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Figure 2. CTGF stimulates the association of p27Kip-1 with Rho A and consequent uncoupling of the Rho A/LIM kinase/cofilin pathway. Mesangial cells were treated with CTGF, and whole cell lysates immunoprecipitated with anti p27Kip-1 antibodies. Western blots were then probed with antibodies to Rho A. CTGF stimulated an increased association between p27Kip-1 and Rho A, (A, upper panels). Similarly, in the reverse immunoprecipitation, mesangial cells were treated with CTGF and whole cell lysates immunoprecipitated with anti Rho A antibodies. Western blots were then probed with antibodies to phospho Thr 157 p27Kip-1. CTGF stimulated an increased association between Rho A and phospho Thr 157 p27Kip-1 (A, middle panels). Immunocytochemistry revealed that 14–3-3 
localized predominantly cytoplasmic and perinuclear in response to CTGF. CTGF also stimulated an increased association between 14–3-3 and p27Kip-1 (A, lower panels). We then investigated the consequences of these events for the Rho A/cofilin/LIM kinase pathway. Cells were treated with CTGF as described, and Western blot analysis was used to investigate the phosphorylation status of cofilin and LIM-kinase. CTGF stimulated an increase in phosphorylation of both LIM-kinase and cofilin, which was again completely abrogated by the addition of the PI3 kinase inhibitor LY294002 and the Akt inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate.
