ABSTRACT Fibrosis in skeletal muscle is associated with a collection of devastating skeletal muscle wasting disorders known as the muscular dystrophies. Although fibrosis, per se, is not causal to the muscular dystrophies it is a significant confounding facet of the disease that contributes to impaired skeletal muscle compliance, contractility and patient morbidity. In dystrophic skeletal muscle, mutations in structural proteins render the muscle architecturally unstable resulting in susceptibility to damage, onset of necrosis, inflammation and repair; the chronicity of which culminates in fibrosis. We have discovered that the mitogen-activated protein kinase (MAPK) phosphatase-5 (MKP5), which is a dual-specificity protein tyrosine phosphatase that directly inactivates p38 MAPK and c-Jun NH2 terminal kinases 1 and 2 (JNK1/2), is a central mediator of skeletal muscle fibrosis. MKP5 is overexpressed in mouse models that develop fibrosis, including skeletal muscle from Duchenne's muscular dystrophy mice and its genetic loss curtails the disease. Mechanistically, MKP5 is essential for the activation of the transforming growth factor-β (TGF-β) cascade which is a major pro-fibrogenic pathway. Activation of p38 MAPK and JNK have been implicated in driving fibrosis. However, our findings suggest a more complex interplay exists between MKP5-mediated MAPK dephosphorylation and TGF-β receptor activation in both fibroblasts and muscle. The contribution of MKP5 to regulate TGF-β signaling and thus fibrosis in these cell types has yet to be explored neither in vivo nor exploited pharmacologically to assess validity of therapeutic potential. The overarching hypothesis is that MKP5 acts as a critical molecular checkpoint for fibrotic skeletal muscle disease. In Aim 1, we will define the cell type of action for MKP5 in skeletal muscle fibrosis by generating tissue-specific knockouts of MKP5. We will test whether MKP5 in either the myofiber and/or fibroblast contributes to disease progression in models of fibrosis for skeletal muscle. In Aim 2, we will identify the molecular basis for the actions of MKP5 in fibrosis by defining how it is involved in mediating the pro-fibrogenic actions of the TGF-β pathway. In Aim 3, we will determine the effectiveness of MKP5 inhibition as an anti-fibrotic therapy using a novel first-in-class allosteric MKP5 inhibitor. Collectively, these studies will define the pathophysiological basis for how MKP5 establishes its role as a regulator of skeletal muscle fibrosis. The successful completion of this project will provide proof-of-principle towards the notion that MKP5 represents a target for the treatment of fibrosis in dystrophic muscle diseases.