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TGFbeta signalling through Smad‐independent mechanisms in human lens cells
Author(s) -
WORMSTONE IM,
ELDRED JA,
DAWES LJ,
REDDAN JR,
WANG L
Publication year - 2008
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2008.4144.x
Subject(s) - smad , mapk/erk pathway , immunocytochemistry , kinase , contraction (grammar) , smad2 protein , transforming growth factor , microbiology and biotechnology , chemistry , population , western blot , blot , biology , medicine , endocrinology , biochemistry , environmental health , gene
Purpose Previous work has shown that TGFß can induce matrix contraction and is likely to be regulated by Smad‐independent pathways. The present study investigated the role of the classical MAPK signalling and Rho kinase signalling pathways in TGFß mediated matrix contraction. Methods Cells from the human lens line FHL 124 were employed. Contraction was assessed using a patch assay, whereby the area covered by cells, was measured using imaging techniques. ERK activation was evaluated using western blot methods. To study SMAD2/3 distribution immunocytochemistry was used. Following a 24hr period of serum starvation, cells were maintained in the following conditions: Control medium +/– 10µM MEK inhibitor (U0126); 10ng/ml TGFß2 +/– U0126; Control medium +/– 10µM Rho kinase Inhibitor (Y–27632); 10ng/ml TGFß2 +/– Y–27632. The experimental duration for Smad2/3 staining was 2 hrs; western blots, 30mins & 24hr; patch contraction assays, 2‐3 days. Results Addition of 10ng/ml TGFß2 lead to a significant elevation of P‐ERK levels following both 30 min and 24hr periods of exposure. Addition of 10ng/ml TGFß2 to patch assays caused a significant contractile event to take place. Maintenance of cultures in the presence of 10µM U0126 or Y–27632 had no significant effect on patch area when added to control medium; however, when added in the presence of TGFß2, contraction was inhibited. With respect to cell population all groups were similar. Immunocytochemistry for SMAD2/3 showed clear nuclear translocation in response to 10ng/ml TGFß2; this signalling event was unaffected by U0126 and Y–27632. Conclusion MAPK and Rho kinase signalling play important roles in TGFß mediated matrix contraction of human lens cells.Commercial interest