A negative feedback loop centered on SMAD3 expression in transforming growth factor 81-induced corneal myofibroblast differentiation

SMAD3 downregulation is documented in transforming growth factor 81 (TGF-81)-induced corneal fibroblasts differentiation to myofibroblasts (fibroTOmyoDiff) or corneal wound healing. However, the exact regulatory mechanism of TGF-81/SMAD3 pathway in this context remains unclear. Here, we investigated the role and related mechanism of SMAD3 down-regulation in TGF-81-induced human corneal fibroTOmyoDiff. By detecting expression changes of SMAD family during this process, we demonstrated that SMAD3 protein expression was dramatically decreased in the process and the decrease occurred mainly in SMAD3 gene transcription. Furthermore, SMAD3 overexpression using lentivirus infection and knockdown using sgRNA lentivirus infection or siRNAs revealed that SMAD3 overexpression enhanced TGF-81-induced corneal fibroTOmyoDiff and vice versa. In addition, specific siRNAs and inhibitors targeting particular signaling pathway were used to figure out the intracellular signaling pathway regulating SMAD3, and the result showed that the decease of SMAD3 induced by TGF-81 stimulation in human corneal fibroblasts (HCFs) was strikingly prevented by SMAD4 knockdown or p38 signaling inhibitor SB203580 treatment. Collectively, these results demonstrate that, in TGF-81 induced corneal fibroTOmyoDiff, down-regulation of SMAD3 expression regulated by SMAD4 and p38 signaling path-ways forms a negative feedback loop of TGF8 signaling to avoid excessive activation of the signaling, which suggest that SMAD3 may be a key target for corneal fibrosis treatment.

Automated summary

This study investigated the role and mechanism of SMAD3 downregulation in TGF-β1-induced corneal fibroblast differentiation. The results showed that SMAD3 expression was dramatically decreased during the process, mainly at the transcription level. Additionally, overexpression or knockdown of SMAD3 enhanced or suppressed TGF-β1-induced fibroblast differentiation, respectively. It was also found that SMAD4 and p38 signaling pathway played a role in regulating SMAD3 downregulation. These findings suggest that targeting SMAD3 may be a potential treatment for corneal fibrosis.