Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B‐mediated phosphorylation and cytoplasmic translocation of p27Kip‐1

Publisher Website
Google Scholar
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 interleukin (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 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 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. since the first description of FISP-12 in 1988 (1)⤻ and the subsequent partial characterization of its human ortholog connective tissue growth factor (CTGF) (2)⤻ , researchers have striven to answer key questions concerning its mechanism of action and the consequences of its increased expression. The rise to prominence of CTGF was initially due to the observation that it was a downstream mediator of TGF-β1, particularly in respect to its profibrotic effects (3)⤻ . It was subsequently implicated in the progression of fibrotic diseases such as atherosclerosis (4)⤻ , pulmonary fibrosis (5)⤻ , and diabetic nephropathy (6; for reviews, see Refs. 7⤻ , 8⤻ , and 9); however, clear signaling and regulatory insights have remained elusive. Recent developments in this field have gone some way toward addressing these questions, revealing divergent signaling pathways that respond to CTGF, controlling processes as diverse as matrix production (10)⤻ , cell proliferation (11⤻ , 12)⤻ , and cell polarization and migration (13)⤻ . In addition significant progress has been made in identifying structure-function correlates that have increased our understanding of how and why CTGF elicits it effects (10⤻ , 11⤻ , 14⤻ 15⤻ 16)⤻ . We first identified CTGF as a profibrotic mediator that was up-regulated in both in vivo and in vitro models of diabetic nephropathy (6)⤻ . We and others subsequently demonstrated that CTGF, like other members of the CCN family, can act through β3 integrins (17⤻ , 18)⤻ to induce fibronectin production in renal mesangial cells via a mechanism that involves recruitment and activation of Src kinase and subsequent activation of both the PI-3 kinase and p42/44 MAPK pathways (10)⤻ . CTGF also induced mesangial cell migration and disassembly of the actin cytoskeleton through an integrated mechanism, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of Rho A activity, activation of Cdc42, and phosphorylation of PKC-ζ and GSK-3β (13)⤻ . Indeed, although a range of studies have described the activation of MAP kinase and PI-3 kinase signaling pathways in various cell types treated with CTGF (19⤻ , 20)⤻ , the downstream consequences remain largely undefined. Here, we have focused particularly on delineating the downstream events resulting from activation of the PI-3 kinase pathway. The PI-3 kinase target, p27Kip1 has previously been implicated in the progressive glomerular hypertrophy of diabetic nephropathy (21⤻ , 22)⤻ . Similarly, p27Kip1 knockout mice fail to develop either renal or glomerular hypertrophy (23)⤻ , suggesting that control of p27Kip1 may ameliorate renal diabetic microvascular complications; however, the mechanism through which p27Kip1 levels are elevated and the consequences of this activation in a diabetic milieu remain unclear. Previously, it has been demonstrated that TGFβ-mediated mesangial cell hypertrophy is CTGF dependent and...

This publication has 51 references indexed in Scilit: