Hyperglycemia stimulates secretion of αVβ3 ligands from vascular cells, including endothelial cells, resulting in activation of the αVβ3 integrin. This study determined whether blocking ligand occupancy of αVβ3 would inhibit the development of diabetic nephropathy. Ten diabetic pigs received an F(ab)2 fragment of an antibody directed against the extracellular domain of the β3-subunit, and 10 received a control IgG F(ab)2 for 18 weeks. Nondiabetic pigs excreted 115 ± 50 μg of protein/mg creatinine compared with control F(ab)2-treated diabetic animals (218 ± 57 μg/mg), whereas diabetic animals treated with the anti-β3 F(ab)2 excreted 119 ± 55 μg/mg (P < .05). Mesangial volume/glomerular volume increased to 21 ± 2.4% in control-treated diabetic animals compared with 14 ± 2.8% (P < .01) in animals treated with active antibody. Diabetic animals treated with control F(ab)2 had significantly less glomerular podocin staining compared with nondiabetic animals, and this decrease was attenuated by treatment with anti-β3 F(ab)2. Glomerular basement membrane thickness was increased in the control, F(ab)2-treated diabetic animals (212 ± 14 nm) compared with nondiabetic animals (170 ± 8.8 nm), but it was unchanged (159.9 ± 16.4 nm) in animals receiving anti-β3 F(ab)2. Podocyte foot process width was greater in control, F(ab)2-treated, animals (502 ± 34 nm) compared with animals treated with the anti-β3 F(ab)2 (357 ± 47 nm, P < .05). Renal β3 tyrosine phosphorylation decreased from 13 934 ± 6437 to 6730 ± 1524 (P < .01) scanning units in the anti-β3-treated group. We conclude that administration of an antibody that inhibits activation of the β3-subunit of αVβ3 that is induced by hyperglycemia attenuates proteinuria and early histologic changes of diabetic nephropathy, suggesting that it may have utility in preventing the progression of this disease complication.

Objective-The goals of this study were to identify the signaling pathway by which sphingosine 1-phosphate (S1P)activates RhoA in smooth muscle cells (SMC) and to evaluate the contribution of this pathway to the regulation of SMC phenotype. Methods and Results-Using a combination of receptor-specific agonists and antagonists we identified S1P receptor 2 (S1PR2) as the major S1P receptor subtype that regulates SMC differentiation marker gene expression. Based on the known coupling properties of S1PR2 and our demonstration that overexpression of G␣ 12 or G␣ 13 increased SMC-specific promoter activity, we next tested whether the effects of S1P in SMC were mediated by the regulator of G protein-signaling-Rho guanine exchange factors (RGS-RhoGEFs) (leukemia-associatedAlthough each of the RGS-RhoGEFs enhanced actin polymerization, myocardinrelated transcription factor-A nuclear localization, and SMC-specific promoter activity when overexpressed in 10T1/2 cells, LARG exhibited the most robust effect and was the only RGS-RhoGEF activated by S1P in SMC. Importantly, siRNA-mediated depletion of LARG significantly inhibited the activation of RhoA and SMC differentiation marker gene expression by S1P. Knockdown of LARG had no effect on SMC proliferation but promoted SMC migration as measured by scratch wound and transwell assays. Conclusion-These data indicate that S1PR2-dependent activation of RhoA in SMC is mediated by LARG and that this signaling mechanism promotes the differentiated SMC phenotype.

Objective-Our goal was to test whether formin homology protein 1 (FHOD1) plays a significant role in the regulation of smooth muscle cell (SMC) differentiation and, if so, whether Rho kinase (ROCK)-dependent phosphorylation in the diaphanous autoinhibitory domain is an important signaling mechanism that controls FHOD1 activity in SMC. Methods and Results-FHOD1 is highly expressed in aortic SMCs and in tissues with a significant SMC component.Exogenous expression of constitutively active FHOD1, but not wild-type, strongly activated SMC-specific gene expression in 10T1/2 cells. Treatment of SMC with the RhoA activator sphingosine-1-phosphate increased FHOD1 phosphorylation at Thr1141, and this effect was completely prevented by inhibition of ROCK with Y-27632. Phosphomimetic mutations to ROCK target residues enhanced FHOD1 activity, suggesting that phosphorylation interferes with FHOD1 autoinhibition. Importantly, knockdown of FHOD1 in SMC strongly inhibited sphingosine-1-phosphate-dependent increases in SMC differentiation marker gene expression and actin polymerization, suggesting that FHOD1 plays a major role in RhoA-dependent signaling in SMC. Key Words: biology, developmental Ⅲ molecular biology Ⅲ signal transduction Ⅲ vascular biology Ⅲ vascular muscle S mooth muscle cell (SMC) differentiation is an important component of vascular development, and defective control of this process in adult animals has been shown to contribute to a variety of cardiovascular pathologies, including atherosclerosis and restenosis (see 1 for review). It is well known that SMC differentiation marker gene expression is regulated by serum response factor binding to conserved CArG cis elements within the SMC-specific promoters. The myocardin family of serum response factor cofactors (myocardin and the myocardin-related transcription factor-[MRTF]-A/ megakaryoblastic leukemia-1 and MRTF-B/megakaryoblastic leukemia-2) are also critical and have been shown to be required for SMC differentiation marker gene expression in a variety of in vitro and in vivo models. 2-5 Thus, identifying the signaling mechanisms by which extrinsic cues regulate serum response factor/myocardin factor activity will be critical for our understanding of the control of SMC phenotype. Conclusion-OurMiralles et al were the first to demonstrate that MRTF-A activity is controlled by the small GTPase RhoA and that MRTF-A nuclear localization was enhanced by RhoAdependent actin polymerization. 6 Studies from our laboratory and others have shown that regulation of MRTF-A and MRTF-B by this mechanism plays an important role in the regulation of SMC phenotype in at least some SMC subtypes. [7][8][9] Of the RhoA effector proteins, Rho kinase (ROCK) is the most well studied and has been shown to enhance actin polymerization through LIM kinase-mediated inhibition of cofilin and to stimulate contractility by inhibiting myosin phosphatase. Although the ROCK inhibitor Y-27632 attenuates SMC-specific transcription, 10,11 it is clear that other RhoA effectors are also involved.Ac...

We have previously shown that RhoA-mediated actin polymerization stimulates smooth muscle cell (SMC)-specific transcription by regulating the nuclear localization of the myocardin-related transcription factors (MRTFs). On the basis of the recent demonstration that nuclear G-actin regulates MRTF nuclear export and observations from our laboratory and others that the RhoA effector, mDia2, shuttles between the nucleus and cytoplasm, we investigated whether nuclear RhoA signaling plays a role in regulating MRTF activity. We identified sequences that control mDia2 nuclearcytoplasmic shuttling and used mDia2 variants to demonstrate that the ability of mDia2 to fully stimulate MRTF nuclear accumulation and SMC-specific gene transcription was dependent on its localization to the nucleus. To test whether RhoA signaling promotes nuclear actin polymerization, we established a fluorescence recovery after photobleaching (FRAP)-based assay to measure green fluorescent proteinactin diffusion in the nuclear compartment. Nuclear actin FRAP was delayed in cells expressing nuclear-targeted constitutively active mDia1 and mDia2 variants and in cells treated with the polymerization inducer, jasplakinolide. In contrast, FRAP was enhanced in cells expressing a nuclear-targeted variant of mDia that inhibits both mDia1 and mDia2. Treatment of 10T1/2 cells with sphingosine 1-phosphate induced RhoA activity in the nucleus and forced nuclear localization of RhoA or the Rho-specific guanine nucleotide exchange factor (GEF), leukemia-associated RhoGEF, enhanced the ability of these proteins to stimulate MRTF activity. Taken together, these data support the emerging idea that RhoA-dependent nuclear actin polymerization has important effects on transcription and nuclear structure. smooth muscle; serum response factor; myocardin-related factors; RhoA; diaphanous formins SMOOTH MUSCLE CELL (SMC) differentiation is critical during vasculogenesis and angiogenesis, and it is well recognized that defective control of this process plays an important role in the progression of atherosclerosis and restenosis (26). Thus identification of mechanisms that control SMC differentiation will be important for our understanding of vascular development and the progression of vascular disease. Serum response factor (SRF) regulates the expression of a number of muscle-specific, cytoskeletal, and early response growth genes by binding to conserved CArG [CC(A/T) 6 GG] cis elements found within their promoters (for a review, see Ref. 37). The cell type-and gene-specific effects of SRF are mediated by direct interactions with additional cofactors, and extensive evidence indicates that the SRF cofactors of the myocardin family (myocardin and the myocardin-related transcription factors, MRTF-A/MKL-1 and MRTF-B/MKL-2) regulate SMC differentiation marker gene expression (46). Indeed, genetic deletion of myocardin or MRTF-B in the mouse resulted in embryonic lethality attributable to defects in SMC differentiation in the dorsal aorta and brachial arches, respectively (22, ...

The GPCR-coupled sphingosine-1-phosphate (S1P) receptors regulate a number of important cell functions, including proliferation, migration, and adhesion. Since these processes require dynamic regulation of the actin cytoskeleton, the ability to monitor S1P-dependent activation of the Rho family GTPases is critical for our understanding of S1P signaling. Herein, we provide methods for the GST pull-down-based assay used to measure Rho, Rac, and Cdc42 activity in cultured cells treated with S1P.