Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B‐mediated phosphorylation and cytoplasmic translocation of p27Kip …

JK Crean, F Furlong, D Mitchell, E McArdle… - The FASEB …, 2006 - Wiley Online Library
JK Crean, F Furlong, D Mitchell, E McArdle, C Godson, F Martin, JK Crean, F Furlong…
The FASEB Journal, 2006Wiley Online Library
Connective tissue growth factor (CTGF/CCN2) is a 38‐kDa secreted protein, a prototypic
member of the CCN family, which is up‐regulated in many diseases, including
atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that
CTGF can cause actin disassembly with concurrent down‐regulation of the small GTPase
Rho A and proposed an integrated signaling network connecting focal adhesion dissolution
and actin disassembly with cell polarization and migration. Here, we further delineate the …
Abstract
Connective tissue growth factor (CTGF/ CCN2) is a 38‐kDa secreted protein, a prototypic member of the CCN family, which is up‐regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down‐regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/ protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27Kip‐1. CTGF stimulated the phosphorylation and cytoplasmic translocation of p27Kip‐1 on serine 10. Addition of the PI‐3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleu‐kin (IL)‐6‐hydroxymethyl‐chiro‐inositol‐2(R)‐2‐methyl‐3‐O‐octadecylcarbonate prevented p27Kip‐1 phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27Kip‐1 colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27Kip‐1, revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27Kip‐1 cyto‐plasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27Kip‐1. Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27Kip‐1 activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF‐stimulated actin depolymerization only in wild‐type mouse embryonic fibroblasts (MEFs) compared to Akt‐1/3 (PKB α/γ) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27Kip‐1 as a mediator of actin rearrangement.—Crean, J. K., Furlong, F., Mitchell, D., McArdle E., Godson, C., and Martin, F. Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B‐mediated phosphorylation and cytoplasmic translocation of p27Kip‐1. FASEB J. 20, E1037‐E1048 (2006)
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