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(The FASEB Journal. 2006;20:1712-1714.)
© 2006 FASEB

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

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

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

{dagger} 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

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 {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 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.


Key Words: CTGF • migration • actin cytoskeleton • p27Kip-1, RhoA • PI-3 kinase • Akt/PKB • LIM kinase • cofilin • diabetic nephropathy

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-ß1, 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 ß3 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-{zeta} and GSK-3ß (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 TGFß-mediated mesangial cell hypertrophy is CTGF dependent and that mesangial cells treated with CTGF have increased levels of p27Kip1 (24) . Here, we show for the first time that the matricellular growth regulator connective tissue growth factor (CCN2) stimulates the phosphorylation of p27Kip1 at the Akt/PKB consensus sequence at threonine 157 in a PI-3 kinase and Akt/PKB-dependent manner. The result of this phosphorylation is increased cytoplasmic and perinuclear localization of p27Kip1 where it interacts with Rho A, resulting in the uncoupling of the Rho A/LIM kinase/Cofilin pathway. We hypothesize that this pathway likely explains the observed disassembly of the mesangial cell actin cytoskeleton on exposure to CTGF and begins to shed mechanistic light on the altered mesangial actin mediated cell contractility observed in diabetic nephropathy.

MATERIALS AND METHODS

Reagents
Anti- Akt/PKB, phospho (Ser-473 Akt/PKB, phospho Akt/PKB substrate, LIM kinase, phospho (Thr 508) LIM kinase, cofilin, phospho (Ser-3 cofilin, FKHR and phospho (Ser-256 FKHR antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti p27Kip1, 14–3-3 {zeta}, and Rho A antibodies were from Santa Cruz; anti phospho (Ser-10 p27Kip1 antibody (Ab) was from Zymed Laboratories (Invitrogen, Carlsbad, CA); anti phospho (Thr 157) p27Kip1 Ab was from R+D Systems (Minneapolis, MN); anti ß actin Ab was from Sigma-Aldrich (St. Louis, MO). The protein kinase inhibitors LY 294002 and PD 98059 and the Akt/PKB inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate were obtained from Calbiochem (San Diego, CA); PP1 was from Biomol (Plymouth Meeting, PA), GF109203X was from AG Scientific Inc. (San Diego, CA) and Y27632 was a kind gift from the Welfide Corp. (Osaka, Japan); FITC- and rhodamine-phalloidin were purchased from Molecular Probes (Leiden, The Netherlands). Recombinant human CTGF/CCN2 (Lot No. CS 950–29) was a kind gift from Fibrogen (San Francisco, CA). Fugene 6 was purchased from Roche Applied Sciences, (Lewes, UK); Optimem was purchased from Invitrogen. p27Kip1 mutant expression plasmids were a kind gift from Professor Joyce Sligerland Columbia University (New York, NY). Akt 1/3 (PKB {alpha}/{gamma}) knockout and wild-type mouse embryonic fibroblasts were a kind gift from Brian Hemmings (Friedrich Miescher Institute, Basel, Switzerland). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

Preparation and analysis of recombinant human CTGF
Recombinant human CTGF (rhCTGF) was produced essentially as described previously (25) Briefly, rh CTGF was produced in a baculovirus expression system using Hi5 cells adapted for suspension growth and virus containing a complete human CTGF cDNA. Cells were cultured in Sf 900 II SFM (Life Technologies) supplemented with 20 µg/ml gentamicin (CellGro) and 1X lipid (Life Technologies) and then scaled up for production in 3L Fernbach culture flasks (Corning, Corning, NY). When cultures reached a density of 1.0–1.5 x 106 cells/ml with a viability of >95%, the cells were infected with human CTGF recombinant baculovirus at a multiplicity of infection of 10. Conditioned media containing rhCTGF was harvested 40 h postinfection, centrifuged (10 K RPM, 10 min), sterile filtered (0.45 µM membrane), and then passed over a heparin-Sepharose affinity column (Amersham-Pharmacia). The heparin column was washed with 10 column volumes (CV) 350 mM NaCl and rhCTGF was eluted using a linear NaCl gradient (350 mM to 1200 mM) for 15 CV, followed by step elution with 5 CV of 1200 mM NaCl. rhCTGF was identified in eluted peak fractions by SDS-PAGE, fractions were pooled, diluted with ddH2O until a conductivity of 5.7 mS was reached, and the pH adjusted to 8.0. Although buffers, columns, and plumbing were all sterilized, any residual endotoxin was removed by purification over Q-Sepharose/carboxymethyl polystyrene (CM) tandem columns. Under these conditions, endotoxin remained on the resin, while the rhCTGF first flowed through the Q-Sepharose (calculated pI of CTGF=8.37) and then readily bound the CM resin. Final sample pools were assayed for total protein content and endotoxin (Quantitative Chromogenic LAL-1000 Assay, BioWhitaker Cat # 50–647U). rhCTGF content and quality were evaluated by ELISA, SDS-PAGE, and Western blot analysis. (see Supplemental Fig. 1).

Cell culture
Primary human mesangial cells (Clonetics, San Diego, CA) (MCs) were cultured at 37°C in an atmosphere of 95% air and 5% CO2 in MCDB 131 (Life Technologies) supplemented with 10% fetal calf serum. Cells were used at the sixth and seventh passage. Before stimulation, cells were rendered quiescent by being maintained in the absence of serum for 24 h. After stimulation, cells were washed twice in ice-cold PBS before being lysed. Cells were lysed on ice for 5 min by adding 1 ml of a lysis buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na2 VO3, 0.2 mM PMSF, 1% Triton X-100, 0.5% Nonidet P-40. Samples were scraped from the dish and collected into 1.5-ml microcentrifuge tubes. Whole cell extracts were prepared by incubating lysates on ice for 30 min, after which time, samples were centrifuged at 12,000 g. The supernatant was then removed for subsequent analysis while the pellet was discarded. Mouse embryonic fibroblasts from Akt-1/3 (PKB {alpha}/{gamma}) knockout and wild-type animals were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Again, before stimulation, cells were rendered quiescent by being maintained in the absence of serum for 24 h. For inhibitor studies, cells were washed once in warm PBS before the addition of pharmacological agents in serum-free media, 30 min before stimulation with CTGF. The following concentrations were used: LY294002, 10 µM; PD98059, 10 µM; PP1, 10 µM; GF109203X, 10 nM; Rottlerin, 5 nM; Y27632 10 µM; and SB203580, 1 µM.

Preparation of nuclear and cytoplasmic fractions
Nuclear and cytoplasmic extracts were prepared using the Nuclear Extract kit from Active Motif. Briefly, cells were washed with 5 ml ice-cold PBS containing a phosphatase inhibitor cocktail and scraped into a prechilled conical tube. After centrifugation, cell were resuspended in a hypotonic buffer solution and incubated on ice for 15 min. Cells were then treated with detergent and centrifuged at 14,000 g for 30 s. The supernatant containing the cytoplasmic fraction was transferred to a clean tube and stored at –80°C until required. The pellet containing the nuclear fraction was resuspended in total lysis buffer and incubated on ice for 30 min with gentle agitation. This solution was then centrifuged at 14,000 g for 10 min, and the supernatant containing the nuclear fraction transferred to a clean microcentrifuge tube and stored at –80°C until required.

Cell migration assay
The effect of CTGF on mesangial cell migration was examined in a scrape-wounding assay, carried out essentially, as described previously (13 , 26) . The inhibitors used were the PI-3 kinase inhibitor LY294002, (10 nM) and the Akt inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate. In all cases, cells were pretreated with the appropriate inhibitor for 30 min before the addition of the growth factor.

Phosphorylation analysis of Akt/PKB, FKHR, p27Kip1, LIM kinase, and cofilin
For assessing the phosphorylation of Akt/PKB, p27Kip-1, LIM kinase, and cofilin whole cell lysates were prepared, as described previously (27) . Samples were normalized for total protein (28) , resuspended in reducing sample buffer, and separated by SDS-PAGE (29) . Proteins were then electrophoretically transferred to PVDF or nitrocellulose (2A, 200V for 90 min) and analyzed with antibodies raised against ß-actin, Akt/PKB, phospho Akt/PKB, phospho Akt/PKB substrate, FKHR, phospho FKHR, p27Kip-1, phospho (Ser-10 p27Kip-1, phospho (Thr 157) p27kip-1, LIM kinase, phospho (Thr 508) LIM kinase, cofilin, and phospho (Ser-3 cofilin. Horseradish peroxidase-conjugated secondary antibodies were used (1:5000) in conjunction with enhanced chemiluminescence duraTM chemiluminescence detection system (Pierce, Rockford, IL).

Immunocytochemistry and confocal microscopy
Mesangial cells were plated as described above onto Permanox 8-well slides (Labtec, Nalgene, Cairo, Egypt). Cells were treated with CTGF as indicated and subsequently stained for p27Kip-1, phospho (Ser-10) p27Kip-1, and phospho (Thr 157) p27Kip-1 using standard techniques. Briefly, chamber slides were rinsed in PBS and fixed in 3.7% paraformaldehyde in PBS for 20 min. After rinsing in PBS, cells were permeabilized with 0.01% triton X-100 for 3 min and then rinsed three times in PBS. Slides were incubated with primary Ab to total p27Kip-1, phospho (Ser-10) p27Kip-1, or phospho (Thr 157) p27Kip-1 for 20 min at room temperature and were rinsed and incubated with FITC-conjugated secondary Ab for an additional 20 min. Controls included the nonimmune IgG and the secondary Ab alone. To visualize F-actin, slides were stained with rhodamine or FITC-phalloidin for 20 min at room temperature. Nuclei were counterstained with 4',6'-diamidino-2-phenylidole. Stained cells were visualized with a Leitz DM40 microscope, and images were captured using the AxioCam system and AxioVision 3.0.6 software (Carl Zeiss, Thornwood, NY). Confocal microscopy was performed on a Zeiss LSM 510 Meta using LSM5 image acquisition software.

Immunoprecipitation
Twenty micrograms of total protein from whole cell lysates was adjusted to 1 ml with immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na2 VO3, 0.2 mM PMSF, 1% Triton X-100, 0.5% Nonidet P-40) and incubated with rolling with 2 µg of the appropriate Ab at 4°C overnight. The immunocomplexes were then captured by the addition of 50 µl of a 50% slurry of preswollen Protein A sepharose beads, followed by incubation again for 1 h again with rotation to ensure sample homogeneity. The complexes were then washed three times by centrifugation at 14,000 g for 5 min followed by gentle resuspension in 1 ml of immunoprecipitation buffer. The final pellet was then resuspended in electrophoresis buffer for Western blot analysis as described previously.

Transfection studies
p27Kip-1 T157A yellow fluorescent protein (YFP) (threonine to alanine mutant), p27Kip-1 T157D YFP (threonine to aspartic acid mutant), and Actin-GFP expression plasmids were transiently transfected or cotransfected into primary human mesangial cells using the Fugene transfection reagent. Briefly, 3 µl of Fugene was added to 97 µl Optimem and incubated at room temperature for 5 min. One microgram of plasmid DNA was added to the Fugene/Optimem mixture and mixed well by tapping. After incubation at room temperature for 15 min, the medium was removed from the cells and replaced with fresh medium; 50 µl of the Fugene/Optimem/DNA mixture were added to each well. Cells were incubated at 37°C for 48 h, after which time they were serum starved for a further 24 h. Cells were then treated with CTGF (25 ng/ml, 24 h) and the integrity of the actin cytoskeleton analyzed by direct immunofluorescent real time microscopy using a Zeiss Aviovert real-time imaging microscope with AxioCam system and AxioVision 4.0 software (Carl Zeiss, Thornwood, NY).

RESULTS

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 (Fig. 1 A). To investigate the downstream consequences of CTGF induced Akt/PKB phosphorylation, cells were treated with recombinant human CTGF in the presence or absence of a range of pharmacological inhibitors, including the PI-3 kinase inhibitor LY294002 and the Akt/PKB inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate.


Figure 1
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  		Figure 1. CTGF stimulates the phosphorylation of Akt/PKB and a number of Akt/PKB substrates in human mesangial cells. Primary human mesangial cells were treated with CTGF (25 ng/ml) for the times indicated and proteins separated by Western blot analysis. Western blots were probed with antibodies to Akt/PKB, phospho Akt/PKB (Ser-473, phospho Akt/PKB substrate, p27Kip-1, and phospho p27Kip-1 (Ser-10. CTGF stimulated phosphorylation of Akt/PKB in a rapid and transient manner (A). Western blot analysis identified a number of putative downstream targets of CTGF signaling with apparent molecular weights of ~70 kDa, 35 kDa and 27 kDa. CTGF stimulated a rapid, yet transient increase in phosphorylation of each of the Akt/PKB substrates, with the maximum phosphorylation at 30 min and declining thereafter. Phosphorylation was completely abrogated by LY294002, indicating that it occurred downstream of PI-3 kinase, while the MEK inhibitor, PD98059, had no discernable effect (B). CTGF also stimulated an increase in both total p27Kip-1 and serine 10 phosphorylated p27Kip-1 levels when normalized to beta actin (C). Subcellular fractionation revealed a time-dependent decrease in the nuclear levels of serine 10 phosphorylated p27Kip-1 with a concomitant increase in the cytoplasmic levels (D) in response to CTGF.

Western blot analysis using a Akt/PKB substrate-specific Ab identified a number of putative downstream targets of CTGF signaling with apparent molecular weights of ~70 kDa, 35 kDa, and 27 kDa. CTGF stimulated a rapid, yet transient, increase in phosphorylation of each of the Akt/PKB substrates, with the maximum phosphorylation at 30 min and declining thereafter. Phosphorylation was completely abrogated by LY294002, indicating that it occurred downstream of PI-3 kinase, while the MEK inhibitor, PD98059, had no discernable effect (Fig. 1B ). Interestingly, phosphorylation was also inhibited by the addition of PP1, implicating Src kinase in CTGF signaling, consistent with our previous findings (10) . The specific protein kinase C inhibitor, GF109203X, inhibited phosphorylation of the p27 protein but had no effect on either the p35 or p70 species. The putative PKC {delta} inhibitor Rottlerin had a similar effect, suggesting a role for protein kinase C in CTGF signaling. The Rho kinase inhibitor Y27632 had no inhibitory effect, indicating that these changes were not downstream of previously observed alterations in Rho kinase activity (13) . Bioinformatic analysis (http://motif.genome.jp) indicated that p27Kip-1 contains a number of Akt/PKB-dependent phosphorylation sites. A previous study (12) had shown that CTGF could induce the expression of p27Kip-1. We therefore considered it a likely target of Akt/PKB and investigated its phosphorylation status and distribution in mesangial cells in response to treatment with CTGF.

CTGF stimulated an increase in both total p27Kip-1 and serine 10 phosphorylated p27Kip-1 levels when normalized to beta actin (Fig. 1C ). Given that previous studies had shown that phosphorylation of p27Kip-1 on serine 10 could alter its subcellular localization (30 31 32) , we determined the phosphorylation status of p27Kip-1 in both cytoplasmic and nuclear fractions in mesangial cells treated with CTGF. CTGF stimulated a time-dependent decrease in the nuclear levels of serine 10 phosphorylated p27Kip-1 with a concomitant increase in the cytoplasmic levels (Fig. 1D ). We also visualized the subcellular localization of p27Kip-1 in response to CTGF by immunocytochemistry (Fig. 2 A). 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. Interestingly, cytoplasmic serine 10 phosphorylated p27Kip-1 appeared to colocalize with the ends of shortened actin fibers in CTGF treated cells (Fig. 2B ) raising the possibility that p27Kip-1 may play a role in the disassembly of the actin cytoskeleton.


Figure 2
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  		Figure 2. CTGF stimulates phosphorylation of p27Kip-1 on serine 10 in an Akt/PKB-dependent manner. We then 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 colocalise 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).

Members of the FOXO subfamily of forkhead transcription factors regulate the expression of a number of genes that control diverse processes such as proliferation and apoptosis. It has previously been shown that transcriptional activation of p27Kip-1 and its subcellular localization was, at least partially, under the control of FKHR (33) Comparison of the molecular weights of our set of putative Akt/PKB targets with known Akt/PKB substrates led us to investigate the phosphorylation of FKHR, which was considered a likely effector of CTGF signaling. CTGF stimulated a time-dependent increase in the phosphorylation of FKHR at serine 256 (Figure 2C ), which was abrogated by the addition of the PI-3 kinase inhibitor LY294002 and the Akt/PKB inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate (Fig. 2C ).

CTGF stimulates phosphorylation of p27Kip-1 on threonine 157 in a Akt/PKB-dependent manner
As stated previously, p27Kip-1 contains a number of Akt/PKB consensus motifs, while phosphorylation of p27Kip-1 on threonine 157 has previously been shown to regulate its cellular localization (34 , 35) . 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 p27Kip-1 (Fig. 3 A), which was abrogated by the addition of the PI-3 kinase inhibitor LY294002 and the PKB inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate. Immunocytochemistry (Fig. 3B ) revealed that CTGF induced nuclear to perinuclear and cytoplasmic translocalization of threonine 157 phosphorylated p27Kip-1, coincident with disruption of the mesangial actin stress fibers.


Figure 3
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  		Figure 3. CTGF stimulates phosphorylation of p27Kip-1 on threonine 157 in a Akt/PKB dependent manner. CTGF further stimulated a rapid and transient increase in the phosphorylation of threonine 157 p27Kip-1 which was abrogated by the addition of the PI-3 kinase inhibitor LY294002 and the Akt/PKB inhibitor IL-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate (A). Immunocytochemistry revealed that CTGF induced nuclear to perinuclear and cytoplasmic translocalization of threonine 157 phosphorylated p27Kip-1, coincident with disruption of the mesangial actin stress fibers (B).

It has recently been shown that phosphorylation of p27Kip-1 on threonine 157 prevents its binding to importin alpha, thereby inhibiting its reimportation into the nucleus (36) . We therefore wished to establish whether p27Kip-1 cytoplasmic localization was required for CTGF-stimulated actin depolymerization. Primary mesangial cells were transfected with T157A and T157D YFP p27Kip-1 mutants or wild-type control and stimulated with CTGF. The expression of both the p27Kip-1 mutants was confined to the nucleus, indicating that phosphorylation was necessary for cytoplasmic localization (Fig. 4 A, T157D not shown).


Figure 4
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  		Figure 4. Phosphorylation of p27 on threonine 157 is required for its cytoplasmic localization and is necessary for CTGF stimulated actin rearrangement. Primary mesangial cells were transfected with T157A and T157D YFP p27Kip-1 mutants or wild-type control and stimulated with CTGF. The expression of both the p27Kip-1 mutants was confined to the nucleus, indicating that phosphorylation was necessary for cytoplasmic localization (A, T157D not shown). To determine the functional significance of this phosphorylation, cells were cotransfected with GFP-actin and p27Kip-1 wild-type or T157A YFP p27Kip-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 in cells transfected with GFP-actin (B, upper panels and Supplemental Movie 1). 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- p27Kip-1 mutants was again confined to the nucleus (B, lower panels and Supplemental Movie 2).

We then used the threonine 157 p27Kip-1 mutants to determine the functional significance of this phosphorylation. Cells were cotransfected with GFP-actin and p27Kip-1 wild-type or T157A YFP p27Kip-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 (13) , in cells transfected with GFP-actin (Fig. 4B , upper panels and Supplemental Movie 1). 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- p27Kip-1 mutants was again confined to the nucleus (Fig. 4B , lower panels and Supplemental Movie 2).

CTGF stimulates the association of p27Kip-1 with Rho A and consequent uncoupling of the Rho A/LIM kinase/cofilin pathway
Until recently, members of the Kip/Cip family of cyclin-dependent kinase inhibitors, including p27Kip-1 were exclusively viewed as nuclear proteins with the principal function of inhibiting cyclin-CDK activity and hence, cell-cycle progression. New data suggest that these proteins play additional roles outside of the nucleus (37) , with a number of reports linking p27Kip-1 to the regulation of actin dynamics and cell migration (38 , 39 , 40) . Given our observation that cells overexpressing the T157A YFP p27Kip-1 mutant do not undergo either actin disassembly or polarization when treated with CTGF, we wished to further investigate the mechanism underlying these changes.

To investigate whether CTGF stimulated a direct interaction between Rho A and p27Kip-1, 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, consistent with the previous observations of Besson et al. (Fig. 5 A, upper panels) (40) . 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. Again, CTGF stimulated an increased association between Rho A and phospho Thr 157 p27Kip-1 (Fig. 5A , middle panels).


Figure 5
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  		Figure 5. 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 {zeta} localized predominantly cytoplasmic and perinuclear in response to CTGF (B). CTGF also stimulated an increased association between 14–3-3 {zeta} 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 (C), 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.

Previous studies suggested that 14–3-3 {zeta} interacts with p27Kip-1 and that this interaction plays a role in suppressing its nuclear localization (35) . In addition, 14–3-3 {zeta} has been shown to interact with the Rho-A GEF AKAP-Lbc, thereby suppressing Rho A activity (41) . Immunocytochemistry revealed that 14–3-3 {zeta} localized predominantly cytoplasmic and perinuclear in response to CTGF (Fig. 5B ), a similar distribution to Thr 157 phosphorylated p27Kip-1. To confirm the in vivo binding of 14–3-3 {zeta} to Thr 157 phosphorylated p27Kip-1, an immunoprecipitation assay was carried out. CTGF stimulated an increased association between 14–3-3 {zeta} and p27Kip-1 (Fig. 5A , lower panels), raising the possibility that CTGF stimulates the formation of a regulatory complex of 14–3-3 {zeta}, p27Kip-1 and Rho A.

We then wished to determine the downstream 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. Surprisingly, CTGF stimulated an increase in phosphorylation of both LIM-kinase and cofilin (Figure 5C ), 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. 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 Akt/PKB-dependent manner and that cytoplasmic p27Kip1 modulates actin dynamics by direct regulation of the small GTPase Rho A pathway. Indeed, further immunocytochemistry studies revealed that cytoplasmic p27kip-1 appears to localize at the ends of actin filaments during the time course of CTGF stimulated actin disassembly (Fig. 2B ).

The data described above suggests a mechanism whereby CTGF can initiate actin depolymerization through Akt/PKB-mediated p27Kip-1 phosphorylation, resulting in cytoplasmic translocation and subsequent interaction with Rho A leading to stress fiber disassembly. We wished to establish the central role of Akt/PKB in controlling actin cytoskeletal integrity. Mouse embryonic fibroblasts (MEFs) from Akt 1/3 (PKB {alpha}/{gamma}) knockout and wild-type mice were treated with CTGF as described. Integrity of the actin cytoskeleton was assessed by immunocytochemistry and cell migration measured using a wound healing assay. In wild-type MEFs, CTGF stimulated approximately a twofold increase in cell migration compared to vehicle-treated cells, consistent with our previous findings for mesangial cells (10 , 13) . In the Akt 1/3 (PKB {alpha}/{gamma}) knockout MEFs however, there was no migration into the wound in either CTGF or vehicle-treated cells. In wild-type cells treated with CTGF, there was an apparent decrease in the number and size of actin stress fibers, concurrent with a significant increase in F-actin containing microspikes and filopodia. In contrast, the Akt 1/3 (PKB {alpha}/{gamma}) knockout MEFs maintained the integrity of their actin stress fibers (Fig. 6 ).


Figure 6
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  		Figure 6. Akt/PKB is a key mediator of CTGF-induced cytoskeletal rearrangements. Mouse embryonic fibroblasts (MEFs) from Akt 1/3 (PKB {alpha}/{gamma}) knockout and wild-type mice were treated with CTGF as described. Integrity of the actin cytoskeleton was assessed by immunocytochemistry. In wild-type cells treated with CTGF, there was an apparent decrease in the number and size of actin stress fibers, concurrent with a significant increase in F-actin containing microspikes and filopodia. In contrast, the Akt 1/3 (PKB {alpha}/{gamma}) knockout MEFs maintained the integrity of their actin stress fibers (A). CTGF also stimulated a significant increase in phospho Thr 157 p27Kip-1 in wild-type MEFs compared to Akt 1/3 (PKB {alpha}/{gamma}) knockout MEFs (B).

DISCUSSION

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 p27Kip-1, which subsequently binds to RhoA, resulting in phosphorylation of LIM kinase and cofilin, indicative of increased actin disassembly. These results are summarized in Fig. 7 .


Figure 7
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  		Figure 7. 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).

We, among others (42 , 43) , demonstrated that at least some of the downstream effects of CTGF could be explained by interactions with integrins; in mesangial cells, particularly {alpha}Vß3. The result of this interaction included Src kinase-mediated activation of both PI-3 kinase and MAP kinase signaling pathways (10) . The downstream consequences of activation of these pathways remain largely unknown, although both CTGF-mediated up-regulation in expression of the extracellular matrix proteins fibronectin and collagen and the metalloproteinase MMP-2 can be at least partially abrogated by the use of specific pharmacological inhibitors to the MAP kinase pathway (10 , 42) . The major aim of this study was to elucidate the downstream signaling events resulting from CTGF activation of the PI-3 kinase pathway and determine the implications of these events.

The recent emergence of new bioinformatic tools, in combination with the commercial availability of an Akt substrate Ab, has led to an upsurge in interest in downstream targets of the PI-3 kinase effector Akt/PKB. We have previously showed that treatment of mesangial cells with CTGF leads to phosphorylation of Akt/PKB and that this phosphorylation can be abrogated by the use of the specific PI-3 kinase inhibitor LY294002 or by using ß3 integrin-blocking antibodies. More recent data have also shown that the PI-3 kinase pathway can be activated by CTGF through interaction with the neurotrophin receptor Trk A (20) . The authors showed that K252a, a selective inhibitor of Trk A blocked CTGF-induced activation of many signaling proteins, adding weight to the suggestion that CTGF is a matricellular protein that can interact with multiple cell surface receptors in a context and cell-specific manner.

Stimulation by numerous growth factors, cytokines, hormones, and neurotransmitters can activate Akt/PKB in a phosphoinositide 3-kinase-dependent manner. Briefly, growth factor stimulation results in activation of phosphoinositide 3-kinase and generation of the membrane phospholipid PtdIns (3 , 4 , 5) P3, which then recruits Akt/PKB to the membrane, where it becomes phosphorylated at Thr 308 and Ser-473 (for Akt1/PKB{alpha}) by two upstream kinases, phosphoinositide-dependent kinase 1 and an unknown Ser-473 kinase. Akt/PKB regulates cellular functions through phosphorylation of serine and threonine residues in the motif RXRXXS/T of targets (43) . Multiple Akt/PKB substrates have been discovered, including transcription factor FOXO proteins, proapoptotic factor Bad, I{kappa}B kinase, mitogenic factor Raf1, p53 negative regulator Mdm2, cyclin-dependent kinase inhibitors p27Kip-1 and p21cip-1 endothelial nitric-oxide synthase, glycogen synthase kinase-3, and the GTPase-activating proteins tuberous sclerosis complex-2 and AS160 (For a review, see ref. 44 ).

In this study, use of the Akt/PKB substrate Ab facilitated the identification of a number of putative Akt/PKB substrates with apparent molecular weights of 70 kDa, 35 kDa, and 27 kDa. These substrates were phosphorylated by CTGF with kinetics similar to Akt/PKB (rapid, transient) and were inhibited or partially inhibited by the PI-3 kinase inhibitor LY294002, indicating that they were likely downstream targets of Akt/PKB. In addition, the Src kinase inhibitor PP1 inhibited their phosphorylation in response to CTGF, consistent with our previous findings that CTGF stimulates the activation of Src kinase upstream of activation of the PI-3 kinase pathway. In contrast, the MAPK inhibitor PD98059 had no effect on substrate phosphorylation. Interestingly, the Rho kinase inhibitor Y27632 had no inhibitory effect on the phosphorylation of the putative p27 Akt/PKB substrate, suggesting that these events were not regulated by altered Rho signaling and instead could play a role upstream of Rho kinase in the regulation of actin rearrangement in response to CTGF.

Cellular hypertrophy in response to elevated levels of glucose is believed to be a key pathophysiological mediator in diabetic nephropathy. Indeed, mesangial cells treated with CTGF actively enter the G1 phase from G0 but do not progress through the cell cycle. An elegant study from Wahab et al. has suggested that this is due to induction of the cyclin-dependent kinase inhibitors p15INK4, p21Cip1, and p27Kip1 and showed that TGF-ß-mediated mesangial cell hypertrophy is CTGF dependent (12) . p27Kip1 has received a great deal of recent attention, following the publication of a study that determined that renal hypertrophy, glomerular hypertrophy, and albuminuria do not develop in diabetic p27Kip-1 –/– mice (45) . Moreover, mesangial expansion was milder in the p27Kip-1 knockouts despite elevated glomerular TGFß, indicating that control of p27Kip1 activity was independent of TGF-ß activity.

The activity of p27Kip1 is controlled by its concentration, phosphorylation status, subcellular localization, and its interactions with other cellular protein complexes and assemblies (46) . Recent data have revealed that p27 is phosphorylated on serine 10 in order to effect nuclear export (31) . It had previously been proposed that cytoplasmic localization of p27Kip-1 allowed phosphorylation of p27Kip-1 on threonine 187, which was then recognized and targeted for ubiquitinylation and subsequent proteolysis by the ubiquitin-proteasome pathway (30) . In contrast, however, Rodier et al. demonstrated that the p27Kip-1 S10A mutant remains in the nucleus and is efficiently degraded on cell cycle re-entry. These results suggested that the cytoplasmic translocation of p27Kip-1 may regulate cellular functions that are cyclin dependent kinase independent, such as cell migration (38) . Subsequent studies revealed that phosphorylation of p27Kip-1 on threonine 157 is mediated by Akt/PKB and impairs its nuclear import (47) . Thus, we have an emerging model, wherein p27Kip-1 localization and activity is 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 a Akt/PKB-dependent manner, leading to cytoplasmic localization. Mutation of the threonine 157 (T157A or T157D) site resulted in nuclear localization of p27Kip-1. Recent data have shown that phosphorylation on Thr 157 prevented binding of p27Kip-1 to importin alpha, thereby inhibiting its reimportation into the nucleus (36) . It is believed that this is facilitated by the interaction of Thr 157 phosphorylated p27Kip-1 with 14–3-3 (35) .

The emerging cytoplasmic regulatory roles for both p27/kip-1 and p21 have raised a number of interesting questions concerning mechanism of turnover and stabilization. The question remains as to whether this apparent increase in the levels of p27 represents decreased turnover (reflecting increased stabilization mediated by CTGF-induced phosphorylation) or increased expression. We suggest it is unlikely due to increased expression. Indeed, a number of studies have addressed this issue, and the current consensus is that phosphorylation on serine 10 facilitates transfer from the nucleus to the cytoplasm where it is ubiquitinated and degraded. Dual phosphorylation at Ser-10 and Thr 157 results in stabilization and hence increased levels. Moreover, a recent study by Shin et al. (34) has concluded that that Akt-mediated phosphorylation of p27 at Thr 157 prevents nuclear re-entry by inhibiting the association of p27 with importin {alpha}. Similarly, Rodier et al., (31) found that p27 is efficiently degraded in the nucleus, and phosphorylation of Ser-10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. They suggest that this cytoplasmic localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin-Cdk2 complexes. In the light of new data highlighting a role for CTGF in regulation of RhoGTPase activity, cytoplasmic localization is now directly implicated in regulation of actin remodeling in response to chemotactic stimuli. Future studies using our phosphoserine and phosphothreonine mutants will probe this mechanistic conundrum.

Our studies have elaborated on the downstream consequences of p27Kip-1 phosphorylation and revealed that cytoplasmic localization, mediated by phosphorylation on Thr 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. Supporting our hypothesis that cytoplasmic p27Kip-1 in response to treatment with CTGF may play a role in regulation of actin rearrangement is the recent study by Besson et al. has shown that p27Kip-1 modulates cell migration through the regulation of Rho A activation (40) . They found that p27Kip-1 binds to Rho A, thereby inhibiting Rho A activation by interfering with the interaction between Rho A and its activators, the guanine-nucleotide exchange factors and suggested that subsequent modulation of the Rho pathway would lead to increased actin disassembly. When we examined this interaction in mesangial cells, we similarly found that CTGF stimulated an increase in the direct association between Rho A and p27Kip-1. Our previous studies had demonstrated that treatment with CTGF leads to loss of integrity of the actin cytoskeleton and showed that this was, in part, due to down-regulation of Rho A activity. Our results in this study identify Akt/PKB mediated phosphorylation and cytoplasmic translocation of p27Kip-1 and its subsequent interaction with Rho A as a mechanism through which CTGF exerts its effect on the cytoskeleton.

Cofilin activity is tightly regulated in cells with high spatial and temporal precision (48) . The ability of cofilin to induce actin depolymerization can be inhibited by phosphorylation on Ser-3 which prevents its interaction with actin. A number of kinases have been implicated in this phosphorylation including the downstream Rho GTPase effectors LIMK-1 and LIMK-2. The mechanism of regulation of cofilin activity by cycles of phosphorylation and dephosphorylation is far from trivial and appears to be both cell type and context specific. For example, in a number of resting cells, cofilin is mostly phosphorylated; induction of motility induces dephosphorylation and activation of cofilin (49 , 50) . In contrast, cofilin is rapidly phosphorylated on cell stimulation with epidermal growth factor (EGF), suggesting a more complex regulatory mechanism than simply phosphatase mediated dephosphorylation (51) . Following EGF stimulation, a PLC-dependent step releases activated cofilin, which then associates with F-actin to promote F-actin severing. This leads both to polymerization and depolymerization, the balance being determined by the relative availability of G-actin. Cofilin is rescued from the cofilin–G-actin heterodimer by two mechanisms (1) . Phosphorylation by LIM kinase (LIMK) or TES kinase (TESK) turns off the actin-binding activity of cofilin, releasing G-actin and phospho-cofilin. Cofilin phosphatases such as PP1, PP2A, or SSH (slingshot) can then replenish the pool of dephosphorylated cofilin (2) . CAP can bind to the cofilin–G-actin heterodimer and release free cofilin and G-actin. The freed cofilin can bind to PtdIns (4 , 5) P2 to form an inhibitory complex that is released locally by EGF-stimulated receptors to begin the activity cycle again (Fig. 7) .

Our studies have 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 (10 , 13) . As with EGF, 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. We previously demonstrated that this disassembly, in combination with induction of polarization, is facilitative of increased cell migration.

Three Akt/PKB isoforms have been identified in mice and humans The differences between corresponding isoforms in humans and mice are subtle (between two and ten amino acid changes), making it feasible to determine functions of Akt/PKB kinases in human physiology by studying them in the mouse. Indeed, a recent study of Akt/PKB expression in mesangial cells revealed the expression of the isoenzymes Akt-1/PKB-{alpha} and Akt-3/PKB-{gamma} but not Akt-2/PKB-ß (52) . Although all three isoforms of Akt are expressed in the kidney, studies have found that only Akt-1 and Akt-3 are expressed in the mesangium. The existence of viable knockout mice for each of the three isoforms of Akt, Akt-1, Akt-2, and Akt-3 suggests functional redundancy. Recent efforts have focused on the generation of mice with combined mutant alleles in order to assess their effect on mouse development. The double knockout (Akt1–/– Akt3–/–) causes embryonic lethality around days 11 and 12 with severe developmental defects in the cardiovascular and nervous systems (53) . Double knockout embryos displayed overt vascular defects with hemorrhages by E11.5. Generally, there was decreased vasculature compared to controls with a striking lack of capillary structures in the neuroepithelium and the trunk. As with the Akt1–/– Akt3–/+ mice, there was a striking hypotrophy of the branchial arches. Evidence suggests that the embryonic lethality of the double knockout may be attributed to these cardiovascular abnormalities. There was also an apparent reduced cell proliferation in the double knockout placenta compared to control. Together with an increased in apoptosis in the neuroendothelium, this suggests defective cell cycle control in the double knockout, implicating regulators of cell cycle, including p27/kip-1 Given that p27/kip-1 is an Akt/PKB substrate, its phosphorylation is likely impaired in these double knockouts, suggesting that Akt/PKB-mediated phosphorylation of p27/kip-1 is essential for CTGF stimulated actin rearrangement. Indeed, preliminary data suggest there was a significant increase in phospho Thr 157 p27Kip-1 in wild-type MEFs compared to Akt 1/3 (PKB {alpha}/{gamma}) knockout MEFs. The central role that Akt/PKB plays in regulation of the actin cytoskeleton is well illustrated by the fact that MEFs from the Akt 1/3 (PKB {alpha}/{gamma}) 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.

In summary, this current study further delineates the role of CTGF in cell migration and actin disassembly, identifying the upstream signaling events that lead to Rho inactivation and the downstream events that regulate actin depolymerization. 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 p27Kip-1, increased association of p27Kip-1 with Rho A, and increased phosphorylation of LIM kinase and cofilin. Two recent influential editorials have laid out the case for therapeutic targeting of CTGF in diabetic nephropathy, yet both have recognized that many questions concerning mechanism of action remain unanswered (54 , 55) . These data extend the rationale for anti-CTGF directed therapies by demonstrating amechanism whereby CTGF contributes to mesangial actin dysregulation.

Received for publication November 7, 2005. Accepted for publication March 20, 2006.

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