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L.J. Dawes, G. Duncan, I.K. Anderson, J.R. Reddan, I.M. Wormstone; Differential Effects of Tgfß1, 2 and 3 on Matrix Contraction, SMAD Signalling and Transdifferentiation of Human Lens Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2878.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the relative influence of TGFß isoforms 1, 2 & 3 on Smad signalling, the transdifferentiation marker α–smooth muscle actin (αSMA) and matrix contraction of a human lens cell line (FHL 124). Methods:FHL 124 cells were routinely cultured and seeded onto glass coverslips (immuno–cytochemistry) or tissue culture dishes (Patch assay and QRT–PCR). Following a 24hr period of serum starvation, cells were exposed to 0, 0.01, 0.1, 1 or 10ng/ml TGFß1, 2 or 3 and analysed as described. Smad2 distribution was observed by immunocytochemistry. Contraction was assessed using a patch growth assay, whereby all area covered by cells was measured using imaging techniques. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. Change in mRNA αSMA expression levels was determined using Real–Time PCR (MJ Opticon 2), with SYBR green as the detection label. Results: All TGFß isoforms induced translocation of Smad2 in to the nucleus, with TGFß2 being the most potent. The EC50 values for TGFß1, ß2 & ß3 were 0.4, 0.12 and 6.3ng/ml respectively. TGFß1 and 2 both induced contraction of cell patches in a concentration–dependent manner, however, TGFß1 showed the greatest potency with an EC50 value of 0.33ng/ml. No such changes were seen with TGFß3 at the concentrations tested. A 24hr exposure to TGFß1 and 2 significantly increased αSMA in a dose–dependent manner. On this occasion TGFß2 was the most potent isoform. TGFß3 had no significant effect on gene expression. Conclusions:TGF beta isoforms have differential effects on signalling, contraction and gene expression. TGFß3 only weakly activated Smad2 translocation and did not influence contraction or αSMA expression. TGFß2 produced the greatest stimulation of Smad signalling and αSMA expression, however TGFß1 is responsible for the greatest degree of contraction. Therefore, both TGFß1 and ß2 can affect human lens cell function.
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