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Xiaoqing Guo, Audrey Hutcheon, James Zieske; Effect of TGF-β1 and 3 on protein expression in human corneal fibroblasts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5247. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
One of the intriguing puzzles of TGF-β signaling is how TGF-β1 and 2 (T1 and T2, respectively) stimulate fibrosis, while TGF-β3 (T3) appears to stimulate regeneration. Previously, we have used RNA arrays to compare the effect of T1 and T3 on human corneal fibroblasts (HCF). In the current experiments, we examined protein expression of genes determined by the arrays to be altered by T1 or T3.
Human corneal fibroblasts cultured in 10% FBS in DMEM were exposed to stabilized Vitamin C (VitC) with/without 2ng/ml T1 or T3. HCF were harvested on day 3 and protein was extracted with RIPA buffer plus protease inhibitors. Equal amount of protein was loaded onto 4-20% gradient gels. Blots were probed with antibodies against collagen I, Smad 3, Smad 7, and thrombospondin-1 (TSP-1). Band intensities were quantified with ImageJ software (v.1.45s). At least four samples were analyzed per condition.
When compared to VitC only samples (control), both T1 and T3 caused a significant increase in collagen I (p<0.05) and dramatically reduced Smad 3 levels (p<0.0001); however, no difference was detected between T1 and T3. Interestingly, T1 decreased Smad 7 significantly (p<0.0001) when compared with control, while T3 had no effect. Lastly, T3 increased TSP-1 levels by 2.5 fold compared to control (p<0.05), while T1 had no effect.
RNA array data indicated that several genes were dramatically altered in HCF treated with T1 and T3 when compared with controls. Analysis of protein expression has confirmed that protein levels were also altered. Most interestingly, we have found that 3-day exposure of HCF to T1 and T3 had significant effects on Smads 3 and 7. Both T1 and T3 lowered Smad 3 protein to nearly undetectable levels, suggesting that proteins dependent on Smad 3 would not be expressed. We also observed a differential effect on Smad 7. Since Smad 7 may prevent fibrosis, it is interesting that a 3-day exposure of HCF to T1, which promotes fibrosis, reduced Smad 7; while T3, which promotes regeneration, maintained levels of Smad 7.
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