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【摘要】 The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors exert modulatory effects on a number of cell signaling cascades by preventing the synthesis of various isoprenoids derived from the mevalonate pathway. In the present study, we describe a novel pleiotropic effect of HMG-CoA reductase inhibitors, also commonly known as statins, on vascular endothelial growth factor (VEGF)-induced type IV collagen accumulation. VEGF is an angiogenic polypeptide that is also known to play a central role in endothelial cell permeability and differentiation. Recently, VEGF has also been implicated in promoting extracellular matrix (ECM) accumulation, although the precise signaling mechanism that mediates VEGF-induced ECM expansion remains poorly characterized. Elucidation of the mechanisms through which VEGF exerts its effect on ECM is clearly a prerequisite for both understanding the complex biology of this molecule as well as targeting VEGF in several pathological processes. To this end, this study explored the underlying molecular mechanisms mediating VEGF-induced ECM expansion in mesangial cells. Our findings show that VEGF stimulation elicits a robust increase in ECM accumulation that involves RhoA activation, an intact actin cytoskeleton, and 1 - integrin activation. Our data also indicate that simvastatin, via mevalonate depletion, reverses VEGF-induced ECM accumulation by preventing RhoA activation.
【关键词】 focal adhesion mevalonate integrins
MANY OF THE CELLS IN TISSUES are embedded in an extracellular matrix (ECM) that provides not only scaffolding support for the cell, but it also presents environmental cues to the cell which affect ultimately many aspects of a cell's fate, including its proliferation, differentiation, motility, and death. In the kidneys, the mesangial cells (MC) are embedded in mesangial matrix, a basement membrane-like PAS-positive ECM. The mesangial matrix, although composed of the same protein macromolecules as the glomerular basement membrane, is more coarsely fibrillar and less electron dense than the latter. Despite the importance of mesangial matrix in health and disease, little is known regarding the cross talk between MC and mesangial matrix and the signaling events that contribute to mesangial ECM expansion at the present time. For instance, expansion of the mesangial matrix is considered to be a major hallmark of diabetic nephropathy ( 23, 26 ). However, the signaling events that mediate mesangial matrix expansion in the diabetic milieu remain controversial. Interestingly, a number of recent observations have suggested that the vascular endothelial growth factor (VEGF), a multifunctional cytokine, may play a central role in the pathogenesis of microvascular complications of diabetes including diabetic nephropathy through its modulatory effects on ECM ( 5, 8, 9, 28, 40, 45 ). Therefore, in the current study, we explored the underlying molecular mechanisms of VEGF-induced ECM expansion in MC. In particular, we tested the role of RhoA activation in actin cytoskeletal remodeling leading to aberrant ECM synthesis and accumulation.
The guanine nucleotide-binding protein Rho family, consisting of Rho, Rac, and Cdc42, are 20- to 40-kDa monomeric G proteins that can cycle between two interconvertible forms: GDP bound (inactive) and GTP bound (active) states ( 34, 46 ). Several growth factors can promote the exchange of GDP to GTP on Rho proteins resulting in membrane translocation and activation of GTP-bound Rho proteins. The Rho family of small GTPases are involved in a number of essential biological activities of the cell including actin stress fiber formation, cell motility, and cell aggregation ( 1, 37 ). More recently, Rho GTPases have also emerged as key regulators in the regulation of integrin-mediated signaling ( 4, 11, 12 ).
Integrins are heterodimeric transmembrane molecules that consist of - and -subunits. The main ligands for integrins are extracellular matrix adhesive proteins and cellular counter receptors. High-affinity ligand binding requires that integrins to become activated by undergoing conformational changes regulated by "inside-out" signaling. In turn, integrin ligation triggers "outside-in" signaling that regulate, among many other functions, gene regulation and cell motility ( 13, 14, 18, 42, 44 ). Integrin -subunit cytoplasmic domains are required for integrin activation, whereas cytoplasmic domain usually plays a regulatory role. Once integrins are activated, one of the earliest changes initiated by their activation is tyrosin phosphorylation of proteins such as talin, paxilin, and the cytosolic focal adhesion kinase (FAK) ( 31, 38 ). FAK phosphorylation is considered to be a critical step in the integrin-mediated signaling events. In this study, we report a critical role for 1 -integrin activation and FAK phosphorylation in VEGF signaling to ECM.
A growing body of evidence is suggesting that HMG-CoA reductase inhibitors (statins), by inhibiting mevalonate (MEV) biosynthesis, may also exert modulatory effects on several signaling pathways beyond their cholesterol-lowering properties ( 27, 36, 47 ). For instance, we and others ( 6, 7, 55 ) previously provided evidence that some of the beneficial effects of statins in the diabetic milieu may be mediated through their modulatory effects on several signaling pathways. In the present study, we extend our previous observations and describe the underlying signaling mechanism through which VEGF leads to increased type IV collagen accumulation in MC. We also explored the effect of MEV depletion by using simvastatin (SMV), a hydrophobic statin, on VEGF-induced signaling pathway.
MATERIALS AND METHODS
Reagents and antibodies. Recombinant rat VEGF 165 was obtained from R&D Systems (Minneapolis, MN). DMEM/F12, FBS, and PBS were purchased from Invitrogen (Carlsbad, CA). MEV was purchased from Sigma (St. Louis, MO). Rho A activation assay kit was obtained from Upstate Biotechnology (Lake Placid, NY). Rho A antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal anti- 1 -integrin subunit (active conformation) antibody, clone HUTS-4, and function-blocking monoclonal mouse anti- 1 integrin antibody were purchased from Chemicon (Temecula, CA). The monoclonal anti- 1 -integrin subunit (active conformation) antibody, clone HUTS-21 was purchased from (BD PharMingen, San Diego, CA), and collagen IV antibody was from Novus Biologicals (Littleton, CO), and cytochalasin D was obtained from Sigma. Rhodamine-phalloidin was purchased from Molecular Probes (Eugene, OR). SMV was kindly provided by Merck (West Point, PA). Both SMV and MEV were chemically activated as described previously ( 6, 7, 55 ).
Cell culture and transfection. Early passaged ( passage 3 - 10 ) rat glomerular MC ( 6, 7, 55 ) were grown in DMEM/F12 medium containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator at 37°C under 5% CO 2. Transfections of RhoA mutants were performed as described previously ( 6, 7, 55 ). Briefly, in transfection studies, cells were grown to 50-60% confluence and then transfected with 1 µg of fusion plasmid DNA using lipofectamine reagent according to the manufacturer's protocol (Invitrogen). Subsequently, the transfected colonies were grown in growth medium containing 800 µg/ml G418 until the cells achieve 70% confluence. Plasmids containing wild-type RhoA (pcDNA3 RhoA) and dominant negative RhoA construct (pcDNA3 RhoA N17) were generous gifts of Dr. J. Sznajder (Northwestern University, Chicago, IL). In transfection studies, control experiments were performed using lipofectamine alone as an additional control group.
Rho A activity pull-down assay. RhoA activity was measured by a pull-down assay according to the instructions by the manufacturer (Rho Activity Assay Kit, Upstate Biotechnology). Briefly, 10 7 cells were grown in 100-mm dishes, washed in ice-cold PBS twice, and lysed in ice-cold MLB buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl 2, 1.0 mM EDTA, and 2% glycerol). The samples were centrifuged and incubated for 45 min at 4°C with 10 µl of rhotekin agarose to precipitate GTP-bound Rho. Precipitated complexes were washed three times in MLB buffer and resuspended in 30 µl of 2 x Laemmli buffer. Total and precipitates were analyzed by performing SDS-PAGE and Western blot analysis using monoclonal anti-RhoA antibody at a dilution of 1:500.
Confocal laser-scanning fluorescence microscopy. MC were grown on glass coverslips. The cells were fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. The cells were incubated with anti-phospho-FAK antibody for 1 h at room temperature and then incubated with FITC-conjugated secondary antibody (Zymed Laboratories, San Francisco, CA). To detect F-actin, cells were incubated with rhodamine-phalloidin (1:100) for 30 min at 37°C after fixation and permeabilization. The coverslips were mounted on glass slides with antifade mounting media (Molecular Probes) and examined using a confocal fluorescence microscope (Zeiss LSM510, Thornwood, NY).
Flow cytometric analysis. Near-confluent growth-arrested MC were pretreated with SMV or vehicle control for 12-18 h, after which VEGF (50 ng/ml) was added to the medium. At the end of the indicated times, cells were harvested by HyQTase (Hyclone, Logan, UT) in their culture media and washed twice with 1 x DPBS without Ca 2+ and Mg 2+ (400 g, 5 min, room temperature). Mesangial ells were then resuspended and fixed with 3.7% formaldehyde. Cells were then incubated with total anti-integrin antibody (Chemicon), monoclonal anti- 1 -integrin subunit (active conformation) antibodies, clones HUTS-21 (BD Pharmingen) and HUTS-4 (Chemicon), in blocking buffer (2% goat serum in DPBS without Ca 2+ and Mg 2+ ) for 45 min on ice. Cells were then incubated with secondary antibodies on ice for 45 min. Flow cytometric analysis was performed using a Beckman Coulter EPICSXL-MCL (Becton, Dickinson, CA).
[ 3 H]proline incorporation. The collagen synthesis was examined by [ 3 H]proline incorporation. For these experiments, 5 x 10 4 cells/well were seeded in 24-well plates in DMEM/F 12 medium containing 10% FBS. At confluence, cells were starved with serum-free medium for 48 h, and then the cells were exposed to various concentrations of VEGF in the presence or absence of SMV for 24 h; 2 µCi of [ 3 H]proline (Amersham, Piscataway, NJ) and ascorbic acid (50 µg/ml) were added to each well for the last 12 h of incubation. The cells were washed twice with ice-cold PBS, precipitated twice with ice-cold 10% TCA (Sigma), solubilized in 2 ml 0.2 N NaOH containing 0.1% Triton X-100. The samples were taken to liquid scintillation counting. Additional cells seeded in parallel were scraped off the plate to detect the protein concentration. Proline incorporation was expressed as counts per minute (cpm) per microgram of protein.
Western blotting. For each experiment, a total of 5 x 10 5 cells were seeded, and at subconfluence ( 70%), cells were made quiescent for 48 h. Cells were rinsed twice with ice-cold PBS and added 0.5 ml of the ice-cold lysis buffer [50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 100 µg/ml PMSF, 0.1% SDS, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 10 µg/ml aprotinin, 2 µg/ml leupeptin, 10 mM EDTA], incubated for 20 min on ice, and then were scraped and centrifuged. Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL), and then separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies diluted in TBS-T containing 5% nonfat milk at 4°C overnight. The membranes were incubated with the appropriate secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized by ECL reaction. Each blot is representative of at least three similar independent experiments.
For detection of occupied 1 integrins, cells were plated on collagen-coated slides before VEGF stimulation. Cells were incubated with HUTS-21 or HUTS-4 antibodies for 30 min at 37°C. Cells were rinsed with PBS, lysed in SDS sample buffer, and bound antibody was detected by Western blot analysis.
Statistical analyses. Data are expressed as means ± SE. ANOVA with a Student-Newman-Keuls test was used to evaluate differences between two or more different experimental groups. A value of P 0.05 was considered significant.
RESULTS
Effects of VEGF and SMV on cell-associated collagen synthesis. VEGF-induced cell-associated collagen synthesis in MC was measured by [ 3 H]proline incorporation. Serum-starved cultured rat MC were exposed to incremental concentrations of VEGF for 24 h. As shown in Fig. 1, VEGF stimulation caused a significant increase in [ 3 H]proline incorporation in a dose-dependent manner. Cotreatment of cells with SMV (1 µM) prevented VEGF-induced (50 ng/ml) increase in [ 3 H]proline uptake. The addition of MEV (300 µM) reversed the effect of SMV on [ 3 H]proline incorporation indicating that the inhibitory effect of SMV on VEGF-induced [ 3 H]proline incorporation was MEV dependent. We next tested whether the inhibitory effect of SMV on VEGF signaling was mediated by isoprenoids derived from the MEV pathway. To this end, VEGF-stimulated MC were incubated with SMV (1 µM) and cotreated with either geranyl geranyl pyrophosphate (GGPP) or squalene (SQ), two important isoprenoids derived from the MEV pathway. GGPP was used since posttranslational modification of GGPP is negatively affected by statin-mediated inhibition of HMG-CoA reductase. We also investigated the effect of SQ, an immediate precursor of cholesterol, to test whether the inhibitory effect of SMV on VEGF signaling is independent of cholesterol biosynthesis. The data on Fig. 1 show that cotreatment of cells with GGPP but not with SQ reversed the inhibitory effect of SMV, suggesting that the modulatory effect of statins on VEGF signaling is geranyl geranyl dependent but independent of cholesterol synthesis since cotreatment of cells with SQ failed to reverse the effect of SMV. Taken together, these findings indicate that the inhibitory effect of SMV on VEGF signaling is MEV and geranyl geranyl dependent.
Fig. 1. Graph illustrating the effects of vascular endothelial growth factor (VEGF) and simvastatin (SMV) on [ 3 H]proline incorporation. Mesangial cells (MC) were exposed to various concentrations of VEGF for 24 h. [ 3 H]proline incorporation was measured as previously described in MATERIALS AND METHODS. The amount of [ 3 H]proline incorporation was measured in a scintillation counter and corrected for the cellular protein content. The results are expressed as percentages of the control and the means ± SE of 3 independent experiments. MEV, mevalonate; GGPP, geranyl geranyl pyrophosphate; SQ, squalene. * P < 0.05, ** P < 0.01 vs. control.
We next examined the effect of VEGF and SMV on type IV collagen protein levels by Western blot analysis. As shown in Fig. 2, cells exposed to incremental concentrations of VEGF exhibited significant increase in type IV collagen protein levels. Cotreatment of VEGF-stimulated cells with SMV (1 µM) abrogated the effect of VEGF (50 ng/ml) on cell layer collagen type IV protein levels. The inhibitory effect of SMV was reversed when cells were cotreated with MEV (300 µM).
Fig. 2. A : protein levels of cell layer type IV collagen were assessed by Western blot analysis. Cells exposed to incremental concentrations of VEGF exhibited significant increase in cell layer collagen type IV protein levels. Cotreatment of VEGF-stimulated cells with SMV abrogated the effect of VEGF on cell layer collagen type IV protein levels, and addition of MEV reversed the inhibitory effect of SMV. B : densitometric analysis of cell-associated type IV collagen synthesis ( n = 3, * P < 0.05 vs. control).
Effects of VEGF and SMV on RhoA activity. We recently reported that SMV modulates the activation of the Rho family of small GTPases in the diabetic milieu ( 6, 7, 55 ). To decipher whether RhoA, a member of the Rho family of small GTPases, mediates VEGF-induced collagen accumulation, MC were exposed to VEGF (50 ng/ml), and RhoA activity was measured by an affinity pull-down assay using GST fusion protein rhotekin, which recognizes only the active form of RhoA (GTP-RhoA). VEGF stimulation significantly increased RhoA activity in MC after 20 min ( Fig. 3 ). To examine the effect of SMV on VEGF-induced RhoA activity, cells stimulated with VEGF were cotreated with SMV (1 µM). As shown in Fig. 3, cotreatment of VEGF-stimulated cells with SMV inhibited VEGF-induced increase in RhoA activity without significantly changing total RhoA protein levels. The inhibitory effect of SMV on RhoA activity was reversed when cells were cotreated with MEV (300 µM), indicating that the effect of SMV on VEGF-induced RhoA activation was MEV dependent.
Fig. 3. A : RhoA-GTP was pulled down using the fusion protein GST-RBD, and samples were then subjected to Western blot analysis using a monoclonal anti-RhoA antibody. RhoA activity significantly increased in cells exposed to VEGF (50 ng/ml) for 20 min. Cotreatment of VEGF-stimulated cells with SMV significantly attenuated the effect of VEGF on RhoA activity, and the addition of MEV reversed the inhibitory effect of SMV. B : densitometric analysis of GTP-RhoA ( n = 3, * P < 0.05 vs. control).
To establish a link between RhoA activation and VEGF-induced type IV collagen protein levels, MC were transfected with dominant-negative mutant (N17RhoA) and wild-type (wt) RhoA. The data in Fig. 4 show that VEGF (50 ng/ml) stimulation failed to increase cell layer collagen IV protein levels in dominant-negative RhoA-transfected cells, indicating a critical role for RhoA activation in VEGF-induced collagen synthesis.
Fig. 4. A : MC were transfected with wild-type (wt) and dominant-negative mutants of RhoA (N19RhoA). Exposure to VEGF (50 ng/ml) in N19RhoA-transfected cells caused a significant reduction in VEGF-induced collagen IV protein levels. However, VEGF stimulation in transfected MC with wtRhoA did not significantly alter type IV collagen protein levels. B : densitometric analysis ( n = 4, * P < 0.05 vs. control).
Effects of VEGF and SMV on the actin cytoskeleton remodeling. To examine whether VEGF signaling in MC involves actin cytoskeleton remodeling and to elucidate the modulatory effect of SMV on VEGF-induced cytoskeletal remodeling, MC were incubated with VEGF (50 ng/ml) for 30 min in the presence or absence of SMV (1 µM) and the actin cytoskeleton was visualized by rhodamine phalloidin staining using scanning confocal electron microscopy. MC treated with VEGF exhibited a significant increase in actin stress fiber formation ( Fig. 5 B ). Upon treatment with SMV (1 µM), the density of stress fibers was significantly decreased ( Fig. 5 C ). Because RhoA is one of the major regulators of actin stress fiber formation, we further examined whether RhoA mediates VEGF-induced stress fiber formation. As shown in Fig. 5 D, MC transfected with dominant-negative RhoA also showed a significant reduction in VEGF-induced density of stress fibers, indicating that VEGF-induced actin stress fiber formation was mediated by a RhoA-dependent pathway.
Fig. 5. Confocal microscopy of the actin cytoskeleton network in MC. A : control cells. B : MC exposed to VEGF exhibited significant stress fiber formation. However, in cells cotreated with SMV ( C ) as well as in dominant-negative RhoA-transfected cells ( D ), VEGF stimulation failed to induce actin cytoskeletal remodeling and significant stress fiber formation. The figure is representative of 3 separate experiments.
Effects of VEGF and SMV on tyrosine phosphorylation of FAK. FAK, a cytoplasmic protein tyrosine kinase, binds directly to the cytoplasmic domain of 1 -integrin subunits and plays an important role in the integrin-mediated signaling pathway ( 31, 38 ). To examine the potential involvement of FAK phosphorylation in VEGF-induced signaling, we performed laser-scanning confocal immunofluorescent microcopy using anti-phosphorylated FAK (Tyr 397 ) antibody. MC stimulated with VEGF (50 ng/ml) exhibited a significant increase in FAK Tyr 397 phosphorylation ( Fig. 6 ). However, in cells cotreated with SMV (1 µM), VEGF-induced FAK phosphorylation was significantly reduced ( Fig. 6 C ). MC transfected with dominant-negative mutant of RhoA also showed a significant decrease in VEGF-induced FAK phosphorylation ( Fig. 6 D ) indicating the central role of RhoA in VEGF-induced FAK phosphorylation.
Fig. 6. FAK-phosphorylation (Tyr 397 ) was evaluated by laser-scanning confocal immunofluorescent microcopy. A : control cells. B : MC stimulated with VEGF exhibited significant increase in FAK tyrosine phosphorylation. However, cotreatment of cells with SMV prevented VEGF-induced FAK phosphorylation ( C ). MC transfected with N19RhoA also showed a significant decrease in VEGF-induced FAK phosphorylation ( D ).
We also assessed VEGF-induced total and phosphorylated FAK protein expression by Western blot analysis. Whereas VEGF stimulation (50 ng/ml) significantly increased phospho-FAK protein levels, SMV (1 µM) and MC transfected with N17RhoA exhibited significantly lower protein levels of phospho-FAK. Total FAK protein levels were unchanged as shown in Fig. 7. Thus our data indicate that VEGF signaling pathway involves FAK phosphorylation, and RhoA mediates VEGF-induced FAK phosphorylation.
Fig. 7. A : VEGF-induced total and phosphorylated FAK protein levels were assessed by Western blot analysis. VEGF stimulation significantly increased phospho-FAK protein levels. Cells cotreated with SMV and MC transfected with N17RhoA exhibited significant decrease in phospho-FAK in response to VEGF. However, total FAK protein levels were unchanged. B : densitometric analysis ( n = 3, * P < 0.05 vs. control).
For determining whether actin cytoskeleton and FAK phosphorylation are involved in VEGF-induced cell-associated collagen synthesis, MC were treated with cytochalasin D (Cyto D; 1.0 µg/ml). Cyto D, a specific inhibitor of filament actin cytoskeleton, has also been shown to abolish FAK phosphorylation ( 25 ). As shown in Fig. 8, VEGF (50 ng/ml) stimulation did not increase the levels of cell layer collagen type IV in MC treated with Cyto D, indicating that VEGF-induced actin cytoskeleton reorganization is necessary for VEGF-induced collagen synthesis.
Fig. 8. Effect of cytochalasin D (cyto D) on cell layer collagen IV protein levels. Cells were treated with either DMSO as control or 1.0 µg/ml of cyto D. VEGF (50 ng/ml) stimulation failed to increase collagen type IV protein levels in MC treated with Cyto D. The figure is representative of 3 separate experiments.
Effects of VEGF and SMV on VEGF-induced 1 -integrin activation. Growing evidence has indicated that 1 -integrins mediate growth factor-dependent expansion of ECM in MC ( 23, 26 ). However, whether VEGF-induced mesangial matrix expansion is also mediated by 1 -integrin activation and whether RhoA activation is necessary for VEGF-induced 1 - integrin activation have not yet been explored. Accordingly, we examined the effect of VEGF and SMV on 1 -integrin activation. Integrins can switch between active and inactive conformations. In the inactive state, integrins have a low affinity for ligands. Intracellular signaling events such as protein kinase C stimulation can prime the integrins, which result in a conformational change that makes the extracellular domain competent for ligand binding by exposing the ligand-binding site. To address the effect of VEGF and SMV on 1 integrin activation, serum-starved MC were stimulated with VEGF (50 ng/ml) and 1 -integrin activation was detected by flow cytometry using an antibody (HUTS-21) specific for the active conformation of 1 -integrin ( 16, 25, 41, 48 ). The data obtained from flow cytometry in Fig. 9 A show that VEGF clearly enhances the expression of activated 1 -integrins in MC. The results in Fig. 9 A further show that SMV ameliorates activation of 1 -integrins by VEGF. To test whether SMV also modulates total 1 -integrin levels, MC were stimulated with VEGF (50 ng/ml) and total 1 -integrin levels were analyzed. The results of flow cytometry indicate that VEGF stimulation increased both the total and activated 1 -integrin. However, SMV only prevented conformational activation of 1 -integrin without significantly altering total 1 -integrin levels ( Fig. 9 B ).
Fig. 9. A : flow cytometric analysis of the active conformation of 1 -integrin activity in MC. Flow cytometry was performed by using HUTS-21, an antibody specific for the active conformation of 1 -integrin. Mean fluorescence intensity (±SE) is calculated from 3 independent experiments (* P < 0.05 vs. control unstimulated cells). B : flow cytometric analysis of the total 1 -integrin levels. Taken together, these data show that VEGF (50 ng/ml) caused a significant increase in both 1 -integrin activity and the total 1 -integrin levels, and SMV reversed the 1 -integrin activation without significantly changing the total 1 -integrin levels (** P < 0.001 vs. control unstimulated cells). C : quantification of integrin ligand occupancy when cells were incubated with HUTS-4 antibody. Bound antibody was assessed by Western blotting. VEGF stimulation failed to increase 1 -integrin activity in cells cotreated with SMV and in cells transfected with N17RhoA. D : densitometric analysis ( n = 3, * P < 0.05 vs. control).
To further explore the role of RhoA activation and to examine the effects of VEGF and SMV on 1 -integrin ligand binding, MC were plated on collagen-coated slides before VEGF (50 ng/ml) stimulation. Cells were incubated with HUTS-21 or HUTS-4 antibodies, which specifically recognize the expression of the active conformation of 1 integrins, for 30 min at 37°C. Cells were then lysed in SDS sample buffer and bound antibody was detected by Western blot analysis as previously described ( 48 ). The results in Fig. 9 C indicate that stimulation of MC with VEGF caused a significant increase in binding of HUTS-4 as well as HUTS-21 (data not shown). Cotreatment of cells with SMV (1 µM) inhibited VEGF-induced increase in 1 integrin activation. The inhibitory effect of SMV was reversed when the cells were cotreated with MEV (300 µM). The data in Fig. 9 C also indicate that RhoA activation is necessary to stimulate 1 -integrin activation since VEGF stimulation failed to activate 1 -integrin in MC transfected with a dominant-negative mutant of RhoA, N17RhoA, indicating that VEGF-induced 1 -integrin activation is mediated by a RhoA-dependent pathway. This experiment also provides strong evidence for the proposed VEGF-induced "inside-out" signaling pathway since our data indicate that RhoA activation is necessary before 1 -integrin activation.
To further ascertain that 1 -integrin activation is necessary for VEGF-induced collagen synthesis, MC were treated with a monoclonal function-blocking anti- 1 integrin antibody (20 µg/ml) and stimulated with VEGF (50 ng/ml). As shown in Fig. 10, VEGF stimulation did not increase the protein levels of cell layer collagen type IV in MC when 1 -integrin activation was blocked, indicating that 1 -integrin activation is necessary for VEGF-induced cell layer collagen synthesis.
Fig. 10. A : 1 -integrin activation was blocked by a functional blocking antibody. MC were treated with either IgG as control or with anti- 1 functional blocking antibody. MC stimulated with VEGF failed to increase cell layer collagen type IV protein levels when 1 -integrin activation was blocked as well as in cells treated with SMV. B : densitometric analysis ( n = 3, * P < 0.05 vs. control).
DISCUSSION
This study describes the signaling events that are responsible for VEGF-induced collagen IV accumulation in MC. Our data suggest that VEGF triggers an inside-out signaling pathway in MC which is characterized by RhoA activation, actin cytoskeleton remodeling, intracellular FAK phosphorylation, and 1 - integrin activation leading to increased type IV collagen synthesis. Thus the results of the present study provide a missing piece of the mechanistic puzzle concerning VEGF-induced mesangial matrix expansion. Furthermore, our data also provide strong evidence that SMV, by preventing RhoA activation, inhibits VEGF signaling pathway and enhanced collagen synthesis in MC.
Statins are commonly used drugs in the treatment of patients with hypercholesterolemia that have been suggested to exert significant pleiotropic effects on cell signaling pathways largely by preventing mevalonic acid biosynthesis ( 27, 36, 47, 50, 51 ). MEV is necessary for the posttranslational lipid modification (isoprenylation) of small GTPase proteins, a process essential for the proper translocation of Rho GTPases from the cytosol to the membrane where activation of these proteins takes place ( 3, 15, 43 ). Previous studies from our laboratory and others indicated that statins modulate several cellular processes by preventing prenylation of small Rho GTPases such as RhoA and Rac1 ( 6, 7, 20, 27, 29, 36, 49, 55 ). In support of a modulatory effect of statins on ECM accumulation, Kim et al. ( 20 ) reported that lovastatin inhibited high glucose-induced overexpression of fibronectin. Similarly, Nishimura et al. ( 29 ) showed that pravastatin prevented serum-induced type IV collagen secretion. The data presented in this study are consistent with these previous results by establishing the inhibitory effects of statins on ECM expansion. In addition, we clearly characterized the sequential activation of various components of VEGF-induced signaling pathway in the current study and identified the modulatory effect of HMG-CoA reductase inhibitors and MEV depletion on VEGF signaling.
To decipher VEGF signaling, we initially explored the role of RhoA activation on VEGF-induced signaling pathway in MC. We showed that RhoA activity is required for VEGF-induced collagen synthesis since MC transfected with dominant-negative RhoA mutant failed to increase cell layer type IV collagen protein levels in response to VEGF. Cotreatment of MC with SMV also inhibited VEGF-induced collagen accumulation by preventing RhoA activation. The addition of MEV reversed the inhibitory effect of SMV on VEGF-induced RhoA activity and collagen accumulation, indicating that the effect of SMV on VEGF-induced collagen accumulation was MEV dependent. Taken together, our data suggest that RhoA mediates VEGF-induced collagen synthesis; and SMV inhibits VEGF signaling to ECM by preventing RhoA inactivation.
The list of cellular events instigated by actin cytoskeletal reorganization is rapidly growing. For instance, Hubchak et al. ( 17 ) showed that TGF- 1 -induced collagen accumulation is associated with cytoskeleton reorganization. Moreover, Cyto D, an inhibitor of actin cytoskeletal assembly, has been previously reported to disrupt the formation of fibronectin networks ( 30, 54 ). In this study, we report that VEGF induces actin cytoskeletal rearrangement in MC, and disruption of actin cytoskeleton with Cyto D reduces VEGF-stimulated collagen type IV synthesis. Furthermore, our data indicate that VEGF-induced actin cytoskeletal remodeling is mediated by RhoA activation which leads to 1 -integrin activation. Thus the results of this study show that actin cytoskeletal remodeling, regulated by RhoA activation, is required for VEGF-induced collagen synthesis.
Integrins, a group of heterodimeric transmembrane receptors, play a pivotal role in ECM assembly and cell-ECM interactions ( 32, 35 ). A critical role of integrins is to provide a link between ECM and the cytoskeleton, whereby ECM ligand-integrins interactions can activate intracellular signaling transduction cascades resulting in enhanced gene expression and cellular differentiation ("outside-in" signaling) ( 13, 42 ). Interestingly, several recent studies indicated that signals from within the cells can also propagate through integrins and regulate ECM remodeling through an, as yet, incompletely understood mechanism termed "inside-out" signaling ( 14, 18, 19, 39, 44 ). Activation of integrins through inside-out signaling seems to be mediated by increased affinity and/or avidity state of integrins ( 44 ). The affinity state of integrin is regulated by its conformational changes induced by a number of signaling pathways or proteins that interact with the cytoplasmic domains of the integrins. Structural studies suggest that modulation of integrin affinity involves changes in the spatial relationship of the cytoplasmic and/or transmembrane domains of the - and -subunits ( 52 ). The cytoplasmic domain is critical for recruitment of integrins to focal contacts since its truncation/mutation impairs this process ( 33 ). Several previous studies suggested a central role for 1 -integrin activation in mesangial matrix accumulation ( 21, 22, 24 ). In this study, we assessed the contribution of 1 -integrin activation on VEGF signaling to ECM. Our data indicate that VEGF stimulation significantly increases 1 -integrins activity. Moreover, the results of this study suggest that SMV inhibits increased affinity of 1 -integrins via a MEV-dependent pathway. Thus the data presented in this study provide strong evidence indicating that 1 -integrins activation is required for VEGF-induced collagen synthesis since a specific 1 -integrin blocker prevented increased protein levels of cell layer collagen type IV. Our study also provides the first evidence that VEGF-induced activation of 1 -integrins in MC is mediated by RhoA activation because VEGF stimulation failed to increase 1 -integrin activity in cells transfected with dominant-negative mutant of RhoA. Furthermore, our data clearly demonstrate that RhoA activation precedes 1 -integrin activation consistent with an inside-out signaling. This adds to the growing body of evidence of the importance of RhoA activation in inside-out signaling.
Several cytoplasmic proteins including talin, -actin, and FAK bind to the 1 cytoplasmic domain of integrins and contribute to integrin cytoskeletal interactions ( 2, 10, 31, 38, 53 ). However, earlier studies on integrin-dependent cell adhesion and signaling demonstrated that integrin clustering could also trigger increased tyrosine phosphorylation of a 120-kDa tyrosine kinase known as FAK ( 2, 10, 53 ). FAK activation as demonstrated by an increase in phosphorylation on Tyr 397 is an integral component of the integrin signaling pathway ( 10, 53 ). To explore whether FAK phosphorylation was involved in VEGF-induced signaling pathway, we examined the effect of VEGF and SMV on FAK phosphorylation. The data presented in this study suggest that FAK phosphorylation is involved in the inside-out VEGF-dependent signaling by transmitting RhoA activation and actin cytoskeletal cues to ECM leading ultimately to collagen accumulation. Our data indicate that VEGF stimulation increased FAK Tyr 397 phosphorylation, and FAK phosphorylation was inhibited by SMV. We also showed that VEGF failed to increase FAK phosphorylation in MC transfected with dominant-negative mutant of RhoA. Thus our results suggest that VEGF, via a RhoA-dependent pathway, mediates tyrosine phosphorylation of FAK.
In conclusion, we demonstrated that VEGF-induced collagen accumulation involves several mediators that include activation of small GTPase protein, RhoA, reorganization of the actin cytoskeleton, FAK phosphorylation, and activation of 1 integrins. Based on these data, we propose an inside-out signaling model for VEGF-induced mesangial matrix expansion. This model provides several novel targets in the therapeutic approach to the abnormal mesangial matrix expansion observed in the diabetic milieu. Our study also provides a new rationale to the use of statins, independent of their cholesterol-lowering properties, in the early stages of DN.
GRANTS
This study was supported by National Institutes of Health Grants DK-67604 and DK-064106 and American Diabetes Association Grant 103-RA-15.
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作者单位：1 Division of Nephrology/Hypertension, 2 Department of Cell and Molecular Biology, and 4 Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois; and 3 Division of Cardiovascular Medicine, Yale School of Medicine, New Haven, Connecticut