Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B‐mediated phosphorylation and cytoplasmic translocation of p27^Kip‐1

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 p27(Kip-1). CTGF stimulated the phosphorylation and cytoplasmic translocation of p27(Kip-1) on serine 10. Addition of the PI-3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate prevented p27(Kip-1) phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27(Kip-1) colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27(Kip-1), revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27(Kip-1) cytoplasmic 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 p27(Kip-1). Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27(Kip-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 alpha/gamma) 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 p27(Kip-1) as a mediator of actin rearrangement.

Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress

“…In fact, it was reported that CCN2 adversely affected actin polymerization through the Rho signaling pathways (Crean et al 2006), which may form a negative feed back loop for CCN2 production …”

Neurofibromatosis-1 regulates neuroglial progenitor proliferation and glial differentiation in a brain region-specific manner

“…In contrast, we found increased expression of the p27 cyclin-dependent kinase inhibitor following Nf1 inactivation in CTX NSCs, but not in BS NSCs. p27 function is regulated by Akt-mediated phosphorylation at residues Thr 198 and Ser 10, resulting in cytoplasmic localization and inhibition of its activity (Fujita et al 2002;Motti et al 2004;Crean et al 2006). Akt also regulates Skp2 (S-phase kinase-associated protein-2), a SCF F-box protein, which binds to p27 and targets it for ubiquitination and degradation (Lin et al 2009).…”

Wnt-induced secreted protein-1 is a prohypertrophic and profibrotic growth factor

“…Interestingly, biglycan, which was shown to be regulated by TGF-␤, may itself be protective since this proteoglycan was reported to enhance the survival of neurons (33,35). CTGF, which is expressed secondarily to TGF-␤ and angiotensin II in the heart post-MI, signals through Akt similarly to WISP-1 (20,55). However, CTGF has been shown to induce apoptosis in several cell types, including human aortic smooth muscle (30) and peritoneal mesothelial cells (53).…”