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J. D. Zieske, X. Q. Guo, A. E. K. Hutcheon; Trx-SARA Inhibits Thrombospondin-1 Synthesis in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5040.
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TGF-β1 stimulates gene expression through pathways that involve Smad proteins and through Smad independent pathways. To dissect TGF-β signaling in human corneal epithelial cells, we constructed a retrovirus encoding a portion of the SARA (Smad Anchor for Receptor Activation) protein linked to a spacer protein Trx (thioredoxin). The Smad pathway is dependent on SARA, which allows for the phosphorylation of Smad 2 and 3 by the TGFβRI/II complex. Trx-SARA has been shown in other systems to competitively inhibit the Smad pathway, while still allowing TGF-β signaling through Smad independent pathways. In the current investigation, we examined the effect of Trx-SARA on thrombospondin-1 (TSP-1) expression.
To determine the best time to look at Trx-SARA effect, Araki-Sasaki human epithelial cell line (HCE-TJ) and primary human epithelial cells (HCE) were serum starved overnight and then grown with 2ng/ml TGFβ1 and collected at 2, 6, 24 and 48 hours. No TGFβ1 cells served as a control. Immunoblotting was performed with anti-TSP-1. To determine the effect of Trx-SARA on TGFβ1 stimulated HCE-TJ and HCE, cells were infected with retroviruses encoding for either 1) no Trx-SARA, 2) NLS (nuclear location sequence)-Trx- GA (glycine/alanine repeat), 3) NLS-Trx-SARA, 4) Trx-GA or 5) Trx-SARA. These cells were then serum starved overnight and grown with or without 2ng/ml of TGFβ1 for 24 hours. These cells were examined for efficiency of infection by looking at the number of cells with GFP. They were also harvested and examined by immunoblotting with anti-TSP-1.
Upon the addition of TGFβ1 to HCE-TJ, the levels of TSP-1 protein increased linearly with time of exposure up to 24 hours, by 48 hours it had decreased back to control level. Upon adding TGFβ1 to HCE, there was an increase of TSP-1, which peaked at 24 hours. However, when the Trx-SARA was introduced, there was no increase of TSP-1 protein levels. NLS-Trx-SARA reduced expression of TSP-1 by 50%. Trx-GA and NLS-Trx-GA had no effect on TSP-1 expression.
TSP-1 is activated through the Smad pathway. Trx-SARA is an effective inhibitor of this pathway and is a useful tool for studying TSP-1 activation.
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