TGF-β-target genes are differentially regulated in corneal epithelial cells and fibroblasts.

PURPOSE

Transforming growth factor-beta (TGF-β) activates the canonical Smad pathway, which includes the Smad family of proteins and SARA (Smad Anchor for Receptor Activation) and other less understood pathways, including one involving p38. The goal of the current research was to determine if corneal epithelial cells and fibroblasts used the classical or alternative TGF-β-signaling pathways. To examine this question, we made use of Trx-SARA, which inhibits native SARA, thus blocking the Smad pathway.

METHODS

A human corneal epithelial cell line (HCE-TJ), and stromal fibroblasts (HCF) were infected with retroviruses (RTV) containing either Trx-SARA or Trx-GA (a control plasmid). The effect of Trx-SARA on thrombospondin-1 (TSP-1) expression in both cell types, p15 expression in HCE-TJ, and cellular fibronectin (cFN) expression in HCF was determined. In addition, the effect of p38 inhibitor on TSP-1 and p15 were examined.

RESULTS

In HCE-TJ with TGF-β1, TSP-1-protein levels increased and peaked at 24 hours. Trx-SARA reduced TSP-1 expression in HCE-TJ, but had no effect on p15. With HCF, Trx-SARA failed to reduce TSP-1 expression; however, cFN expression decreased and proliferation was inhibited. By blocking the p38 pathway, TSP-1 expression was reduced in HCF and p15 expression was decreased in HCE-TJ.

CONCLUSIONS

Surprisingly, TSP-1 was regulated through the Smad pathway in HCE-TJ and the p38 pathway in HCF. The p38 pathway also induced p15 in HCE-TJ. Our results indicate that not all TGF-β-target proteins require the Smad pathway, and it may be possible to block certain TGF-β-target proteins without blocking the expression of all the TGF-β-target proteins.