TGFβ 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. TGFβ stimulates activation of myofibroblasts, and here, we demonstrate that cultured Col3-deficient fibroblasts have increased TGFβ 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 TGFβ1, while a CR control peptide with mutated cysteines does not, suggesting that Col3 attenuates TGFβ signaling in part through the N-propeptide CR domain. Consistent with this hypothesis, the CR peptide attenuates TGFβ 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 TGFβ signaling targets, consistent with its extracellular binding to TGFβ. Taken together, our results suggest that the Col3 N-propeptide CR domain binds TGFβ1 and attenuates (but importantly does not eliminate) TGFβ 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.

TGFβ 是细胞与细胞外基质之间动态相互作用的关键调节因子，可驱动组织修复和肿瘤微环境中的生理和病理反应。我们的研究将 III 型胶原 (Col3) 定义为疤痕形成和结缔组织形成的抑制剂，部分是通过其对肌成纤维细胞的影响。 TGFβ 刺激肌成纤维细胞的活化，在这里，我们证明，与野生型成纤维细胞相比，培养的 Col3 缺陷型成纤维细胞具有增强的 TGFβ 信号传导。此外，动力学结合研究表明，在其 N 前肽中发现的含有 Col3 富含半胱氨酸 (CR) 结构域的合成肽以剂量依赖性方式与 TGFβ1 结合，而具有突变半胱氨酸的 CR 对照肽则不会，这表明 Col3部分通过 N-前肽 CR 结构域减弱 TGFβ 信号传导。与这一假设一致的是，CR 肽减弱成纤维细胞和 4T1 乳腺癌细胞中的 TGFβ 信号传导，并抑制成纤维细胞活化和收缩，通过 α-平滑肌肌动蛋白染色、可变形硅胶的细胞起皱和受压成纤维细胞填充的胶原晶格收缩进行评估化验。最后，与 CR 对照相比，原位注射乳腺癌细胞 (4T1) 的 CR 肽治疗可抑制瘤内成纤维细胞活化并抑制原发性肿瘤生长。 CR 肽治疗可降低肿瘤内典型和非典型下游 TGFβ 信号传导靶标，这与其细胞外与 TGFβ 的结合一致。综上所述，我们的结果表明 Col3 N-前肽 CR 结构域结合 TGFβ1 并减弱（但重要的是不会消除）成纤维细胞和癌细胞中的 TGFβ 信号传导。 这项研究扩展了我们之前的工作，展示了 Col3 在损伤后和肿瘤微环境中调节细胞行为的另一种机制，并表明新型 Col3 靶向策略可以有效控制体内生物反应并改善抗疤痕/纤维化和肿瘤治疗。