Vasodilator-stimulated Phosphoprotein Ser157 Research Articles

Oncogenic BRAF noncanonically promotes tumor metastasis by mediating VASP phosphorylation and filopodia formation.

BRAF is frequently mutated in various cancer types and contributes to tumorigenesis and metastasis. As an important switch in RAS signaling pathway, BRAF typically enables the activation of MEK and ERK, and its mutation significantly promotes metastasis. However, whether BRAF could stimulate metastasis via a distinct manner is still unknown. Herein, we found that a portion of the BRAF protein localized at the plasma membrane and that the BRAFV600E mutation led to abundant formation of filopodia, which is a hallmark of invasive cancer cells. Mechanistically, BRAF physically interacts with the pseudopod formation-related protein Vasodilator-stimulated phosphoprotein (VASP), and BRAF specifically catalyzes VASP phosphorylation at Ser157. VASP depletion or disruption of Ser157 phosphorylation preferentially reduced the motility, invasion and metastasis of tumor cells harboring oncogenic BRAF or KRAS. Moreover, in clinical cancer tissues, BRAFV600E was positively correlated with the extent of invasion, and tissues with BRAFV600E expression exhibited elevated levels of VASP Ser157 phosphorylation. Our study therefor reveals a noncanonical mechanism by which oncogenic BRAF or KRAS promotes metastasis, suggests that VASP Ser157 phosphorylation might serve as a valuable therapeutic target in BRAF or KRAS mutant cancers.

Effect of Different Extraction Methods on Anthocyanin Content in Hibiscus sabdariffa L. and their Antiplatelet and Vasorelaxant Properties.

Hibiscus sabdariffa L. is a worldwide component for tea and beverages, being a natural source of anthocyanins, which are associated with cardiovascular activities. To investigate this relationship, we explored different methods of aqueous extraction on the anthocyanin content and antioxidant activity of H. sabdariffa L. calyx extract (HSCE). Pharmacological effects via platelet aggregation, calcium mobilization, cyclic nucleotide levels, vasodilator-stimulated phosphoprotein Ser157 and Ser239, and on the vasomotor response of aortic rings isolated from mice are studied herewith. We found that the application of ultrasonic turbolization, 20min, combined with acidified water was significantly more effective in the extraction process, providing extracts with the highest levels of anthocyanins (8.73 and 9.63mg/100g) and higher antioxidant activity (6.66 and 6.78μM trolox/g of sample). HSCE significantly inhibited (100-1000μg/mL) arachidonic acid-induced platelet aggregation, reduced calcium mobilization, and increased cAMP and cGMP levels with VASPSer157 and VASPSer239 phosphorylation. Vasorelaxation reduction was confirmed by the aortic rings and endothelium assays treated with nitric oxide synthase inhibitors, soluble guanylyl cyclase (sGC) oxidizing agent, or Ca2+-activated K+ channel inhibitor. The increasing of cGMP levels could be understood considering the sGC stimulation by HSCE compounds in the specific stimulus domain, which allows an understanding of the observed antiplatelet and vasorelaxant properties of H. sabdariffa L. calyx extract.

Rutin present in Alibertia edulis extract acts on human platelet aggregation through inhibition of cyclooxygenase/thromboxane.

Alibertia edulis leaf extract is commonly used in folk medicine, with rutin caffeic and vanillic acids being its major compounds. The Alibertia edulis leaf extract was investigated for its pharmacological effects via platelet aggregation, calcium mobilization, cyclic nucleotides levels, vasodilator-stimulated phosphoprotein Ser157 and Ser239 and protein kinase Cβ2 phosphorylation, thromboxane B2, cyclooxygenases 1 and 2, docking and molecular dynamics. Alibertia edulis leaf extract significantly inhibited (100-1000 μg mL-1) platelet aggregation induced by different agonists. Arachidonic acid increased levels of calcium and thromboxane B2, phosphorylation of vasodilator-stimulated phosphoprotein Ser157 and Ser239, and protein kinase Cβ, which were significantly reduced by Alibertia edulis leaf extract, rutin, and caffeic acid as well mixtures of rutin/caffeic acid. Cyclooxygenase 1 activity was inhibited for Alibertia edulis leaf extract, rutin and caffeic acid. These inhibitions were firsrtly explored by specific stabilization of rutin and caffeic acid compared to diclofenac at the catalytic site from docking score and free-energy dissociation profiles. Then, simulations detailed the rutin interactions close to the heme group and Tyr385, responsible for catalyzing the conversion of arachidonic acid to its products. Our results reveal the antiplatelet aggregation properties of Alibertia edulis leaf extract, rutin and caffeic acid providing pharmacological information about its origin from cyclooxygenase 1 inhibition and its downstream pathway.

Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls. Intriguingly, FL-VASP/239D abrogated the stimulatory effects of FL-VASP/WT and FL-VASP/239A cells on PKG activity. In turn, pharmacologic blockade of PKG in the presence of BAY60 reversed the inhibitory effect of BAY60 on naïve ASM cell migration. Taken together, we demonstrate for the first time that BAY60 inhibits ASM cell migration through cGMP/PKG/VASP signaling yet through mechanisms independent of pVASP·S239 and that FL-VASP overexpression regulates PKG activity in rat ASM cells. These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity.

Serine phosphorylation of vasodilator-stimulated phosphoprotein (VASP) regulates colon cancer cell survival and apoptosis

AimsIn colon cancer, disease recurrence and death are associated with abnormal tumor cell survival. Vasodilator-stimulated phosphoprotein (VASP) is an actin binding protein regulating cell shape and polarity through the F-actin cytoskeleton, whose activity is controlled by cAMP-dependent phosphorylation at Ser157 and cGMP-dependent phosphorylation at Ser239. This study examined the role of differential VASP Ser phosphorylation in regulating cell survival and apoptosis in human colon carcinoma cells. Main methodsSelective inhibition of VASP Ser157 or Ser239 phosphorylation in colon cancer cells was performed with specific phosphomutant constructs. F-actin organization was examined by confocal microscopy, and the balance of cell survival and death assessed by measuring acridine orange and ethidium bromide staining, caspase-3 and BAD-pS112 expression and DNA fragmentation. Key findingsIn human colon carcinoma cells suppression of VASP Ser157 phosphorylation reduced F-actin content and survival and increased apoptosis, while inhibition of VASP Ser239 phosphorylation increased F-actin content and survival and reduced cell death. Also, while 8Br-cAMP induced VASP Ser157 phosphorylation and reduced cell death, treatments with 8CPT-cGMP elevated VASP Ser239 phosphorylation and promoted apoptosis. SignificanceThese findings suggest that differential VASP Ser phosphorylation represents a unique therapeutic target to control cell survival and death behavior in colon cancer. In particular, pharmacological manipulation of VASP Ser phosphorylation could be exploited to affect the malignant actin cytoskeleton and induce apoptosis in colorectal cancer cells.

Effects of peroxisome proliferator-activated receptor γ in simvastatin antiplatelet activity: Influences on cAMP and mitogen-activated protein kinases

Statins are widely used as hypolipidemic drugs, and have beneficial effects in reducing cardiovascular events. In addition, recent studies on the pleiotropic effects of statins (i.e., simvastatin) reveal that these drugs have many additional anti-atherogenic effects, including antiplatelet activity. The mechanisms may be partly related to activation of peroxisome proliferator-activated receptors (PPARs), which are present in human platelets, and whose activation inhibits platelet aggregation. However, the details of the signaling pathway by which simvastatin inhibits platelet activation via PPARs have not yet been completely established. The aim of this study was to examine the mechanisms by which the PPAR-mediated pathways contribute to the antiplatelet activity of simvastatin. Simvastatin (3-50μM) induced PPARα and PPARγ activation in a dose-dependent manner in washed platelets. Additionally, simvastatin inhibited collagen-induced platelet aggregation, expression of CD62 and PAC-1, and Ca2+ mobilization. These effects of simvastatin on platelet responses were strongly reduced by adding a selective PPARγ antagonist (GW9662), but not PPARα antagonist (GW6471). Moreover, in the presence of GW9662, simvastatin-mediated increase of cyclic adenosine monophosphate (cAMP) production, vasodilator-stimulated phosphoprotein (VASP) Ser157 phosphorylation and inhibition of Akt phosphorylation were markedly reversed. Furthermore, simvastatin was found to inhibit phosphorylation of mitogen-activated protein kinases (MAPKs, i.e., p38 MAPK, ERK) by increasing the association between PPARγ and the components of MAPKs after platelet activation. Taken together, the present results confirm that simvastatin inhibition of platelet activation is mediated by PPARγ-dependent processes, which involves mediating MAPKs signaling, increase of cAMP formation and VASP Ser157 phosphorylation, inhibition of Akt phosphorylation and intracellular Ca2+ mobilization.

Abstract 3944: Serine phosphorylation of vasodilator-stimulated phosphoprotein regulates the tumorigenic potential of colon cancer cells.

Abstract Colorectal cancer is one of the most common and deadly cancers in developed nations. Molecular mechanisms precisely defining colorectal tumorigenesis and cancer progression remain unclear, an obstacle for innovative research applications. The vasodilator-stimulated phosphoprotein (VASP) is an actin-binding protein which critically regulates the cytoskeleton and its dependent functions, including cell shape, adhesion and migration. Of relevance, VASP Ser phosphorylation becomes deregulated in colorectal transformation and promotes invasive protrusion dynamics. Here, VASP Ser phosphorylation was selectively inhibited in colon carcinoma cells employing dominant negative mutants. Effects on cell tumorigenic potential were examined with two mouse models of colorectal cancer. Compared to control conditions, suppression of VASP Ser239 phosphorylation resulted in an enhanced cancer cell capability to establish tumor colonies in both the subcutaneous and peritoneal xenograft model. Increased tumorigenicity reflected, in part, hyper proliferative kinetics of cancer cells bearing VASP Ser239 mutants, which exhibited accelerated growth and DNA synthesis rates. In contrast, selective inhibition of VASP Ser157 slowed cell proliferation and reduced the in vivo tumorigenic potential of colon carcinoma cells, compared to respective controls. These observations support the suggestion that VASP Ser phosphorylation represents a fine signaling mechanism to control intestinal tumorigenesis and invasion, a novel paradigm for targeted anticancer strategies in patients with colorectal cancer. Citation Format: Mehboob Ali, Inna Chervoneva, Giovanni M. Pitari. Serine phosphorylation of vasodilator-stimulated phosphoprotein regulates the tumorigenic potential of colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3944. doi:10.1158/1538-7445.AM2013-3944

Effects of testosterone and 17β-oestradiol on expression of the G protein-coupled receptor P2Y12 in megakaryocytic DAMI cells

P2Y12 is an important G protein-coupled receptor that is involved in ADP-induced platelet aggregation, which is essential for normal haemostasis. Gender differences in the incidence of cardiovascular disease have been proposed to be linked to the effects of sex hormones on cardiovascular-related genes. We examined the influences of testosterone and 17β-oestradiol on P2Y12 gene expression in megakaryocytic DAMI cell line. Altered levels of P2Y12 mRNA, protein and the cAMP-dependent vasodilator-stimulated phosphoprotein-Ser157 (VASP-Ser157) phosphorylation were investigated after treatment with 17β-oestradioal or testosterone as compared to the control groups. Quantitative real-time PCR revealed that the P2Y12 mRNA levels were increased by testosterone in a dose-dependent manner, whereas 17β-oestrodiol had no effect on P2Y12 gene expression. Induction of the P2Y12 protein by testosterone was found in Western blots of the proteins isolated from testosterone-treated cells. Testosterone-mediated P2Y12 expression was repressed at both the transcriptional and translational levels by the anti-androgen receptor bicalutamide. Treatment with testosterone also resulted in a decrease in the level of VASP-Ser157 phosphorylation, as compared to the control group. The decrease in the level of VASP-Ser157 phosphorylation was reversed by bicalutamide. These findings suggest a novel pathway for testosterone regulation of P2Y12 expression in a megakaryocytic DAMI cell line. Further studies using primary human megakaryocytes and platelets could be necessary to know the effect of hormones on the P2Y12 expression in circulating platelets.

Abstract 483: Serine phosphorylation of vasodilator-stimulated phosphoprotein defines the malignant cell phenotype in colon cancer

Abstract Metastasis is the principal pathological process underlying mortality for colorectal cancer, the third deadly cancer worldwide. During tumor invasion, cancer cells assume a mesenchymal morphology that confers migratory and proliferative advantages. One of the mechanisms regulating malignant cell shape is the activity of the vasodilator-stimulated phosphoprotein (VASP) on the actin cytoskeleton. Here, human colon carcinoma cells were treated with cell permeant analogs of cAMP or cGMP, to preferentially induce VASP Ser157 (pVASP-Ser157) or Ser239 (pVASP-Ser239) phosphorylation, and examined by confocal microscopy, western blotting and colony formation. Confirming previous results, cancer dynamic membrane regions formed by the actin cytoskeleton were differentially modulated by VASP Ser phosphorylation. However, treatments with cAMP, which promoted a mesenchymal cell phenotype, reduced E-cadherin at tumor cell-cell junctions and enhanced vimentin and vinculin levels. In contrast cGMP induced an opposite cell morphology with increased E-cadherin-rich cell-cell contacts and decreased cellular vimentin and vinculin. Moreover, the cAMP-dependent phenotype increased the cancer cell ability to form tumor colonies, while cGMP compromised that metastatic potential. Importantly, cyclic nucleotide effects were mediated by selective VASP Ser phosphorylation, because VASP phosphomutants at Ser157 and Ser239 blocked cAMP or cGMP actions, respectively. Together, these findings suggest that in colon cancer cells pVASP-Ser157 and pVASP-Ser239 are critical determinants of the malignant phenotype, which define the function of actin cytoskeletal regulators, invasive cell morphology and tumorigenic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 483. doi:1538-7445.AM2012-483

Phosphorylation of vasodilator‐stimulated phosphoprotein Ser239 suppresses filopodia and invadopodia in colon cancer

In colorectal cancer, the antitumorigenic guanylyl cyclase C (GCC) signalome is defective reflecting ligand deprivation from downregulation of endogenous hormone expression. Although the proximal intracellular mediators of that signal transduction system, including cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG), are well characterized, the functional significance of its distal effectors remain vague. Dysregulation of ligand-dependent GCC signaling through vasodilator-stimulated phosphoprotein (VASP), an actin-binding protein implicated in membrane protrusion dynamics, drastically reduced cGMP-dependent VASP phosphorylation levels in colorectal tumors from patients. Restoration of cGMP-dependent VASP phosphorylation by GCC agonists suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin-based organelles in human colon cancer cells. Membrane organelle disassembly reflected specific phosphorylation of VASP Ser239, the cGMP/PKG preferred site, and rapid VASP removal from tumor cell protrusions. Importantly, VASP Ser239 phosphorylation inhibited the proteolytic function of invadopodia, reflected by suppression of the cancer cell ability to digest DQ-collagen IV embedded in Matrigel. These results demonstrate a previously unrecognized role for VASP Ser239 phosphorylation, a single intracellular biochemical reaction, as an effective mechanism which opposes tumor cell shape promoting colon cancer invasion and metastasis. Reconstitution of physiological cGMP circuitry through VASP, in turn, represents an attractive targeted approach for patients with colorectal cancer.

Riboflavin and ultraviolet light treatment potentiates vasodilator‐stimulated phosphoprotein Ser‐239 phosphorylation in platelet concentrates during storage

Pathogen reduction technologies (PRTs) were developed to improve the safety of platelet concentrates (PCs) for transfusion purposes; however, several studies report a negative impact on the in vitro and in vivo platelet (PLT) quality. Therefore, analyses of the underlying molecular processes triggered by PRT treatments are necessary to understand their effects on PLT function. In two separate two-arm studies PCs prepared in plasma for storage either by the leukoreduced buffy coat (BC-PCs) or by the leukoreduced apheresis (AP-PCs) method were treated with or without riboflavin and ultraviolet (UV) light (Mirasol; 6.24 J/mL; 265-375 nm). Samples were drawn after treatment and after 1, 4, and 6 days of storage with subsequent analyses performed using in vitro measurements for PLT quality monitoring. Semiquantitative proteomic studies identified proteins that changed in band intensities in response to treatment or storage. Protein validation and subsequent biochemical studies were carried out by immunoblot analyses. The proteomic results identified changes mainly of proteins associated with the structure and regulation of the cytoskeleton. Focusing on the vasodilator-stimulated phosphoprotein (VASP) in AP-PCs revealed a storage-dependent, but treatment-independent, delocalization and a strong treatment-dependent phosphorylation at Ser-239 that was also present, but to a much lesser degree in BC-PCs. This modification correlated exponentially with PLT activation as determined by P-selectin expression. Treatment of PCs with Mirasol leads to the amplification of VASP Ser-239 phosphorylation, which is linked to actin dynamics and regulation of integrin α(IIb) β(3) activation. This change offers one explanation for Mirasol's impact on PLT in vitro quality measures. The Ser-239 phosphorylation level of VASP might be a useful protein marker for riboflavin and UV light-mediated PLT compromise.

Abstract 1653: Vasodilator-stimulated phosphoprotein ser157 and ser239 are antimetastatic targets in colon cancer

Abstract Colorectal cancer is the third deadly cancer worldwide. Patient mortality is largely the result of metastatic disease progression, in which tumor cells undergo dramatic morphological changes characterized by the formation of membrane protrusive structures mediating invasion (lamellipodia, filopodia, invadopodia). These events are driven by the activity of actin-regulatory proteins, including the vasodilator-stimulated phosphoprotein (VASP), which reorganize the actin cytoskeleton to promote cell migration and invasion. In colon cancer, cAMP- and cGMP-dependent VASP phosphorylation at Ser157 (pVASP-Ser157) and Ser239 (pVASP-Ser239) represent opposing molecular switches, which regulate the invasive cell shape. Here, pVASP-Ser157 and pVASP-Ser239 differentially affected the metastatic potential of colon cancer cells, including tumor proliferation, colony formation, migration, adhesion and metastasis in vivo. Investigations employed intestinal cancer cells, human tumor cells expressing serine phosphoresistant VASP mutants, cGMP or cAMP analogs, agonists selectively elevating intracellular cGMP or cAMP levels, and orthotopic mouse models of colorectal cancer. Collectively, results demonstrated pVASP-Ser239 suppresses, while pVASP-Ser157 promoted, the metastatic phenotype of colon cancer cells. Thus, differential VASP serine phosphorylation is a novel targeted strategy to prevent colon cancer metastasis. Clinical translation of these findings may provide original therapeutic interventions to reduce mortality in patients with colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1653. doi:10.1158/1538-7445.AM2011-1653

Phosphorylation of Vasodilator‐Stimulated Phosphoprotein (VASP) Regulates Contractility of Airway Smooth Muscle (ASM) Tissues by Regulating Actin Dynamics

VASP is an actin regulatory protein that plays a role in numerous cellular processes. VASP is a substrate for protein kinase A, which catalyzes its phosphorylation at ser157. Contractile stimulation of ASM initiates actin polymerization which is required for force development. We hypothesized that VASP might contribute to the regulation of actin dynamics and contraction of ASM tissues. Acetylcholine (ACh) stimulation dramatically increased VASP phosphorylation on Ser157, which was inhibited by a PKC inhibitor. The expression of EGFP-VASP Ser157A mutant protein in ASM tissues decreased endogenous VASP ser157 phosphorylation, depressed contractile force in response to ACh by 50%, and inhibited ACh-induced actin polymerization. MLC phosphorylation was not affected. Immunofluorescence studies of dissociated ASM cells showed that ACh stimulated the recruitment of endogenous VASP protein to the cell membrane. Ser157 phosphorylated VASP was localized at the cell membrane and increased at the membrane after ACh stimulation. Expression of VASP ser157A mutant inhibited recruitment of endogenous VASP protein to the membrane. We conclude that ACh regulates VASP ser157 phosphorylation in ASM tissues through a PKC-dependent pathway, and that VASP phosphorylation at ser 157 affects contractile force development by participating in the regulation of ACh-stimulated cortical actin polymerization.

Abstract A8: Vasodilator-stimulated phosphoprotein Ser239 regulates matrix metalloproteinase-9 in colon cancer

Abstract Cancer invasion and spreading is promoted by membrane protrusion formation driven by actin polymerization and filament (F)-actin elongation, a process controlled by the actin-binding protein vasodilator-stimulated phosphoprotein (VASP). Tumor epithelial cell matrix metalloproteinase-9 (MMP-9) is a key promoter of carcinogenesis and invasion in colon cancer. Interestingly, both VASP and MMP-9 activities are regulated by intracellular cGMP signaling in colon cancer cells. However, direct functional relationships between VASP and MMP-9 are unrecognized. Here, human colon carcinoma cells were examined by immunoblotting, sucrose-gradient fractionation, confocal microscopy and imaging analysis. MMP-9 localizes at membrane protrusions and lipid rafts, cell surface microdomains specialized in signal transduction, in a cGMP-dependent manner. Activation of cGMP signaling disrupted membrane protrusion dynamics and induced intracellular MMP-9 redistribution. Strikingly, cancer cells expressing the serine-to-alanine VASP phosphomutant at Ser239, resistant to cGMP-dependent VASP phosphorylation, were insensitive to cGMP effects on membrane protrusions and MMP-9. Importantly, cGMP signaling through VASP Ser239, but not Ser157, critically regulates the ability of cancer cells to release MMP-9 in the extracellular environment. Together, these observations indicate tumor epithelial cell MMP-9, a key promoter of cancer invasion, is regulated by VASP Ser239 phosphorylation, a previously unappreciated paradigm in colon cancer biology. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A8.

Carbon monoxide and nitric oxide mediate cytoskeletal reorganization in microvascular cells via vasodilator-stimulated phosphoprotein phosphorylation: evidence for blunted responsiveness in diabetes.

OBJECTIVE— We examined the effect of the vasoactive agents carbon monoxide (CO) and nitric oxide (NO) on the phosphorylation and intracellular redistribution of vasodilator-stimulated phosphoprotein (VASP), a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation.RESEARCH DESIGN AND METHODS— We examined the effect of donor-released CO and NO in endothelial progenitor cells (EPCs) and platelets from nondiabetic and diabetic subjects and in human microvascular endothelial cells (HMECs) cultured under low (5.5 mmol/l) or high (25 mmol/l) glucose conditions. VASP phosphorylation was evaluated using phosphorylation site-specific antibodies.RESULTS— In control platelets, CO selectively promotes phosphorylation at VASP Ser-157, whereas NO promotes phosphorylation primarily at Ser-157 and also at Ser-239, with maximal responses at 1 min with both agents on Ser-157 and at 15 min on Ser-239 with NO treatment. In diabetic platelets, neither agent resulted in VASP phosphorylation. In nondiabetic EPCs, NO and CO increased phosphorylation at Ser-239 and Ser-157, respectively, but this response was markedly reduced in diabetic EPCs. In endothelial cells cultured under low glucose conditions, both CO and NO induced phosphorylation at Ser-157 and Ser-239; however, this response was completely lost when cells were cultured under high glucose conditions. In control EPCs and in HMECs exposed to low glucose, VASP was redistributed to filopodia-like structures following CO or NO exposure; however, redistribution was dramatically attenuated under high glucose conditions.CONCLUSIONS— Vasoactive gases CO and NO promote cytoskeletal changes through site- and cell type–specific VASP phosphorylation, and in diabetes, blunted responses to these agents may lead to reduced vascular repair and tissue perfusion.

Atrial Natriuretic Peptide-initiated cGMP Pathways Regulate Vasodilator-stimulated Phosphoprotein Phosphorylation and Angiogenesis in Vascular Endothelium

Nitric oxide (NO)- and atrial natriuretic peptide (ANP)-initiated cGMP signaling cascades are important in the maintenance of cardiovascular homeostasis. The molecular signaling mechanisms downstream of cGMP are not well understood, however. We have used small interfering RNA (siRNA) approaches to specifically knock down a series of signaling proteins in bovine aortic endothelial cells, and we have combined biochemical analyses with physiological assays to investigate cGMP-mediated signal transduction pathways. Activation of particulate guanylate cyclase (GC-A) by ANP leads to a substantial, dose-dependent, rapid, and sustained increase in intracellular cGMP. In contrast, stimulation of soluble guanylate cyclase by NO yields only a weak and transient increase in cGMP. ANP-induced cGMP production is selectively suppressed by siRNA-mediated knockdown of GC-A. ANP greatly enhances the phosphorylation at Ser-239 of the vasodilator-stimulated phosphoprotein (VASP), a major substrate of cGMP-dependent protein kinase (PKG) that significantly influences actin dynamics. Moreover, the ANP-induced phosphorylation of VASP at Ser-239 is accompanied by increased actin stress fiber formation and enhanced endothelial tube formation. siRNA-mediated knockdown of GC-A, VASP, or PKG abolishes ANP-induced VASP Ser-239 phosphorylation, stress fiber formation, and endothelial tube formation. We have demonstrated similar findings in human umbilical vein endothelial cells, where ANP substantially enhances intracellular cGMP content, phosphorylation of VASP at Ser-239, and endothelial tube formation. Taken together, our findings suggest that ANP-mediated cGMP signal transduction pathways regulate PKG phosphorylation of VASP Ser-239 in endothelial cells, resulting in reorganization of the actin cytoskeleton and enhancement of angiogenesis.

Overexpression of WAVE‐1 inhibits cell migration induced by PDGF via PKA signaling without affecting VASP ser157 phosphorylation

WAVE-1 protein acts as a member of AKAPs family, which serve to associate PKA with various components of actin cytoskeleton so as to modulate cell shape and movement. Vasodilator-stimulated phosphoprotein (VASP) is a major substrate for PKA, which appears to be critical for its function during cell migration. To investigate the effect of WAVE-1 on cell migration and the underlying regulation mechanism, we developed stable WAVE-1 overexpressing ECV304 cell line. The study show PDGF stimulate cell migration of empty vector control through transwell migration assay, accompanied with enhanced PKA activity and increased VASP ser157site phosphorylation. In contrast,cells overexpressed WAVE-1 show diminished cell migration and 3-fold higher PKA in response to PDGF. However, inhibition of cell migration in this condition was partly reversed in the presence of St-Ht31(PKA RII-AKAP selectivity anchoring inhibitor peptide). Meanwhile, PKA activity was attenuated by St-Ht31.Cells overexpressed WAVE-1 and control show very similar VASP phosphorylation after PDGF stimulation, which did not obviously changed by adding St-Ht31 yet. Nevertheless, We observed transfecting VASP ser157 site mutant into cells overexpressed WAVE-1 lead to further diminution of migration under PDGF treatment. These findings suggest that overexpressed WAVE-1 inhibits cell migration induced by PDGF,which is correlated with hyper-activation of PKA. It seems that PKA- mediated VASP phosphorylation was not involved in negative regulation of cell migration but help to maintain stable cell motility ability in part. (The research is supported by national natural science foundation of China granted NO.30470680)

Regulation of cGMP-dependent Protein Kinase Expression by Rho and Krüppel-like Transcription Factor-4

Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury.

Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser157 by protein kinase C-dependent and -independent mechanisms in thrombin-stimulated human platelets

VASP (vasodilator-stimulated phosphoprotein) is an actin- and profilin-binding protein that is expressed in platelets at high levels and plays a major role in negatively regulating secretory and adhesive events in these cells. VASP is a major substrate for cAMP- and cGMP-regulated protein kinases and it has been shown to be directly phosphorylated on Ser157 by PKC (protein kinase C). In the present paper, we show that, in human platelets, VASP is phosphorylated by PKC on Ser157, but not Ser239, in response to phorbol ester stimulation, in a manner blocked by the PKC inhibitor BIM I (bisindolylmaleimide I). In response to thrombin, VASP was also phosphorylated on Ser157, but this response was only partially inhibited by BIM I, indicating PKC-dependent and -independent pathways to VASP phosphorylation by thrombin. Using inhibitors, we have ruled out the possibility that the PKC-independent pathway acts through guanylate cyclase generation of cGMP, or through a phosphoinositide 3-kinase-dependent kinase. Inhibition of Rho kinase, however, substantially reduced Ser157 VASP phosphorylation, and its effects were additive with BIM I. This implicates Rho kinase and PKC as the major kinases that phosphorylate VASP Ser157 in response to thrombin in platelets.

Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide

AbstractThe thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistive-particle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PET-cGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-Br-cAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPT-cGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change. (Blood. 2004;104:2775-2782)