Decorin developmental expression and function in the early avian embryo

Decorin, a proteoglycan, interacts with extracellular matrix proteins, growth factors and receptors. Decorin expression and spatio-temporal distribution were studied by RT-PCR and immunofluorescence, while decorin function was examined by blocking antibodies in the early chick embryo. Decorin was first detectable at stage XIII (late blastula). During gastrulation (stage HH3-4), decorin fluorescence was intense in epiblast cells immediately adjacent to the streak, and in migrating cells. Decorin fluorescence was intense in endoderm and strong at mesoderm-neural plate surfaces at stage HH5-6 (neurula). At stage HH10-11 (12 somites), decorin fluorescence was intense in myelencephalon and then showed distinct expression patterns along the myelencephalon axes by stage HH17. Decorin fluorescence was intense in neural crest cells, dorsal aorta, heart, somite and neuroepithelial cells apposing the somite, nephrotome, gut and in pancreatic and liver primordia. Antibody-mediated inhibition of decorin function affected the head-to-tail embryonic axis extension, indicating that decorin is essential for convergent extension cell movements during avian gastrulation. Decorin was also essential for retinal progenitor cell polarization, neural crest migration, somite boundary formation and cell polarization, mesenchymal cell polarization and primary endoderm displacement to the embryo periphery. The embryonic blood vessels were deformed, the dorsal mesocardium was thinned and the cardiac jelly was abnormally thickened in the heart. Decorin is known to modulate collagen fibrillogenesis, a key mechanism of matrix assembly, and cell proliferation. Decorin also appears to be essential for the coordination of cell and tissue polarization, which is an important feature in organ patterning of the embryo.     

Effect of adenovirus-mediated overexpression of decorin on metalloproteinases, tissue inhibitors of metalloproteinases and cytokines secretion by human gingival fibroblasts.

Decorin is a small leucine-rich proteoglycan that plays a role in control of cell proliferation, cell migration, collagen fibrillogenesis and modulation of the activity of TGF-beta. In the present study, we investigated the effects of decorin on the production of metalloproteinases (MMP-1, -2, -3, -9 and -13), tissue inhibitors of metalloproteinases (TIMP-1, -2) and cytokines (TGF-beta, IL-1beta, IL-4 and TNF-alpha). Decorin was overexpressed in cultured human gingival fibroblasts using adenovirus-mediated gene transfer. Decorin infection resulted in decreased protein levels of MMP-1 and MMP-3 whereas MMP-2 and TIMP-2 secretion was increased. MMP-9, MMP-13 and TIMP-1 were not affected by decorin infection. Cytokine measurements by ELISA showed that decorin overexpression reduced TGF-beta and IL-1beta. In contrast, IL-4 and TNF-alpha levels were markedly increased in decorin-infected cells. These results suggest that decorin could modulate the expression of certain metalloproteinases and their inhibitors, as well as the production of cytokines. Altogether, our data suggest that decorin might play a pivotal role in tissue remodeling by acting on the balance between extracellular matrix synthesis and degradation.     

Effects of rhDecorin on TGF-beta1 induced human hepatic stellate cells LX-2 activation

Decorin is a small leucine-rich extracellular matrix proteoglycan composed of a core protein with a single glycosaminoglycan (GAG) chain near the N-terminus and N-glycosylated at three potential sites. Decorin is involved in the regulation of formation and organization of collagen fibrils, modulation of the activity of growth factors such as transforming growth factor beta (TGF-beta), and exerts other effects on cell proliferation and behavior. Increasing evidences show that decorin plays an important role in fibrogenesis by regulating TGF-beta, a key stimulator of fibrosis, and by directly modulating the degradation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). In this study, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) significantly inhibited the proliferation of LX-2 cells, a human HSC cell line, stimulated by TGF-beta1. RT-PCR result showed that the expression of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced by rhDecorin in LX-2 cells stimulated by TGF-beta1. Furthermore, the protein expression of smooth muscle-alpha-actin (alpha-SMA), collagen type III and phosphorylated Smad2 (p-Smad2) was significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-beta1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray technique and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells.     

Effects of rhDecorin on TGF-β1 induced human hepatic stellate cells LX-2 activation

Decorin is a small leucine-rich extracellular matrix proteoglycan composed of a core protein with a single glycosaminoglycan (GAG) chain near the N-terminus and N-glycosylated at three potential sites. Decorin is involved in the regulation of formation and organization of collagen fibrils, modulation of the activity of growth factors such as transforming growth factor β (TGF-β), and exerts other effects on cell proliferation and behavior. Increasing evidences show that decorin plays an important role in fibrogenesis by regulating TGF-β, a key stimulator of fibrosis, and by directly modulating the degradation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). In this study, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) significantly inhibited the proliferation of LX-2 cells, a human HSC cell line, stimulated by TGF-β1. RT-PCR result showed that the expression of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced by rhDecorin in LX-2 cells stimulated by TGF-β1. Furthermore, the protein expression of smooth muscle-α-actin (α-SMA), collagen type III and phosphorylated Smad2 (p-Smad2) was significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-β1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray technique and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells.     

Granzyme B cleavage of fibronectin disrupts endothelial cell adhesion,migration and capillary tube formation

Dysregulated angiogenesis contributes to the pathogenesis of chronic inflammatory diseases. Modulation of the extracellular matrix by immune-derived proteases can alter endothelial cell–matrix interactions as well as endothelial cell sprouting, migration and capillary formation. Granzyme B is a serine protease that is expressed by a variety of immune cells, and accumulates in the extracellular milieu in many chronic inflammatory disorders that are associated with dysregulated angiogenesis. Although granzyme B is known to cleave fibronectin, an essential glycoprotein in vascular morphogenesis, the role of granzyme B in modulating angiogenesis is unknown. In the present study, granzyme B cleaved both plasma fibronectin and cell-derived fibronectin, resulting in the release of multiple fibronectin fragments. Granzyme B cleavage of fibronectin resulted in a dose-dependent reduction in endothelial cell adhesion to fibronectin as well as reduced endothelial cell migration and tubular formation. These events were prevented when granzyme B activity was inhibited by a small molecule inhibitor. In summary, granzyme B-mediated cleavage of fibronectin contributes to attenuated angiogenesis through the disruption of endothelial cell — fibronectin interaction resulting in impaired endothelial cell migration and tubular formation.     

Factor VIII-related antigen : A pericellular matrix component of cultured human endothelial cells

We studied the extracellular localization of factor VIII-related antigen (VIIIR: Ag) in cultures of human endothelial cells. The cells deposited both VIIIR: Ag and fibronectin already during their initial adhesion phase and in immunofluorescence microscopy of spread cells extracellular VIIIR: Ag was localized to fibrils coaligning with pericellular fibronectin. When human fibroblasts, which do not synthesize VIIIR: Ag, were cultured in endothelial cell post-culture medium, a fibrillar matrix localization of VIIIR: Ag was seen, comparable to that of endothelial cell cultures. A fibrillar VIIIR: Ag-specific staining was also seen in cell-free pericellular matrices of endothelial cells, produced by deoxycholate treatment. In immunoelectron microscopy, VIIIR: Ag was seen in fibrillar extracellular material between and underneath the cells and in cell-free matrices of endothelial cells as well.In immunofluorescence microscopy of cell-free matrices, VIIIR: Ag codistributed with both fibronectin and type III procollagen. Digestion of the matrices with purified bacterial collagenase abolished the type III procollagen-specific fluorescence, whereas the fibrillar VIIIR: Ag-specific staining, codistributing with fibronectin, remained unaffected. In electrophoresis of isolated, metabolically labelled endothelial cell matrices, major polypeptides with M_r 220–240; 180; 160; 80 and 45 kD and some minor polypeptides were resolved. In addition, immunoblotting revealed fibronectin, VIIIR: Ag and type III procollagen as components of cell-free matrices of endothelial cells. Direct overlay of iodinated cellular fibronectin on electrophoretically separated polypeptides of cultured endothelial cells, transferred to nitrocellulose, suggested that fibronectin binds directly to VIIIR: Ag. Our results indicate that VIIIR: Ag produced by human endothelial cells is a component of the pericellular matrix and is not bound to collagen but may directly associate with fibronectin.     

Peptides derived from human decorin leucine-rich repeat 5 inhibit angiogenesis

Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 3(10) helix and beta-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs.     

Expression of decorin by sprouting bovine aortic endothelial cells exhibiting angiogenesis in vitro.

In our recent studies, we have demonstrated that monolayer cultures of bovine aortic endothelial (BAE) cells that do not express type I collagen also fail to express and synthesize decorin, a small chondroitin/dermatan sulfate proteoglycan that interacts with type I collagen and regulates collagen fibrillogenesis in vitro. However, BAE cells exhibiting a spontaneous sprouting phenotype and a predisposition toward the formation of cords and tube-like structures (an in vitro model for angiogenesis) initiate the synthesis of type I collagen during their morphological transition from a polygonal monolayer to an angiogenic phenotype. In the present study, we examined whether BAE cells also initiate the synthesis of the proteoglycan decorin during this morphological transition. We show by Northern blot analysis and by immunochemical methods that BAE cell cultures containing sprouting cells and cords, but not monolayer cultures of these cells, express and synthesize decorin (M(r) approximately 100,000). We also show that type I collagen expression by BAE cell cultures is initiated concomitantly. However, the localization of decorin and type I collagen in cord and tube-forming BAE cell cultures is not completely identical. Type I collagen is detected only in sprouting BAE cells and in endothelial cords, whereas decorin is also apparent in BAE cells surrounding the cords and tubes. Our results indicate that the synthesis of decorin as well as type I collagen is associated with endothelial cord and tube formation in vitro.     

Retinal capillary endothelial cells prefer different substrates for growth and migration

Recent studies have shown that the extracellular matrix modifies the behaviour of endothelial cells. We have studied the effects of extracellular matrix components on retinal capillary endothelial cell migration and proliferation. Bovine retinal capillary endothelial cells were selectively cultured from collagenase-digested microvessel fragments. In a filter system for the assessment of migration, endothelial cells responded to substrate-bound fibronectin but not to soluble fibronectin. Cell migration on collagen- or gelatin-coated filters was minimal, and these cells failed to adopt a spread morphology, remaining instead as round cells. Cell replication was quantified using a protein dye binding assay for adherent cells in 96 well plates. Serum was essential for growth irrespective of the substrate. Cells harvested from microvessel cultures proliferated more rapidly on collagen- and gelatin-coated plastic than on fibronectin and were unaffected by additions to the medium such as endothelial cell conditioned medium, whereas cells proliferating directly from the microvessels grew at a faster rate on fibronectin and also responded to conditioned medium supplement. When cultured on collagen gels, initial microvessel cells and harvested cells required surface fibronectin in order to adopt a cobblestone morphology. These results show that fibronectin is a requirement for bovine retinal capillary endothelial cell migration, but proliferation of these cells can be supported, with slight differences, by both fibronectin and collagen provided serum growth factors are present. These findings are relevant to the early phase of angiogenesis in which migration and proliferation of endothelial cells occurs.     

Matricellular hevin regulates decorin production and collagen assembly

Matricellular proteins such as SPARC, thrombospondin 1 and 2, and tenascin C and X subserve important functions in extracellular matrix synthesis and cellular adhesion to extracellular matrix. By virtue of its reported interaction with collagen I and deadhesive activity on cells, we hypothesized that hevin, a member of the SPARC gene family, regulates dermal extracellular matrix and collagen fibril formation. We present evidence for an altered collagen matrix and levels of the proteoglycan decorin in the normal dermis and dermal wound bed of hevin-null mice. The dermal elastic modulus was also enhanced in hevin-null animals. The levels of decorin protein secreted by hevin-null dermal fibroblasts were increased by exogenous hevin in vitro, data indicating that hevin might regulate both decorin and collagen fibrillogenesis. We also report a decorin-independent function for hevin in collagen fibrillogenesis. In vitro fibrillogenesis assays indicated that hevin enhanced fibril formation kinetics. Furthermore, cell adhesion assays indicated that cells adhered differently to collagen fibrils formed in the presence of hevin. Our observations support the capacity of hevin to modulate the structure of dermal extracellular matrix, specifically by its regulation of decorin levels and collagen fibril assembly.     

Transforming growth factor-β and p-21: multiple molecular targets of decorin-mediated suppression of neoplastic growth

Decorin is a member of the small leucine-rich proteoglycan (SLRP) gene family that has recently become a focus in various areas of cancer research. The decorin protein consists of a core protein and a covalently linked glycosaminoglycan chain. Decorin binds to collagens type I, II and IV in vivo and promotes the formation of fibers with increased stability and changes in solubility. Further, the decorin core protein binds to growth factors, including transforming growth factor-beta (TGF-beta), to other intercellular matrix molecules such as fibronectin and thrombospondin, and to the decorin endocytosis receptor. Decorin may directly interfere with the cell cycle via the induction of p21WAF1/CIP1 (p21), a potent inhibitor of cyclin-dependent kinases (CDKs). Here, we discuss interactions of decorin with TGF-beta and with p21, both of which are relevant to carcinogenesis and tumor progression. TGF-beta is released by tumors of various histogenetic origins and promotes immunosuppression in the host and tumor immune escape by induction of growth arrest and apoptosis in immune cells, by downregulation of MHC II antigen expression and by changes in the cytokine release profiles of immune and tumor cells. Moreover, TGF-beta may modulate tumor growth in an autocrine and paracrine fashion, may mediate drug resistance, and may facilitate tumor angiogenesis. Decorin binds to TGF-beta, thus inhibiting its bioactivity, and is a direct or indirect negative modulator of TGF-beta synthesis. Ectopic expression of decorin results in the regression of rat C6 gliomas, an antineoplastic effect attributed to the reversal of TGF-beta-induced immunosuppression. On the other hand, de novo expression of decorin in colon cancer cells and some other tumor cells, even though not in glioma cells, results in an upregulation of p21 expression and a cell cycle arrest, presumably in a TGF-beta-independent manner. Decorin expression is downregulated in many tumors but upregulated in the peritumoral stroma. By virtue of its growth regulatory and immunomodulatory properties, decorin promises to become a novel target for the experimental therapy of human cancers.     

Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions

Remodeling of extracellular matrices occurs during development, wound healing, and in a variety of pathological processes including atherosclerosis, ischemic injury, and angiogenesis. Thus, identifying factors that control the balance between matrix deposition and degradation during tissue remodeling is essential for understanding mechanisms that regulate a variety of normal and pathological processes. Using fibronectin-null cells, we found that fibronectin polymerization into the extracellular matrix is required for the deposition of collagen-I and thrombospondin-1 and that the maintenance of extracellular matrix fibronectin fibrils requires the continual polymerization of a fibronectin matrix. Further, integrin ligation alone is not sufficient to maintain extracellular matrix fibronectin in the absence of fibronectin deposition. Our data also demonstrate that the retention of thrombospondin-1 and collagen I into fibrillar structures within the extracellular matrix depends on an intact fibronectin matrix. An intact fibronectin matrix is also critical for maintaining the composition of cell-matrix adhesion sites; in the absence of fibronectin and fibronectin polymerization, neither alpha5beta1 integrin nor tensin localize to fibrillar cell-matrix adhesion sites. These data indicate that fibronectin polymerization is a critical regulator of extracellular matrix organization and stability. The ability of fibronectin polymerization to act as a switch that controls the organization and composition of the extracellular matrix and cell-matrix adhesion sites provides cells with a means of precisely controlling cell-extracellular matrix signaling events that regulate many aspects of cell behavior including cell proliferation, migration, and differentiation.     

Inhibition of myoblast migration via decorin expression is critical for normal skeletal muscle differentiation

During limb skeletal muscle formation, committed muscle cells proliferate and differentiate in the presence of extracellular signals that stimulate or repress each process. Proteoglycans are extracellular matrix organizers and modulators of growth factor activities, regulating muscle differentiation in vitro. Previously, we characterized proteoglycan expression during early limb muscle formation and showed a spatiotemporal relation between the onset of myogenesis and the expression of decorin, an important muscle extracellular matrix component and potent regulator of TGF-beta activity. To evaluate decorin's role during in vivo differentiation in committed muscle cells, we grafted wild type and decorin-null myoblasts onto chick limb buds. The absence of decorin enhanced the migration and distribution of myoblasts in the limb, correlating with the inhibition of skeletal muscle differentiation. Both phenotypes were reverted by de novo decorin expression. In vitro, we determined that both decorin core protein and its glycosaminoglycan chain were required to reverse the migration phenotype. Results presented here suggest that the enhanced migration observed in decorin-null myoblasts may not be dependent on chemotactic growth factor signaling nor the differentiation status of the cells. Decorin may be involved in the establishment and/or coordination of a critical myoblast density, through inhibition of migration, that permits normal muscle differentiation during embryonic myogenesis.     

Decorin regulates endothelial cell-matrix interactions during angiogenesis

Interactions between endothelial cells and the surrounding extracellular matrix are continuously adapted during angiogenesis, from early sprouting through to lumen formation and vessel maturation. Regulated control of these interactions is crucial to sustain normal responses in this rapidly changing environment, and dysfunctional endothelial cell behaviour results in angiogenic disorders. The proteoglycan decorin, an extracellular matrix component, is upregulated during angiogenesis. While it was shown previously that the absence of decorin leads to dysregulated angiogenesis in vivo, the molecular mechanisms were not clear. These abnormal endothelial cell responses have been attributed to indirect effects of decorin; however, our recent data provides evidence that decorin directly regulates endothelial cell-matrix interactions. This data will be discussed in conjunction with findings from previous studies, to better understand the role of this proteoglycan in angiogenesis.     

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.     

Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

Therapeutic angiogenesis may be applied in medical conditions to promote stimulation of angiogenesis. Angiogenesis is a multistep process, which includes endothelial cell proliferation, migration, and tube formation, which is mediated by various angiogenic polypeptides. Thus, studies that elucidate the cellular mechanisms involved in these processes are necessary to develop novel therapeutic strategies. This study investigated the in vitro effects of the pro-angiogenic factors, insulin-like growth factor-1 (IGF-1) and/or chemokine (CC motif) ligand 2 (CCL2), on endothelial cells. Flow cytometry analysis showed that IGF-1 and CCL2 treatment did not interfere with IGF-1 receptor (IGF-1R) expression, but CCL2 treatment increased CCL2 receptor (CCR2) expression. Immunofluorescence analysis revealed that the IGF-1/CCL2 combination induced a greater increase in fibronectin deposition, but the treatments did not alter the expression of the fibronectin receptors, CD49e and CD44. The interaction of fibronectin with cytokines demonstrated that IGF-1/CCL2 promoted changes in intermediate F-actin remodeling that may result in increased endothelial cell adhesion and cell migration mediated by fibronectin. Furthermore, IGF-1/CCL2 stimulated endothelial cells, grown on fibronectin, to form capillary-like structures and intercellular lumina with greater luminal area. These data suggest that IGF-1/CCL2 combination and a fibronectin matrix may contribute to the angiogenesis process to stimulate adhesion, migration, and tube formation by endothelial cells as a result of F-actin remodeling.     

Decorin enhances the proliferation and differentiation of myogenic cells through suppressing myostatin activity

Decorin, a small leucine-rich proteoglycan, plays an important role in the regulation of cell growth. Our recent study has shown that immobilized decorin in the collagen matrix sequesters myostatin into the extracellular matrix and prevents its inhibitory action to myoblast proliferation in vitro. However, it still remains unclear whether free decorin could affect the proliferation and differentiation of myogenic cells by regulating myostatin activity. In the present study, we generated stable clonal C2C12 myoblasts that were over-expressing decorin, and showed that decorin over-expressing cells had an increased rate of proliferation as compared to control cells. Decorin over-expressing cells formed multi-giant hypertrophic myotubes with an elongated morphology and larger size as compared to control cells, although the initiation of differentiation in decorin over-expressing cells was somewhat delayed as compared to control cells. Western blot analysis demonstrated that MyoD expression in decorin over-expressing cells was lower than that in control cells until 12 h after induction to differentiate. At 48-h differentiation, the expressions of MyoD, p21 and myogenin were dramatically increased in cells that over-expressed decorin. Furthermore, we revealed that over-expression of decorin suppressed the activity of myostatin endogenously synthesized in C2C12 myoblasts and attenuated the signaling of exogenous myostatin. Consistent with these results, knock-down of decorin impairs C2C12 myoblast growth by increasing the sensitivity to exogenous myostatin. These results clearly show that decorin enhances the proliferation and differentiation of C2C12 myoblasts through suppressing myostatin activity.     

14-3-3beta-Rac1-p21 activated kinase signaling regulates Akt1-mediated cytoskeletal organization, lamellipodia formation and fibronectin matrix assembly

Akt1 belongs to the three-gene Akt family and functions as a serine-threonine kinase regulating phosphorylation of an array of substrates and mediating cellular processes such as cell migration, proliferation, survival, and cell cycle. Our previous studies have established the importance of Akt1 in angiogenesis and absence of Akt1 resulted in impaired integrin activation, adhesion, migration, and extracellular matrix assembly by endothelial cells and fibroblasts. In this study, we identify the downstream signaling pathways activated by Akt1 in the regulation of these cellular events. We demonstrate here that Akt1 is necessary for the growth factor stimulated activation of 14-3-3beta-Rac1-p21 activated kinase (Pak) pathway in endothelial cells and fibroblasts. While activation of Akt1 resulted in translocation of Rac1 to membrane ruffles, enhanced Rac1 activity, Pak1 phosphorylation, and lamellipodia formation, resulting in enhanced adhesion and assembly of fibronectin, inhibition of Akt1 resulted in inhibition of these processes due to impaired Rac1-Pak signaling. Formation of lamellipodia, adhesion, and fibronectin assembly by myristoylated Akt1 expression in NIH 3T3 fibroblasts was inhibited by co-expression with either dominant negative Rac1 or dominant negative Pak1. In contrast, impaired lamellipodia formation, adhesion, and fibronectin assembly by dominant negative-Akt1 expression was rescued by co-expression with either constitutively active-Rac1 or -Pak1. Moreover, previously reported defects in adhesion and extracellular matrix assembly by Akt1(-/-) fibroblasts could be rescued by expression with either active-Rac1 or -Pak1, implying the importance of Rac1-Pak signaling in growth factor stimulated cytoskeletal assembly, lamellipodia formation and cell migration in endothelial cells and fibroblasts downstream of Akt1 activation.     

Decorin modulates collagen I-stimulated, but not fibronectin-stimulated, migration of C2C12 myoblasts

Extracellular matrix factors, specifically fibronectin and collagen I, are essential for structural support during muscle regeneration. Decorin has been identified as an anti-fibrotic agent with binding sites located on both fibronectin and collagen I. Upon injury, activated myoblasts are required to migrate through the extracellular matrix factors deposited by the myofibroblasts to facilitate skeletal muscle regeneration. In this study we looked at the effects decorin on fibronectin- and collagen I-stimulated myoblast migration. Dose response studies demonstrated 10 μg/ml, 5 μg/ml and 25 μg/ml as the optimal stimulatory concentrations of decorin (1.2 fold increase), fibronectin (3.5 fold increase) and collagen I (2.4 fold increase), when compared with control respectively. A synergistic effect was identified when decorin and collagen I were added in combination; this effect was not evident when decorin was added with fibronectin. The effects of these factors on the ROCK signalling pathway were also analyzed. ROCK-2 was identified as the key Rho-activated kinase isoform involved in migration, due to its higher expression levels and localisation to focal points within migrating C2C12 myoblasts. Decorin and collagen I in combination stimulated an increase in the number of ROCK-2 localized focal points when compared with control, decorin and collagen I added separately. Fibronectin did not show any increase in ROCK-2 focal points when compared with control. These results show for the first time that decorin can modify collagen I-stimulated, but not fibronectin-stimulated myoblast migration in vitro. Furthermore, the synergistic, rather than additive, effect observed suggests a direct modification of collagen I signalling by decorin mediated, at least in part, by ROCK-2 rather than ROCK-1.