Cysteine-rich domain of type III collagen N-propeptide inhibits fibroblast activation by attenuating TGFß signaling.

TGFß is a key regulator of the dynamic reciprocity between cells and the extracellular matrix that drives physiologic and pathologic responses in both tissue repair and tumor microenvironments. Our studies define type III Collagen (Col3) as a suppressor of scar formation and desmoplasia through its effects, in part, on myofibroblasts. TGFß stimulates activation of myofibroblasts, and here, we demonstrate that cultured Col3-deficient fibroblasts have increased TGFß signaling compared to wild-type fibroblasts. Moreover, kinetic binding studies show that a synthetic peptide containing a Col3 cysteine-rich (CR) domain found within its N-propeptide binds in a dose-dependent manner to TGFß1, while a CR control peptide with mutated cysteines does not, suggesting that Col3 attenuates TGFß signaling in part through the N-propeptide CR domain. Consistent with this hypothesis, the CR peptide attenuates TGFß signaling in fibroblasts and 4T1 breast cancer cells and suppresses fibroblast activation and contraction, as assessed by α-smooth-muscle actin staining, cell wrinkling of deformable silicone, and stressed-fibroblast populated collagen lattice contraction assays. Finally, CR peptide treatment of orthotopically injected breast cancer cells (4T1) suppresses intratumoral fibroblast activation and inhibits primary tumor growth compared to CR control. Treatment with the CR peptide decreases both intratumoral canonical and non-canonical downstream TGFß signaling targets, consistent with its extracellular binding to TGFß. Taken together, our results suggest that the Col3 N-propeptide CR domain binds TGFß1 and attenuates (but importantly does not eliminate) TGFß signaling in fibroblasts and cancer cells. Expanding on our previous work, this study demonstrates an additional mechanism by which Col3 regulates cell behaviors in post-injury and tumor microenvironments and suggests that novel Col3-targeted strategies could effectively control biologic responses in vivo and improve anti-scarring/fibrosis and oncologic therapies.