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Xiaoqing Q Guo, Jennifer A Tran, Audrey E K Hutcheon, James D Zieske; Blockage of Smad-Signaling Pathway in Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4906.
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© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor beta (TGFβ) affects both corneal epithelial and stromal wound healing mechanisms by activating several signaling pathways. One of these being the Smad pathway, which, in addition to TGFβ, requires the presence of Smad Anchor for Receptor Activation (SARA) to become activated. Previous studies in human corneal epithelial cells (HCE) have shown that Trx-SARA, an inhibitor of SARA, can effectively inhibit the Smad pathway. In the current study, we investigated the use of Trx-SARA in primary human corneal fibroblasts (HCF) to discern the relationship between the Smad pathway and the activation of a known TGFβ-target protein, thrombospondin-1 (TSP-1), and HCF proliferation.
Retroviruses (RTV) were made with either Trx-SARA or Trx-GA, a control plasmid. HCF ± Trx-SARA or -GA were serum starved and then grown with 2ng/ml TGFβ1 overnight. Samples with no TGFβ1-treatment served as controls. Samples were collected and then processed to examine the effects of Trx-SARA on TSP-1 expression by western blotting (WB) or on Ki67 expression by indirect-immunofluorescence (IF).
Inhibition of Smad signaling reduced HCF proliferation by almost 50%; however, blocking Smad signaling had no effect on TSP-1 expression in HCF, which is in contrast to HCE data where TSP-1 expression was reduced by 90%.
Trx-SARA is an effective inhibitor of the Smad pathway and a useful tool for studying TSP-1 activation. TSP-1 response to TGFβ1 was not inhibited by Trx-SARA in HCF, suggesting that different signaling pathways are used to activate TSP-1 in HCF and HCE. Interestingly, Trx-SARA inhibited HCF proliferation indicating that proliferation is dependent on Smad signaling.
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