Protein kinase G signaling disrupts Rac1-dependent focal adhesion assembly in liver specific pericytes

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMP-dependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1.     

Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKG(I)). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKG(I) barrier protection because VASP is a cytoskeletal phosphorylation target of PKG(I) expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKG(I)-mediated phosphorylation of Ser(157) and Ser(239). To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H(2)O(2) in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKG(I) expression introduced by adenoviral infection (Ad.PKG). In the absence of PKG(I) expression, H(2)O(2) (100-250 microM) caused a transient increased permeability and pSer(157)-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser(157) phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H(2)O(2). With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H(2)O(2)-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated PKG(I). Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKG(I) to attenuate H(2)O(2)-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKG(I) on H(2)O(2)-induced endothelial barrier dysfunction in HPAEC.     

Pro-survival effects of 17β-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.     

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells.

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.     

Vimentin expression influences flow dependent VASP phosphorylation and regulates cell migration and proliferation

The cytoskeleton plays a central role for the integration of biochemical and biomechanical signals across the cell required for complex cellular functions. Recent studies indicate that the intermediate filament vimentin is necessary for endothelial cell morphogenesis e.g. in the context of leukocyte transmigration. Here, we present evidence, that the scaffold provided by vimentin is essential for VASP localization and PKG mediated VASP phosphorylation and thus controls endothelial cell migration and proliferation. Vimentin suppression using siRNA technique significantly decreased migration velocity by 50% (videomicroscopy), diminished transmigration activity by 42.5% (Boyden chamber) and reduced proliferation by 43% (BrdU-incorporation). In confocal microscopy Vimentin colocalized with VASP and PKG in endothelial cells. Vimentin suppression was accompanied with a translocation of VASP from focal contacts to the perinuclear region. VASP/Vimentin and PKG/Vimentin colocalization appeared to be essential for proper PKG mediated VASP phosphorylation because we detected a diminished expression of PKG and p^Ser239-VASP in vimentin-suppressed cells, Furthermore, the induction of VASP phosphorylation in perfused arteries was markedly decreased in vimentin knockout mice compared to wildtypes. A link is proposed between vimentin, VASP phosphorylation and actin dynamics that delivers an explanation for the important role of vimentin in controlling endothelial cell morphogenesis.     

VEGF-E activates endothelial nitric oxide synthase to induce angiogenesis via cGMP and PKG-independent pathways

Vascular endothelial growth factor-A (VEGF), which binds to both VEGF receptor-1 (Flt1) and VEGFR-2 (KDR/Flk-1), requires nitric oxide (NO) to induce angiogenesis in a cGMP-dependent manner. Here we show that VEGF-E, a VEGFR-2-selective ligand stimulates NO release and tube formation in human umbilical vein endothelial cells (HUVEC). Inhibition of phospholipase Cgamma (PLCgamma) with U73122 abrogated VEGF-E induced endothelial cell migration, tube formation and NO release. Inhibition of endothelial nitric oxide synthase (eNOS) using l-NNA blocked VEGF-E-induced NO release and angiogenesis. Pre-incubation of HUVEC with the soluble guanylate cyclase inhibitor, ODQ, or the protein kinase G (PKG) inhibitor, KT-5823, had no effect on angiogenesis suggesting that the action of VEGF-E is cGMP-independent. Our data provide the first demonstration that VEGFR-2-mediated NO signaling and subsequent angiogenesis is through a mechanism that is dependent on PLCgamma but independent of cGMP and PKG.     

Activation of cGMP-dependent protein kinase by protein kinase C

The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.     

Regulation of human endothelial cell focal adhesion sites and migration by cGMP-dependent protein kinase I

cGMP-dependent protein kinase type I (cGK I), a major constituent of the atrial natriuretic peptide (ANP)/nitric oxide/cGMP signal transduction pathway, phosphorylates the vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins involved in regulation of the actin cytoskeleton. Here we demonstrate that stimulation of human umbilical vein endothelial cells (HUVECs) by both ANP and 8-(4-chlorophenylthio)guanosine 3':5'-monophosphate (8-pCPT-cGMP) activates transfected cGK I and causes detachment of VASP and its known binding partner (zyxin) from focal adhesions in >60% of cells after 30 min. The ANP effects, but not the 8-pCPT-cGMP effects, reversed after 3 h of treatment. In contrast, a catalytically inactive cGK Ibeta mutant (cGK Ibeta-K405A) was incapable of mediating these effects. VASP mutated (Ser/Thr to Ala) at all three of its established phosphorylation sites (vesicular stomatitis virus-tagged VASP-AAA mutant) was not phosphorylated by cGK I and was resistant to detaching from HUVEC focal adhesions in response to 8-pCPT-cGMP. Furthermore, activation of cGK I, but not of mutant cGK Ibeta-K405A, caused a 1.5-2-fold inhibition of HUVEC migration, a dynamic process highly dependent on focal adhesion formation and disassembly. These results indicate that cGK I phosphorylation of VASP results in loss of VASP and zyxin from focal adhesions, a response that could contribute to cGK alteration of cytoskeleton-regulated processes such as cell migration.     

Phosphorylation of the PKG substrate, vasodilator-stimulated phosphoprotein (VASP), in human cultured prostatic stromal cells.

Nitric oxide (NO) is known to regulate contractility and proliferation of cells within the prostate, however, the mechanism by which this occurs is unknown. The cGMP-dependent protein kinase (PKG) signalling pathway may be involved, and recent work has shown that activation of this pathway can be assessed by analysis of phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The aim of the current study is to characterise the expression of VASP in the human prostate and human cultured prostatic stromal cells (HCPSCs), and to investigate whether NO activates PKG in these cells. Our studies revealed that VASP is expressed, and that incubation of HCPSCs with PKG-activating cGMP-analogues or the NO-donor, SNP, caused a significant PKG-dependent increase in VASP serine-239 phosphorylation. In addition, SNP elicited a reduction in intracellular K(+) in a time frame consistent with the phosphorylation of VASP and activation of PKG. These data demonstrate that VASP can be used to assess the NO/cGMP/PKG signalling pathway in HCPSCs. In addition, we demonstrate for the first time that SNP, probably via NO release, leads to phosphorylation of VASP in a manner consistent with PKG activation.     

The Slow Cyclic GMP Increase Caused by Serotonin in NG108-15 Cells Is Not Inhibited by Antagonists of Known Serotonin Receptors: Possible Existence of a New Receptor Subtype Coupled with Membrane-Bound Guanylate Cyclase

Characterization of the serotonin (5-HT)-induced cyclic GMP (cGMP) elevation was investigated in comparison with bradykinin- and ANP-induced elevations in NG108-15 cells. At 20 s, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM, 100 microM), a membrane-permeabilized Ca2+ chelator, or N-monomethyl-L-arginine (NMMA, 300 microM), an inhibitor of L-arginine-derived nitric oxide (NO) synthesis, inhibited 5-HT-induced elevation by approximately 40%, and completely inhibited bradykinin-induced response. Neither 5-HT- nor ANP-induced cGMP elevation at 10 min was affected by BAPTA-AM or NMMA. The cGMP elevated by 5-HT as well as by ANP was effluxed to the extracellular medium. These results and our previous report suggest that 5-HT stimulates two subtypes of 5-HT receptors in NG108-15: first, 5-HT3 subtype stimulating Ca(2+)-sensitive cytosolic guanylate cyclase through NO derived from L-arginine and second, a probably novel 5-HT receptor subtype involved in activation of membrane-bound guanylate cyclase.     

Natriuretic peptides inhibit G protein activation. Mediation through cross-talk between cyclic GMP-dependent protein kinase and regulators of G protein-signaling proteins

Atrial natriuretic peptide (ANP) inhibits the proliferation of many cells, in part through interfering with signal transduction enacted by G protein-coupled growth factor receptors. Signaling interactions between ANP and the G protein-coupled growth factor receptor ligand, endothelin-3 (ET-3), regulate astrocyte proliferation at a very proximal but undefined point. Here, we find that ANP inhibits the ability of ET-3 to activate Galpha(q) and Galpha(i) in these cells. ANP stimulated the translocation of endogenous regulators of G protein-signaling (RGS) proteins 3 and 4 from the cytosol to the cell membrane, and enhanced their association with Galpha(q) and Galpha(i). ANP effects were significantly blocked by HS-142-1, an inhibitor of guanylate cyclase activation, or by ET-3. KT5823, an inhibitor of cyclic GMP-dependent protein kinase (PKG) reversed the RGS translocation induced by ANP; conversely, expression of an active catalytic subunit of PKG-I, or 8-bromo-cyclic GMP stimulated RGS translocation. ANP caused the phosphorylation of both RGS proteins in a PKG-dependent fashion, and the expressed PKG (in the absence of ANP) also stimulated RGS phosphorylation. A novel cross-talk between PKG and RGS proteins is stimulated by ANP and leads to the increased translocation and association of RGS proteins with Galpha. The rapid inactivation of G proteins provides a mechanism by which ANP inhibits downstream signaling to the cell proliferation program.     

VASP phosphorylation at serine239 regulates the effects of NO on smooth muscle cell invasion and contraction of collagen

Nitric oxide triggers cGMP‐dependent kinase‐mediated phosphorylation of the actin regulator vasodilator‐stimulated phosphoprotein (VASP) at residue serine239. The function of this phosphorylation for smooth muscle cell (SMC) adhesion, spreading, matrix contraction, and invasion is not well understood. We reconstituted VASP deficient SMC with wild‐type VASP (wt‐VASP) or VASP mutants that mimic “locked” serine239 phosphorylation (S239D‐VASP) or “blocked” serine239 phosphorylation (S239A‐VASP). Collagen gel contraction was reduced in S239D‐VASP compared to S239A‐VASP and wt‐VASP expressing cells and nitric oxide (NO) stimulation decreased gel contraction of wt‐VASP reconstituted SMC. Invasion of collagen was enhanced in S239D‐VASP and NO‐stimulated wild‐type SMCs compared to S239A‐VASP expressing cells. Expression of S239D‐VASP impaired SMC attachment to collagen, reduced the number of membrane protrusions, and caused cell rounding compared to expression of S239A‐VASP. Treatment of wt‐VASP reconstituted SMCs with NO exerted similar effects as expression of S239D‐VASP. As unstimulated cells were spreading on collagen S239A‐VASP and wt‐VASP localized to actin fibers whereas S239D‐VASP was enriched in the cytosol. NO interferes with SMC invasion and contraction of collagen matrices. This requires phosphorylation of VASP on serine239, which reduces VASP binding to actin fibers. These findings support the conclusion that VASP phosphorylation at serine239 regulates cytoskeleton remodeling. J. Cell. Physiol. 222:230–237, 2010. © 2009 Wiley‐Liss, Inc.     

Regulation of the atrial natriuretic peptide receptor by heat shock protein 90 complexes

Heat shock protein 90 (hsp90) is a chaperone required for the proper folding and trafficking of many proteins involved in signal transduction. We tested whether hsp90 plays a role as a chaperone for GC-A, the membrane guanylate cyclase that acts as a receptor for atrial natriuretic peptide (ANP). When cultured cells expressing recombinant GC-A were treated with geldanamycin, an inhibitor of hsp90 function, the ANP-stimulated production of cyclic GMP was inhibited. This suggested that hsp90 was required for GC-A processing and/or stability. A physical association between hsp90 and GC-A was demonstrated in coimmunoprecipitation experiments. Treatment with geldanamycin disrupted this association and led to the accumulation of complexes containing GC-A and heat shock protein 70 (hsp70). Protein folding pathways involving hsp70 and hsp90 include several pathway-specific co-chaperones. Complexes between GC-A and hsp90 contained the co-chaperone p50(cdc37), typically found associated with protein kinase.hsp90 heterocomplexes. GC-A immunoprecipitates did not contain detectable amounts of Hop, FKBP51, FKBP52, PP5, or p23, all co-chaperones found in hsp90 complexes with other signaling proteins. The association of hsp90 and p50(cdc37) with GC-A was dependent on the kinase homology domain of this receptor but not on its ANP-binding, transmembrane, or guanylate cyclase domains. The data suggest that GC-A is regulated by hsp90 complexes similar to those involved in the maturation of protein kinases.     

Vasodilator-stimulated phosphoprotein is a substrate for protein kinase C

Vasodilator-stimulated phosphoprotein (VASP), an actin binding protein localized to areas of focal contacts, is a substrate for the cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP)-dependent protein kinases (PKA, PKG). In this study, we show that serum stimulation of vascular smooth muscle cells (SMCs) induces VASP phosphorylation on Ser157, in a mechanism not dependent on PKA or PKG. We tested the possibility that protein kinase C (PKC), a regulator of cytoskeletal function, is involved. PKC inhibition or down-regulation prevented serum-induced phosphorylation of VASP at Ser157 in rat vascular SMCs. Additionally, recombinant PKCalpha directly phosphorylated Ser157 on VASP. In summary, our data support the hypothesis that PKC phosphorylates VASP and mediates serum-induced VASP regulation.     

Notch activation augments nitric oxide/soluble guanylyl cyclase signaling in immortalized ovarian surface epithelial cells and ovarian cancer cells

Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.     

Deciphering vascular endothelial cell growth factor/vascular permeability factor signaling to vascular permeability. Inhibition by atrial natriuretic peptide

Vascular endothelial cell growth factor (VEGF) was originally described as a potent vascular permeability factor (VPF) that importantly contributes to vascular pathobiology. The signaling pathways that underlie VEGF/VPF-induced permeability are not well defined. Furthermore, endogenous vascular peptides that regulate this important VPF function are currently unknown. We report here that VPF significantly enhances permeability in aortic endothelial cells via a linked signaling pathway, sequentially involving Src, ERK, JNK, and phosphatidylinositol 3-kinase/AKT. This leads to the serine/threonine phosphorylation and redistribution of actin and the tight junction (TJ) proteins, zona occludens-1 and occludin, and the loss of the endothelial cell barrier architecture. Atrial natriuretic peptide (ANP) inhibited VPF signaling, TJ protein phosphorylation and localization, and VPF-induced permeability. This involved both guanylate cyclase and natriuretic peptide clearance receptors. In vivo, transgenic mice that overexpress ANP showed significantly less VPF-induced kinase activation and vascular permeability compared with non-transgenic littermates. Thus, ANP acts as an anti-permeability factor by inhibiting the signaling functions of VPF that we define here and by preserving the endothelial cell TJ functional morphology.