosaccharide chain in relation with the observed bio- Uemura Y, Miyabe M, Ishii M, Yamamoto T, Shimizu Y, Walsh logical effects of FSTL1 is needed and essential to forward K, Murohara T (2012) Therapeutic impact of follistatin-like 1 the use of FSTL1 in clinics. on myocardial ischemic injury in preclinical models. Circula- tion 126(14):1728–1738. https ://doi.or g/10.1161/cir cu latio Acknowledgements The authors thank Dr. Jan Ruijter for critically naha.112.11508 9 reading the manuscript. Andrea Mattiotti and Stuti Prakash were finan- 12. Shimano M, Ouchi N, Nakamura K, van Wijk B, Ohashi K, cially supported by CardioNeT, an EU FP7-Marie Curie-ITN actions Asaumi Y, Higuchi A, Pimentel DR, Sam F, Murohara T, van ITN-GA-2011-289600 Grant. den Hoff MJ, Walsh K (2011) Cardiac myocyte follistatin-like 1 functions to attenuate hypertrophy following pressure over- load. 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Follistatin-like 1 in development and human diseases

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Abstract

Follistatin-like 1 (FSTL1) is a secreted glycoprotein displaying expression changes during development and disease, among which cardiovascular disease, cancer, and arthritis. The cardioprotective role of FSTL1 has been intensively studied over the last years, though its mechanism of action remains elusive. FSTL1 is involved in multiple signaling pathways and biological processes, including vascularization and regulation of the immune response, a feature that complicates its study. Binding to the DIP2A, TLR4 and BMP receptors have been shown, but other molecular partners probably exist. During cancer progres- sion and rheumatoid arthritis, controversial data have been reported with respect to the proliferative, apoptotic, migratory, and inflammatory effects of FSTL1. This controversy might reside in the extensive post-transcriptional regulation of FSTL1. The FSTL1 primary transcript also encodes for a microRNA (miR-198) in primates and multiple microRNA-binding sites are present in the 3′UTR. The switch between expression of the FSTL1 protein and miR-198 is an important regulator of tumour metastasis and wound healing. The glycosylation state of FSTL1 is a determinant of biological activity, in cardiomyocytes the glycosylated form promoting proliferation and the non-glycosylated working anti-apoptotic. Moreover, the glycosylation state shows die ff rences between species and tissues which might underlie the die ff rences observed in in vitro studies. Finally, regulation at the level of protein secretion has been described. Keywords Cardiovascular disease · Cancer · Immune disease · Inflammation · Fibrosis · Obesity · Pulmonary disease · Signal transduction · Glycosylation · miRNA Introduction 308 Aa) and mouse (Genbank Q62356; 306 Aa) protein sequences shows that the secretion signal (human: Aa 1–20 Follistatin-like 1 (FSTL1) is a glycoprotein of the secreted and mouse: Aa 1–18) is most species variable, whereas the protein acid and rich in cysteine (SPARC) family. In the remaining 272 Aa shows a very high degree of similarity literature, FSTL1 is referred to by many different names [ 1]. (94.4%). In the sequence of mouse Fstl1, three potential sites Two groups independently discovered FSTL1 and named for N-glycosylation and two for O-glycosylation are present it (I) transforming growth factor (TFG)-β-induced clone and in vitro studies have shown that only the three aspar- 142 173 178 36 (TSC36) isolated from mouse osteoblasts [2] and (II) tate residues Asp, Asp , and Asp are N-glycosylated. Follistatin-related protein (FRP) secreted from rat glioma Moreover, glycosylation at these sites shows cell-type speci- [3]. FSTL1 comprises a secretion signal, a Follistatin- and a ficity [ 4]. N-glycoproteome analysis on human blood plasma Kazal-like domain, two EF-hand domains, and a von Wille- identified only one glycosylated form of FSTL1 in which 175 180 brand factor type C domain (http://www.unipr ot.org/unipr two (Asp and Asp ) of the three sites are used [5]. From ot/Q12841 ). Comparison of the human (Genbank: Q12841, gastrulation onward, Fstl1 mRNA is broadly expressed throughout the entire mouse embryo and its expression becomes restricted to the mesenchymal component of most Electronic supplementary material The online version of this tissues at the end of gestation [6]. In the adult mouse, the article (https ://doi.org/10.1007/s0001 8-018-2805-0) contains highest levels of Fstl1 mRNA are found in heart, lung, and supplementary material, which is available to authorized users. subcutaneous white adipose tissue [7]. Interestingly, the * Maurice J. B. van den Hoff expression of FSTL1 changes with respect to its level and m.j.vandenhoff@amc.uva.nl pattern during various diseases, including cardiovascular disease [8–15], cancer progression [16–22], and systemic Department of Medical Biology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Vol.:(0123456789) 1 3 2340 A. Mattiotti et al. autoimmune diseases [23–26]. Analysis of the exomes of Cardiovascular system over 60,000 individuals revealed that the estimated probabil- ity of loss-of-function intolerance is 0.96 (http://exac.broad Cardiovascular disease (CVD), such as heart failure (HF) institute.or g/gene/ENSG0 00001 63430 ), r eflected in the find- and coronary artery disease, is a group of disorders that ing that homozygous loss-of-function mutations is never affect the heart and blood vessels. CVD is a leading cause observed and heterozygous ones have only been described of death in Western countries [34]. For the sake of clarity, in 35 individuals. In line with this, functional disruption of we have included a summary figure to accompany the text Fstl1 in mice was found to result in respiratory distress and below (Fig. 1). death within hours after birth. Gene knock-out mice display a phenotype that appears to suggest an important inhibitory Clinical relevance role of FSTL1 in BMP signaling [27, 28]. Multiple TGFβ superfamily receptors as well as disco-interacting protein In humans, circulating concentrations of FSTL1 increase 2 homolog A (DIP2A) have been shown to interact with during cardiac and vascular diseases, such as HF [9, 35], FSTL1 [28–30]. However, using other transgenic models, HF with preserved ejection fraction (HFpEF) [15], acute FSTL1 has been implicated in multiple signaling pathways coronary syndrome (ACS) [36], and chronic obstructive and its role during diseases remains unclear [1]. Recently, a pulmonary disease [37]. In patients with ACS, the increase study on the role of FSTL1 in cardiac regeneration showed in the serum level of FSTL1 correlates with mortality [36]. different effects on cardiomyocyte proliferation and pro- Moreover, the levels of FSTL1 in the circulating blood tection from apoptosis depending on the post-translational were found to correlate with the severity of chronic HF [35, modification of the protein [13], opening a new perspective 38]. Although FSTL1 has a negative prognostic value with on the interpretation of previously seemingly contradict- respect to HF, it was found that patients with end-stage HF ing data. Moreover, post-transcriptional regulation plays an who received a combination of a left ventricle assist device important role in the expression of the protein [31] and mul- (LVAD) and pharmacological therapy, who had high levels tiple functional miRNA-binding sites have been identified of FSTL1 at the time of LVAD implantation, showed recu- in the 3′UTR of FSTL1 mRNA [24, 32, 33]. For this reason, peration and recovery of ejection fraction [9]. This finding we present a review of the studies that have reported various suggests that FSTL1 could be a therapeutic target for drug roles of FSTL1 in development and disease while trying to development and serum concentration of FSTL1 can be used clarify the sometimes contradictory results. as a prognostic biomarker for CVD [35, 39]. Fig. 1 Follistatin-like 1 in car- diovascular disease. Schematic Fa�y acid oxidaon Glucoseoxidaon representation of the known signaling pathways interacting Cardiomyocytes Hypertrophy with FSTL1 in cardiovascular hypertrophy disease. Grey components Smooth muscle indicate unknown receptors. AMPK ACC cells proliferaon The coloured arrows denote the Pathological and migraon secreted Fstl1 (width relates remodeling amount). Coloured area defines Fibroblasts different conditions. Image MEK1/2 ERK1/2 proliferaon and adjusted from http://smart .servi migraon er.com Ischemia FSTL1 mTOR Cardiomyocytes DIP2A FOXO1/3 apoptosis PI3K AKT1 Endothelial cells eNOS survival, migraon ALK3/6 and differenaon BMP4 BMPR-2 / Inflammatory SMAD1/5/8 ACTR-IIA/B response Cardiomyocytes proliferaon 1 3 Follistatin-like 1 in development and human diseases 2341 using a recombinant adenovirus, have positive effects on sur - Cardiac development vival and regeneration leading to a significant reduction of the infarct size [8, 13]. To study the beneficial role of FSTL1, RNA-Seq data (https ://www.ncbi.nlm.nih.gov/gene/11167 ) show that FSTL1 mRNA is expressed in the adult human E. coli produced human FSTL1 was intravenously adminis- trated in a murine model either before inducing ischemia or heart, but the expression pattern of the mRNA has not been established. However, immunohistochemical data showed after reperfusion; to validate a therapeutic effect in a larger animal, a porcine model was subjected to 45 min of ischemia that FSTL1 is present in vascular endothelial cells of ves- sels located within the myocardium, in smooth muscle cells and 24 h of reperfusion and intracoronary administration of FSTL1 during the first 10 min after ischemia. In both of larger vessels and at a low, but significant levels in car - diomyocytes [9]. Furthermore, the pattern of expression models, administration of FSTL1 reduced ischemic damage and improved cardiac performance after reperfusion [11]. of FSTL1 during human cardiac development remains to be established. In mouse and chicken, Fstl1 is expressed Applying a collagen patch to the infarcted heart ameliorated recovery, which was even further improved when this patch throughout the entire heart during early development and with expression subsequently becoming largely restricted was enriched with epicardium-derived medium or bacteri- ally produced recombinant FSTL1 protein. The addition to the non-myocardial component, with low levels being expressed in the cardiomyocytes [6, 40]. Homozygous dele- of bacterially produced FSTL1 significantly decreases the infarct area and as a consequence increases survival after tion of Fstl1 in mice results in an overall enlargement of the heart of neonates [27]. Whether this enlargement is due to MI [13]. In pig, a collagen patch loaded with FSTL1 applied to the ventricle 1 week after ischemia/reperfusion was also hyperplasia or hypertrophy of the cardiomyocytes is not yet known, and is currently being evaluated. found to promote the regenerative response [13]. In cul- tured neonatal rat cardiomyocytes (NRCM), recombinant Myocardial infarction protein reduced hypoxia/reoxygenation-induced apoptosis both directly via (I) the MEK1/2 (mitogen-activated protein During a myocardial infarction (MI), blood flow to a portion kinase kinase) and ERK1/2 (extracellular signal-regulated kinase) signaling pathway, (II) the DIP2A, PI3K (phospho- of the heart muscle is interrupted causing local ischemia when prolonged results in cardiomyocyte death [41]. The inositide 3-kinase) and AKT1 (RAC-alpha serine/threo- nine–protein kinase) pathway with activation of downstream dying cardiomyocytes trigger an inflammatory response which is followed by a reparative process that results in the effectors mTOR and FOXO1/3 [8 , 44] and (III) AMPK (AMP-activated protein kinase) phosphorylation, and indi- formation of non-muscular scar tissue, thus reducing cardiac contractility and output [42]. rectly via (IV) inhibition of BMP4-induced apoptosis and (V) reduction of pro-inflammatory cytokines expression After inducing an MI in mice by permanent occlusion of the left anterior descending artery (LAD), Fstl1 is transiently [11]. Interestingly, a difference in the effect between bac- terially and eukaryotically produced FSTL1 was observed highly expressed in the heart, especially in the ischemic zone. Fstl1 reaches its highest level of mRNA expres- in the study of Wei and colleagues [13]. It should be noted that bacterially produced proteins are not glycosylated, sion 1 week after MI and normalizes in the remote zone of the heart at 1 month after MI, while low levels of expression while eukaryotically produced proteins are. The non-gly- cosylated FSTL1 increased NRCM proliferation, while the persist in the infarct zone [10, 14]. Non-myocardial cells have been shown to be the major source of Fstl1 in the heart, glycosylated protein, in accordance with previous reports [8, 11], protected NRCM from peroxidase-induced apoptosis with low levels also being detectable in the cardiomyocytes [13, 14]. Because Fstl1 is predominantly expressed in the [13]. In vitro experiments showed that glycosylated FSTL1 promotes fibroblast proliferation and migration via ERK1/2 ischemic zone, the serum levels seem to be a reflection of the infarct size. This might also underlie the observed correla- phosphorylation [14]. Deletion of Fstl1 from a part of the fibroblast population using the S100A4–Cre mouse line did tion between elevated FSTL1 levels and mortality in ACS patients [36]. However, other organs that serve as a source of not affect cardiac function compared to control littermates. Upon the induction of an MI, however, the number of mice FSTL1 cannot be excluded. During the healing process after an MI, the epicardium plays an important role as a source dying due to cardiac rupture within the acute phase, i.e., first 7 day post-MI, doubled from a 25% in wild type to 50% of signaling molecules and provides cells to the infarct zone [10, 43]. Fstl1 is transiently expressed in the epicardium in Fstl1-depleted animals. Analysis of cardiac function did not reveal any significant differences, though the number of overlaying the infarct tissue and in the derived mesenchymal cells that populate the infarct scar [10, 13]. myob fi roblasts was decreased and the synthesis and matura - tion of the extracellular matrix proteins were reduced [14], In both permanent and transient LAD ligation mouse models, it was found that locally produced and circulating indicating that the initial reparative response is abrogated. Fstl1 protein, assessed by overexpression of Fstl1 in the liver 1 3 2342 A. Mattiotti et al. Fstl1 expression was found to be induced in skeletal muscle Cardiac hypertrophy cells [48]. Adenoviral-mediated overexpression of Fstl1 in the latter mouse model was found to stimulate revasculariza- Cardiac hypertrophy is characterized by the abnormal thick- ening of the wall of the heart as a result of an increase in the tion. Interestingly, adenoviral-mediated overexpression of Fstl1 in non-ischemic muscle did not alter its vasculariza- volume of the individual cardiomyocytes [45]. Fstl1 expression is upregulated in mice after transverse tion. In this model of hind limb ischemia, glycosylated Fstl1 was found to promote endothelial cell survival, migration, aortic constriction (TAC), which results in pressure over- load-induced hypertrophy [12] or by aldosterone infusion and differentiation into a vascular network-like structure via phosphatidylinositol-3 kinase (PI3K), AKT, and endothelial which causes hypertension-induced HFpEF [15]. In both these models, cardiomyocytes are the major source of Fstl1 nitric-oxide synthase (eNOS) activation [48]. At that time, the receptor conveying the extracellular Fstl1 signal into the [12, 15]. Treatment of adult rat ventricular cardiomyocytes with recombinant glycosylated human FSTL1 produced in cell was unknown, but recently, DIP2A has been identified as a potential cell-surface receptor upstream of PI3K and Sf9 insect cells abrogates the increase in protein synthesis and Nppa (also known as: ANF or ANP) expression induced AKT1 phosphorylation [44]. Interestingly, an opposite effect of FSTL1 has been observed in smooth muscle cells (SMCs) by aldosterone stimulation, indicating that FSTL1 prevents hypertrophy in cardiomyocytes. In HFpEF mice, increased during pulmonary hypertension [37] and vascular injury [49, 50]. In the latter two models, FSTL1 prevents pathological levels of circulating Fstl1, mediated by overexpression in the liver via adenoviral delivery, significantly reduce car - vascular remodelling, as a result of a decrease in SMC pro- liferation and migration which is mediated via the induction diomyocyte hypertrophy and ameliorate cardiac functions [15]. Specific deletion of Fstl1 from cardiomyocytes using of AMPK and inhibition of ERK phosphorylation [37, 49, 50]. Studies on HUVEC cells showed that FSTL1 affects the alpha myosin heavy chain (αMHC)–Cre mouse line did not show structural or functional differences compared to vascular endothelial cell polarization, but not migration and tube formation [51]. control littermates [12, 15]. However, when these mice were challenged by TAC, cardiac hypertrophy was enhanced and Mitral valve disease ventricular performance decreased [12]. On the other hand, when these mice were challenged by uninephrectomy in Mitral valve disease is a major cause of morbidity, heart combination with 4 weeks of contiguous infusion of aldos- terone, HFpEF ensued [15]. Interestingly, in the latter model, failure, and death worldwide [52]. Deletion of Fstl1 from the endothelial/endocardial lineage using the Tie2–Cre mouse the role of Fstl1 in the development of HFpEF was inde- pendent of the changes in cardiac fibrosis but crucial in the line resulted in dysfunctional mitral valves, HFpEF and death [53]. The dysfunctional mitral valve leaflets became development of cardiac hypertrophy [15]. In vitro experi- ments showed that the inhibitory effect of Fstl1 on cardio- long and thick, suggestive of enhanced proliferation of the valve cells and formation of mesenchymal cells as a result myocyte hypertrophy is mediated by AMPK and acetyl-CoA carboxylase (ACC) phosphorylation [12]. of prolonged endocardial to mesenchymal transition, valve enlargement itself probably being due to enhanced TGF-β During cardiac disease, the energy consumption of car- diomyocytes changes from fatty acid to glucose [46]. When and BMP signaling. Whether the observed development of HFpEF is a direct consequence of deletion of Fstl1 or heart failure is induced by tachy-pacing in dogs, this meta- bolic switch is also observed. Treatment of these dogs with a secondary effect of the dysfunctional mitral valve or the enhanced TGF-β and BMP signaling, is not yet clear [53]. a single dose or long-term infusion (14 days) with glyco- sylated human FSTL1 (CHO cells) inhibited the pathologi- It should be noted at this point that FSTL1 was not found in a meta-analysis of genome-wide association studies identi- cal switch from fatty acid to glucose oxidation. This effect was transient, because after clearing FSTL1 from the blood, fying 23 loci with suggestive evidence of association with mitral valve prolapse in the human population [54]. consumption of glucose increased and fatty acid consump- tion decreased. Moreover, the infusion of AMPK inhibitor In conclusion neutralized the effect of FSTL1 [47]. FSTL1 is a secreted glycoprotein with both protective and Vascular system regenerative capacities of which the circulating concen- tration increases during CVD. Overexpression or lowered In some of the previously mentioned studies on MI and cardiac hypertrophy, mice also displayed a pro-angiogenic expression (hypomorphic or heterozygous KO) of Fstl1 is tolerated in healthy animal models, whereas during patho- effect of Fstl1, though the underlying mechanism was not investigated [12–14]. A similar pro-angiogenic effect was logical conditions, additional Fstl1 prevents extensive car- diac damage and abnormal vascular remodelling. A lack of also found in a mouse model of ischemic hind limb, in which 1 3 Follistatin-like 1 in development and human diseases 2343 Fstl1 exacerbates cardiac injury. Most importantly, Fstl1 In clear-cell renal cell carcinoma (ccRCC), high levels of affects multiple pathways in a cell-type specific manner and FSTL1 expression correlate with a favorable post-operative different effects are observed depending on the glycosylation survival (hazard ratio = 0.325; p = 0.030). Knock-down of state of the protein. These results suggest that FSTL1 may FSTL1 using retrovirus mediated short hairpin RNA in not just represent a biomarker, but could also be an interest- ccRCC cell lines resulted in anchorage independent growth ing candidate for the development of new therapies in CVD. and invasion. Further analysis showed that the tumour sup- pressor function of FSTL1 is mediated through repression of the NF-κB and HIF-2α signaling pathways [57]. Moreover, Cancer and tumours in line with these novel findings, a polymorphism in the second intron of the FSTL1 gene, associated with downregu- Cancer is characterized by an imbalance in growth regula- lation of its expression, correlates with an increased risk of tion caused by genetic changes. When cancer cells do not renal cell carcinoma and poor prognosis [21]. Interestingly, migrate into the surrounding tissues, the tumour is referred in nasopharyngeal carcinoma (NPC) cell lines and tumour to as a benign. Cancer cells become malignant when they biopsies, decreased levels of FSTL1 mRNA correlate with acquire the additional property of migration, allowing them hypermethylation of CpG islands in its proximal promoter to invade other tissues, this being referred to as metastasis. (− 166 to + 332 bp) and also with increased tumourigenic- When left untreated, tumour metastasis is the leading cause ity. Restoring of FSTL1 mRNA levels using demethylat- of death in cancer patients [1, 55, 56]. For the sake of clarity, ing agents decreased tumourigenicity. The reduced levels we have included a summary figure to accompany the text of expression of Fstl1 in the NPC tumours are reflected in below (Fig. 2). decreased FSTL1 serum levels [22]. A low level of expres- sion of FSTL1 in lung adenocarcinoma patients has a poor Clinical relevance prognostic value (hazard ratio = 2.09; p = 0.022), but no correlation was observed between FSTL1 expression and Compared to healthy tissue, the expression level of FSTL1 survival in squamous cell carcinoma [58]. was found to be reduced in biopsies of various types of On the other hand, increased expression of FSTL1 com- cancers, such as prostate [16], ovarian, endometrial [19], pared to healthy control tissue was observed in brain cancer kidney [20, 21, 57], nasopharyngeal carcinoma (NPC) [22], cells [18], in fibroblasts but not in epithelial cells in colon and lung adenocarcinoma [58]. In endometrial and ovar- tumours [59], in castration-recurrent prostate cancer [60], ian tumours, a trend between poorer prognostic character- in most cases of hepatocellular carcinomas (HCC) [61], in istics and decreasing levels of FSTL1 was observed [19]. head and neck squamous cell carcinoma (HNSCC) [62], and Fig. 2 Follistatin-like 1 in FSTL1 cancer and tumour. Schematic representation of the known signaling pathways interact- ing with FSTL1 during cancer growth and metastasis. Grey FASLG Smad3 proliferaon components indicate unknown AKT ERK1/2 FAS receptors. Helical structures GSK-3β G2/M represent gene expression. arrest BIM BIM-EL/L/S p/uBIM Dashed arrow indicates transla- BAX tion from mRNA to protein. The BCL2 Caspase3/9 Caspase3/7 dashed line separates different CDC2 processes: tumour growth and apoptosis PARP metastasis. Image adjusted from http://smart .servi er.com CCL2 CCR2 MMP9 CXCL12 CXCR4 migraon invasion ERK1/2 FSTL1 MMP2 WNT7a CX43 FSTL1 EGF 1 3 Metastasis Growth CyclinA/B1, CDK1 accumulaon 2344 A. Mattiotti et al. esophageal squamous cell carcinoma (ESCC) [63]. FSTL1 breast cancer [64] cells promotes proliferation. Phosphoryla- is higher expressed in metastatic brain tumours compared tion of Smad2/3 is one of the pathways that mediate FSTL1 to primary breast cancer [64]. FSTL1 is not expressed at a inhibition of proliferation [64]. Injection of NPC cells sta- detectable level in normal brain tissue and in diffuse-infil- bly transfected with FSTL1 in nude mice showed a similar trating astrocytes of grade II or III gliomas, but is expressed tumour incidence compared to non-transfected cells. How- at high levels in grade IV gliomas, referred to as glioblas- ever, the tumour growth rate was lower in FSTL1-transfected toma [18]. The level of FSTL1 was found to correlate with NPC cells compared to control cells [22]. Similar in vivo the malignancy of the astrocytoma and its coexpression results were observed in NSCLC; tumour growth was inhib- with p53 has a negative prognostic value [18]. In HCC, high ited when FSTL1 was overexpressed in the CL1-5 cell line levels of FSTL1 correlate with larger tumours, advanced and promoted when FSTL1 expression was downregulated tumour/node/metastasis (TNM) stages, metastasis, and poor in the CL1-0 cell line [58]. post-surgical outcomes (hazard ratio = 1.84; p = 0.015) [61]. Decreasing the level of FSTL1 in human NSCLC cell The high levels of Fstl1 correlate with lower survival per- lines (NCI-H460 and A549) using an siRNA induced the spective in HNSCC and ESCC patients [62, 63]. accumulation of cell cycle proteins, such as cyclin A, cyclin Most cell lines derived from human tumours express B1, CDK1, and phosphorylated CDC2 and caused G2/M lower levels of FSTL1 than immortalized non-tumourigenic arrest [69]. In this same study, it was also reported that apop- fibroblasts [65]. In vitro transfection of fibroblasts (mouse tosis increased. The increase in apoptosis was mediated by NIH3T3 and rat 208F) using oncogenes like ras, myc, or fos a decrease in ERK1/2 phosphorylation, an increase in both induces a tumourigenic phenotype and downregulation of cleaved (activated) Caspase-3, Caspase-9, and PARP, and Fstl1 expression, suggesting a tumour suppressor role [2, 65, an accumulation of the pro-apoptotic factor BIM-EL [69]. 66]. Comparison of human cancer cell lines with a differ - Overexpression of FSTL1 in a human hepatocellular car- ence in aggressiveness of their phenotype showed opposite cinoma (Huh7) cell line promoted its expansion, being the outcomes: the prostate cancer cell line LNCaP shows lower result of increased proliferation and inhibited apoptosis. The FSTL1 expression levels compared to the more aggressive inhibition of apoptosis was found to be the result of activated C4-2 variant [17], while less aggressive non-small cell lung AKT/GSK-3β signaling, with as consequence an increase carcinoma (NSCLC) cells express on average higher levels in the anti-apoptotic protein BCL-2 and a decrease in the of FSTL1 compared to more malignant small cell lung can- pro-apoptotic BIM and BAX proteins [61]. Similar results cer (SCLC) cells [67]. However, high variability in FSTL1 were reported in ESCC cells (KYSE-150), where inhibitory expression levels is observed in NSCL cell lines from both effect of FSTL1 on the BMP-signaling pathway and chem- adeno- or squamous carcinomas. oresistance could be demonstrated [63]. Overexpression of Taken together, the data on the expression levels of FSTL1 in mouse MC3T3 osteoblast precursor cells [65] or FSTL1 in relation with its effect in different tumours are rat 208F fibroblasts [66], on the other hand, did not affect highly variable from respect to growth inhibition or induc- cell morphology or proliferation. Although downregulation tion and from invasiveness to immobility. As a consequence, of FSTL1 in a squamous cell carcinoma (SCC12) cell line the expression level of FSTL1 should be regarded with cau- did not affect the proliferation rate in vitro, they were found tion, and not be associated with a specific phenotype. to form larger tumours when injected in nude mice [62]. Tumour growth Metastasis With respect to imbalanced tumour growth, contradictory Overexpression of FSTL1 in lung (PC-14 line and H446) results have also been reported upon in vitro manipulation [67, 68], ovarian, endometrial [19], or NPC [22] cell lines of the level of expression of FSTL1 in cell lines. not only decreased their proliferative capacity, but also their Transfection of lung NSCLC cell lines (PC-14 and H446) ability to migrate and invade flanking tissues. This reduction [58, 67, 68], ovarian, endometrial [19], NPC [22], and breast in migratory capacity was accompanied by a reduction in cancer [64] cell lines with FSTL1 reduces their growth rate metalloproteinase-2 (MMP-2) expression [19, 66]. In line via an unknown signaling pathway. Overexpression of with these results, downregulation of FSTL1 in ccRCC cells FSTL1 in ovarian or endometrial cells was also found to was shown to lead to a reduced migratory capacity and tissue increase the rate of apoptosis via a death receptor-initiated invasion [57]. pathway. In these cells, an increase in the mRNA of Fas Downregulation of FSTL1 in human melanoma cells cell-surface death receptor (FAS or First Apoptotic Signal using a siRNA results in the inhibition of the expression receptor) and its ligand (FASLG) was found to result in an of genes associated with migration, such as CCL2 and increase in cleaved (activated) PARP, Caspase-3, and Cas- CXCL12, and with the formation of bone metastasis, such pase-7 [19]. Downregulation of FSTL1 in ccRCC [57] and as CCR2 and CXCR4 [70]. In line with these observations, 1 3 Follistatin-like 1 in development and human diseases 2345 stimulation of these melanoma cells with recombinant Immune diseases glycosylated FSTL1 induced their migratory capacity and differentiation into a bone phenotype. FSTL1 is also lower The immune system is the collective of tissues, cells, and expressed in the breast cancer cell line MDA-MB-231 com- processes that provide a specific response to protect the pared to its metastatic version 231-BR [64]. In vivo studies, organism upon invasion by organisms, foreign cells, and tox- further showed that injection of an FSTL1 siRNA into a ins. Dysfunction of components of the immune system often subcutaneous tumour suppressed tumour growth and the for- results in immune disorders. Systemic autoimmune diseases mation of bone metastasis, increasing mouse survival com- (SADs) are disorders characterized by loss of function or pared to injections with a control siRNA [70]. Overexpres- destruction of normal tissues due to autoimmunity which is sion of FSTL1 in ESCC cells (KYSE-150) promoted tumour specific to each disease. The role of FSTL1 during inflam- growth and metastasis in vivo. In vitro analysis showed that matory processes has been studied in several models and inflammation (Fig.  3) and epithelial-to-mesenchymal transi- reported to be both pro- and anti-inflammatory. For the sake tion processes were strongly affected by FSTL1 [57, 63]. A of clarity, we have included a summary figure to accompany recent study on the metastatic effect of FSTL1 showed that the text below (Fig. 3). FSTL1 specifically interacted with WNT7a and antagonized its inhibitory effect on endothelial growth factor-mediated ERK phosphorylation which in turn induced the expression Clinical relevance of MMP9 [62], a prerequisite for cell migration. Overex- pression of Connexin-43 in pulmonary giant carcinoma cell High serum concentrations of FSTL1 are found in many inhibited their metastatic capacity. This inhibitory effect SADs, such as rheumatoid arthritis (RA) [23, 24], in par- could be reversed by adding antibodies against FSTL1 pro- ticular juvenile RA with systemic onset [25], osteoarthritis tein [71]. These observations need to be regarded with cau- (OA) [26], and Sjögren’s syndrome [23]. Synovial tissue of tion, as they might be pointing to two independent signaling RA patients expresses FSTL1 at high levels, which correlate pathways regulating cell invasion. positively with clinical and serological parameters of disease [72]. In serum of RA patients, antibodies directed against In conclusion FSTL1 appear more frequently (30%) than in other SADs, such as systemic sclerosis (17%), systemic lupus erythema- Due to the heterogeneity in the different cell lines and can- tosus (10%), and Sjögren’s syndrome (10%). Antibodies cers and the complexity of the multiple mechanisms under- against FSTL1 were not found in patients with OA or poly- lying tumour development, our knowledge on the role of myositis/dermatomyositis or healthy controls [73]. Besides FSTL1 during cancer development and progression is still the elevated serum levels of FSTL1 in RA, no association of fragmented and limited. The origin of the tumourigenic cell polymorphisms in Fstl1 with susceptibility to RA has been line appears to be a main determinant for the different, some- found to date [74], suggesting that overexpression of FSTL1 times opposite, effect of FSTL1. is a consequence of RA rather than a cause. Fig. 3 Follistatin-like 1 in FSTL1 immune diseases. Schematic representation of the known signaling pathways interacting with FSTL1 during inflamma- CD14 tory processes. Grey compo- DIP2A nents indicate unknown recep- TLR4 tors. Helical structures represent gene expression. Dashed arrow ACC AMPK Inflammatory factors indicates translation from FSTL1 mRNA to protein. The dashed ERK1/2 line separates the two opposite IKKβ Transcrip­on factors effects of FSTL1: pro- and anti- IKBα (ETS-2, c-FOS) inflammatory. Image adjusted JNK Proinflammatory from http://smart .servi er.com NF-Kβ cytokines Matrix metalloproteases IL-1β IL-6 (MMP-2, MMP-3, MMP-13) miR-27a IFN-γ MCP-1 miR-32-5p TNF-α Pro-inflammatory effects in chronic disease An -inflammatory effect during acute phase 1 3 2346 A. Mattiotti et al. Patients with Kawasaki disease present with inflamma- inflammatory response and Fstl1 expression. In CIA mice, tion of blood vessels throughout their body. Plasma levels of Ad-mFstl1 exacerbates severity of arthritis, while admin- FSTL1 are significantly elevated in acute Kawasaki disease istration of anti-mFstl1 IgG neutralizes endogenous Fstl1 patients compared to control subjects. Upon intravenous and reduces the severity of disease [81, 82]. A similar pro- immunoglobulin therapy, levels of FSTL1 were found to inflammatory effect, mediated via the CD14-TLR4 pathway, slowly decrease over time [75]. Interestingly, FSTL1 lev- was also found in CIA mice in which Fstl1 was delivered by els correlate with a high risk of developing coronary artery genetically modie fi d-T cells [ 83]. Moreover, it was observed aneurysm [75], which is a major cause of morbidity and that FSTL1 functioned in a species-specific manner: recom- mortality [76]. binant human but not mouse protein, both produced in Lumbar disc herniation (LDH) is a medical condition human cells (HEK293 and HT-1080), induces IL-6 expres- affecting the spine in which a rupture in the outer fibrous sion in cultured synovial cells from RA patients but not in ring of an intervertebral disc allows the soft central por- mouse NIH-3T3 cells and vice versa [83]. Interestingly, the tion to bulge out beyond the damaged outer rings causing mouse and human proteins are very similar (94.4%), and inflammation. Serum levels of FSTL1 are higher in LDH although the effect difference may lie in this small differ - patients compared to scoliosis patients and healthy controls ence, perhaps, a differing glycosylation state of the two and correlate with the amount of pain on a visual analogue proteins plays a major role in determining their observed scale [77]. biological activities. In the CAIA mouse model, an increase in Fstl1 expression Arthritis is observed during early stages of arthritis. In this model, the administration of E. coli-derived recombinant human To evaluate the role of FSTL1 in arthritis, two different ani- FSTL1 showed anti-destructive and anti-inflammatory mal models are used that result in apparently contradictory effects, reducing synovial cellular infiltration and retaining effects. In the collagen-induced arthritis (CIA) model, the cartilage proteoglycan. Treatment of synovial cells and joints mice are immunized by intradermal administration of an with FSTL1 resulted in downregulation of the transcription emulsion of complete Freund’s adjuvant and bovine type II factors c-Fos and Ets-2 and downstream matrix metallopro- collagen, and 21 days later, a booster or secondary immu- teases, such as Mmp-3 [84, 85]. In line with these obser- nization is given [78]; in this mouse model, FSTL1 was vations, upregulation of these genes was observed when found to promote inflammation. In the anti-type II collagen cells were treated with antibodies directed against FSTL1 antibody-induced arthritis (CAIA) model, the mice are intra- that were obtained from mouse or from RA patients [84, peritoneally injected with a cocktail of monoclonal antibod- 85]. Interestingly, both c-FOS and ETS-2 are expressed at ies directed against type II collagen, and 72 h later, they are higher levels in RA patients than in healthy controls [86]. injected with endotoxin (LPS) [79]; in this acute, destructive In vitro experiments showed that downregulation of c-FOS arthritis model FSTL1 was found to be anti-inflammatory. is mediated by FSTL1 binding to DIP2A receptor [84]. Curi- The different effects reported in these two models of arthri- ously, during differentiation of bone-marrow-derived mac- tis are likely due to the differences in disease development rophages into osteoclasts, Fstl1 induces the transcription of and in the evaluated therapeutic potential of FSTL1; in the c-Fos [87], suggesting a different role of Fstl1 during normal CIA model, adenovirus mediated overexpression of mouse development and in pathological conditions. In this CAIA Fstl1 (Ad-mFstl1) and in the CAIA model administration of model, like in the CIA model, the expression of IL-6 was recombinant non-glycosylated FSTL1. reduced [85], but it is important to note that in the CAIA Fstl1 is normally expressed in synovium, but in early mouse model, IL-6 is not responsible for the progression of stage of CIA, Fstl1 becomes highly overexpressed in fibro- arthritis [88]. blasts at the margin of the eroding bone and in cells of the mesenchymal lineage including osteocytes, chondrocytes, Pro‑inflammatory effect and adipocytes but not in cells of the hematopoietic line- age, like macrophages, neutrophils, or T cells [25, 80, 81]. In a mouse model for bacterially induced septic shock, endo- Ad-mFstl1 increased the secretion of pro-inflammatory toxin (LPS) administration in the rear footpads induces the cytokines, IFN-γ, tumour necrosis factor alpha (TNF-α), expression of both Fstl1 and IL-1β. In control mice and interleukin-1β (IL-1β), and 6 (IL-6), resulting in the induc- in transgenic mice, where Fstl1 was removed by tamox- tion of synovitis (inflammation of the synovial membrane) ifen administration, IL-1β remained low or undetectable with infiltration of inflammatory cells in the synovium [89]. Overexpression of FSTL1 in cultured monocytes and and surrounding tissue. Both in vitro and in vivo experi- macrophages or in the septic shock mouse model induces ments showed that IL-1β induces Fstl1 expression through expression of caspase-1 and NLRP3 (nucleotide-binding NF-κB [82], suggesting a positive feedback between the domain leucine-rich repeat containing (NLR) family, pyrin 1 3 Follistatin-like 1 in development and human diseases 2347 domain containing 3), confirming that FSTL1 mediates probably as a result of infiltrating CD8 T cells. It is of rel- pro-inflammatory events [89]. In cultured adipocytes, mac- evance to note that the infiltration of T cells was not related rophages, and nucleus pulposus cells, the pro-inflammatory to an inflammatory response due to surgery. Intravenous effect of FSTL1 is mediated via the signaling cascade IKKβ, administration of an adenovirus expressing FSTL1 leads IκBα, NF-κB, JNK, and ERK1/2 resulting in the induction to a reduction in expression of pro-inflammatory cytokines of expression of IL-1β, IL-6, TNF-α, MCP (monocyte (IL-17A, IL-6, and IFN-γ) and prolonging survival of trans- chemotactic protein)-1, COX (cyclooxygenase)-2, MMP- planted patients [93]. A similar effect is also observed dur - 13, and iNOS (inducible nitric-oxide synthase) [22, 77, ing cancer progression, in which FSTL1 plays an important 87, 90]. Moreover, stimulation of nucleus pulposus cells role in immune dysfunction regulating thymocyte matura- with TNF-α induces FSTL1 secretion [77], again suggest- tion: in vivo inhibition of Fstl1 increases the tumour-specific ing a positive feedback loop on the inflammatory response. CD8 T-cell response [70]. Treatment of bone-marrow-derived macrophages with gly- cosylated recombinant FSTL1 increases cell proliferation in In conclusion a dose-dependent manner [87]. Together. these data suggest that FSTL1 promotes inflammation by inducing not only Taken together FSTL1 was observed to have a dual function cytokine production, but also proliferation of inflammatory during inflammatory processes, acting as an anti-inflamma- cells. tory factor in the acute phase but having a pro-inflamma- tory effect in the long term and in chronic diseases. This Anti‑inflammatory effect is likely due to activation of different signaling pathways: initially, FSTL1 binds the DIP2A receptor and prevents tis- Observing lowered expression levels of Fstl1 using a hypo- sue degradation by MMPs through the downregulation of morphic mouse model revealed no effect on the levels of the transcription factors c-FOS and ETS-2; subsequently, pro-inflammatory cytokines, such as TNF-α, IL-6, and FSTL1 activates the inflammatory response via the TLR4/ IL-1β, in kidney [91]. However, in renal injury mouse CD14 pathway, the activation of the AMPK pathway, and models, circulating Fstl1 appeared to regulate the immune the inhibition of BMP-signaling pathway. However, it cannot response: in a cisplatin nephrotoxicity model Fstl1 inhib- be excluded that additional endogenous or exogenous factors its the synthesis of pro-inflammatory cytokines, like IL-1β are involved in the regulation. [91], and in a subtotal nephrectomy model Fstl1 decreases TNF-α, IL-6, and MCP-1 (monocyte chemotactic protein-1) expression [92]. In vitro experiments on human, mesangial FSTL1 in other diseases cells showed that this anti-inflammatory effect is mediated by phosphorylation of AMPK and activation of acetyl-CoA Fibrosis carboxylase (ACC) [92]. Upon cardiovascular injury, administration of recom- Fibrosis refers to the formation of excessive connective tis- binant glycosylated human FSTL1 produced in Sf9 insect sue in an organ or tissue during a reactive and/or repara- cells reduced pro-inflammatory cytokine expression. In vitro tive process. Compared to healthy tissue, FSTL1 expression studies on NRCM and macrophages showed that this ina fl m- increases in patients with idiopathic pulmonary fibrosis and matory response is mediated both (I) by inducing AMPK in mouse models such as bleomycin-induced lung injury phosphorylation and downstream ACC activation and (II) [94–96], CCl -induced liver injury and in kidney after uni- by inhibiting BMP4 signaling which otherwise increases lateral ureteral obstruction [97]. Haplodeficiency of Fstl1 or TNF-α and IL-6 expression via pSmad1/5/8 [11]. After liga- reduced expression of Fstl1 using siRNA results in a reduc- tion of the femoral artery in mice, in which Fstl1 expres- tion in collagen accumulation in both lung and liver injury sion from the muscle cells was deleted using the muscle [95, 97]. The effect on fibrosis is most probably due to dis- creatine kinase (MCK)–Cre mouse line, the inflammatory ruption of the TGF-β/BMP balance by FSTL1 because of response was enhanced. This was evidenced by an increase the ability of FSTL1 to inhibit Smad1/5/8-mediated BMP4 in the expression of the pro-inflammatory cytokines TNF-α, signaling and of the stimulation of expression of FSTL1 IL-1β, and MCP-1 and in the infiltration of monocytes and via Smad2/3 mediated TGF-β1 signaling [28, 95]. During macrophages [50]. nephrectomy, overexpression of circulating Fstl1 by adeno- In host-versus-graft disease, FSTL1 was also found to virus mediated delivery reduces kidney fibrosis formation play a role. An upstream morphogen of FSTL1, TGF-β, is and the expression of fibrosis markers, such as TGF-β1, known to play a central role in allograft tolerance [2, 93]. collagen-I, collagen-III, and connective tissue growth fac- In line with this, FSTL1 has been shown to be induced in tor [92]. It is important to note that unlike initial studies, in donor specific blood transfusions after heart transplantation which the mice where analyzed 1–2 weeks after induction of 1 3 2348 A. Mattiotti et al. fibrosis [95, 97], later studies analyzed mice 2 months after and microtubule-associated protein 1A/1B-light chain 3). surgery [92]. This opposite effect on fibrosis development FSTL1 stimulation of these cells induced the switch from is in line with studies in immune diseases, in which FSTL1 E-cadherin to N-cadherin expression, pointing endothe- also shows an opposite effect during the acute or chronic lial-to-mesenchymal transition. Interestingly, when these phases of disease. cells were also treated with an autophagy inhibitor, not only autophagy but also endothelial-to-mesenchymal tran- Lung development and asthma sition was attenuated [102]. During mouse development and in the adult mouse, Fstl1 mRNA is expressed in mesenchymal cells of the lung, vas- Obesity cular and airway smooth muscle cells, goblet cells of the airway epithelium and in the endothelial cells [6, 98]. Upon Serum levels of FSTL1 were found to correlate with the functional disruption of Fstl1, neonates die at birth due to body mass index with FSTL1 higher in overweight and respiratory distress, displaying tracheomalacia, hypoplastic, obese subjects than in controls [90]. During differentia- and absence of tracheal cartilage rings. Within the lung tis- tion of pre-adipocytes (3T3–L1) into adipocytes, Fstl1 sue, a thickening of the alveolar walls and a reduction in showed a transient short high level expression to become airspace were found. Moreover, differentiation of the airway subsequently downregulated to background levels at both epithelium seems to be impaired as seen in the reduced level the mRNA and protein level [7, 104]. Either by prevent- in mature surfactant protein [27, 28]. ing this initial peak in Fstl1 expression or by maintaining Asthma, a chronic respiratory disease, is characterized high levels of Fstl1 in the culture medium during induced by airway inflammation, remodelling, and hyper-respon- adipogenesis, the differentiation of 3T3–L1 cells was siveness. Proteomic analysis of sputum of patients with blocked [104]. Two different mechanisms have been iden- asthma revealed that FSTL1 is one of the highest expressed tified linked directly to the regulation of Fstl1 expression. proteins [99]. Histological analysis of post-mortem human Recently, it was discovered that the secretion of Fstl1 was lungs of asthma patients showed expression of FSTL1 in regulated via cilia. When genes essential for ciliogenesis, alveolar macrophages [100]. Moreover, the elevated levels BBS4 or IFT88, were knocked-down in 3T3–L1 pre- of FSTL1 were found in blood plasma and bronchoalveolar adipocytes, the levels of Fstl1 mRNA and protein were lavage fluid in asthma patients compared to healthy con- downregulated and the cells failed to undergo correct adi- trols [101]. The FSTL1 concentration negatively correlates pogenesis [104]. with lung function parameters and positively with airway The second mechanism involved the downregulation of remodelling markers. Interestingly, plasma levels of FSTL1 the pro-inflammatory cytokines, IL-6, IL-8, and MCP-1, significantly decrease and return to control levels 1 month during the differentiation of 3T3–L1 cells into adipocytes after treatment with inhaled corticosteroids and long-acting [90]. The addition of TNFα to 3T3–L1 adipocytes induced β-agonist therapy and/or oral leukotriene receptor antagonist their de-differentiation and was accompanied by a re-expres- therapy [101]. Along the same line, mice chronically, but sion of Fstl1 mRNA and protein [7] and an upregulation not acutely, challenged with an allergen showed an increase of the pro-inflammatory cytokines [90]. The changes in the in Fstl1 expression [100]. Haplodeficiency of Fstl1 [ 102] or ratio of pro- and anti-inflammatory cytokines are thought to deletion of Fstl1 from macrophages/myeloid cells using the underlie the chronic inflammation observed in obesity, which Lys–Cre mouse line [100] reduced inflammation (Fig.  3) and leads to insulin resistance and other obesity-associated dis- airway remodelling in OVA-challenged mice, while opposite eases [105]. This closely resembles the affects reported in effects are observed when mice were treated with recombi- arthritis, pointing to a role of FSTL1 in the regulation of the nant glycosylated FSTL1, though in this study, it is not clear balance of pro- and anti-inflammatory cytokines. whether human or mouse protein was used [100]. Increased Another, as yet, unexplored alternative mechanism could expression of Fstl1 in a chronic but not in an acute mouse be via micro RNA (miRNA) regulation. Like in the differ - model of asthma is in line with previously reported results entiation of adipocytes, a similar downregulation of Fstl1 is suggesting different roles of FSTL1 during acute and chronic observed during in vitro myogenesis of C2C12 myoblasts. In inflammation in arthritis [100]. myoblasts, the downregulation of Fstl1 mRNA is regulated Autophagy and epithelial-to-mesenchymal transi- by muscle-specific miR-206 [32]. Furthermore, during adi- tion are two associated biological processes [103]. pogenesis, regulation of gene expression levels by microR- Autophagosome formation was found to be increased in NAs has been reported [106], and candidate miRNA-binding human bronchial epithelial cell line 16HBE stimulated sites can be found when the 3′UTR of FSTLl1 is scanned with recombinant FSTL1 protein using electron micro- for potential binding sites of microRNAs (Supplemental scopical analysis and autophagy biomarkers (Beclin-1 Table 1). 1 3 Follistatin-like 1 in development and human diseases 2349 no FSTL1 protein expression is observed [113]. Disregula- Central nervous system tion of the ratio FSTL1 versus miR-198 is observed in head and neck squamous cell carcinoma, in which FSTL1 pro- During mouse development, Fstl1 is locally expressed in all the component of the central nervous system [107] and tein persists with the afore-mentioned consequences [62]. MiR-198 is also involved in suppressing colorectal cancer it is involved in the radial glial scaffold formation [108]. In the dorsal root ganglia, Fstl1 is involved in maintenance growth [114] and lung adenocarcinoma A549 cell prolifera- tion [115], but the relation between FSTL1 and miR-198 in of the normal threshold of somatic sensation: it is secreted from afferent axons and it activates the α1 subunit of the this cancer type has not yet been studied. + + MiR-206 is one of the most abundant miRNAs expressed Na ,K -ATPase, suppressing synaptic transmission. Neural deletion of Fstl1 using Na 1.8–Cre mice causes hypersensi- during skeletal myogenesis [116]. In chicken, the expression of FSTL1 (also referred to as Flik) is downregulated in the tivity of both wide dynamic range neurons and nociceptive neurons [109]. embryo during myotome formation suggesting a role during myogenesis [117]; however, the role of FSTL1 during this FSTL1 and microRNAs process has not been investigated. The group of Tapscott showed that skeletal muscle cell differentiation is coordi- MicroRNAs (miRNAs) are short (~ 22  nt) endogenous nated by the transcription factor MyoD, and expression of FSTL1 is reduced [118]. This process appears to be medi- noncoding RNAs that regulate messenger RNA (mRNA) degradation and/or translational repression [110]. Several ated by the induction of miR-206 that binds to the 3′UTR of FSTL1 mRNA [32]. studies have highlighted the relevance of the miRNA and miRNA-binding sites during human disease [111]. miR-198 During mycobacterial infection, miR-32-5p negatively regulates the inflammatory response [119] and promotes is encoded in the 3′UTR of human FSTL1 primary transcript [112]. The FSTL1 mRNA, therefore, not only encodes the the survival of infected macrophages. MiR-32-5p binds to the 3′UTR of FSTL1 decreasing mRNA and protein levels FSTL1 protein, but also an miRNA. Though the FSTL1 protein has been highly conserved during evolution from (Fig. 3). Re-expression of FSTL1 completely reverses the inhibitory effects of miR-32-5p on secretion of inflammatory tick to human [29], the encoded miRNA is only found in primates. Moreover, in silico analysis revealed multiple cytokines, indicating that inhibition of FSTL1 is a mediator of the anti-inflammatory effects [33]. However, the increased miRNA-binding sites in the 3′UTR of the FSTL1 mRNA of which three have been functionally analyzed (miR-206 level of Fslt1 protein seems to have no effect on the level of released cytokines. [32], miR-32-5p [33], and miR-27a [24]) (Table 1). A list of predicted miRNA-binding sites in the 3′UTR of human Significant lower levels of miR-27a and higher levels of FSTL1 are found in serum, synovial tissue, and fibroblast- FSTL1 with associated clinical relevance is provided in Sup- plemental Table 1. like synoviocytes of RA patients compared to healthy con- trols. Transfection of fibroblast-like synoviocytes with miR- In the normal healthy human epidermis, FSTL1 mRNA is expressed, but the protein is present at low to almost unde- 27a inhibits cell migration and invasion and downregulates TLR4, NF-κB, and MMPs. Further analysis showed that tectable, levels. Contrary to this, miR-198 is expressed in this tissue. During wound healing, a switch from miR-198 these effects are the result of miR-27a-mediated downregu- lation of FSTL1 expression via its target sequence in the to FSTL1 protein expression is observed without a change in the FSTL1 mRNA level. TGF-β1 indirectly regulates this 3′UTR (Fig.  3). Moreover, overexpression of FSTL1 via adenoviral vector rescues the miR-27a effects [24]. switch via KH-type splicing regulatory protein (KHSRP). In the healing skin, FSTL1 protein promotes keratinocyte migration during re-epithelization. Interestingly, in chronic non-healing ulcer wounds in patients with diabetes mellitus, Concluding remarks From a clinical point of view, it is important to consider that Table 1 MicroRNA-binding sites in FSTL1 gene during pathological conditions, such as cancer and cardio- Human microRNA Binding position in Biological relevance vascular disease, not only individual cell behaviors such as 3′UTR proliferation and migration are important but also paracrine miR-27a 1537 Inflammation [24] communication between cells, inflammation, and vasculari- miR-32-5p 142 Inflammation [33] zation. Because FSTL1 has been shown to be involved in miR-206 2101; 2375 Myogenesis [32] multiple signaling pathways and processes, results observed in specific cell types and in defined conditions should be List, position, and biological processes of the verified microRNA- regarded on their specific merits and not generalized, as yet. binding sites in the 3′UTR of FSTL1 gene 1 3 2350 A. Mattiotti et al. 5. 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Cellular and Molecular Life SciencesSpringer Journals

Published: Mar 29, 2018

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