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

Xiaoqing Guo,James D Zieske,Jennifer A Tran,Audrey E K Hutcheon

doi:10.15761/nfo.1000151

Abstract

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 p38MAPK. 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, p15ink4b expression in HCE-TJ, and cellular fibronectin (cFN) expression in HCF was determined. In addition, the effect of p38MAPK inhibitor on TSP-1 and p15ink4b 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 p15ink4b. With HCF, Trx-SARA failed to reduce TSP-1 expression; however, cFN expression decreased and proliferation was inhibited. By blocking the p38MAPK pathway, TSP-1 expression was reduced in HCF and p15ink4b expression was decreased in HCE-TJ. Surprisingly, TSP-1 was regulated through the Smad pathway in HCE-TJ and the p38MAPK pathway in HCF. The p38MAPK pathway also induced p15ink4b 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.

Highlights

Corneal wound repair is an ordered complex process that is regulated by many growth factors
No apparent difference was noted between Trx-SARA (Figure 1A and C) and NLS-Trx-SARA (Figure 1B1 and D1), and no fluorescence was seen in the uninfected HCE-TJ (1B2) and HCF (D2), which served as the negative controls for GFP
To determine the optimal exposure time to TGF-β1, we examined the following question: At what time does TGF-β1 have its maximal effect on TSP-1 in HCE-TJ and Primary human corneal epithelial cells (pHCE) cells? As seen in Figure 2A, even with starving the cells overnight (Control: basic media (BM) only with no TGF-β1), the HCE-TJ produced low levels of TSP-1

Summary

Introduction

Corneal wound repair is an ordered complex process that is regulated by many growth factors. One such growth factor is transforming growth factor beta (TGF-β), which is a multifunctional cytokine that plays a pivotal role in regulating growth, differentiation, proliferation, adhesion, apoptosis, and function of numerous cell types, including human corneal tissue [1,2,3]. Smad and 3 are TGF-β-specific intracellular signal transducers, which, upon phosphorylation, interact with the common Smad (Smad4) and form heterodimeric and heterotrimeric complexes. These complexes translocate into the nucleus and bind to transcription factors, coactivators, or corepressors, to activate or inhibit the expression of TGF-β-response genes [4,5,7,8]

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