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

J. K. Crean^*,1, F. Furlong^*, D. Mitchell, E. McArdle^*, C. Godson and F. Martin^*

^* UCD School of Biomolecular and Biomedical Science and UCD School of Medicine and Medical Science;

UCD Conway Institute, University College Dublin; and The Dublin Molecular Medicine Centre, Belfield, Dublin, Ireland

¹Correspondence: School of Biomolecular and Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. E-mail: john.crean{at}ucd.ie

SPECIFIC AIMS

Here, we examined the role of CTGF in actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. Explicitly, we wished to determine how CTGF mediates actin rearrangement through PI-3 Kinase, Akt/Protein Kinase B (PKB) and p27^Kip-1 and examine the implications for mesangial cell dysfunction in diabetic nephropathy.

PRINCIPAL FINDINGS

1. CTGF stimulates the phosphorylation of Akt/PKB and a number of Akt/PKB substrates in human mesangial cells
Previously, we identified PI-3 kinase-mediated Akt/PKB phosphorylation as a signal transduction pathway in human mesangial cells stimulated with recombinant CTGF. Western blot analysis using a Akt/PKB substrate-specific antibody identified a number of putative downstream targets of CTGF signaling with apparent molecular weights of 70 kDa, 35 kDa, and 27 kDa. Phosphorylation was completely abrogated by LY294002, indicating that it occurred downstream of PI-3 kinase, while the MEK inhibitor PD98059 had no discernable effect. We tentatively identified two of these targets as FKHR and p27^kip-1 based on bioinformatic analyses. Further studies revealed that CTGF stimulated a time-dependent increase in the phosphorylation of FKHR at serine 256, which was abrogated by the addition of the PI-3 kinase inhibitor LY294002 and the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate (Fig. 1 C) Similarly, CTGF stimulated an increase in both total p27^kip-1 and serine 10 phosphorylated p27^kip-1. CTGF treatment also resulted in a time-dependent decrease in the nuclear levels of serine 10 phosphorylated p27^kip-1 with a concomitant increase in the cytoplasmic levels. Serine 10 phosphorylated p27^kip-1 was primarily localized in the nucleus in untreated cells. Stimulation with CTGF led to a clear cytoplasmic accumulation of Ser-10 phosphorylated p27^kip-1 in an Akt/PKB-sensitive manner; addition of the Akt/PKB inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate resulted in nuclear localization (Figure 1A ).

View larger version (49K): [in this window] [in a new window]   Figure 1. CTGF stimulates phosphorylation of p27Kip-1 on serine 10 in an Akt/PKB-dependent manner. We visualized the subcellular localization of p27Kip-1 in response to CTGF by immunocytochemistry. Serine 10 phosphorylated p27Kip-1 was primarily localized in the nucleus in untreated cells. Stimulation with CTGF led to a clear cytoplasmic accumulation of serine 10-phosphorylated p27Kip-1 in a Akt/PKB-sensitive manner; addition of the Akt/PKB inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate resulted in nuclear localization (A). Cytoplasmic serine 10-phosphorylated p27Kip-1 appeared to colocalize with the ends of shortened actin fibers in CTGF treated cells (B). CTGF also stimulated the Akt/PKB dependent phosphorylation of the p27Kip-1 transcriptional regulator FKHR (C).

2. CTGF stimulates phosphorylation of p27 on threonine 157 in an Akt/PKB-dependent manner
Phosphorylation of p27^kip-1 on threonine 157 has previously been shown to regulate its cellular localization. We therefore examined its phosphorylation status on Thr 157 and whether this affected its subcellular localization in response to CTGF. CTGF stimulated a rapid and transient increase in the phosphorylation of threonine 157 p27^kip-1, which was PI3-kinase and Akt/PKB dependent. Immunocytochemistry revealed that CTGF-induced perinuclear and cytoplasmic localization of threonine 157 phosphorylated p27^kip-1, coincident with disruption of the mesangial actin stress fibers (Fig. 1B ).

We then wished to establish if p27^kip-1 cytoplasmic localization was required for CTGF-stimulated actin depolymerization. Mesangial cells were transfected with a T157A and T157D yellow fluorescent protein (YFP) p27^kip-1 mutants and stimulated with CTGF. The expression of the p27^kip-1 mutants was confined to the nucleus, indicating that phosphorylation was necessary for cytoplasmic localization. We then used the threonine 157 p27^kip-1 mutants to determine the functional significance of this phosphorylation. Cells were cotransfected with GFP-actin and p27^kip-1 wild-type or T157A YFP- p27^kip-1 mutant, or with GFP-actin alone, stimulated with CTGF and monitored by real-time fluorescence microscopy. CTGF stimulated a rapid disassembly of the actin cytoskeleton accompanied by the adoption of a polarized morphology with clear accumulation of actin at the leading edge, consistent with our previous findings in cells transfected with GFP-actin. In contrast, the actin cytoskeleton remained intact, and there was no polarized morphology in cells cotransfected with GFP-actin and either mutant; expression of the YFP-p27^kip-1 mutants was again confined to the nucleus.

3. CTGF stimulates the association of p27 with RhoA and consequent uncoupling of the Rho A/LIM kinase/Cofilin pathway
Given our observation that cells overexpressing the T157A YFP p27^kip-1 mutant do not undergo either actin disassembly or polarization when treated with CTGF, we wished to further investigate the mechanism underlying these changes.

Using immunoprecipitation assays, we found that CTGF stimulated an increased association between RhoA and phospho Thr 157 p27. CTGF also stimulated an increased association between 14–3-3 and p27^kip-1, raising the possibility that CTGF stimulates the formation of a regulatory complex of 14–3-3 , p27^kip-1 and Rho A (Fig. 2 A).

View larger version (39K): [in this window] [in a new window]   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.

We then wished to determine the downstream consequences of these events for the RhoA/cofilin/LIM kinase pathway. Surprisingly, 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/PKB inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate (Figure 2B ). These results suggest that CTGF mediated decreases in Rho A activity results in uncoupling of the LIM-kinase/cofilin pathway, thereby facilitating increased actin disassembly in PKB dependent manner and that cytoplasmic p27^kip1 modulates actin dynamics by direct regulation of the small GTPase RhoA pathway. Indeed, further immunocytochemistry studies revealed that cytoplasmic p27^kip-1 appears to localize at the ends of actin filaments during the time course of CTGF stimulated actin disassembly (Fig. 1B ). 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 /) knockout MEFs.

CONCLUSIONS AND SIGNIFICANCE

This study examines the consequences of CTGF stimulated activation of the PI-3 kinase pathway in human mesangial cells, leading to actin depolymerization. We show that this is mediated by Akt/PKB-dependent phosphorylation and cytoplasmic translocation of p27^kip-1, which subsequently binds to RhoA, resulting in phosphorylation of LIM kinase and cofilin, indicative of increased actin disassembly. These results are summarized in the schematic (Fig. 3 ).

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic showing the matricellular growth regulator CTGF as it interacts with cell surface receptors in a context-dependent manner, activating the PI-3 kinase pathway (1). Akt/PKB then phosphorylates p27 at two sites, serine 10 and threonine 157, resulting in cytoplasmic translocation and stabilization (2), where it binds to and inactivates RhoA (3), resulting in uncoupling of RhoA from the Lim kinase/cofilin pathway and increased actin severing activity leading to actin depolymerization (4).

Recent findings suggest an emerging model wherein p27^kip-1 localization and activity are ultimately controlled by its phosphorylation, mediated by Akt/PKB. Our results confirm this hypothesis; stimulation with CTGF resulted in phosphorylation on Ser-10 and Thr 157 in an Akt/PKB-dependent manner, leading to cytoplasmic localization. Mutation of the threonine 157 (T157A or T157D) site resulted in nuclear localization of p27^kip-1. Recent data have shown that phosphorylation on threonine 157 prevented binding of p27^kip-1 to importin alpha, thereby inhibiting its reimportation into the nucleus. It is believed that this is facilitated by the interaction of threonine 157 phosphorylated p27^kip-1 with 14–3-3. Our studies have further elaborated on the downstream consequences of p27 phosphorylation and revealed that cytoplasmic localization, mediated by phosphorylation on threonine 157 is necessary for CTGF-mediated actin rearrangement, as evidenced by the fact that the actin cytoskeleton remained intact in cells overexpressing the T157A mutant compared to wild-type.

We have also demonstrated that there is both an increase in the phosphorylation of LIM kinase and cofilin in response to treatment with CTGF, most probably reflecting increased actin disassembly, consistent with our previous observations. As with epidermal growth factor, this suggests that the accumulation of phosphorylated cofilin in cells stimulated with CTGF is an attempt to redress the balance between polymerization and depolymerization, resulting from the gross actin disassembly and directly reflects increased actin severing activity. The central role that Akt/PKB plays in regulation of the actin cytoskeleton is well illustrated by the fact that MEFs from the Akt/PKB / knockout treated with CTGF did not undergo actin rearrangement and maintained their stress fibers, contrasting with the dramatic cytoskeletal alterations observed in embryonic fibroblasts from the wild-type mouse.

This is the first study to describe the consequences of Akt /PKB activation by CTGF. These include the phosphorylation of FKHR, phosphorylation and cytoplasmic translocation of p27^kip-1, increased association of p27^kip-1 with RhoA, and increased phosphorylation of LIM kinase and cofilin. These data extend the rationale for anti-CTGF directed therapies by demonstrating a mechanism whereby CTGF contributes to mesangial actin dysregulation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5010fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-5010fjev1 20/10/1712    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Crean, J. K. Articles by Martin, F. Search for Related Content PubMed PubMed Citation Articles by Crean, J. K. Articles by Martin, F.