Fibrosis is a hallmark of skeletal muscular dystrophies. Several inducers of fibrosis have been described for many fibrotic diseases, including hypoxia , endothelin , angiotensin II , and the growth factors TGF-β  and CTGF .
TGF-β levels are increased in DMD [20, 46, 57-60]. Increased TGF-β activity regulates miR-21 expression and fibrosis in DMD patients and mdx muscles . Mdx macrophages produce elevated levels of TGF-β in response to fibrinogen. This TGF-β acts on fibroblasts, increasing the accumulation of collagen, which is exacerbated by fibrinogen .
Several attempts to inhibit TGF-β activity during skeletal muscle damage in muscular dystrophies have been reported. These comprise expression of TGF-β-sequestering proteins by gene transfer , inhibition of ligand binding to the receptors , targeting of TGF-β receptor expression , the use of inhibitory plant-derived drugs , and the use of decorin to inhibit TGF-β [61, 65-68].
Of particular interest is the SLRP decorin, because of its ability to bind TGF-β and regulate the cell response to TGF-β [26, 69, 70] and other ligands [3, 8, 71, 4]. Decorin is upregulated during skeletal muscle differentiation, and this seems to be necessary to sequester TGF-β and myostatin, two potent myogenic inhibitors [45, 72, 73]; this removal by decorin seems to be essential for successful skeletal muscle differentiation. However, TGF-β regulation by decorin is more complex; under proliferative conditions, the undifferentiated myoblasts require decorin for a full TGF-β cell response, by a mechanism that is dependent on the giant receptor lipoprotein receptor-related protein (LRP)1 [74, 75]. Decorin also seems to be necessary for myogenesis, as antisense inhibition of its expression in myoblasts accelerates skeletal muscle differentiation by decreasing the sensitivity to TGF-β signaling ; further analysis is required to clearly establish the role of decorin in each step of muscle differentiation.
As mentioned, the full cell response to TGF-β-mediated signaling [74, 76] depends on its canonical transducing receptors (TGF-β-RI and TGF-β receptor type II) and the cell surface complex of decorin and LRP-1, which is an endocytic receptor for decorin [74, 76]. This novel mechanism of signaling requires the Smad canonical pathway and AKT . Decorin contains 12 leucine-rich repeats (LRRs), LRR1–LRR12 [1, 3, 4]. The decorin region responsible for the interaction between TGF-β–decorin and LRP-1 was determined by the use of decorin deletion mutants and peptides derived from internal LRR regions. LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β  (Fig. 1). Remarkably, the LRR6 internal region, composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling . Furthermore, with an in vivo approach, the LRR6 region of decorin can inhibit TGF-β-mediated action in response to skeletal muscle injury . As already mentioned, decorin localized in the ECM is able to immobilize TGF-β  and myostatin [68, 77], thus concentrating these growth factors at the ECM and acting as growth factor reservoirs; these factors could be released under pathological conditions. Interestingly, improved muscle healing with reduced fibrosis was observed in myostatin-null mice .
Figure 1. The profibrotic growth factors TGF-β and CTGF share several elements, suggesting a common signaling/regulatory complex. Full TGF-β signaling activity depends on binding to the canonical transducing receptors (TGF-β receptors), activating the Smad pathway (red signals), and to the decorin–LRP-1 receptor complex, which, upon endocytosis, activates the phosphoinositide 3-kinase-dependent pathway (purple signals). The role of β-glycan in canonical Smad signaling is omitted for simplicity (see text for details). CTGF binds to several proteins, among them integrins that would activate the fibrotic signaling response (green signals). Furthermore, TGF-β and CTGF bind to decorin, probably stabilizing their interaction and potentiating their signaling. This decorin–TGF-β–CTGF complex can be sequestered in the ECM, acting as a reservoir of the ligands, or be associated with other cell surface proteins. In the latter scenario, the decorin–TGF-β–CTGF complex would interact with LRP-1, which also interacts with the HSPG β-glycan through GAG chains. β-Glycan also binds TGF-β via its core protein and CTGF via its GAG chains. All of these proteins might form a regulatory signaling complex that is critical for modulating the biological activity of these two profibrotic growth factors. The top part of the figure shows a magnification of the indicated box indicating in detail the interaction between decorin, CTGF and TGF-β.
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Besides the TGF-β receptors and the decorin–LRP-1 complex, TGF-β has another cell surface receptor, the HSPG β-glycan or TGF-β receptor type III (see above). This HSPG has two independent high-affinity binding domains for TGF-β, one in the membrane-distal half and the other in the membrane-proximal half of the β-glycan ectodomain . Thus, β-glycan can present TGF-β to the type II signaling receptor to activate the canonical Smad signaling pathway , but also, when it is overexpressed, is able to activate TGF-β signaling by a mechanism that is independent of the ligand but dependent on the kinase activity of TGF-β-RI .
CTGF induces fibrosis in skeletal muscle both in vitro and in vivo. Increased levels of CTGF mRNA have been reported in the skeletal muscle from dystrophic dogs  and mdx mice . Increased amounts of CTGF are found in skeletal muscle biopsies from DMD patients . Myoblasts and myotubes produce and respond to CTGF, increasing ECM accumulation and ERK-1/2 phosphorylation . This CTGF-mediated ERK-1/2 phosphorylation is strongly inhibited by heparin, suggesting a role for HSPG in its mode of action [49, 50]. Overexpression of CTGF in normal mice, by use of an adenovirus containing the CTGF mouse sequence, induced extensive skeletal muscle damage followed by regeneration, with an increase in the levels of fibrotic markers (fibronectin, collagen, and α-smooth muscle actin) . This overexpressed CTGF also caused a decrease in the specific isometric contractile muscle force. When CTGF overexpression stopped, the entire phenotype proved to be reversible, in parallel with normalization of CTGF levels . Overexpression of CTGF in skeletal muscle, together with infusion of an angiotensin receptor blocker (losartan), decreased the CTGF-mediated increases in the levels of ECM molecules and α-smooth muscle actin, and ERK-1/2 phosphorylation levels. Notably, losartan was able to prevent the loss of contractile force of muscles overexpressing CTGF .
CTGF is a complex protein and, as mentioned, does not have a traditional high-affinity receptor to activate a signal transduction pathway. However, it is able to interact with several proteins present on the cell surface, such the ECM proteins, fibronectin , HSPGs , and several growth factors, TGF-β among them . Also, CTGF interacts with plasma membrane-bound proteins and receptors such as TrkA , LRP-1 , LRP-6 , and integrins , participating in many mechanisms at the same time [31, 48, 56, 89]. Because of this complex network of interactions, the cell response to CTGF is highly context-dependent and environment-dependent ; thus, it has been reported that CTGF induces the expression of fibronectin, but, when the amount of fibronectin reaches a certain level, the cell response to CTGF is notably affected [43, 88], apparently by a mechanism that depends on the competition between fibronectin and CTGF for αv-subunit-containing integrin .
Efforts have been concentrated on inhibiting CTGF profibrotic activity. Inhibition of CTGF by small interfering RNA prevents liver fibrosis in rats . Specific downregulation of CTGF in the kidney with antisense oligonucleotides attenuates the progression of nephropathy in mouse models of type 1 and type 2 diabetes , and CTGF antisense inhibition decreases hypertrophic scarring . The use of mAbs (FG-3019) has given positive results in diminishing fibrosis in a model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-β  and in diabetic nephropathy . Dystrophic mice treated with FG-3019 showed decreased fibrosis, less skeletal muscle damage, and an improvement in the capacity to generate skeletal muscle strength. Moreover, if fibrosis is diminished by targeting CTGF, the efficiency of muscle stem cell therapy in the dystrophic mdx mouse is augmented (manuscript under preparation).
Decorin-null myoblasts are more sensitive to CTGF, producing more fibronectin and collagen III than wild-type myoblasts, suggesting that decorin is an endogenous inhibitor of the fibrotic effects of CTGF. Furthermore, decorin exogenously added to myoblasts and fibroblasts negatively regulated CTGF profibrotic activity. CTGF interacts with decorin in a saturable manner, with a Kd of 4.4 nm. By the use of decorin deletion mutants, the region where decorin binds to CTGF has been determined; thus, a mutant form of decorin without LRR10–LRR12 is unable to bind CTGF and inhibit its fibrotic effects. Moreover, a peptide derived from LRR12 was able to inhibit CTGF–decorin complex formation and directly inhibit CTGF activity, indicating that decorin binds to CTGF via decorin's LRR12, and suggesting that this CTGF domain could be somehow involved in its recognition by one of the CTGF receptors related to the induction of the fibrotic response . All of these findings suggest that decorin interacts with CTGF and regulates its biological activity .