Egr-1 increases angiogenesis in cartilage via binding Netrin-1 receptor DCC promoter

Egr-1 increases angiogenesis in cartilage via binding Netrin-1 receptor DCC promoter Background: Osteoarthritis (OA) is a joint disease characterized by degradation of cartilage. The etiology of OA is still unclear. Vascular endothelial growth factor (VEGF) plays a key role of angiogenesis in the pathogenesis of OA and contributes to the angiogenesis of NT-1/DCC. Whether or not NT-1/DCC and VEGF interact in regulating angiogenesis of OA cartilage is not known. Methods: Histological studies for CD34, VEGF, and safranin-O staining were performed to determine angiogenesis and cartilage tissue injury. ELISA indicated the level of pro-inflammation cytokines. Immunoblotting, immunoprecipitation, and electrophoretic mobility shift assay (EMSA) were performed to assay the expression and function of NT-1/DCC-VEGF signaling pathway. Results: Our data indicated that VEGF expression was increased in cartilage tissue from OA rats, while the chondrocytes were disorganized, and cartilage degeneration was increasing in OA rats. The inflammation factors in articular cavity fluid were higher in the OA rats than in the sham. The protein expression of NT-1, DCC, and VEGF were increased in osteoarthritic cartilage. DCC was involved in the positive regulation of osteoarthritic angiogenesis by VEGF. Egr-1 expression was higher in OA rats than in sham rats. Egr-1 is a regulator of DCC promoter activity, and the binding is higher in OA rats than in sham rats. Conclusion: Our present study provides a mechanism by which Egr-1 induced angiogenesis via NT-1/DCC-VEGF pathway. Keywords: Egr-1, Angiogenesis, Osteoarthritis, Netrin-1 receptor, DDC promoter Background found the contribution of angiogenesis of osteophyte Osteoarthritis (OA) is an age-dependent, chronic, incur- formation in OA [6]. Angiogenesis is closely associated able, and destructive joint disease characterized by deg- with the pathogenesis of OA [7]. Neovascularization radation of cartilage, hypertrophy of chondrocyte, and modulates chondrocyte functions and contributes to- sclerosis of subchondral bone [1] and is a main cause of wards abnormal tissue growth and perfusion, ossifica- pain and disability of older individuals [2]. It seems that tion, and endochondral bone development [8] and leads no pharmacological agents could prevent and treat OA, to oxidative stress and inflammation, which results to and alleviating joint pain could be the only medical op- matrix degradation [9]. Angiogenesis is regulated by a tion for OA, which is often unsuccessful, leading to total delicate balance between endogenous angiogenic and joint replacement [3, 4]. The lack of treatment may be anti-angiogenic factors. It has been demonstrated that ascribed to the unclear etiology of OA. Netrin-1 (NT-1) is the factor regulating patterning of Articular cartilage is a highly specialized connective the vascular system [10], and NT-1 induced prolifera- tissue with an avascular structure. However, cartilage tion, migration, and tube formation of endothelial cells loses the ability to stay avascular in the osteoarthritic en- and human aortic smooth muscle cells, via Netrin-1 re- vironment [5], indicating that angiogenesis could con- ceptor DCC (Deleted in Colorectal Cancer) [11]. The tribute to the pathogenesis of OA. Previous researches vascular endothelial growth factor (VEGF) might con- tribute to the angiogenesis of NT-1/DCC [12]. VEGF * Correspondence: zhengweipaper@sina.com plays a key role of angiogenesis in the pathogenesis Jun Sheng and Da Liu contributed equally to this work. of OA and is essential for establishing epiphyseal Department of Orthopedics, Chengdu Military General Hospital, 270 Rongdu Avenue, Jinniu District, Chengdu 610083, Sichuan, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 2 of 7 vascularization and remodeling hypertrophic cartilage, pulverized in liquid nitrogen, and lysed [20]. The ho- which finally leads to endochondral bone formation [13]. mogenates (20 μg of protein) were separated by 8% Whether or not NT-1/DCC and VEGF interact in regu- SDS-PAGE and blotted on polyvinylidene fluori (PVDF) lating angiogenesis of OA cartilage is not known. And membranes (Bio–Rad Laboratories). Transblots were the upstream signaling that regulates NT-1/DCC-VEGF probed with the rabbit anti-Egr-1 (Cell Signaling to angiogenesis in cartilage is still unclear. technology Inc., 1:1000), rabbit anti-VEGF antibody Early growth response-1 (Egr-1), also called NGFI-A, (Santa Cruz Biotechnology, 1:400), rabbit anti-DCC anti- is a zinc finger transcription factor and immediate early body (Santa Cruz Biotechnology, 1:400), or rabbit anti- gene, which plays an important role in angiogenesis [14, Netrin-1 antibody (Santa Cruz Biotechnology, 1:400) to 15]. Meanwhile, Egr-1 may be involved in the pathogen- examine the protein expression in the lysates. The esis of OA [16, 17]. Few studies report the effect of Egr- amount of protein transferred onto the membranes was 1 to angiogenesis in osteoarthritic cartilage. Thus, our verified by immunoblotting for GAPDH (Santa Cruz present study provides a mechanism by which Egr-1 in- Biotechnology, 1:500). duced angiogenesis via NT-1/DCC-VEGF pathway. Immunoprecipitation Methods Equal amounts of homogenates (400 μgofprotein) were Animals incubated with affinity-purified anti-DCC antibody for 1 h Lewis rats (260–280 g) were purchased from Chengdu and protein-G agarose at 4 °C for 12 h. The immunopre- Da Shuo Biotech Co. Ltd. (Chengdu, China) and ran- cipitates were suspended in a sample buffer and subjected domized to the sham group and OA group, n = 8 per to immunoblotting with the VEGF antibody. To determine group. The rat OA model was performed as anterior the specificity of the bands found on the immunoblots, cruciate ligament transection (ACLT). The ACLT was IgG (negative control) and anti-DCC antibody (positive conducted as described in the previous study [18], which control) were used as the immunoprecipitants. induced mechanical instability-associated OA. The rats were euthanized 2 months after ACLT. And siRNA or recombinant netrin 1 were injected into knee joints 48 h Small interfering RNA before euthanasia. The limbs of OA rats were dissected Small interfering RNA (siRNA) against rat DCC (NM_ for staining, and articular cavity fluid were collected for 012841) and Egr-1 (NM_012551) mRNA was synthesized further research. and purified by Qiagen (Hilden, Germany). The effects of All experiments conformed to the guidelines of the 50-nM siRNA were compared with scrambled RNA ethical use of animals, and all efforts were made to (negative control, Qiagen). Briefly, 10-nM siRNA or minimize animal suffering and to reduce the number of control RNA were mixed with 6 μL of oligofectamine in animals used. Optimem medium (Invitrogen Life Technologies) and in- cubated with cells grown in six-well plates for 24 h and Histological study then switched to growth medium and incubated for an- Cartilage tissues from rats in each group were fixed with other 24 h. 4% paraformaldehyde for 24 h and decalcified for 2 weeks with 20% EDTA at 4 °C. After embedding in paraffin, the tissues were sectioned to 4-μm slices and mounted on EMSA slides. The slides were incubated with anti-CD34 (1:100) Electrophoretic mobility shift assays (EMSAs) were for determining microvessel density (MVD) or anti-VEGF performed as described before [21, 22], by the Light-shift (1:100) antibody (Santa Cruz Biotechnology, CA) over- Chemiluminescent EMSA Kit (Pierce Chemical Co., night at 4 °C followed by immunofluorescence and immu- Rockford, IL). A synthetic DNA double-stranded nohistochemistry, or stained with 0.1% safranin-O for oligonucleotide probe (5′-biotin-CGGTACATGACACAG 30 min (Sigma Aldrich, St. Louis, MN). To assess micro- GCTGAC-5′) containing the sequence of the rat DCC vessel density in epiphysis tissues, the number of new gene promoter between nucleotides -101 and -91 bp blood vessels visualized by CD34 vascular endothelial (5′-CCAGCTCGCA-3′) was labeled with biotin and incu- cells was counted per high-power field (× 400) [19]. bated with the nuclear extracts. Rabbit anti-Egr-1 (Cell Signaling technology Inc.) was used for supershift experi- ELISA and immunoblotting ments. A chemiluminescent detection method with a lu- The concentration of IL-1β and TNF-α in articular minal/enhancer solution and stable peroxide solution cavity fluid was determined by the ELISA kit (R&D (Pierce Chemical Co., Rockford, IL) was used, as described Systems) according to the manufacturer’s instructions. by the manufacturer, and the membranes were exposed to And tissues were washed three times with PBS, X-ray films for 30 s to 5 min before development. Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 3 of 7 Statistical analysis cells and vascular smooth muscle [23]. However, the role The data are expressed as mean ± SEM. Comparison of NT-1/DCC in angiogenesis of osteoarthritic cartilage within groups was made by t test or one-way ANOVA is still unclear. Thus, our studies checked the expression for repeated measures. A value of P < 0.05 was consid- of NT-1 and DCC in cartilage and found that the pro- ered significant. tein expression of NT-1 and DCC were increased in osteoarthritic cartilage (Fig. 2a, b). Results Besides, VEGF protein expression was significantly Angiogenesis in cartilage of OA rat higher in the OA rats than in the sham, while the DCC To check the angiogenesis in OA rat, the microvessel siRNA decreased the increasing VEGF expression (Fig. 2c) density in epiphysis tissues of the proximal epiphysis of , indicating DCC was involved in the positive regulation of the tibia was quantified via CD34 staining, and found osteoarthritic angiogenesis by VEGF. microvessel density significantly increased in OA cartil- An additional study found a co-immunoprecipitation age tissue (Fig. 1a, b). The data showed that VEGF (Fig. 2d) between DCC and VEGF; the co-immunoprecipitation expression was increased in cartilage tissue from OA of DCCand VEGF washighinOAratsthaninsham rats (Fig. 1c), indicating that proliferation and migration rats, which could be a factor in the increased expres- of endothelial cell were promoted in OA. Moreover, the sion of VEGF in OA rats. chondrocytes were disorganized, and cartilage degener- ation was increasing in OA rats (Fig. 1d). The TNF-α The binding of Egr-1 at DCC promoter regulates the and IL-1β levels in articular cavity fluid were higher in angiogenesis the OA rats than in the sham (Fig. 1e). These data To investigate whether or not Egr-1 was involved in the suggest that angiogenesis could contribute to the patho- regulation of DCC on angiogenesis, we measured the genesis of OA. Egr-1 expression in cartilage firstly. Egr-1 protein expression was higher in OA rats than in sham rats DCC regulates the angiogenesis in OA via VEGF (Fig. 3a). The Egr-1 siRNA decreased the increasing DCC Previous study demonstrated that NT-1 and its receptor, expression in osteoarthritic cartilage (Fig. 3b) and de- DCC, stimulate growth of umbilical vein endothelial creased the NT-1-induced VEGF expression (Fig. 3c). Fig. 1 Angiogenesis in cartilage of OA rat. a, b Immunofluorescence staining of CD34. CD34 immunofluorescence showed the stained microvessels. The number of new blood vessels visualized by CD34 vascular endothelial cells was counted per high-power field (× 400). (*P = 0.0004 vs. sham, n =5, scale bar = 20 μm). c, d Immunohistochemical staining of VEGF. The severity of proteoglycan depletion in OA cartilage was demonstrated by safranin-O staining. e The levels of IL-1β and TNF-α were measured by ELISA (*P = 0.0001 vs. others, n =5) Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 4 of 7 Fig. 2 DCC regulates the angiogenesis in OA via VEGF. a Netrin-1 expression in OA cartilage. Results are expressed as the ratio of Netrin-1 and GAPDH (*P = 0.00009 vs. the sham, n = 5 in each group). b DCC expression in OA cartilage. Results are expressed as the ratio of DCC and GAPDH (*P = 0.0012 vs. the sham, n = 5 in each group). c VEGF expression in DCC siRNA-treated OA cartilage. Results are expressed as the ratio of VEGF and GAPDH (*P =0.0002 vs. the others, P = 0.0003 vs. OA group and scramble siRNA-treated group, n = 5 in each group). d Co-immunoprecipitation of DCC and VEGF in OA cartilage. The lysates were immunoprecipitated with DCC antibody and immunoblotted with VEGF antibodies (*P =0.0008 vs. sham, n = 4, lane 1 = positive control, lane 2 = negative control, lane 3 = sham, lane 4 = OA group. For the positive control, VEGF antibody was used, and for the negative control, IgG was used instead of DCC antibody as the immunoprecipitants) To identify the hypothesis that Egr-1 is a regulator of mediators of the catabolism of OA cartilage and stimula- DCC promoter activity, we measured Egr-1 binding to tor for osteoarthritic chondrocyte [31–33]. Thus, VEGF the DCC promoter and found it higher in OA rats than seems to induce osteoarthritis via angiogenesis and in- in sham rats (Fig. 3d), indicating that the regulation of flammatory. And whether or not NT-1/DCC and VEGF DCC by Egr-1 occurred at transcriptional level. interact in regulating angiogenesis of OA cartilage is not known. Discussion Further studies identified the increasing expression of The balance between angiogenic and anti-angiogenic NT-1/DCC in osteoarthritic cartilage and increasing factors is an important regulator of cartilage homeostasis co-immunoprecipitation between DCC and VEGF. [24–26]. And healthy adult human articular cartilage is Meanwhile, the DCC siRNA decreased the increasing avascular, and blood vessels could penetrate newly VEGF expression in OA. These data indicated DCC was formed cartilage at the joint margins in OA and increase involved in the positive regulation of osteoarthritic osteophyte formation, which may contribute to symp- angiogenesis by VEGF directly in OA. The mechanisms toms and joint damage [5, 27, 28]. In the present study, of DCC in the regulation of VEGF are still unclear. we found that the endothelial cell proliferation, vascular Previous studies found that DCC, on NT-1 binding, density, and VEGF abundance were increased in osteo- activates the extracelluar signal-regulated kinase-1/2 arthritic cartilage which lead to the cartilage degener- (ERK-1/2) and mitogen-activated protein kinase (MAPK) ation. Besides the stimulation of angiogenesis, VEGF [34, 35]. And DCC was also shown to activate the ERK- may also contribute to inflammation [29, 30]. Indeed, 1/2-eNOS pathway [36]. It was reported earlier that NO our data show a significant upregulation in osteoarthritic mediates VEGF-induced angiogenesis [37]. MAPK/ERK articular cavity fluid of cytokines such as TNF-α and signaling pathway also plays an important role in the IL-1β. The pro-inflammatory factors are the major regulation of VEGF [38, 39]. We infer that the DCC Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 5 of 7 Fig. 3 The binding of Egr-1 at DCC promoter regulates the angiogenesis. a Egr-1 expression in OA cartilage. Results are expressed as the ratio of Egr-1 and GAPDH (*P = 0.0003 vs. the sham, n = 5 in each group). b DCC expression in Egr-1 siRNA-treated OA cartilage. Results are expressed as the ratio of DCC and GAPDH (*P = 0.0001 vs. the others, P = 0.0005 vs. OA group and scramble siRNA-treated group, n = 5 in each group). c VEGF expression in Netrin-1 and Egr-1 siRNA-treated cartilage. Results are expressed as the ratio of VEGF and GAPDH (*P = 0.00005 vs. the others, P = 0.00009 vs. OA group and scramble siRNA-treated group, n = 5 in each group). d EMSA of nuclear protein from cartilage tissue. Binding activity of DCC gene promoter, containing an Egr-1 site, was examined in nuclear protein (*P = 0.0002 vs. the sham, n = 5 in each group) could upregulate VEGF via MAPK/ERK or eNOS/NO The current study has several limitations. First, chro- signaling pathway. However, more data from further matin immunoprecitation (ChIP) may be used to identify studies are necessary to support the hypothesis. the increased binding of Egr-1 to the DCC promoter in As an active transcription factor, Egr-1 regulates pro- OA cartilage, rather than EMSA. Second, while our moter activity of various proteins. We believe the Egr-1 present study focused on the role of Egr-1/DCC path- could be an upstream regulator of DCC/VEGF pathway. way, whether or not Egr-1 or DCC could be a therapy The expression of Egr-1 in OA cartilage has been con- target is another topic of future study. troversial. Despite Wang et al. found the Egr-1 expres- sion in OA cartilage is decreased [40], we demonstrated Conclusion that Egr-1 protein expression was higher in OA rats than In conclusion, the present study reinforces the role of Egr-1 in sham rats. This is consistent with previous studies in OA and shows that an increased expression of Egr-1 in- showing that Egr-1 expression increases in OA cartilage creases cartilage DCC expression, which may be involved in [17]. The Egr-1-mediated regulation of DCC expression the abnormalities of angiogenesis in OA. The results imply might be complicated, as in our present study, we found that Egr-1 may be an effective therapeutic target for OA. that Egr-1 siRNA decreased the increasing DCC expres- sion in osteoarthritic cartilage and NT-1-induced VEGF Abbreviations ACLT: Anterior cruciate ligament transection; DCC: Deleted in Colorectal expression. And our data uncover a possible mechanism: Cancer; Egr-1: Early growth response-1; ERK-1/2: Extracelluar signal-regulated The activity of Egr-1, a regulator of DCC promoter ac- kinase-1/2; MAPK: Mitogen-activated protein kinase; MVD: Determining tivity, is increased, accompanied by an increase in its microvessel density; NT-1: Netrin-1; OA: Osteoarthritis; PVDF: Polyvinylidene fluori; VEGF: Vascular endothelial growth factor binding to the DCC promoter in OA cartilage. And the pathway leading to the higher binding of Egr-1 with Funding DCC promoter is not known, which needs to be eluci- The research reported was supported by the Foundation of Department of dated in the future. Science and Technology of Sichuan Province (Grant no. 2014JY0009). Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 6 of 7 Availability of data and materials 14. Wang LF, Liu YS, Yang B, et al. The extracellular matrix protein mindin All data and materials were in full compliance with the journal’s policy. attenuates colon cancer progression by blocking angiogenesis via Egr-1- mediated regulation. Oncogene. 2018;37(5):601–15. https://doi.org/10.1038/ Authors’ contributions onc.2017.359. JS and DL designed, directed, and carried out the experiments; analyzed the 15. Yoon YJ, Kim DK, Yoon CM, et al. Egr-1 activation by cancer-derived data; and wrote the manuscript. 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Egr-1 increases angiogenesis in cartilage via binding Netrin-1 receptor DCC promoter

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Abstract

Background: Osteoarthritis (OA) is a joint disease characterized by degradation of cartilage. The etiology of OA is still unclear. Vascular endothelial growth factor (VEGF) plays a key role of angiogenesis in the pathogenesis of OA and contributes to the angiogenesis of NT-1/DCC. Whether or not NT-1/DCC and VEGF interact in regulating angiogenesis of OA cartilage is not known. Methods: Histological studies for CD34, VEGF, and safranin-O staining were performed to determine angiogenesis and cartilage tissue injury. ELISA indicated the level of pro-inflammation cytokines. Immunoblotting, immunoprecipitation, and electrophoretic mobility shift assay (EMSA) were performed to assay the expression and function of NT-1/DCC-VEGF signaling pathway. Results: Our data indicated that VEGF expression was increased in cartilage tissue from OA rats, while the chondrocytes were disorganized, and cartilage degeneration was increasing in OA rats. The inflammation factors in articular cavity fluid were higher in the OA rats than in the sham. The protein expression of NT-1, DCC, and VEGF were increased in osteoarthritic cartilage. DCC was involved in the positive regulation of osteoarthritic angiogenesis by VEGF. Egr-1 expression was higher in OA rats than in sham rats. Egr-1 is a regulator of DCC promoter activity, and the binding is higher in OA rats than in sham rats. Conclusion: Our present study provides a mechanism by which Egr-1 induced angiogenesis via NT-1/DCC-VEGF pathway. Keywords: Egr-1, Angiogenesis, Osteoarthritis, Netrin-1 receptor, DDC promoter Background found the contribution of angiogenesis of osteophyte Osteoarthritis (OA) is an age-dependent, chronic, incur- formation in OA [6]. Angiogenesis is closely associated able, and destructive joint disease characterized by deg- with the pathogenesis of OA [7]. Neovascularization radation of cartilage, hypertrophy of chondrocyte, and modulates chondrocyte functions and contributes to- sclerosis of subchondral bone [1] and is a main cause of wards abnormal tissue growth and perfusion, ossifica- pain and disability of older individuals [2]. It seems that tion, and endochondral bone development [8] and leads no pharmacological agents could prevent and treat OA, to oxidative stress and inflammation, which results to and alleviating joint pain could be the only medical op- matrix degradation [9]. Angiogenesis is regulated by a tion for OA, which is often unsuccessful, leading to total delicate balance between endogenous angiogenic and joint replacement [3, 4]. The lack of treatment may be anti-angiogenic factors. It has been demonstrated that ascribed to the unclear etiology of OA. Netrin-1 (NT-1) is the factor regulating patterning of Articular cartilage is a highly specialized connective the vascular system [10], and NT-1 induced prolifera- tissue with an avascular structure. However, cartilage tion, migration, and tube formation of endothelial cells loses the ability to stay avascular in the osteoarthritic en- and human aortic smooth muscle cells, via Netrin-1 re- vironment [5], indicating that angiogenesis could con- ceptor DCC (Deleted in Colorectal Cancer) [11]. The tribute to the pathogenesis of OA. Previous researches vascular endothelial growth factor (VEGF) might con- tribute to the angiogenesis of NT-1/DCC [12]. VEGF * Correspondence: zhengweipaper@sina.com plays a key role of angiogenesis in the pathogenesis Jun Sheng and Da Liu contributed equally to this work. of OA and is essential for establishing epiphyseal Department of Orthopedics, Chengdu Military General Hospital, 270 Rongdu Avenue, Jinniu District, Chengdu 610083, Sichuan, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 2 of 7 vascularization and remodeling hypertrophic cartilage, pulverized in liquid nitrogen, and lysed [20]. The ho- which finally leads to endochondral bone formation [13]. mogenates (20 μg of protein) were separated by 8% Whether or not NT-1/DCC and VEGF interact in regu- SDS-PAGE and blotted on polyvinylidene fluori (PVDF) lating angiogenesis of OA cartilage is not known. And membranes (Bio–Rad Laboratories). Transblots were the upstream signaling that regulates NT-1/DCC-VEGF probed with the rabbit anti-Egr-1 (Cell Signaling to angiogenesis in cartilage is still unclear. technology Inc., 1:1000), rabbit anti-VEGF antibody Early growth response-1 (Egr-1), also called NGFI-A, (Santa Cruz Biotechnology, 1:400), rabbit anti-DCC anti- is a zinc finger transcription factor and immediate early body (Santa Cruz Biotechnology, 1:400), or rabbit anti- gene, which plays an important role in angiogenesis [14, Netrin-1 antibody (Santa Cruz Biotechnology, 1:400) to 15]. Meanwhile, Egr-1 may be involved in the pathogen- examine the protein expression in the lysates. The esis of OA [16, 17]. Few studies report the effect of Egr- amount of protein transferred onto the membranes was 1 to angiogenesis in osteoarthritic cartilage. Thus, our verified by immunoblotting for GAPDH (Santa Cruz present study provides a mechanism by which Egr-1 in- Biotechnology, 1:500). duced angiogenesis via NT-1/DCC-VEGF pathway. Immunoprecipitation Methods Equal amounts of homogenates (400 μgofprotein) were Animals incubated with affinity-purified anti-DCC antibody for 1 h Lewis rats (260–280 g) were purchased from Chengdu and protein-G agarose at 4 °C for 12 h. The immunopre- Da Shuo Biotech Co. Ltd. (Chengdu, China) and ran- cipitates were suspended in a sample buffer and subjected domized to the sham group and OA group, n = 8 per to immunoblotting with the VEGF antibody. To determine group. The rat OA model was performed as anterior the specificity of the bands found on the immunoblots, cruciate ligament transection (ACLT). The ACLT was IgG (negative control) and anti-DCC antibody (positive conducted as described in the previous study [18], which control) were used as the immunoprecipitants. induced mechanical instability-associated OA. The rats were euthanized 2 months after ACLT. And siRNA or recombinant netrin 1 were injected into knee joints 48 h Small interfering RNA before euthanasia. The limbs of OA rats were dissected Small interfering RNA (siRNA) against rat DCC (NM_ for staining, and articular cavity fluid were collected for 012841) and Egr-1 (NM_012551) mRNA was synthesized further research. and purified by Qiagen (Hilden, Germany). The effects of All experiments conformed to the guidelines of the 50-nM siRNA were compared with scrambled RNA ethical use of animals, and all efforts were made to (negative control, Qiagen). Briefly, 10-nM siRNA or minimize animal suffering and to reduce the number of control RNA were mixed with 6 μL of oligofectamine in animals used. Optimem medium (Invitrogen Life Technologies) and in- cubated with cells grown in six-well plates for 24 h and Histological study then switched to growth medium and incubated for an- Cartilage tissues from rats in each group were fixed with other 24 h. 4% paraformaldehyde for 24 h and decalcified for 2 weeks with 20% EDTA at 4 °C. After embedding in paraffin, the tissues were sectioned to 4-μm slices and mounted on EMSA slides. The slides were incubated with anti-CD34 (1:100) Electrophoretic mobility shift assays (EMSAs) were for determining microvessel density (MVD) or anti-VEGF performed as described before [21, 22], by the Light-shift (1:100) antibody (Santa Cruz Biotechnology, CA) over- Chemiluminescent EMSA Kit (Pierce Chemical Co., night at 4 °C followed by immunofluorescence and immu- Rockford, IL). A synthetic DNA double-stranded nohistochemistry, or stained with 0.1% safranin-O for oligonucleotide probe (5′-biotin-CGGTACATGACACAG 30 min (Sigma Aldrich, St. Louis, MN). To assess micro- GCTGAC-5′) containing the sequence of the rat DCC vessel density in epiphysis tissues, the number of new gene promoter between nucleotides -101 and -91 bp blood vessels visualized by CD34 vascular endothelial (5′-CCAGCTCGCA-3′) was labeled with biotin and incu- cells was counted per high-power field (× 400) [19]. bated with the nuclear extracts. Rabbit anti-Egr-1 (Cell Signaling technology Inc.) was used for supershift experi- ELISA and immunoblotting ments. A chemiluminescent detection method with a lu- The concentration of IL-1β and TNF-α in articular minal/enhancer solution and stable peroxide solution cavity fluid was determined by the ELISA kit (R&D (Pierce Chemical Co., Rockford, IL) was used, as described Systems) according to the manufacturer’s instructions. by the manufacturer, and the membranes were exposed to And tissues were washed three times with PBS, X-ray films for 30 s to 5 min before development. Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 3 of 7 Statistical analysis cells and vascular smooth muscle [23]. However, the role The data are expressed as mean ± SEM. Comparison of NT-1/DCC in angiogenesis of osteoarthritic cartilage within groups was made by t test or one-way ANOVA is still unclear. Thus, our studies checked the expression for repeated measures. A value of P < 0.05 was consid- of NT-1 and DCC in cartilage and found that the pro- ered significant. tein expression of NT-1 and DCC were increased in osteoarthritic cartilage (Fig. 2a, b). Results Besides, VEGF protein expression was significantly Angiogenesis in cartilage of OA rat higher in the OA rats than in the sham, while the DCC To check the angiogenesis in OA rat, the microvessel siRNA decreased the increasing VEGF expression (Fig. 2c) density in epiphysis tissues of the proximal epiphysis of , indicating DCC was involved in the positive regulation of the tibia was quantified via CD34 staining, and found osteoarthritic angiogenesis by VEGF. microvessel density significantly increased in OA cartil- An additional study found a co-immunoprecipitation age tissue (Fig. 1a, b). The data showed that VEGF (Fig. 2d) between DCC and VEGF; the co-immunoprecipitation expression was increased in cartilage tissue from OA of DCCand VEGF washighinOAratsthaninsham rats (Fig. 1c), indicating that proliferation and migration rats, which could be a factor in the increased expres- of endothelial cell were promoted in OA. Moreover, the sion of VEGF in OA rats. chondrocytes were disorganized, and cartilage degener- ation was increasing in OA rats (Fig. 1d). The TNF-α The binding of Egr-1 at DCC promoter regulates the and IL-1β levels in articular cavity fluid were higher in angiogenesis the OA rats than in the sham (Fig. 1e). These data To investigate whether or not Egr-1 was involved in the suggest that angiogenesis could contribute to the patho- regulation of DCC on angiogenesis, we measured the genesis of OA. Egr-1 expression in cartilage firstly. Egr-1 protein expression was higher in OA rats than in sham rats DCC regulates the angiogenesis in OA via VEGF (Fig. 3a). The Egr-1 siRNA decreased the increasing DCC Previous study demonstrated that NT-1 and its receptor, expression in osteoarthritic cartilage (Fig. 3b) and de- DCC, stimulate growth of umbilical vein endothelial creased the NT-1-induced VEGF expression (Fig. 3c). Fig. 1 Angiogenesis in cartilage of OA rat. a, b Immunofluorescence staining of CD34. CD34 immunofluorescence showed the stained microvessels. The number of new blood vessels visualized by CD34 vascular endothelial cells was counted per high-power field (× 400). (*P = 0.0004 vs. sham, n =5, scale bar = 20 μm). c, d Immunohistochemical staining of VEGF. The severity of proteoglycan depletion in OA cartilage was demonstrated by safranin-O staining. e The levels of IL-1β and TNF-α were measured by ELISA (*P = 0.0001 vs. others, n =5) Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 4 of 7 Fig. 2 DCC regulates the angiogenesis in OA via VEGF. a Netrin-1 expression in OA cartilage. Results are expressed as the ratio of Netrin-1 and GAPDH (*P = 0.00009 vs. the sham, n = 5 in each group). b DCC expression in OA cartilage. Results are expressed as the ratio of DCC and GAPDH (*P = 0.0012 vs. the sham, n = 5 in each group). c VEGF expression in DCC siRNA-treated OA cartilage. Results are expressed as the ratio of VEGF and GAPDH (*P =0.0002 vs. the others, P = 0.0003 vs. OA group and scramble siRNA-treated group, n = 5 in each group). d Co-immunoprecipitation of DCC and VEGF in OA cartilage. The lysates were immunoprecipitated with DCC antibody and immunoblotted with VEGF antibodies (*P =0.0008 vs. sham, n = 4, lane 1 = positive control, lane 2 = negative control, lane 3 = sham, lane 4 = OA group. For the positive control, VEGF antibody was used, and for the negative control, IgG was used instead of DCC antibody as the immunoprecipitants) To identify the hypothesis that Egr-1 is a regulator of mediators of the catabolism of OA cartilage and stimula- DCC promoter activity, we measured Egr-1 binding to tor for osteoarthritic chondrocyte [31–33]. Thus, VEGF the DCC promoter and found it higher in OA rats than seems to induce osteoarthritis via angiogenesis and in- in sham rats (Fig. 3d), indicating that the regulation of flammatory. And whether or not NT-1/DCC and VEGF DCC by Egr-1 occurred at transcriptional level. interact in regulating angiogenesis of OA cartilage is not known. Discussion Further studies identified the increasing expression of The balance between angiogenic and anti-angiogenic NT-1/DCC in osteoarthritic cartilage and increasing factors is an important regulator of cartilage homeostasis co-immunoprecipitation between DCC and VEGF. [24–26]. And healthy adult human articular cartilage is Meanwhile, the DCC siRNA decreased the increasing avascular, and blood vessels could penetrate newly VEGF expression in OA. These data indicated DCC was formed cartilage at the joint margins in OA and increase involved in the positive regulation of osteoarthritic osteophyte formation, which may contribute to symp- angiogenesis by VEGF directly in OA. The mechanisms toms and joint damage [5, 27, 28]. In the present study, of DCC in the regulation of VEGF are still unclear. we found that the endothelial cell proliferation, vascular Previous studies found that DCC, on NT-1 binding, density, and VEGF abundance were increased in osteo- activates the extracelluar signal-regulated kinase-1/2 arthritic cartilage which lead to the cartilage degener- (ERK-1/2) and mitogen-activated protein kinase (MAPK) ation. Besides the stimulation of angiogenesis, VEGF [34, 35]. And DCC was also shown to activate the ERK- may also contribute to inflammation [29, 30]. Indeed, 1/2-eNOS pathway [36]. It was reported earlier that NO our data show a significant upregulation in osteoarthritic mediates VEGF-induced angiogenesis [37]. MAPK/ERK articular cavity fluid of cytokines such as TNF-α and signaling pathway also plays an important role in the IL-1β. The pro-inflammatory factors are the major regulation of VEGF [38, 39]. We infer that the DCC Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 5 of 7 Fig. 3 The binding of Egr-1 at DCC promoter regulates the angiogenesis. a Egr-1 expression in OA cartilage. Results are expressed as the ratio of Egr-1 and GAPDH (*P = 0.0003 vs. the sham, n = 5 in each group). b DCC expression in Egr-1 siRNA-treated OA cartilage. Results are expressed as the ratio of DCC and GAPDH (*P = 0.0001 vs. the others, P = 0.0005 vs. OA group and scramble siRNA-treated group, n = 5 in each group). c VEGF expression in Netrin-1 and Egr-1 siRNA-treated cartilage. Results are expressed as the ratio of VEGF and GAPDH (*P = 0.00005 vs. the others, P = 0.00009 vs. OA group and scramble siRNA-treated group, n = 5 in each group). d EMSA of nuclear protein from cartilage tissue. Binding activity of DCC gene promoter, containing an Egr-1 site, was examined in nuclear protein (*P = 0.0002 vs. the sham, n = 5 in each group) could upregulate VEGF via MAPK/ERK or eNOS/NO The current study has several limitations. First, chro- signaling pathway. However, more data from further matin immunoprecitation (ChIP) may be used to identify studies are necessary to support the hypothesis. the increased binding of Egr-1 to the DCC promoter in As an active transcription factor, Egr-1 regulates pro- OA cartilage, rather than EMSA. Second, while our moter activity of various proteins. We believe the Egr-1 present study focused on the role of Egr-1/DCC path- could be an upstream regulator of DCC/VEGF pathway. way, whether or not Egr-1 or DCC could be a therapy The expression of Egr-1 in OA cartilage has been con- target is another topic of future study. troversial. Despite Wang et al. found the Egr-1 expres- sion in OA cartilage is decreased [40], we demonstrated Conclusion that Egr-1 protein expression was higher in OA rats than In conclusion, the present study reinforces the role of Egr-1 in sham rats. This is consistent with previous studies in OA and shows that an increased expression of Egr-1 in- showing that Egr-1 expression increases in OA cartilage creases cartilage DCC expression, which may be involved in [17]. The Egr-1-mediated regulation of DCC expression the abnormalities of angiogenesis in OA. The results imply might be complicated, as in our present study, we found that Egr-1 may be an effective therapeutic target for OA. that Egr-1 siRNA decreased the increasing DCC expres- sion in osteoarthritic cartilage and NT-1-induced VEGF Abbreviations ACLT: Anterior cruciate ligament transection; DCC: Deleted in Colorectal expression. And our data uncover a possible mechanism: Cancer; Egr-1: Early growth response-1; ERK-1/2: Extracelluar signal-regulated The activity of Egr-1, a regulator of DCC promoter ac- kinase-1/2; MAPK: Mitogen-activated protein kinase; MVD: Determining tivity, is increased, accompanied by an increase in its microvessel density; NT-1: Netrin-1; OA: Osteoarthritis; PVDF: Polyvinylidene fluori; VEGF: Vascular endothelial growth factor binding to the DCC promoter in OA cartilage. And the pathway leading to the higher binding of Egr-1 with Funding DCC promoter is not known, which needs to be eluci- The research reported was supported by the Foundation of Department of dated in the future. Science and Technology of Sichuan Province (Grant no. 2014JY0009). Sheng et al. Journal of Orthopaedic Surgery and Research (2018) 13:125 Page 6 of 7 Availability of data and materials 14. Wang LF, Liu YS, Yang B, et al. The extracellular matrix protein mindin All data and materials were in full compliance with the journal’s policy. attenuates colon cancer progression by blocking angiogenesis via Egr-1- mediated regulation. Oncogene. 2018;37(5):601–15. https://doi.org/10.1038/ Authors’ contributions onc.2017.359. JS and DL designed, directed, and carried out the experiments; analyzed the 15. Yoon YJ, Kim DK, Yoon CM, et al. Egr-1 activation by cancer-derived data; and wrote the manuscript. 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Journal of Orthopaedic Surgery and ResearchSpringer Journals

Published: May 29, 2018

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