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Sarcospan: a small protein with large potential for Duchenne muscular dystrophy

Sarcospan: a small protein with large potential for Duchenne muscular dystrophy Purification of the proteins associated with dystrophin, the gene product responsible for Duchenne muscular dystrophy, led to the discovery of the dystrophin-glycoprotein complex. Sarcospan, a 25-kDa transmembrane protein, was the last component to be identified and its function in skeletal muscle has been elusive. This review will focus on progress over the last decade revealing that sarcospan is an important regulator of muscle cell adhesion, strength, and regeneration. Investigations using several transgenic mouse models demonstrate that overexpression of sarcospan in the mouse model for Duchenne muscular dystrophy ameliorates pathology and restores muscle cell binding to laminin. Sarcospan improves cell surface expression of the dystrophin- and utrophin-glycoprotein complexes as well as α7β1 integrin, which are the three major laminin-binding complexes in muscle. Utrophin and α7β1 integrin compensate for the loss of dystrophin and the finding that sarcospan increases their abundance at the extra-synaptic sarcolemma supports the use of sarcospan as a therapeutic target. Newly discovered phenotypes in sarcospan-deficient mice, including a reduction in specific force output and increased drop in force in the diaphragm muscle, result from decreased utrophin and dystrophin expression and further reveal sarcospan’s role in determining abundance of these complexes. Dystrophin protein levels and the specific force output of the diaphragm muscle are further reduced upon genetic removal of α7 integrin (Itga7) in SSPN-deficient mice, demonstrating that interactions between integrin and sarcospan are critical for maintenance of the dystrophin-glycoprotein complex and force production of the diaphragm muscle. Sarcospan is a major regulator of Akt signaling pathways and sarcospan-deficiency significantly impairs muscle regeneration, a process that is dependent on Akt activation. Intriguingly, sarcospan regulates glycosylation of a specific subpopulation of α- dystroglycan, the laminin-binding receptor associated with dystrophin and utrophin, localized to the neuromuscular junction. Understanding the basic mechanisms responsible for assembly and trafficking of the dystrophin- and utrophin-glycoprotein complexes to the cell surface is lacking and recent studies suggest that sarcospan plays a role in these essential processes. Keywords: Akt, Cell adhesion, Duchenne, Dystrophin, Integrin, Laminin-binding, mdx, Muscular dystrophy, Neuromuscular junction, Regeneration, Sarcolemma, Sarcospan, Utrophin Review that result in loss of dystrophin, a protein that is nor- Identification of sarcospan mally localized to the subsarcolemma [1-5]. Discovery of Muscular dystrophies represent a group of progressive dystrophin-associated proteins, referred to as the muscle disorders characterized by extensive muscle dystrophin-glycoprotein complex (DGC), represent a wasting and weakness. Duchenne muscular dystrophy major advancement in the understanding of the DGC’s (DMD) is caused by mutations in the dystrophin gene function in skeletal muscle and provide further support for the contraction-induced sarcolemma injury model underlying DMD pathogenesis [1,2,4,5]. In addition to * Correspondence: rcrosbie@physci.ucla.edu Department of Integrative Biology and Physiology, University of California dystrophin, the DGC is composed of α/β-dystroglycan Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, (DG), the sarcoglycans (SGs), the syntrophins, and dys- Los Angeles, CA 90095, USA trobrevin (for review, [6]). One of the last components Center for Duchenne Muscular Dystrophy, University of California Los Angeles, Los Angeles, CA 90095, USA of the DGC to be identified was a 25-kDa dystrophin- Full list of author information is available at the end of the article © 2013 Marshall and Crosbie-Watson; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 2 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 associated protein (DAP), which was resistant to identifi- DGC-containing fractions providing additional evidence cation, in part due to lack of polyclonal antibodies that that SSPN is an integral component of the DGC cross-reacted with the 25-kDa DAP (also called A5) [5,7,13,14]. In contrast, while a fraction of caveolin-3 from goats and sheep, immunized with the DGC [7]. maintains association with the DGC during purification The hydrophobic probe, 3-trifluoromethyl-3-(m-[ I] by sWGA lectin affinity chromatography, it is localized iodophenyl) diazirine or TID, bound very strongly to the to heavier fractions during sucrose gradient centrifuga- 25-kDa DAP, suggesting that it might be an integral tion [7,13]. For the third criterion, the laminin binding membrane protein [5]. In fact, TID binding to the capacity of α-dystroglycan (α-DG) was exploited to 25-kDa DAP was greater than its binding to the SGs or separate the DGC from other membrane-associated β-DG, which possess a single transmembrane span, pro- proteins. Application of sWGA enrichments from ske- viding strong evidence that the 25-kDa DAP contained letal muscle reveals that SSPN is entirely retained on multiple membrane-spanning regions and was unlikely laminin-sepharose columns, but caveolin-3 is found only to be a protein degradation product, as had been specu- in the void fraction [13,15-19]. Finally, it is well estab- lated based on its weak staining with Coomassie- lished that core components of the DGC depend on Brilliant blue [5,7]. Identification of the 25-kDa DAP dystrophin for localization to the sarcolemma. In was accomplished by in-gel digestion and sequencing of dystrophin-deficient DMD patients and the mdx mouse two amino acid peptides, leading to isolation of the model, SSPN is absent from the sarcolemma while mem- corresponding human cDNA [7] that was previously brane expression of caveolin-3 is not affected by loss of identified in part as Kirsten ras associated gene (krag), a dystrophin [7,13]. gene that is co-amplified with Ki-ras in the Y1 murine adrenal carcinoma cell line [8,9]. The gene was renamed Structural analysis of sarcospan provides insight into to sarcospan (SSPN) for its multiple sarcolemma span- function ning helices predicted from hydropathy analysis [7]. Topology algorithms predict that SSPN possesses four While SSPN is expressed in many non-muscle tissues transmembrane domains with a small extracellular loop [10,11], its biochemical characterization was performed (between transmembrane domains 1 and 2), a large extra- in skeletal muscle where it is most abundant. cellular loop (LEL; between transmembrane domains 3 and 4), and intracellular N- and C-termini (Figure 1) [7]. Rigorous criteria define components of the dystrophin- Although several protein families contain four transmem- glycoprotein complex brane domains, dendrogram analysis suggests that SSPN Although characterization of the 25-kDa DAP led to its is related to the tetraspanins, although not all characteris- identification, there was much speculation that SSPN tics are conserved. Like other tetraspanins, the LEL of was a contaminant of the purified complex rather than a SSPN contains conserved Cys residues that are important bona fide member of the DGC. Integral components of for tertiary structure, although SSPN lacks the hallmark the DGC are defined by four biochemical characteristics Cys Cys Gly motifs within the LEL and conserved sites for and SSPN was rigorously tested with these established N-linked glycosylation and palmitolyation that are charac- criteria. First, purification of the DGC from skeletal teristic of tetraspanins [20]. Tetraspanins facilitate protein muscle membranes enriches proteins that are associated interactions by forming tetraspanin-enriched microdo- in a complex with dystrophin. Campbell and colleagues mains within the membrane to regulate intracellular cell exploited the presence of several glycoproteins within signaling (for review, [20-23]). Similarly, SSPN forms the DGC to enrich the complex using succinylated higher ordered homo-oligomers by laterally associating wheat germ agglutinin (sWGA) lectin chromatography with one another in the sarcolemma of skeletal muscle of digitonin-solubilized skeletal muscle membranes (Figure 1) [24]. [1-5]. sWGA enrichments containing the DGC can be Reconstitution of SSPN oligomerization using a heter- further purified by diethylaminoethyl (DEAE)-cellulose ologous cell expression system and muscle lysates from ion exchange chromatography, which separates the DGC SSPN transgenic mice reveals the presence of pentamers from abundant calcium channels. It was discovered that that were maintained during high-speed ultracentrifuga- SSPN elutes from DEAE columns at 175 mM NaCl, tion through non-reducing sucrose gradients (Figure 1) along with purified DGC components [7]. A second [24]. Using a site-directed mutagenesis approach, SSPN- characteristic of DGC proteins is their migration as a SSPN interfaces were defined within the intracellular complex during high-speed centrifugation through (N- and C-termini) and extracellular regions of SSPN, sucrose gradients. Only proteins that bind with high suggesting that the formation of SSPN oligomers occurs affinity and specificity will be retained with dystrophin through a complex set of protein interactions (Figure 1). during sucrose gradient fractionation where it migrates Alanine replacement of cysteine residues reveals that as an 18S complex [2-4,12]. SSPN co-migrates with peak intramolecular thiol bridges between Cys 162 and Cys Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 3 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Figure 1 SSPN interacts with the sarcoglycans and forms oligomers characteristic of tetraspanins. SSPN is a tetraspanin-like protein, with four transmembrane domains, which complexes with the DGC and UGC at the sarcolemmal membrane of skeletal muscle. Site directed mutagenesis of SSPN revealed that the N- and C-termini as well as regions of the large extracellular loop (LEL, between transmembrane domains 3 and 4) are necessary for SSPN-SSPN and SSPN-SG interactions, respectively [24]. Deletion mutagenesis, in which regions of six amino acids were removed at a time, was performed to identify regions within SSPN that are important for protein interactions (left). The N-terminus and C- terminus (green) are critical for SSPN dimer formation and the LEL is important for trimer and tetramer oligomers. SSPN-SG interactions were identified in the LEL (purple) and mutations in the LEL disrupt SSPN monomer formation, likely due to disruption of thiol bonds (orange) critical for stabilizing the structure of SSPN. Immunoblot analysis of skeletal muscle lysates from SSPN transgenic mice demonstrates that SSPN forms homo-oligomers under non-reducing (NR) conditions (middle) [24]. In reducing conditions (R), SSPN exists solely in monomeric form. Similar to all tetraspanins, SSPN forms higher ordered structures through homo-oligomerization. We propose a model whereby SSPN-SSPN oligomers form a scaffold on which the DGC and UGC complexes are assembled (right). DGC, dystrophin-glycoprotein complex; DG, α/β dystroglycan; SG, sarcoglycans; UGC, utrophin-glycoprotein complex. 164 within the LEL are critical determinants of SSPN to tetraspanins, CD20 forms multimeric oligomers structure [24]. In fact, mutation of any cysteine within within the plasma membrane and is unlikely to exist the LEL disrupts cysteine packing within the LEL, lea- solely in a monomeric state [30]. The specific function ding to destabilization of SSPN monomer formation of CD20 has not been elucidated, but CD20 localizes to [24]. Based on structural and functional analysis of SSPN lipid rafts where it may play a role in regulating cell overexpression in several mouse models, it is reasonable cycle progression, tyrosine kinase-dependent signaling, that multiple SSPN proteins may interact with each and B-cell differentiation (for review, [31]). adhesion complex, thereby mediating cross-talk between transmembrane glycoprotein complexes. The biological Sarcospan and the sarcoglycans form a subcomplex significance of SSPN oligomers mediating protein- The SGs are single pass transmembrane glycoproteins protein interactions between adhesion complexes is referred to as α-, β-, γ-, and δ-SG (for review, [6]). The appealing, but requires further investigation. first evidence that the DGC is composed of bioche- Although SSPN exhibits many tetraspanin-like charac- mically distinct subcomplexes came from experiments in teristics, it may be more structurally similar to the CD20 which purified DGC was subjected to alkaline conditions family of proteins, which includes the beta subunit of that dissociate pH-sensitive protein interactions [4]. The the high affinity receptor for IgE Fc [25]. Members of finding that SSPN tightly associates with the SG sub- the CD20 family span the plasma membrane four times complex is supported by sucrose gradient analysis of and possess a LEL (between transmembrane domains 3 alkaline-treated preparations revealing that SSPN and 4) as well as intracellular N- and C-termini [26-29]. co-sediments exclusively with the SGs [32]. Further- The regions of homology that define the CD20 family more, treatments with denaturing agents such as sodium are largely within the transmembrane domains. Similar dodecyl sulfate [33] and n-octyl β-D-glucoside [34] fail Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 4 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 to disrupt the integrity of the SG-SSPN subcomplex. an autosomal homolog of dystrophin and forms a func- Finally, the SG-SSPN subcomplex can be reconstituted tionally similar utrophin-glycoprotein complex (UGC) in an in vivo cell culture model system lacking expres- where utrophin replaces dystrophin [54-56]. In normal sion of DGs and dystrophin [32]. SSPN also associates muscle, the UGC is located at the postsynaptic mem- with the SGs in smooth muscle purified from kidney brane of neuromuscular junctions (NMJ) [55,57,58]. and lung tissues [35-38] as well as epididymal white adi- Overexpression of both full-length utrophin and mini- pose tissue [11]. constructs ameliorates dystrophic pathology in the mdx Mutations in the α-, β-, γ-, or δ-SG genes cause auto- mouse model of DMD [59-68]. Mice engineered to over- somal recessive limb-girdle muscular dystrophy (AR- express threefold levels of human SSPN driven by the LGMD) type 2D, 2E, 2C, and 2F, respectively (for review, muscle-specific human skeletal actin promoter increased [39]) leading to absence or a significant reduction in the ectopic expression of utrophin, dystrophin, and α7β1 SG subcomplex from the sarcolemma (for review, [40]). integrin at the sarcolemma (Figure 2) [19,69]. In fact, Genetic ablation of individual SG genes in mice has gen- analysis of SSPN transgenic mice overexpressing 0.5- erated robust animal models for the SG-deficient AR- and 1.5-fold levels of SSPN reveals that these molecular LGMDs in which the entire SG complex is absent from events occur in a SSPN dose-dependent manner the sarcolemma (for review, [40]). Similarly, a large dele- (Figure 2). Introduction of SSPN (threefold) ameliorates tion in the δ-SG gene causes cardiomyopathic and myo- dystrophy in the mdx mouse model of DMD by reducing pathic features in the BIO14.6 hamster model [41,42]. cycles of degeneration/regeneration and preventing Consistent with its tight biochemical association with sarcolemma damage (Figure 3). Biochemical purification the SGs, SSPN is absent from the sarcolemma of mice of the UGC complex using lectin affinity chromatog- deficient in α-, β-, and δ-SG as well as the δ-SG deficient raphy followed by sucrose gradient ultracentrifugation BIO14.6 hamster [32,36,43-46]. SSPN expression is revealed that SSPN is a component of the UGC [19,69]. restored to normal levels in BIO14.6 muscle after deli- Consistent with the role of SSPN in regulating adhesion very of an adenovirus encoding δ-SG [32]. Furthermore, complexes at the cell surface, overexpression of 10-fold investigation of over 30 AR-LGMD muscle biopsies with levels of SSPN causes formation of insoluble protein primary mutations in α-, β-, or γ-SG genes that result in aggregates at the sarcolemma, resulting in muscle path- either complete or partial absence of the SGs revealed ology [70]. To date, thirteen human SSPN transgenic that SSPN was absent from the sarcolemma [46]. The lines have been created and only one line expressed levels of SSPN expression were not analyzed in γ-SG tenfold levels of SSPN. In both 3- and 1.5-fold lines of deficient mice [47], but it would be interesting to deter- SSPN expression, internal down regulation of endoge- mine if the trend was similar to the observations made nous SSPN was observed, suggesting that the levels of in human AR-LGMD biopsies. Interaction between the SSPN are tightly controlled within the cell [19]. Based SGs and SSPN is very sensitive to structural perturba- on this data, it may be unlikely to achieve tenfold levels tions within the LEL of SSPN, as revealed by alanine of SSPN in a clinical setting. Future studies are needed scanning and deletion mutagenesis within the LEL to determine whether SSPN amelioration of dystrophic (Figure 1) [24]. SSPN interaction with the SGs is not pathology occurs in aged mdx mice and whether SSPN unique to skeletal muscle. The SSPN-SG subcomplex ameliorates disease in mouse models of laminin- has been characterized in many tissues, including the deficient congenital muscular dystrophy and SG- smooth muscle from lung and kidney [11,35,48-51]. In deficient LGMD. this context, SSPN interacts with a modified SG sub- complex consisting of β-, γ-, and ε-SG (a homolog of Sarcospan affects glycosylation of α-dystroglycan α-SG). Interestingly, the SSPN-SG subcomplex does not The cytotoxic T cell (CT) GalNAc transferase (Galgt2) is co-migrate in sucrose gradient fractions of sWGA puri- confined to the NMJ and catalyzes addition of the ter- fied DG from epithelial cells derived from lung or kidney minal β1,4 GalNAc residues onto the CT carbohydrate tissue [35]. SSPN is not conserved in Drosophila mela- of a subset of α-DG proteins [71,72]. α-DG is the pre- nogaster and Caenorhabditis elegans, thus the resulting dominant glycoprotein modified with the CT carbohy- DGC equivalent lacks SSPN and is predicted to be com- drate in skeletal muscle where it is enriched at the posed of DGs, SGs, and dystrophin [52,53]. postsynaptic membrane of the NMJ [73]. Overexpression of Galgt2 in mdx mice increases abundance and extrasy- Sarcospan uniquely increases abundance of laminin- naptic expression of α-DG modified with the CT anti- binding complexes gen, resulting in improved laminin-binding activity The functional replacement of utrophin for dystrophin is [72,73]. Overexpression of SSPN in mdx mice increases one of many attractive therapeutic strategies for the GalNAc modifications in a similar manner to the over- treatment of Duchenne muscular dystrophy. Utrophin is expression of Galgt2, as revealed by the increased cell Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 5 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Figure 2 SSPN increases abundance of laminin-binding complexes at the sarcolemma. Several lines of SSPN-transgenic mice with 0.5-, 1.5-, and 3-fold levels of SSPN overexpression were generated to investigate the dose-dependent effects of SSPN expression. SSPN transgenic mice on 0.5 1.5 3.0 0.5 1.5 3.0 C57/Bl6 background (WT, WT , WT ,WT ) as well as 0.5-, 1.5-, and 3-fold SSPN transgenic on mdx background (mdx, mdx , mdx , mdx ) were analyzed [19,70]. (A) Transverse cryosections of quadriceps muscle from six-week old SSPN-transgenic mice were stained with antibodies to dystrophin (Dys) and hSSPN to reveal exogenous SSPN expression (hSSPN Tg). (B) Muscle sections from SSPN-transgenic mdx mice were also stained with utrophin (Utr) and hSSPN. Sections overlayed with Wisteria floribunda agglutinin (WFA) lectin reveal increased cell surface glycosylation with elevations in SSPN overexpression [19]. WFA lectin binds terminal GalNAc residues and serves as a marker for the CT antigen modification of α-DG that normally occurs at the NMJ [19]. Note that SSPN increases cell surface expression of dystrophin, utrophin, and glycosylation in a manner dependent on SSPN abundance. Bar, 50 μm. CT, cytotoxic T cell; NMJ, neuromuscular junction. surface binding of the lectin Wisteria floribunda agglu- laminin binding or reduce muscle degeneration/regener- tinin (WFA) [19], which is a marker for NMJ-specific ation, revealing a minimum (threefold) level of SSPN CT carbohydrate modification of α-DG (Figure 2) needed for ‘rescue’ (Figure 2) [19]. [72,74-77]. WFA binding is localized to NMJs in normal The ‘dystroglycanopathies’ are a group of disorders resul- muscle and is increased around the extra-synaptic sarco- ting from hypoglycosylation of α-DG that abolishes its lemma of mdx muscle cryosections [19,78]. WFA bind- laminin-binding function. A spontaneous mutation in the 3.0 ing to SSPN-transgenic mdx (mdx ) muscle was LARGE gene, which encodes an enzyme with xylosyltransfe- significantly increased around the extra-synaptic sarco- rase and glucuronyltransferase activities, causes muscular lemma similar to utrophin expression [19]. Increased dystrophy in the myodystrophy (myd) mouse [83]. In myd Galgt2 activity in mdx mice results from a two-fold ele- muscle, α-DG is hypoglycosylated and exhibits severely vation of Galgt2 mRNA levels in mdx muscle relative to reduced ligand binding activity due to loss of the glycan- wild-type controls [73]. However, SSPN does not affect laminin binding domain on α-DG [84,85]. LARGE elongates Galgt2 transcript abundance, raising the possibility that phosphorylation dependent glycosylaminoglycan modifica- SSPN increases Galgt2 activity or improves α-DG as a tions on the central mucin domain of α-DG by direct inter- substrate for Galgt2 [19]. SSPN also increases laminin at action with α-DG [86,87]. Loss of LARGE increases the sarcolemma as well as levels of plectin-1, which utrophin and SSPN staining and WFA binding around the binds cytoskeletal proteins including β-DG, dystrophin, extra-synaptic sarcolemma of myd muscle [19]. Introduction utrophin, and F-actin [79-82], supporting the conclusion of the SSPN transgene into skeletal muscle of myd mice fur- that SSPN strengthens the structural connection be- ther elevated WFA binding along with broad, extra-synaptic tween actin and laminin across the sarcolemma [19]. localization of utrophin, while removal of SSPN from myd Furthermore, laminin binding to α-DG was restored to muscle reduced utrophin and GalNAc-glycan modification normal levels in threefold SSPN overexpressing mdx of α-DG [19]. Pathology of myd muscle was unaffected by muscle [19]. Transgenic overexpression of 0.5- and 1.5- the loss of SSPN or SSPN overexpression, demonstrating fold levels of SSPN increased glycosylation of α-DG, Akt that alterations in GalNAc glycosylation of α-DG or utrophin signaling, and utrophin levels, but failed to restore abundance do not affect absence of the main laminin- Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 6 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Figure 3 SSPN overexpression ameliorates dystrophic pathology in mdx mice. Transverse cryosections of quadriceps muscle from six-week 3.0 old mdx and SSPN transgenic mdx (mdx ) mice were stained with hematoxylin and eosin (H&E) and visualized at low magnification to reveal the extensive areas of necrosis, denoted in white, in mdx muscle (A). Necrosis was significantly diminished in SSPN transgenic mdx muscle [69]. Sections were also viewed at higher magnification for evaluation of degeneration/regeneration, which is marked by central nucleation of myofibers (B). Overexpression of SSPN in mdx muscle dramatically reduced central nucleation [69]. Bar, 50 μm. binding domain on α-DG [19]. The conclusion from these SSPN knockdown in a cultured glioma cell line experiments is that Galgt2 (or enzyme with similar function) (LN-229) did not affect cell division as determined by modifies α-DG in the absence of ‘LARGE’ glycans, demon- bromodeoxyuridine (BrdU) incorporation, but did in- strating that GalNAc modification of α-DG can occur inde- crease vulnerability of glioma cells to hypoxia [92]. pendently of O-mannose-linked glycans. Additionally, these Genetic ablation of SSPN did not appear to alter data reveal that GalNAc carbohydrate structures on α-DG muscle physiology or strength in young mice [93]. How- are unable to compensate for the loss of LARGE glycans, ever, when SSPN-null mice were analyzed at older ages which constitute the major laminin-binding motif. (4.5 month old), several deficiencies emerged. Reduction in the levels of the UGC and DGC as well as diminished A newly discovered phenotype for sarcospan-null mice NMJ-specific glycosylation of α-DG and decreased Evidence that SSPN loss may affect skeletal muscle was laminin-binding was observed in aged SSPN-nulls [14]. first suggested from comparative microarray analysis re- Diaphragms from older SSPN-null mice exhibited dimi- vealing a mild decrease in mRNA levels of dystrophin nished specific force generating capacity and were more and α-SG in SSPN-null muscle [88]. Furthermore, the susceptible to eccentric-contraction induced damage as authors reported increased expression of two genes, evidenced by the increased percentage drop in force osteopontin and the S100 calcium-binding protein cal- compared to wild-type controls [14]. These physiological granulin B, that have been implicated in immunological phenotypes were not observed in extensor digitorum function and fibrosis [89-91]. A second gene expression longus (EDL) or soleus muscles, suggesting that decrea- study compared hippocampus and cortex of mice sed DGC expression in EDL or soleus muscles is insuffi- exposed to chronic constant hypoxia (CCH) and chronic cient to manifest in loss of muscle strength or sarcolemma intermittent hypoxia (CIH). CCH occurs in chronic lung damage [14,93]. diseases or at high altitudes while CIH develops from disorders such as sleep apnea or sickle cell disease. SSPN Sarcospan genetically interacts with integrins was one of two identified genes down-regulated in the It is well established that all tetraspanins interact with hippocampus and cortex after both treatments [92]. integrin partners to regulate cell signaling, adhesion, and Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 7 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 laminin-binding capacity of integrins (for review, [94,95]). The table summarizes phenotypic and biochemical data The primary integrin expressed in adult skeletal muscle from 4.5-month old SSPN-null, Itga7-null, and SSPN-null: is α7β1 integrin, whichis localized at the NMJ and myo- Itga7-null double knockout (DKO) mice. All comparisons tendinous (MTJ) regions within the sarcolemma [96-99]. are relative to age-matched wild-type mice. Survival stu- The relatively mild phenotype of mdx mice has been at- dies were carried out to eight months of age. At this time tributed to increased, compensatory expression of both point, 40% of the DKO mice had succumbed to death utrophin and α7β1 integrin in response to loss of dys- compared to less than 5% of the controls. The extent of trophin that, when ablated, exacerbates mdx pathology kyphosis was documented in 4.5-month and 6-month old [100-102]. Overexpression of Itga7 in mdx:utrophin-null mice. Itga7-null mice exhibited minor kyphosis and the mice and β1 integrin in mdx mice ameliorates pathology additional loss of SSPN caused severe kyphosis in DKO [103-105]. The recent observation that α7β1integrin mice. Myofiber cross-sectional area (CSA) quantified from levels are increased in response to SSPN deficiency is the quadriceps and diaphragm muscles at 4.5 months of intriguing as it suggests that α7β1integrincompen- age are represented. DKO muscles exhibited an increase sates for SSPN function and moderates the severity of in myopathy and increase in very small (0 to 500 μm ) muscle phenotypes in SSPN-nulls [14]. Analysis of myofibers. Central nucleation is provided as an indicator SSPN-deficient and Itga7-deficient double knockout of overall muscle phenotype. Analysis of sarcolemmal (DKO) mice supports this hypothesis. In comparison damage (Evans blue dye assay) and fibrosis (Van Geison) to controls at 4.5-months of age, DKO mice exhibit of the quadriceps and diaphragm muscles at 4.5 months of increased kyphosis and premature lethality at one month age are exacerbated in DKOs [14]. The levels of utrophin, of age, which is significant given that the single-nulls dystrophin, laminin-binding to α-DG, and β1 integrin display no overt signs of pathology or lethality (Table 1) were analyzed by densitometry of sWGA eluates from [14]. Furthermore, DKO muscle appears severely dys- digitonin-solubilized total skeletal muscle lysates. SSPN- trophic with extensive fibrosis surrounding individual deficient mice exhibit a reduction in the UGC, DGC, and hypertrophic muscle fibers, in a manner identical to laminin-binding to α-DG and a corresponding compensa- histological images of DMD biopsies [14]. Genetic re- tory increase in β1 integrin [14]. Loss of Itga7 results in a moval of Itga7 from SSPN-nulls further reduced levels reduction in the levels of the DGC and laminin-binding to of the DGC at the sarcolemma, diminished laminin- α-DG. Importantly, combined loss of Itga7 and SSPN binding to α-DG, and consequently decreased specific causes further reduction of the DGC, UGC, and laminin- force output in the diaphragm (Table 1) [14]. The con- binding to α-DG compared to all controls. The relative clusion from this work is that SSPN is a necessary com- levels of P-Akt/Akt and P-IGFR/IGFR are provided. Spe- ponent of dystrophin and utrophin function and that cific force measurements of the diaphragm muscles reveal SSPN modulation of integrin signaling is required for a loss of specific force in SSPN-null and Itga7-null that is growth, extracellular matrix attachment, and muscle force additive in DKO mice. Interestingly, specific force produc- development. tion of the EDL and soleus muscles are unaffected by the Table 1 Sarcospan- and α7 integrin-double nulls display severe growth and muscle phenotypes a a a a a a Genotype Survival Kyphosis Myofiber Central Utrophin Dystrophin Integrin Laminin- Signaling Specific Force a a a a Analysis CSA Nucleation Binding Wild type 100% No Normal Normal 100% 100% 100% 100% P-Akt/Akt: Normal (EDL, Soleus, 100% Diaphragm) P-IGFR/IGFR: 100% SSPN null 100% No Normal Normal 31% 47% 293% 69% P-Akt/Akt: Normal (EDL, Soleus); 49% Decrease (Diaphragm) P-IGFR/IGFR: 80% Itga7 null 95% Minor Normal Normal 102% 48% Absent 85% P-Akt/Akt: Decrease (Diaphragm) 95% P-IGFR/IGFR: 99% DKO 60% Severe Decrease Increase 44% 34% Absent 28% P-Akt/Akt: Severe Decrease 40% (Diaphragm) P-IGFR/IGFR: 35% Measurements represented relative to wild type. DKO, double knockout; Itga7, α7 integrin. Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 8 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 loss of SSPN suggesting that, in these muscles, integrin [108,109]. Interestingly, overexpression of threefold may successfully compensate for DGC or that reduction levels of SSPN results in amelioration of mdx dystrophic of the DGC does not affect EDL and soleus muscle pathology through stabilization of the UGC and α7β1 physiology. integrin at the sarcolemma, and activation of Akt and downstream p70S6K [19,70]. Conversely, the Akt/ Sarcospan activates Akt signaling pathways to facilitate p70S6K pathway and activation of the IGF receptor is regeneration depressed in SSPN-deficient mice, rendering the muscle Modulation of the phosphatidylinositol-3 kinase (PI3K)/ unable to repair efficiently after cardiotoxin-induced Akt signaling pathway leading to downstream activation injury (Figure 4) [19]. Pretreatment of SSPN-null muscle of p70S6K protein synthesis pathways is important for with adenovirus expressing constitutively active Akt regulation of muscle strength, hypertrophy, and patho- increased the UGC to normal levels and restored muscle physiology (for review, [106,107]). The Akt signaling cas- regeneration after cardiotoxin-injury (Figure 4) [19]. The cade through p70S6K is activated by several receptors, conclusion from these experiments is that SSPN modu- including: insulin like growth factor 1 (IGF-1) and β1 lates utrophin protein levels at least in part through integrin associated integrin-linked kinase (ILK) (for re- Akt/p70S6K signaling pathways (Figure 5). view, [106,107]). Overexpression of constitutively active Akt in skeletal muscles of dystrophin-deficient mdx mice A chaperone-like function for sarcospan is emerging results in increased abundance of utrophin and α7β1 in- It has been assumed that the prematurely truncated dys- tegrin, which leads to improvements in force generation trophin protein produced from the mdx mutation is Figure 4 Muscle recovery from cardiotoxin injury requires SSPN-dependent Akt activation. (A) An acute injury model was used to investigate muscle repair in wild-type and SSPN-null mice. Quadriceps injected with equivalent volumes of saline (mock) and cardiotoxin dissolved in saline (CTX) were evaluated. CTX causes localized regions of myofiber necrosis followed by regeneration (for review, [125]). Mice at six weeks of age were injected with CTX (or mock) and analyzed after seven days. To test the dependency of muscle repair on Akt signaling, a subset of mice was pre-treated with adenovirus containing constitutively active Akt (Ad-caAkt) two days prior to CTX treatment. (B) Analysis was performed using H&E, laminin/eMHC, and laminin/EBD stained sections from quadriceps muscle. SSPN-deficient mice exhibit increased active regeneration (eMHC, 30%) seven days after CTX injury when wild-type mice have already undergone successful repair (black). Administration of Ad-caAkt prior to CTX treatment rescued the repair defect in SSPN-deficient mice (grey). (C) Immunoblot analysis of RIPA quadriceps lysates revealed reductions in the levels of dystrophin (Dys), utrophin (Utr), integrin (β1 integrin), phosphorylated Akt (P-Akt), and phosphorylated p70S6K (P-p70S6K) in SSPN-null mice compared to wild-type. Quantification of utrophin and P-Akt is provided. Coomassie blue (CB) serves as a loading control. (D) Immunoblot analysis of RIPA lysates from quadriceps pre-treated with Ad-caAkt revealed reductions in the levels of dystrophin (Dys) and integrin (β1 integrin) in SSPN-null mice compared to wild-type. Utrophin levels were restored in SSPN-null mice compared to wild-type mice with supplementation of active Akt (right). These data reveal that SSPN is upstream of the Akt signaling pathway regulating utrophin expression. The Ad-caAkt contains the HA-tag for detection [19]. RIPA, radioimmunoprecipitation assay. Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 9 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Figure 5 Sarcospan is a critical regulator of laminin-binding receptors in muscle. (A) The DGC/UGC and α7β1 integrin at the sarcolemma 3.0 in mdx (SSPN-Tg:mdx) muscle is depicted. The dystroglycans (DGs; pink), sarcoglycans (SGs; yellow), sarcospan (SSPN; blue), dystrophin (grey) and integrins (purple) are shown. Overexpression of SSPN in mdx muscle elicits a series of molecular events that lead to restoration of laminin binding, amelioration of pathology, and restoration of membrane integrity [19]. As shown in the illustration, SSPN activates Akt, which stabilizes utrophin, and increases the abundance of integrin and WFA-reactive α-DG at the cell surface. SSPN facilitates increased CT antigen modification of α-DG and enhances transportation of utrophin-DG at the sarcolemma [19]. Collectively, these events lead to stabilization of the sarcolemmal membrane and amelioration of dystrophic pathology. (B) SSPN-null muscle exhibits decreased dystrophin and Akt activation followed by decreased expression of utrophin, resulting in the reduction of laminin-binding to α-DG (middle panel) [19]. Acute muscle injury by cardiotoxin injection into SSPN-null muscle impairs muscle regeneration (right) [19]. However, pre-treatment of SSPN-null mice with adenovirus containing constitutively active Akt (Ad-caAkt) restored the activation of downstream p70S6K, utrophin expression, and improved muscle regeneration (right panel). These studies reveal the importance of sarcospan and Akt in regulating utrophin expression that is critical for muscle repair. CT, cytotoxic T cell; DGC, dystrophin-glycoprotein complex; UGC, utrophin-glycoprotein complex; WFA, Wisteria floribunda agglutinin. rapidly degraded in muscle based on lack of its detection sarcolemma, suggesting that SSPN possesses chaperone- in whole skeletal muscle extracts. However, recent work like functions to improve protein folding and/or traffick- has revealed that truncated dystrophin protein is synthe- ing to the cell surface (Figure 5). sized in mdx mice [19]. In fact, truncated dystrophin proteins are produced and detected in high abundance Sarcospan as a candidate disease gene in ER/golgi compartments in mdx muscle, suggesting The sarcospan gene is localized to human chromosome that they accumulate in intracellular compartments due 12p11.2 and is encoded by three small exons that are to insufficient transportation to the cell surface [19]. separated by very large introns [8,9]. A novel exon 4 was These data are exciting as they reveal for the first time recently identified to encode for an alternative C- that truncated dystrophin fragments are synthesized in terminal region in humans. In fact, alternate mRNA spli- mdx muscle, but then retained in intracellular mem- cing of human SSPN exons 1 and 2 to exon 4 generates brane compartments rather than properly transported to a protein called microspan (μSPN) that lacks transmem- the sarcolemma. brane domains 3 and 4 as well as the LEL so that the re- In addition to its role at the cell surface, a role for sultant protein has only two transmembrane spans and a SSPN within the ER/golgi is suggested from recent stu- novel intracellular C-terminus [10]. μSPN does not dies. Biochemical analysis of ER/golgi membranes iso- interact with the DGC and its expression is maintained lated from mdx muscle revealed abundant levels of in dystrophin-deficient muscle. Although μSPN is not utrophin and α-DG relative to wild-type [19]. Interes- localized to the sarcolemma, it is enriched in the sarco- tingly, utrophin and α-DG are reduced in ER/golgi pre- plasmic reticulum (SR) [10]. Overexpression of μSPN in parations from SSPN transgenic mdx muscle while these skeletal muscle of transgenic mice reduces levels of rya- same proteins are increased in abundance at the nodine receptor, dihydropyridine receptor as well as Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 10 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 SERCA-1 resulting in aberrant triad morphology [10]. required for SSPN’s ‘rescue’ effect. It is also critical to in- μSPN is also reduced in isolated SR membranes of vestigate whether Itga7 levels are affected by the ma- δ-SG-null muscle contributing to SERCA dysfunction ny other genes that increase utrophin expression, which [110]. Given that both SSPN and μSPN interact with would reveal important mechanisms regulating laminin- proteins that are critical to skeletal muscle function, it binding receptors in skeletal muscle. SSPN is a promising can be hypothesized that genetic mutations affecting therapeutic target, particularly for adeno-associated virus SSPN function would have significant consequences for delivery due to its small size and low potential for un- muscle. PCR-based approaches to screen muscular dys- wanted immune reaction. Future studies will reveal the trophy patients for possible abnormalities within the potential of this small protein to alleviate the significant SSPN gene have not yet produced any disease-causing problem of DMD. mutations, although several single-nucleotide poly- Abbreviations morphisms were identified in exons 2 and 3 [111]. SSPN BTX: bungarotoxin; CCH: chronic constant hypoxia; CIH: chronic intermittent was also excluded as a candidate disease gene for con- hypoxia; CSA: cross-sectional area; CT: cytotoxic T cell; DAP: dystrophin- associated protein; DEAE: diethylaminoethyl; DG: dystroglycan; genital fibrosis of the extraocular muscle (CFEOM), DGC: dystrophin-glycoprotein complex; DKO: double knockout; which is an autosomal dominant disorder linked to the DMD: Duchenne muscular dystrophy; Dys: dystrophin; EBD: Evans blue dye; pericentromere of chromosome 12 [112,113]. These EDL: extensor digitorum longus; IGF-1: insulin-like growth factor; ILK: integrin- linked kinase; Intg: integrin; LEL: large extracellular loop; NMJ: neuromuscular findings may come as no surprise given the apparently junction; PCR: polymerase chain reaction; SDS: sodium dodecyl sulfate; normal phenotype of SSPN-deficient mice [93]. How- SG: sarcoglycan; SSPN: sarcospan; UGC: utrophin-glycoprotein complex; ever, re-analysis of SSPN-deficient mice revealed signifi- Utr: utrophin; WFA: Wisteria floribunda agglutinin; sWGA: succinylated wheat germ agglutinin; μSPN: microspan. cant phenotypes in muscle of aged mice and after exposure to conditions of cellular stress that may have Competing interests implications for disease (Figure 5) [14,19]. Furthermore, The authors declare that they have no competing interests. the idea that SSPN may serve as a chaperone to improve Authors’ contributions cell surface expression of the DGC and UGC make it an Both authors contributed to writing and preparation of figures for the excellent candidate as a genetic modifier of disease. manuscript. All authors have read and approved the final manuscript. Authors’ information Conclusions Rachelle H. Crosbie-Watson, Ph.D. is a Professor of Integrative Biology and Overexpression of many proteins and compounds amelio- Physiology at the University of California Los Angeles and her research group rates dystrophic pathology in the mdx mouse by increasing is focused on investigation of macromolecular adhesion complexes at the cell surface in normal and dystrophic skeletal muscle. Professor Crosbie- UGC abundance at the extrasynaptic sarcolemma. A sam- Watson earned a Ph.D. in biochemistry from the University of California Los pling of these includes: CT GalNAc transferase (Galgt2) Angeles investigating structure-function relationships of contractile proteins [73], ADAM12 [114], heregulin [115], L-arginine [116,117], and she identified sarcospan during her MDA-sponsored postdoctoral fellowship at the University of Iowa College of Medicine with Professor Kevin activated calcineurin-A alpha [118], N-acetylcysteine P. Campbell (HHMI). [119-121], activated Akt [108,109], GW501516 (activates Jamie L. Marshall, Ph.D. is a postdoctoral researcher in Dr. Crosbie-Watson’s PPAR beta/delta) [122], artificial gene Jazz [123], and big- laboratory at the University of California Los Angeles. Dr. Marshall earned a Ph.D. in Molecular, Cellular, and Integrative Physiology from the University of lycan [124]. Several studies have now revealed that SSPN California Los Angeles investigating the role of sarcospan in the adhesion can be added to this list of secondary proteins that modify glycoprotein complexes at the sarcolemma. Dr. Marshall pioneered utrophin expression [19,69]. The mechanism by which purification the utrophin-glycoprotein complex [69] and discovered novel deficits of this complex in sarcospan-deficient muscle [14,19,]. Dr. Marshall SSPN increases expression of utrophin involves activation has received numerous predoctoral fellowships as well as a postdoctoral of Akt signaling and increased glycosylation of α-DG, fellowship based on these studies. Dr. Marshall has extensive experience in likely by increased modification of Galgt2 [19]. Intro- genetics, analysis of dystrophic mouse models, and detailed biochemical investigation of the dystrophin- and utrophin-glycoprotein complexes. duction of constitutively active Akt or Galgt2 alone also improves extrasynaptic utrophin expression, strongly sug- Acknowledgements gesting that SSPN, Akt, and Galgt2 may act via a common This work was supported by grants from the Genetic Mechanisms Pre- or overlapping pathway(s). It will be important to de- doctoral Training Fellowship USPHS National Research Service Award GM07104, the Edith Hyde Fellowship, the Eureka Pre-doctoral Training termine whether every gene that increases utrophin ex- Fellowship, and the Ruth L. Kirschstein National Research Service Award pression also alters Akt and α-DG glycosylation, which T32AR059033 from the National Institute of Arthritis and Musculoskeletal and would provide further evidence for a common post- Skin Diseases to J.L.M.; NIH/NIAMS (R01 AR048179) to R.C.W. transcriptional mechanism controlling utrophin abun- Author details dance. Furthermore, these data reveal that there are Department of Integrative Biology and Physiology, University of California multiple targets that affect utrophin, which is encou- Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, Los Angeles, CA 90095, USA. Center for Duchenne Muscular Dystrophy, raging for pharmacological and gene-based therapies. University of California Los Angeles, Los Angeles, CA 90095, USA. Molecular SSPN also increases expression of Itga7, and future Biology Institute, University of California Los Angeles, Los Angeles, CA 90095, studies will determine whether Itga7 and/or utrophin are USA. Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 11 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Received: 16 September 2012 Accepted: 27 November 2012 25. Hupp K, Siwarski D, Mock BA, Kinet JP: Gene mapping of the three Published: 3 January 2013 subunits of the high affinity FcR for IgE to mouse chromosomes 1 and 19. J Immunol 1989, 143:3787–3791. 26. Einfeld DA, Brown JP, Valentine MA, Clark EA, Ledbetter JA: Molecular References cloning of the human B cell CD20 receptor predicts a hydrophobic 1. Campbell KP, Kahl SD: Association of dystrophin and an integral protein with multiple transmembrane domains. EMBO J 1988, membrane glycoprotein. Nature 1989, 338:259–262. 7:711–717. 2. 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K, Crosbie RH: Myogenic Akt signaling upregulates the utrophin- glycoprotein complex and promotes sarcolemma stability in muscular dystrophy. Hum Mol Genet 2009, 18:318–327. 109. Kim MH, Kay DI, Rudra RT, Chen BM, Hsu N, Izumiya Y, Martinez L, Spencer MJ, Walsh K, Grinnell AD, Crosbie RH: Myogenic Akt signaling attenuates muscular degeneration, promotes myofiber regeneration and improves http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Sarcospan: a small protein with large potential for Duchenne muscular dystrophy

Skeletal Muscle , Volume 3 (1) – Jan 3, 2013

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Copyright © 2013 by Marshall and Watson; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Purification of the proteins associated with dystrophin, the gene product responsible for Duchenne muscular dystrophy, led to the discovery of the dystrophin-glycoprotein complex. Sarcospan, a 25-kDa transmembrane protein, was the last component to be identified and its function in skeletal muscle has been elusive. This review will focus on progress over the last decade revealing that sarcospan is an important regulator of muscle cell adhesion, strength, and regeneration. Investigations using several transgenic mouse models demonstrate that overexpression of sarcospan in the mouse model for Duchenne muscular dystrophy ameliorates pathology and restores muscle cell binding to laminin. Sarcospan improves cell surface expression of the dystrophin- and utrophin-glycoprotein complexes as well as α7β1 integrin, which are the three major laminin-binding complexes in muscle. Utrophin and α7β1 integrin compensate for the loss of dystrophin and the finding that sarcospan increases their abundance at the extra-synaptic sarcolemma supports the use of sarcospan as a therapeutic target. Newly discovered phenotypes in sarcospan-deficient mice, including a reduction in specific force output and increased drop in force in the diaphragm muscle, result from decreased utrophin and dystrophin expression and further reveal sarcospan’s role in determining abundance of these complexes. Dystrophin protein levels and the specific force output of the diaphragm muscle are further reduced upon genetic removal of α7 integrin (Itga7) in SSPN-deficient mice, demonstrating that interactions between integrin and sarcospan are critical for maintenance of the dystrophin-glycoprotein complex and force production of the diaphragm muscle. Sarcospan is a major regulator of Akt signaling pathways and sarcospan-deficiency significantly impairs muscle regeneration, a process that is dependent on Akt activation. Intriguingly, sarcospan regulates glycosylation of a specific subpopulation of α- dystroglycan, the laminin-binding receptor associated with dystrophin and utrophin, localized to the neuromuscular junction. Understanding the basic mechanisms responsible for assembly and trafficking of the dystrophin- and utrophin-glycoprotein complexes to the cell surface is lacking and recent studies suggest that sarcospan plays a role in these essential processes. Keywords: Akt, Cell adhesion, Duchenne, Dystrophin, Integrin, Laminin-binding, mdx, Muscular dystrophy, Neuromuscular junction, Regeneration, Sarcolemma, Sarcospan, Utrophin Review that result in loss of dystrophin, a protein that is nor- Identification of sarcospan mally localized to the subsarcolemma [1-5]. Discovery of Muscular dystrophies represent a group of progressive dystrophin-associated proteins, referred to as the muscle disorders characterized by extensive muscle dystrophin-glycoprotein complex (DGC), represent a wasting and weakness. Duchenne muscular dystrophy major advancement in the understanding of the DGC’s (DMD) is caused by mutations in the dystrophin gene function in skeletal muscle and provide further support for the contraction-induced sarcolemma injury model underlying DMD pathogenesis [1,2,4,5]. In addition to * Correspondence: rcrosbie@physci.ucla.edu Department of Integrative Biology and Physiology, University of California dystrophin, the DGC is composed of α/β-dystroglycan Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, (DG), the sarcoglycans (SGs), the syntrophins, and dys- Los Angeles, CA 90095, USA trobrevin (for review, [6]). One of the last components Center for Duchenne Muscular Dystrophy, University of California Los Angeles, Los Angeles, CA 90095, USA of the DGC to be identified was a 25-kDa dystrophin- Full list of author information is available at the end of the article © 2013 Marshall and Crosbie-Watson; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 2 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 associated protein (DAP), which was resistant to identifi- DGC-containing fractions providing additional evidence cation, in part due to lack of polyclonal antibodies that that SSPN is an integral component of the DGC cross-reacted with the 25-kDa DAP (also called A5) [5,7,13,14]. In contrast, while a fraction of caveolin-3 from goats and sheep, immunized with the DGC [7]. maintains association with the DGC during purification The hydrophobic probe, 3-trifluoromethyl-3-(m-[ I] by sWGA lectin affinity chromatography, it is localized iodophenyl) diazirine or TID, bound very strongly to the to heavier fractions during sucrose gradient centrifuga- 25-kDa DAP, suggesting that it might be an integral tion [7,13]. For the third criterion, the laminin binding membrane protein [5]. In fact, TID binding to the capacity of α-dystroglycan (α-DG) was exploited to 25-kDa DAP was greater than its binding to the SGs or separate the DGC from other membrane-associated β-DG, which possess a single transmembrane span, pro- proteins. Application of sWGA enrichments from ske- viding strong evidence that the 25-kDa DAP contained letal muscle reveals that SSPN is entirely retained on multiple membrane-spanning regions and was unlikely laminin-sepharose columns, but caveolin-3 is found only to be a protein degradation product, as had been specu- in the void fraction [13,15-19]. Finally, it is well estab- lated based on its weak staining with Coomassie- lished that core components of the DGC depend on Brilliant blue [5,7]. Identification of the 25-kDa DAP dystrophin for localization to the sarcolemma. In was accomplished by in-gel digestion and sequencing of dystrophin-deficient DMD patients and the mdx mouse two amino acid peptides, leading to isolation of the model, SSPN is absent from the sarcolemma while mem- corresponding human cDNA [7] that was previously brane expression of caveolin-3 is not affected by loss of identified in part as Kirsten ras associated gene (krag), a dystrophin [7,13]. gene that is co-amplified with Ki-ras in the Y1 murine adrenal carcinoma cell line [8,9]. The gene was renamed Structural analysis of sarcospan provides insight into to sarcospan (SSPN) for its multiple sarcolemma span- function ning helices predicted from hydropathy analysis [7]. Topology algorithms predict that SSPN possesses four While SSPN is expressed in many non-muscle tissues transmembrane domains with a small extracellular loop [10,11], its biochemical characterization was performed (between transmembrane domains 1 and 2), a large extra- in skeletal muscle where it is most abundant. cellular loop (LEL; between transmembrane domains 3 and 4), and intracellular N- and C-termini (Figure 1) [7]. Rigorous criteria define components of the dystrophin- Although several protein families contain four transmem- glycoprotein complex brane domains, dendrogram analysis suggests that SSPN Although characterization of the 25-kDa DAP led to its is related to the tetraspanins, although not all characteris- identification, there was much speculation that SSPN tics are conserved. Like other tetraspanins, the LEL of was a contaminant of the purified complex rather than a SSPN contains conserved Cys residues that are important bona fide member of the DGC. Integral components of for tertiary structure, although SSPN lacks the hallmark the DGC are defined by four biochemical characteristics Cys Cys Gly motifs within the LEL and conserved sites for and SSPN was rigorously tested with these established N-linked glycosylation and palmitolyation that are charac- criteria. First, purification of the DGC from skeletal teristic of tetraspanins [20]. Tetraspanins facilitate protein muscle membranes enriches proteins that are associated interactions by forming tetraspanin-enriched microdo- in a complex with dystrophin. Campbell and colleagues mains within the membrane to regulate intracellular cell exploited the presence of several glycoproteins within signaling (for review, [20-23]). Similarly, SSPN forms the DGC to enrich the complex using succinylated higher ordered homo-oligomers by laterally associating wheat germ agglutinin (sWGA) lectin chromatography with one another in the sarcolemma of skeletal muscle of digitonin-solubilized skeletal muscle membranes (Figure 1) [24]. [1-5]. sWGA enrichments containing the DGC can be Reconstitution of SSPN oligomerization using a heter- further purified by diethylaminoethyl (DEAE)-cellulose ologous cell expression system and muscle lysates from ion exchange chromatography, which separates the DGC SSPN transgenic mice reveals the presence of pentamers from abundant calcium channels. It was discovered that that were maintained during high-speed ultracentrifuga- SSPN elutes from DEAE columns at 175 mM NaCl, tion through non-reducing sucrose gradients (Figure 1) along with purified DGC components [7]. A second [24]. Using a site-directed mutagenesis approach, SSPN- characteristic of DGC proteins is their migration as a SSPN interfaces were defined within the intracellular complex during high-speed centrifugation through (N- and C-termini) and extracellular regions of SSPN, sucrose gradients. Only proteins that bind with high suggesting that the formation of SSPN oligomers occurs affinity and specificity will be retained with dystrophin through a complex set of protein interactions (Figure 1). during sucrose gradient fractionation where it migrates Alanine replacement of cysteine residues reveals that as an 18S complex [2-4,12]. SSPN co-migrates with peak intramolecular thiol bridges between Cys 162 and Cys Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 3 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Figure 1 SSPN interacts with the sarcoglycans and forms oligomers characteristic of tetraspanins. SSPN is a tetraspanin-like protein, with four transmembrane domains, which complexes with the DGC and UGC at the sarcolemmal membrane of skeletal muscle. Site directed mutagenesis of SSPN revealed that the N- and C-termini as well as regions of the large extracellular loop (LEL, between transmembrane domains 3 and 4) are necessary for SSPN-SSPN and SSPN-SG interactions, respectively [24]. Deletion mutagenesis, in which regions of six amino acids were removed at a time, was performed to identify regions within SSPN that are important for protein interactions (left). The N-terminus and C- terminus (green) are critical for SSPN dimer formation and the LEL is important for trimer and tetramer oligomers. SSPN-SG interactions were identified in the LEL (purple) and mutations in the LEL disrupt SSPN monomer formation, likely due to disruption of thiol bonds (orange) critical for stabilizing the structure of SSPN. Immunoblot analysis of skeletal muscle lysates from SSPN transgenic mice demonstrates that SSPN forms homo-oligomers under non-reducing (NR) conditions (middle) [24]. In reducing conditions (R), SSPN exists solely in monomeric form. Similar to all tetraspanins, SSPN forms higher ordered structures through homo-oligomerization. We propose a model whereby SSPN-SSPN oligomers form a scaffold on which the DGC and UGC complexes are assembled (right). DGC, dystrophin-glycoprotein complex; DG, α/β dystroglycan; SG, sarcoglycans; UGC, utrophin-glycoprotein complex. 164 within the LEL are critical determinants of SSPN to tetraspanins, CD20 forms multimeric oligomers structure [24]. In fact, mutation of any cysteine within within the plasma membrane and is unlikely to exist the LEL disrupts cysteine packing within the LEL, lea- solely in a monomeric state [30]. The specific function ding to destabilization of SSPN monomer formation of CD20 has not been elucidated, but CD20 localizes to [24]. Based on structural and functional analysis of SSPN lipid rafts where it may play a role in regulating cell overexpression in several mouse models, it is reasonable cycle progression, tyrosine kinase-dependent signaling, that multiple SSPN proteins may interact with each and B-cell differentiation (for review, [31]). adhesion complex, thereby mediating cross-talk between transmembrane glycoprotein complexes. The biological Sarcospan and the sarcoglycans form a subcomplex significance of SSPN oligomers mediating protein- The SGs are single pass transmembrane glycoproteins protein interactions between adhesion complexes is referred to as α-, β-, γ-, and δ-SG (for review, [6]). The appealing, but requires further investigation. first evidence that the DGC is composed of bioche- Although SSPN exhibits many tetraspanin-like charac- mically distinct subcomplexes came from experiments in teristics, it may be more structurally similar to the CD20 which purified DGC was subjected to alkaline conditions family of proteins, which includes the beta subunit of that dissociate pH-sensitive protein interactions [4]. The the high affinity receptor for IgE Fc [25]. Members of finding that SSPN tightly associates with the SG sub- the CD20 family span the plasma membrane four times complex is supported by sucrose gradient analysis of and possess a LEL (between transmembrane domains 3 alkaline-treated preparations revealing that SSPN and 4) as well as intracellular N- and C-termini [26-29]. co-sediments exclusively with the SGs [32]. Further- The regions of homology that define the CD20 family more, treatments with denaturing agents such as sodium are largely within the transmembrane domains. Similar dodecyl sulfate [33] and n-octyl β-D-glucoside [34] fail Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 4 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 to disrupt the integrity of the SG-SSPN subcomplex. an autosomal homolog of dystrophin and forms a func- Finally, the SG-SSPN subcomplex can be reconstituted tionally similar utrophin-glycoprotein complex (UGC) in an in vivo cell culture model system lacking expres- where utrophin replaces dystrophin [54-56]. In normal sion of DGs and dystrophin [32]. SSPN also associates muscle, the UGC is located at the postsynaptic mem- with the SGs in smooth muscle purified from kidney brane of neuromuscular junctions (NMJ) [55,57,58]. and lung tissues [35-38] as well as epididymal white adi- Overexpression of both full-length utrophin and mini- pose tissue [11]. constructs ameliorates dystrophic pathology in the mdx Mutations in the α-, β-, γ-, or δ-SG genes cause auto- mouse model of DMD [59-68]. Mice engineered to over- somal recessive limb-girdle muscular dystrophy (AR- express threefold levels of human SSPN driven by the LGMD) type 2D, 2E, 2C, and 2F, respectively (for review, muscle-specific human skeletal actin promoter increased [39]) leading to absence or a significant reduction in the ectopic expression of utrophin, dystrophin, and α7β1 SG subcomplex from the sarcolemma (for review, [40]). integrin at the sarcolemma (Figure 2) [19,69]. In fact, Genetic ablation of individual SG genes in mice has gen- analysis of SSPN transgenic mice overexpressing 0.5- erated robust animal models for the SG-deficient AR- and 1.5-fold levels of SSPN reveals that these molecular LGMDs in which the entire SG complex is absent from events occur in a SSPN dose-dependent manner the sarcolemma (for review, [40]). Similarly, a large dele- (Figure 2). Introduction of SSPN (threefold) ameliorates tion in the δ-SG gene causes cardiomyopathic and myo- dystrophy in the mdx mouse model of DMD by reducing pathic features in the BIO14.6 hamster model [41,42]. cycles of degeneration/regeneration and preventing Consistent with its tight biochemical association with sarcolemma damage (Figure 3). Biochemical purification the SGs, SSPN is absent from the sarcolemma of mice of the UGC complex using lectin affinity chromatog- deficient in α-, β-, and δ-SG as well as the δ-SG deficient raphy followed by sucrose gradient ultracentrifugation BIO14.6 hamster [32,36,43-46]. SSPN expression is revealed that SSPN is a component of the UGC [19,69]. restored to normal levels in BIO14.6 muscle after deli- Consistent with the role of SSPN in regulating adhesion very of an adenovirus encoding δ-SG [32]. Furthermore, complexes at the cell surface, overexpression of 10-fold investigation of over 30 AR-LGMD muscle biopsies with levels of SSPN causes formation of insoluble protein primary mutations in α-, β-, or γ-SG genes that result in aggregates at the sarcolemma, resulting in muscle path- either complete or partial absence of the SGs revealed ology [70]. To date, thirteen human SSPN transgenic that SSPN was absent from the sarcolemma [46]. The lines have been created and only one line expressed levels of SSPN expression were not analyzed in γ-SG tenfold levels of SSPN. In both 3- and 1.5-fold lines of deficient mice [47], but it would be interesting to deter- SSPN expression, internal down regulation of endoge- mine if the trend was similar to the observations made nous SSPN was observed, suggesting that the levels of in human AR-LGMD biopsies. Interaction between the SSPN are tightly controlled within the cell [19]. Based SGs and SSPN is very sensitive to structural perturba- on this data, it may be unlikely to achieve tenfold levels tions within the LEL of SSPN, as revealed by alanine of SSPN in a clinical setting. Future studies are needed scanning and deletion mutagenesis within the LEL to determine whether SSPN amelioration of dystrophic (Figure 1) [24]. SSPN interaction with the SGs is not pathology occurs in aged mdx mice and whether SSPN unique to skeletal muscle. The SSPN-SG subcomplex ameliorates disease in mouse models of laminin- has been characterized in many tissues, including the deficient congenital muscular dystrophy and SG- smooth muscle from lung and kidney [11,35,48-51]. In deficient LGMD. this context, SSPN interacts with a modified SG sub- complex consisting of β-, γ-, and ε-SG (a homolog of Sarcospan affects glycosylation of α-dystroglycan α-SG). Interestingly, the SSPN-SG subcomplex does not The cytotoxic T cell (CT) GalNAc transferase (Galgt2) is co-migrate in sucrose gradient fractions of sWGA puri- confined to the NMJ and catalyzes addition of the ter- fied DG from epithelial cells derived from lung or kidney minal β1,4 GalNAc residues onto the CT carbohydrate tissue [35]. SSPN is not conserved in Drosophila mela- of a subset of α-DG proteins [71,72]. α-DG is the pre- nogaster and Caenorhabditis elegans, thus the resulting dominant glycoprotein modified with the CT carbohy- DGC equivalent lacks SSPN and is predicted to be com- drate in skeletal muscle where it is enriched at the posed of DGs, SGs, and dystrophin [52,53]. postsynaptic membrane of the NMJ [73]. Overexpression of Galgt2 in mdx mice increases abundance and extrasy- Sarcospan uniquely increases abundance of laminin- naptic expression of α-DG modified with the CT anti- binding complexes gen, resulting in improved laminin-binding activity The functional replacement of utrophin for dystrophin is [72,73]. Overexpression of SSPN in mdx mice increases one of many attractive therapeutic strategies for the GalNAc modifications in a similar manner to the over- treatment of Duchenne muscular dystrophy. Utrophin is expression of Galgt2, as revealed by the increased cell Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 5 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Figure 2 SSPN increases abundance of laminin-binding complexes at the sarcolemma. Several lines of SSPN-transgenic mice with 0.5-, 1.5-, and 3-fold levels of SSPN overexpression were generated to investigate the dose-dependent effects of SSPN expression. SSPN transgenic mice on 0.5 1.5 3.0 0.5 1.5 3.0 C57/Bl6 background (WT, WT , WT ,WT ) as well as 0.5-, 1.5-, and 3-fold SSPN transgenic on mdx background (mdx, mdx , mdx , mdx ) were analyzed [19,70]. (A) Transverse cryosections of quadriceps muscle from six-week old SSPN-transgenic mice were stained with antibodies to dystrophin (Dys) and hSSPN to reveal exogenous SSPN expression (hSSPN Tg). (B) Muscle sections from SSPN-transgenic mdx mice were also stained with utrophin (Utr) and hSSPN. Sections overlayed with Wisteria floribunda agglutinin (WFA) lectin reveal increased cell surface glycosylation with elevations in SSPN overexpression [19]. WFA lectin binds terminal GalNAc residues and serves as a marker for the CT antigen modification of α-DG that normally occurs at the NMJ [19]. Note that SSPN increases cell surface expression of dystrophin, utrophin, and glycosylation in a manner dependent on SSPN abundance. Bar, 50 μm. CT, cytotoxic T cell; NMJ, neuromuscular junction. surface binding of the lectin Wisteria floribunda agglu- laminin binding or reduce muscle degeneration/regener- tinin (WFA) [19], which is a marker for NMJ-specific ation, revealing a minimum (threefold) level of SSPN CT carbohydrate modification of α-DG (Figure 2) needed for ‘rescue’ (Figure 2) [19]. [72,74-77]. WFA binding is localized to NMJs in normal The ‘dystroglycanopathies’ are a group of disorders resul- muscle and is increased around the extra-synaptic sarco- ting from hypoglycosylation of α-DG that abolishes its lemma of mdx muscle cryosections [19,78]. WFA bind- laminin-binding function. A spontaneous mutation in the 3.0 ing to SSPN-transgenic mdx (mdx ) muscle was LARGE gene, which encodes an enzyme with xylosyltransfe- significantly increased around the extra-synaptic sarco- rase and glucuronyltransferase activities, causes muscular lemma similar to utrophin expression [19]. Increased dystrophy in the myodystrophy (myd) mouse [83]. In myd Galgt2 activity in mdx mice results from a two-fold ele- muscle, α-DG is hypoglycosylated and exhibits severely vation of Galgt2 mRNA levels in mdx muscle relative to reduced ligand binding activity due to loss of the glycan- wild-type controls [73]. However, SSPN does not affect laminin binding domain on α-DG [84,85]. LARGE elongates Galgt2 transcript abundance, raising the possibility that phosphorylation dependent glycosylaminoglycan modifica- SSPN increases Galgt2 activity or improves α-DG as a tions on the central mucin domain of α-DG by direct inter- substrate for Galgt2 [19]. SSPN also increases laminin at action with α-DG [86,87]. Loss of LARGE increases the sarcolemma as well as levels of plectin-1, which utrophin and SSPN staining and WFA binding around the binds cytoskeletal proteins including β-DG, dystrophin, extra-synaptic sarcolemma of myd muscle [19]. Introduction utrophin, and F-actin [79-82], supporting the conclusion of the SSPN transgene into skeletal muscle of myd mice fur- that SSPN strengthens the structural connection be- ther elevated WFA binding along with broad, extra-synaptic tween actin and laminin across the sarcolemma [19]. localization of utrophin, while removal of SSPN from myd Furthermore, laminin binding to α-DG was restored to muscle reduced utrophin and GalNAc-glycan modification normal levels in threefold SSPN overexpressing mdx of α-DG [19]. Pathology of myd muscle was unaffected by muscle [19]. Transgenic overexpression of 0.5- and 1.5- the loss of SSPN or SSPN overexpression, demonstrating fold levels of SSPN increased glycosylation of α-DG, Akt that alterations in GalNAc glycosylation of α-DG or utrophin signaling, and utrophin levels, but failed to restore abundance do not affect absence of the main laminin- Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 6 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Figure 3 SSPN overexpression ameliorates dystrophic pathology in mdx mice. Transverse cryosections of quadriceps muscle from six-week 3.0 old mdx and SSPN transgenic mdx (mdx ) mice were stained with hematoxylin and eosin (H&E) and visualized at low magnification to reveal the extensive areas of necrosis, denoted in white, in mdx muscle (A). Necrosis was significantly diminished in SSPN transgenic mdx muscle [69]. Sections were also viewed at higher magnification for evaluation of degeneration/regeneration, which is marked by central nucleation of myofibers (B). Overexpression of SSPN in mdx muscle dramatically reduced central nucleation [69]. Bar, 50 μm. binding domain on α-DG [19]. The conclusion from these SSPN knockdown in a cultured glioma cell line experiments is that Galgt2 (or enzyme with similar function) (LN-229) did not affect cell division as determined by modifies α-DG in the absence of ‘LARGE’ glycans, demon- bromodeoxyuridine (BrdU) incorporation, but did in- strating that GalNAc modification of α-DG can occur inde- crease vulnerability of glioma cells to hypoxia [92]. pendently of O-mannose-linked glycans. Additionally, these Genetic ablation of SSPN did not appear to alter data reveal that GalNAc carbohydrate structures on α-DG muscle physiology or strength in young mice [93]. How- are unable to compensate for the loss of LARGE glycans, ever, when SSPN-null mice were analyzed at older ages which constitute the major laminin-binding motif. (4.5 month old), several deficiencies emerged. Reduction in the levels of the UGC and DGC as well as diminished A newly discovered phenotype for sarcospan-null mice NMJ-specific glycosylation of α-DG and decreased Evidence that SSPN loss may affect skeletal muscle was laminin-binding was observed in aged SSPN-nulls [14]. first suggested from comparative microarray analysis re- Diaphragms from older SSPN-null mice exhibited dimi- vealing a mild decrease in mRNA levels of dystrophin nished specific force generating capacity and were more and α-SG in SSPN-null muscle [88]. Furthermore, the susceptible to eccentric-contraction induced damage as authors reported increased expression of two genes, evidenced by the increased percentage drop in force osteopontin and the S100 calcium-binding protein cal- compared to wild-type controls [14]. These physiological granulin B, that have been implicated in immunological phenotypes were not observed in extensor digitorum function and fibrosis [89-91]. A second gene expression longus (EDL) or soleus muscles, suggesting that decrea- study compared hippocampus and cortex of mice sed DGC expression in EDL or soleus muscles is insuffi- exposed to chronic constant hypoxia (CCH) and chronic cient to manifest in loss of muscle strength or sarcolemma intermittent hypoxia (CIH). CCH occurs in chronic lung damage [14,93]. diseases or at high altitudes while CIH develops from disorders such as sleep apnea or sickle cell disease. SSPN Sarcospan genetically interacts with integrins was one of two identified genes down-regulated in the It is well established that all tetraspanins interact with hippocampus and cortex after both treatments [92]. integrin partners to regulate cell signaling, adhesion, and Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 7 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 laminin-binding capacity of integrins (for review, [94,95]). The table summarizes phenotypic and biochemical data The primary integrin expressed in adult skeletal muscle from 4.5-month old SSPN-null, Itga7-null, and SSPN-null: is α7β1 integrin, whichis localized at the NMJ and myo- Itga7-null double knockout (DKO) mice. All comparisons tendinous (MTJ) regions within the sarcolemma [96-99]. are relative to age-matched wild-type mice. Survival stu- The relatively mild phenotype of mdx mice has been at- dies were carried out to eight months of age. At this time tributed to increased, compensatory expression of both point, 40% of the DKO mice had succumbed to death utrophin and α7β1 integrin in response to loss of dys- compared to less than 5% of the controls. The extent of trophin that, when ablated, exacerbates mdx pathology kyphosis was documented in 4.5-month and 6-month old [100-102]. Overexpression of Itga7 in mdx:utrophin-null mice. Itga7-null mice exhibited minor kyphosis and the mice and β1 integrin in mdx mice ameliorates pathology additional loss of SSPN caused severe kyphosis in DKO [103-105]. The recent observation that α7β1integrin mice. Myofiber cross-sectional area (CSA) quantified from levels are increased in response to SSPN deficiency is the quadriceps and diaphragm muscles at 4.5 months of intriguing as it suggests that α7β1integrincompen- age are represented. DKO muscles exhibited an increase sates for SSPN function and moderates the severity of in myopathy and increase in very small (0 to 500 μm ) muscle phenotypes in SSPN-nulls [14]. Analysis of myofibers. Central nucleation is provided as an indicator SSPN-deficient and Itga7-deficient double knockout of overall muscle phenotype. Analysis of sarcolemmal (DKO) mice supports this hypothesis. In comparison damage (Evans blue dye assay) and fibrosis (Van Geison) to controls at 4.5-months of age, DKO mice exhibit of the quadriceps and diaphragm muscles at 4.5 months of increased kyphosis and premature lethality at one month age are exacerbated in DKOs [14]. The levels of utrophin, of age, which is significant given that the single-nulls dystrophin, laminin-binding to α-DG, and β1 integrin display no overt signs of pathology or lethality (Table 1) were analyzed by densitometry of sWGA eluates from [14]. Furthermore, DKO muscle appears severely dys- digitonin-solubilized total skeletal muscle lysates. SSPN- trophic with extensive fibrosis surrounding individual deficient mice exhibit a reduction in the UGC, DGC, and hypertrophic muscle fibers, in a manner identical to laminin-binding to α-DG and a corresponding compensa- histological images of DMD biopsies [14]. Genetic re- tory increase in β1 integrin [14]. Loss of Itga7 results in a moval of Itga7 from SSPN-nulls further reduced levels reduction in the levels of the DGC and laminin-binding to of the DGC at the sarcolemma, diminished laminin- α-DG. Importantly, combined loss of Itga7 and SSPN binding to α-DG, and consequently decreased specific causes further reduction of the DGC, UGC, and laminin- force output in the diaphragm (Table 1) [14]. The con- binding to α-DG compared to all controls. The relative clusion from this work is that SSPN is a necessary com- levels of P-Akt/Akt and P-IGFR/IGFR are provided. Spe- ponent of dystrophin and utrophin function and that cific force measurements of the diaphragm muscles reveal SSPN modulation of integrin signaling is required for a loss of specific force in SSPN-null and Itga7-null that is growth, extracellular matrix attachment, and muscle force additive in DKO mice. Interestingly, specific force produc- development. tion of the EDL and soleus muscles are unaffected by the Table 1 Sarcospan- and α7 integrin-double nulls display severe growth and muscle phenotypes a a a a a a Genotype Survival Kyphosis Myofiber Central Utrophin Dystrophin Integrin Laminin- Signaling Specific Force a a a a Analysis CSA Nucleation Binding Wild type 100% No Normal Normal 100% 100% 100% 100% P-Akt/Akt: Normal (EDL, Soleus, 100% Diaphragm) P-IGFR/IGFR: 100% SSPN null 100% No Normal Normal 31% 47% 293% 69% P-Akt/Akt: Normal (EDL, Soleus); 49% Decrease (Diaphragm) P-IGFR/IGFR: 80% Itga7 null 95% Minor Normal Normal 102% 48% Absent 85% P-Akt/Akt: Decrease (Diaphragm) 95% P-IGFR/IGFR: 99% DKO 60% Severe Decrease Increase 44% 34% Absent 28% P-Akt/Akt: Severe Decrease 40% (Diaphragm) P-IGFR/IGFR: 35% Measurements represented relative to wild type. DKO, double knockout; Itga7, α7 integrin. Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 8 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 loss of SSPN suggesting that, in these muscles, integrin [108,109]. Interestingly, overexpression of threefold may successfully compensate for DGC or that reduction levels of SSPN results in amelioration of mdx dystrophic of the DGC does not affect EDL and soleus muscle pathology through stabilization of the UGC and α7β1 physiology. integrin at the sarcolemma, and activation of Akt and downstream p70S6K [19,70]. Conversely, the Akt/ Sarcospan activates Akt signaling pathways to facilitate p70S6K pathway and activation of the IGF receptor is regeneration depressed in SSPN-deficient mice, rendering the muscle Modulation of the phosphatidylinositol-3 kinase (PI3K)/ unable to repair efficiently after cardiotoxin-induced Akt signaling pathway leading to downstream activation injury (Figure 4) [19]. Pretreatment of SSPN-null muscle of p70S6K protein synthesis pathways is important for with adenovirus expressing constitutively active Akt regulation of muscle strength, hypertrophy, and patho- increased the UGC to normal levels and restored muscle physiology (for review, [106,107]). The Akt signaling cas- regeneration after cardiotoxin-injury (Figure 4) [19]. The cade through p70S6K is activated by several receptors, conclusion from these experiments is that SSPN modu- including: insulin like growth factor 1 (IGF-1) and β1 lates utrophin protein levels at least in part through integrin associated integrin-linked kinase (ILK) (for re- Akt/p70S6K signaling pathways (Figure 5). view, [106,107]). Overexpression of constitutively active Akt in skeletal muscles of dystrophin-deficient mdx mice A chaperone-like function for sarcospan is emerging results in increased abundance of utrophin and α7β1 in- It has been assumed that the prematurely truncated dys- tegrin, which leads to improvements in force generation trophin protein produced from the mdx mutation is Figure 4 Muscle recovery from cardiotoxin injury requires SSPN-dependent Akt activation. (A) An acute injury model was used to investigate muscle repair in wild-type and SSPN-null mice. Quadriceps injected with equivalent volumes of saline (mock) and cardiotoxin dissolved in saline (CTX) were evaluated. CTX causes localized regions of myofiber necrosis followed by regeneration (for review, [125]). Mice at six weeks of age were injected with CTX (or mock) and analyzed after seven days. To test the dependency of muscle repair on Akt signaling, a subset of mice was pre-treated with adenovirus containing constitutively active Akt (Ad-caAkt) two days prior to CTX treatment. (B) Analysis was performed using H&E, laminin/eMHC, and laminin/EBD stained sections from quadriceps muscle. SSPN-deficient mice exhibit increased active regeneration (eMHC, 30%) seven days after CTX injury when wild-type mice have already undergone successful repair (black). Administration of Ad-caAkt prior to CTX treatment rescued the repair defect in SSPN-deficient mice (grey). (C) Immunoblot analysis of RIPA quadriceps lysates revealed reductions in the levels of dystrophin (Dys), utrophin (Utr), integrin (β1 integrin), phosphorylated Akt (P-Akt), and phosphorylated p70S6K (P-p70S6K) in SSPN-null mice compared to wild-type. Quantification of utrophin and P-Akt is provided. Coomassie blue (CB) serves as a loading control. (D) Immunoblot analysis of RIPA lysates from quadriceps pre-treated with Ad-caAkt revealed reductions in the levels of dystrophin (Dys) and integrin (β1 integrin) in SSPN-null mice compared to wild-type. Utrophin levels were restored in SSPN-null mice compared to wild-type mice with supplementation of active Akt (right). These data reveal that SSPN is upstream of the Akt signaling pathway regulating utrophin expression. The Ad-caAkt contains the HA-tag for detection [19]. RIPA, radioimmunoprecipitation assay. Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 9 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Figure 5 Sarcospan is a critical regulator of laminin-binding receptors in muscle. (A) The DGC/UGC and α7β1 integrin at the sarcolemma 3.0 in mdx (SSPN-Tg:mdx) muscle is depicted. The dystroglycans (DGs; pink), sarcoglycans (SGs; yellow), sarcospan (SSPN; blue), dystrophin (grey) and integrins (purple) are shown. Overexpression of SSPN in mdx muscle elicits a series of molecular events that lead to restoration of laminin binding, amelioration of pathology, and restoration of membrane integrity [19]. As shown in the illustration, SSPN activates Akt, which stabilizes utrophin, and increases the abundance of integrin and WFA-reactive α-DG at the cell surface. SSPN facilitates increased CT antigen modification of α-DG and enhances transportation of utrophin-DG at the sarcolemma [19]. Collectively, these events lead to stabilization of the sarcolemmal membrane and amelioration of dystrophic pathology. (B) SSPN-null muscle exhibits decreased dystrophin and Akt activation followed by decreased expression of utrophin, resulting in the reduction of laminin-binding to α-DG (middle panel) [19]. Acute muscle injury by cardiotoxin injection into SSPN-null muscle impairs muscle regeneration (right) [19]. However, pre-treatment of SSPN-null mice with adenovirus containing constitutively active Akt (Ad-caAkt) restored the activation of downstream p70S6K, utrophin expression, and improved muscle regeneration (right panel). These studies reveal the importance of sarcospan and Akt in regulating utrophin expression that is critical for muscle repair. CT, cytotoxic T cell; DGC, dystrophin-glycoprotein complex; UGC, utrophin-glycoprotein complex; WFA, Wisteria floribunda agglutinin. rapidly degraded in muscle based on lack of its detection sarcolemma, suggesting that SSPN possesses chaperone- in whole skeletal muscle extracts. However, recent work like functions to improve protein folding and/or traffick- has revealed that truncated dystrophin protein is synthe- ing to the cell surface (Figure 5). sized in mdx mice [19]. In fact, truncated dystrophin proteins are produced and detected in high abundance Sarcospan as a candidate disease gene in ER/golgi compartments in mdx muscle, suggesting The sarcospan gene is localized to human chromosome that they accumulate in intracellular compartments due 12p11.2 and is encoded by three small exons that are to insufficient transportation to the cell surface [19]. separated by very large introns [8,9]. A novel exon 4 was These data are exciting as they reveal for the first time recently identified to encode for an alternative C- that truncated dystrophin fragments are synthesized in terminal region in humans. In fact, alternate mRNA spli- mdx muscle, but then retained in intracellular mem- cing of human SSPN exons 1 and 2 to exon 4 generates brane compartments rather than properly transported to a protein called microspan (μSPN) that lacks transmem- the sarcolemma. brane domains 3 and 4 as well as the LEL so that the re- In addition to its role at the cell surface, a role for sultant protein has only two transmembrane spans and a SSPN within the ER/golgi is suggested from recent stu- novel intracellular C-terminus [10]. μSPN does not dies. Biochemical analysis of ER/golgi membranes iso- interact with the DGC and its expression is maintained lated from mdx muscle revealed abundant levels of in dystrophin-deficient muscle. Although μSPN is not utrophin and α-DG relative to wild-type [19]. Interes- localized to the sarcolemma, it is enriched in the sarco- tingly, utrophin and α-DG are reduced in ER/golgi pre- plasmic reticulum (SR) [10]. Overexpression of μSPN in parations from SSPN transgenic mdx muscle while these skeletal muscle of transgenic mice reduces levels of rya- same proteins are increased in abundance at the nodine receptor, dihydropyridine receptor as well as Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 10 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 SERCA-1 resulting in aberrant triad morphology [10]. required for SSPN’s ‘rescue’ effect. It is also critical to in- μSPN is also reduced in isolated SR membranes of vestigate whether Itga7 levels are affected by the ma- δ-SG-null muscle contributing to SERCA dysfunction ny other genes that increase utrophin expression, which [110]. Given that both SSPN and μSPN interact with would reveal important mechanisms regulating laminin- proteins that are critical to skeletal muscle function, it binding receptors in skeletal muscle. SSPN is a promising can be hypothesized that genetic mutations affecting therapeutic target, particularly for adeno-associated virus SSPN function would have significant consequences for delivery due to its small size and low potential for un- muscle. PCR-based approaches to screen muscular dys- wanted immune reaction. Future studies will reveal the trophy patients for possible abnormalities within the potential of this small protein to alleviate the significant SSPN gene have not yet produced any disease-causing problem of DMD. mutations, although several single-nucleotide poly- Abbreviations morphisms were identified in exons 2 and 3 [111]. SSPN BTX: bungarotoxin; CCH: chronic constant hypoxia; CIH: chronic intermittent was also excluded as a candidate disease gene for con- hypoxia; CSA: cross-sectional area; CT: cytotoxic T cell; DAP: dystrophin- associated protein; DEAE: diethylaminoethyl; DG: dystroglycan; genital fibrosis of the extraocular muscle (CFEOM), DGC: dystrophin-glycoprotein complex; DKO: double knockout; which is an autosomal dominant disorder linked to the DMD: Duchenne muscular dystrophy; Dys: dystrophin; EBD: Evans blue dye; pericentromere of chromosome 12 [112,113]. These EDL: extensor digitorum longus; IGF-1: insulin-like growth factor; ILK: integrin- linked kinase; Intg: integrin; LEL: large extracellular loop; NMJ: neuromuscular findings may come as no surprise given the apparently junction; PCR: polymerase chain reaction; SDS: sodium dodecyl sulfate; normal phenotype of SSPN-deficient mice [93]. How- SG: sarcoglycan; SSPN: sarcospan; UGC: utrophin-glycoprotein complex; ever, re-analysis of SSPN-deficient mice revealed signifi- Utr: utrophin; WFA: Wisteria floribunda agglutinin; sWGA: succinylated wheat germ agglutinin; μSPN: microspan. cant phenotypes in muscle of aged mice and after exposure to conditions of cellular stress that may have Competing interests implications for disease (Figure 5) [14,19]. Furthermore, The authors declare that they have no competing interests. the idea that SSPN may serve as a chaperone to improve Authors’ contributions cell surface expression of the DGC and UGC make it an Both authors contributed to writing and preparation of figures for the excellent candidate as a genetic modifier of disease. manuscript. All authors have read and approved the final manuscript. Authors’ information Conclusions Rachelle H. Crosbie-Watson, Ph.D. is a Professor of Integrative Biology and Overexpression of many proteins and compounds amelio- Physiology at the University of California Los Angeles and her research group rates dystrophic pathology in the mdx mouse by increasing is focused on investigation of macromolecular adhesion complexes at the cell surface in normal and dystrophic skeletal muscle. Professor Crosbie- UGC abundance at the extrasynaptic sarcolemma. A sam- Watson earned a Ph.D. in biochemistry from the University of California Los pling of these includes: CT GalNAc transferase (Galgt2) Angeles investigating structure-function relationships of contractile proteins [73], ADAM12 [114], heregulin [115], L-arginine [116,117], and she identified sarcospan during her MDA-sponsored postdoctoral fellowship at the University of Iowa College of Medicine with Professor Kevin activated calcineurin-A alpha [118], N-acetylcysteine P. Campbell (HHMI). [119-121], activated Akt [108,109], GW501516 (activates Jamie L. Marshall, Ph.D. is a postdoctoral researcher in Dr. Crosbie-Watson’s PPAR beta/delta) [122], artificial gene Jazz [123], and big- laboratory at the University of California Los Angeles. Dr. Marshall earned a Ph.D. in Molecular, Cellular, and Integrative Physiology from the University of lycan [124]. Several studies have now revealed that SSPN California Los Angeles investigating the role of sarcospan in the adhesion can be added to this list of secondary proteins that modify glycoprotein complexes at the sarcolemma. Dr. Marshall pioneered utrophin expression [19,69]. The mechanism by which purification the utrophin-glycoprotein complex [69] and discovered novel deficits of this complex in sarcospan-deficient muscle [14,19,]. Dr. Marshall SSPN increases expression of utrophin involves activation has received numerous predoctoral fellowships as well as a postdoctoral of Akt signaling and increased glycosylation of α-DG, fellowship based on these studies. Dr. Marshall has extensive experience in likely by increased modification of Galgt2 [19]. Intro- genetics, analysis of dystrophic mouse models, and detailed biochemical investigation of the dystrophin- and utrophin-glycoprotein complexes. duction of constitutively active Akt or Galgt2 alone also improves extrasynaptic utrophin expression, strongly sug- Acknowledgements gesting that SSPN, Akt, and Galgt2 may act via a common This work was supported by grants from the Genetic Mechanisms Pre- or overlapping pathway(s). It will be important to de- doctoral Training Fellowship USPHS National Research Service Award GM07104, the Edith Hyde Fellowship, the Eureka Pre-doctoral Training termine whether every gene that increases utrophin ex- Fellowship, and the Ruth L. Kirschstein National Research Service Award pression also alters Akt and α-DG glycosylation, which T32AR059033 from the National Institute of Arthritis and Musculoskeletal and would provide further evidence for a common post- Skin Diseases to J.L.M.; NIH/NIAMS (R01 AR048179) to R.C.W. transcriptional mechanism controlling utrophin abun- Author details dance. Furthermore, these data reveal that there are Department of Integrative Biology and Physiology, University of California multiple targets that affect utrophin, which is encou- Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, Los Angeles, CA 90095, USA. Center for Duchenne Muscular Dystrophy, raging for pharmacological and gene-based therapies. University of California Los Angeles, Los Angeles, CA 90095, USA. Molecular SSPN also increases expression of Itga7, and future Biology Institute, University of California Los Angeles, Los Angeles, CA 90095, studies will determine whether Itga7 and/or utrophin are USA. Marshall and Crosbie-Watson Skeletal Muscle 2013, 3:1 Page 11 of 13 http://www.skeletalmusclejournal.com/content/3/1/1 Received: 16 September 2012 Accepted: 27 November 2012 25. Hupp K, Siwarski D, Mock BA, Kinet JP: Gene mapping of the three Published: 3 January 2013 subunits of the high affinity FcR for IgE to mouse chromosomes 1 and 19. J Immunol 1989, 143:3787–3791. 26. Einfeld DA, Brown JP, Valentine MA, Clark EA, Ledbetter JA: Molecular References cloning of the human B cell CD20 receptor predicts a hydrophobic 1. Campbell KP, Kahl SD: Association of dystrophin and an integral protein with multiple transmembrane domains. EMBO J 1988, membrane glycoprotein. Nature 1989, 338:259–262. 7:711–717. 2. 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Skeletal MuscleSpringer Journals

Published: Jan 3, 2013

References