AbstractDuchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. Prior work has shown that DMD progression can vary, depending on the genetic makeup of the patient. Several modifier alleles have been identified including LTBP4 and SPP1. We previously showed that Spp1 exacerbates the DMD phenotype in the mdx mouse model by promoting fibrosis and by skewing macrophage polarization. Here, we studied the mechanisms involved in Spp1's promotion of fibrosis by using both isolated fibroblasts and genetically modified mice. We found that Spp1 upregulates collagen expression in mdx fibroblasts by enhancing TGFβ signaling. Spp1's effects on TGFβ signaling are through induction of MMP9 expression. MMP9 is a protease that can release active TGFβ ligand from its latent complex. In support for activation of this pathway in our model, we showed that treatment of mdx fibroblasts with MMP9 inhibitor led to accumulation of the TGFβ latent complex, decreased levels of active TGFβ and reduced collagen expression. Correspondingly, we found reduced active TGFβ in Spp1−/−mdxB10 and Mmp9−/−mdxB10 muscles in vivo. Taken together with previous observations of reduced fibrosis in both models, these data suggest that Spp1 acts upstream of TGFβ to promote fibrosis in mdx muscles. We found that in the context of constitutively upregulated TGFβ signaling (such as in the mdxD2 model), ablation of Spp1 has very little effect on fibrosis. Finally, we performed proof-of-concept studies showing that postnatal pharmacological inhibition of Spp1 reduces fibrosis and improves muscle function in mdx mice.