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

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^MAPK. 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.

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^ink4b expression in HCE-TJ, and cellular fibronectin (cFN) expression in HCF was determined. In addition, the effect of p38^MAPK inhibitor on TSP-1 and p15^ink4b were examined.

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^ink4b. With HCF, Trx-SARA failed to reduce TSP-1 expression; however, cFN expression decreased and proliferation was inhibited. By blocking the p38^MAPK pathway, TSP-1 expression was reduced in HCF and p15^ink4b expression was decreased in HCE-TJ.

Surprisingly, TSP-1 was regulated through the Smad pathway in HCE-TJ and the p38^MAPK pathway in HCF. The p38^MAPK pathway also induced p15^ink4b 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.