A novel regulator of angiogenesis in endothelial cells: 5-hydroxytriptamine 4 receptor.

The 5-hydroxytryptamine type 4 receptor (5-HT(4)R) regulates many physiological processes, including learning and memory, cognition, and gastrointestinal motility. Little is known about its role in angiogenesis. Using mouse hindlimb ischemia model of angiogenesis, we observed a significant reduction of limb blood flow recovery 14 days after ischemia and a decrease in density of CD31-positive vessels in adductor muscles in 5-HT(4)R(-/-) mice compared to wild type littermates. Our in vitro data indicated that 5-HT(4)R endogenously expressed in endothelial cells (ECs) may promote angiogenesis. Inhibition of the receptor with 5-HT(4)R antagonist RS 39604 reduced EC capillary tube formation in the reconstituted basement membrane. Using Boyden chamber migration assay and wound healing "scratch" assay, we demonstrated that RS 39604 treatment significantly suppressed EC migration. Transendothelial resistance measurement and immunofluorescence analysis showed that a 5-HT(4)R agonist RS 67333 led to an increase in endothelial permeability, actin stress fiber and interendothelial gap formation. Importantly, we provided the evidence that 5-HT(4)R-regulated EC migration may be mediated by Gα13 and RhoA. Our results suggest a prominent role of 5-HT(4)R in promoting angiogenesis and identify 5-HT(4)R as a potential therapeutic target for modulating angiogenesis under pathological conditions.

Vasodilator-stimulated phosphoprotein deficiency potentiates PAR-1-induced increase in endothelial permeability in mouse lungs.

Vasodilator-stimulated phosphoprotein (VASP) is implicated in the protection of the endothelial barrier in vitro and in vivo. The function of VASP in thrombin signaling in the endothelial cells (ECs) is not known. For the first time we studied the effects of VASP deficiency on EC permeability and pulmonary vascular permeability in response to thrombin receptor stimulation. We provided the evidence that VASP deficiency potentiates the increase in endothelial permeability induced by activation of thrombin receptor in cultured human umbilical vein endothelial cells (HUVECs) and isolated mouse lungs. Using transendothelial resistance measurement, we showed that siRNA-mediated VASP downregulation in HUVECs leads to a potentiation of thrombin- and protease-activated receptor 1 (PAR-1) agonist-induced increase in endothelial permeability. Compared to control cells, VASP-deficient HUVECs had delayed endothelial junctional reassembly and abrogated VE-cadherin cytoskeletal anchoring in the recovery phase after thrombin stimulation, as demonstrated by immunofluorescence studies and cell fractionation analysis, respectively. Measurement of the capillary filtration coefficient in isolated mouse lungs demonstrated that VASP(-/-) mice have increased microvascular permeability in response to infusion with PAR-1 agonist compared to wild type mice. Lack of VASP led to decreased Rac1 activation both in VASP-deficient HUVECs after thrombin stimulation and VASP(-/-) mouse lungs after PAR-1 agonist infusion, indicating that VASP effects on thrombin signaling may be correlated with changes in Rac1 activity. This study demonstrates that VASP may play critical and complex role in the regulation of thrombin-dependent disruption of the endothelial barrier function.

LIM kinase 1 promotes endothelial barrier disruption and neutrophil infiltration in mouse lungs.

RATIONALE: Disruption of endothelial barrier function and neutrophil-mediated injury are two major mechanisms underlying the pathophysiology of sepsis-induced acute lung injury (ALI). Recently we reported that endotoxin induced activation of RhoA in mice lungs that led to the disruption of endothelial barrier and lung edema formation; however, the molecular mechanism of this phenomenon remained unknown. OBJECTIVE: We reasoned that LIMK1, which participates in the regulation of endothelial cell contractility and is activated by RhoA/Rho kinase pathway, could mediate RhoA-dependent disruption of endothelial barrier function in mouse lungs during ALI. And if that is the case, then attenuation of endothelial cell contractility by downregulating LIMK1 may lead to the enhancement of endothelial barrier function, which could protect mice from endotoxin-induced ALI. METHODS AND RESULTS: Here we report that LIMK1 deficiency in mice significantly reduced mortality induced by endotoxin. Data showed that lung edema formation, lung microvascular permeability, and neutrophil infiltration into the lungs were suppressed in limk1(-/-) mice. CONCLUSIONS: We identified that improvement of endothelial barrier function along with impaired neutrophil chemotaxis were the underlying mechanisms that reduced severity of ALI in limk1(-/-) mice, pointing to a new therapeutic target for diseases associated with acute inflammation of the lungs.

Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cGMP-dependent protein kinase pathway.

Bacterial LPS induces rapid thrombocytopenia, hypotension, and sepsis. Although growing evidence suggests that platelet activation plays a critical role in LPS-induced thrombocytopenia and tissue damage, the mechanism of LPS-mediated platelet activation is unclear. In this study, we show that LPS stimulates platelet secretion of dense and alpha granules as indicated by ATP release and P-selectin expression, and thus enhances platelet activation induced by low concentrations of platelet agonists. Platelets express components of the LPS receptor-signaling complex, including TLR (TLR4), CD14, MD2, and MyD88, and the effect of LPS on platelet activation was abolished by an anti-TLR4-blocking Ab or TLR4 knockout, suggesting that the effect of LPS on platelet aggregation requires the TLR4 pathway. Furthermore, LPS-potentiated thrombin- and collagen-induced platelet aggregation and FeCl(3)-induced thrombus formation were abolished in MyD88 knockout mice. LPS also induced cGMP elevation and the stimulatory effect of LPS on platelet aggregation was abolished by inhibitors of NO synthase and the cGMP-dependent protein kinase (PKG). LPS-induced cGMP elevation was inhibited by an anti-TLR4 Ab or by TLR4 deficiency, suggesting that activation of the cGMP/protein kinase G pathway by LPS involves the TLR4 pathway. Taken together, our data indicate that LPS stimulates platelet secretion and potentiates platelet aggregation through a TLR4/MyD88- and cGMP/PKG-dependent pathway.

T-cadherin modulates endothelial barrier function.

T-cadherin is an atypical member of the cadherin family, which lacks the transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. Unlike canonical cadherins, it is believed to function primarily as a signaling molecule. T-cadherin is highly expressed in endothelium. Using transendothelial electrical resistance measurements and siRNA-mediated depletion of T-cadherin in human umbilical vein endothelial cells, we examined its involvement in regulation of endothelial barrier. We found that in resting confluent monolayers adjusted either to 1% or 10% serum, T-cadherin depletion modestly, but consistently reduced transendothelial resistance. This was accompanied by increased phosphorylation of Akt and LIM kinase, reduced phosphorylation of p38 MAP kinase, but no difference in tubulin acetylation and in phosphorylation of an actin filament severing protein cofilin and myosin light chain kinase. Serum stimulation elicited a biphasic increase in resistance with peaks at 0.5 and 4-5 h, which was suppressed by a PI3 kinase/Akt inhibitor wortmannin and a p38 inhibitor SB 239063. T-cadherin depletion increased transendothelial resistance between the two peaks and reduced the amplitude of the second peak. T-cadherin depletion abrogated serum-induced Akt phosphorylation at Thr308 and reduced phosphorylation at Ser473, reduced phosphorylation of cofilin, and accelerated tubulin deacetylation. Adiponectin slightly improved transendothelial resistance irrespectively of T-cadherin depletion. T-cadherin depletion also resulted in a reduced sensitivity and delayed responses to thrombin. These data implicate T-cadherin in regulation of endothelial barrier function, and suggest a complex signaling network that links T-cadherin and regulation of barrier function.

Zyxin is involved in thrombin signaling via interaction with PAR-1 receptor.

Protease-activated receptor 1 (PAR-1) mediates thrombin signaling in human endothelial cells. As a G-protein-coupled receptor, PAR-1 transmits thrombin signal through activation of the heterotrimeric G proteins, Gi, Gq, and G12/13. In this study, we demonstrated that zyxin, a LIM-domain-containing protein, is involved in thrombin-mediated actin cytoskeleton remodeling and serum response element (SRE)-dependent gene transcription. We determined that zyxin binds to the C-terminal domain of PAR-1, providing a possible mechanism of involvement of zyxin as a signal transducer in PAR-1 signaling. Data showing that disruption of PAR-1-zyxin interaction inhibited thrombin-induced stress fiber formation and SRE activation supports this hypothesis. Similarly, depletion of zyxin using siRNA inhibited thrombin-induced actin stress fiber formation and SRE-dependent gene transcription. In addition, depletion of zyxin resulted in delay of endothelial barrier restoration after thrombin treatment. Notably, down-regulation of zyxin did not affect thrombin-induced activation of RhoA or Gi, Gq, and G12/13 heterotrimeric G proteins, implicating a novel signaling pathway regulated by PAR-1 that is not mediated by G-proteins. The observation that zyxin targets VASP, a partner of zyxin in regulation of actin assembly and dynamics, to focal adhesions and along stress fibers on thrombin stimulation suggests that zyxin may participate in thrombin-induced cytoskeletal remodeling through recruitment of VASP. In summary, this study establishes a crucial role of zyxin in thrombin signaling in endothelial cells and provides evidence for a novel PAR-1 signaling pathway mediated by zyxin.

T-cadherin is located in the nucleus and centrosomes in endothelial cells.

T-cadherin (H-cadherin, cadherin 13) is upregulated in vascular proliferative disorders and in tumor-associated neovascularization and is deregulated in many cancers. Unlike canonical cadherins, it lacks transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. T-cadherin is thought to function in signaling rather than as an adhesion molecule. Some interactive partners of T-cadherin at the plasma membrane have recently been identified. We examined T-cadherin location in human endothelial cells using confocal microscopy and subcellular fractionation. We found that a considerable proportion of T-cadherin is located in the nucleus and in the centrosomes. T-cadherin colocalized with a centrosomal marker gamma-tubulin uniformly throughout the cell cycle at least in human umbilical vein endothelial cells. In the telophase, T-cadherin transiently concentrated in the midbody and was apparently degraded. Its overexpression resulted in an increase in the number of multinuclear cells, whereas its downregulation by small interfering RNA led to an increase in the number of cells with multiple centrosomes. These findings indicate that deregulation of T-cadherin in endothelial cells may lead to disturbances in cytokinesis or centrosomal replication.

Galpha13 regulates MEF2-dependent gene transcription in endothelial cells: role in angiogenesis.

The alpha subunit of heterotrimeric G13 protein is required for the embryonic angiogenesis (Offermanns et al., Science 275:533-536, 1997). However, the molecular mechanism of Galpha13-dependent angiogenesis is not understood. Here, we show that myocyte-specific enhancer factor-2 (MEF2) mediates Galpha13-dependent angiogenesis. Our data showed that constitutively activated Galpha13Q226L stimulated MEF2-dependent gene transcription. In addition, downregulation of endogenous Galpha13 inhibited thrombin-stimulated MEF2-dependent gene transcription in endothelial cells. Both Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) and histone deacetylase 5 (HDAC5) were involved in Galpha13-mediated MEF2-dependent gene transcription. Galpha13Q226L also increased Ca(2+)/calmodulin-independent CaMKIV activity, while dominant negative mutant of CaMKIV inhibited MEF2-dependent gene transcription induced by Galpha13Q226L. Furthermore, Galpha13Q226L was able to derepress HDAC5-mediated repression of gene transcription and induce the translocation of HDAC5 from nucleus to cytoplasm. Finally, downregulation of endogenous Galpha13 and MEF2 proteins in endothelial cells reduced cell proliferation and capillary tube formation. Decrease of endothelial cell proliferation that was caused by the Galpha13 downregulation was partially restored by the constitutively active MEF2-VP16. Our studies suggest that MEF2 proteins are an important component in Galpha13-mediated angiogenesis.

G alpha12 is targeted to the mitochondria and affects mitochondrial morphology and motility.

G alpha12 constitutes, along with G alpha13, one of the four families of alpha subunits of heterotrimeric G proteins. We found that the N terminus of G alpha12, but not those of other G alpha subunits, contains a predicted mitochondrial targeting sequence. Using confocal microscopy and cell fractionation, we demonstrated that up to 40% of endogenous G alpha12 in human umbilical vein endothelial cells colocalize with mitochondrial markers. N-terminal sequence of G alpha12 fused to GFP efficiently targeted the fusion protein to mitochondria. G alpha12 with mutated mitochondrial targeting sequence was still located in mitochondria, suggesting the existence of additional mechanisms for mitochondrial localization. Lysophosphatidic acid, one of the known stimuli transduced by G alpha12/13, inhibited mitochondrial motility, while depletion of endogenous G alpha12 increased mitochondrial motility. G alpha12Q229L variants uncoupled from RhoGEFs (but not fully functional activated G alpha12Q229L) induced transformation of the mitochondrial network into punctate mitochondria and resulted in a loss of mitochondrial membrane potential. All examined G alpha12Q229L variants reduced phosphorylation of Bcl-2 at Ser-70, while only mutants unable to bind RhoGEFs also decreased cellular levels of Bcl-2. These G alpha12 mutants were also more efficient Hsp90 interactors. These findings are the first demonstration of a heterotrimeric G protein alpha subunit specifically targeted to mitochondria and involved in the control of mitochondrial morphology and dynamics.

RhoGDI-1 modulation of the activity of monomeric RhoGTPase RhoA regulates endothelial barrier function in mouse lungs.

Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1(-/-) mice. We observed that basal endothelial permeability in lungs of RhoGDI-1(-/-) mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1(-/-) lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1-mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.

Downregulation of LIM kinase 1 suppresses ocular inflammation and fibrosis.

PURPOSE: The purpose of this study was to determine if downregulation of LIM kinase 1 (LIMK1) by genetic deletion or direct application of LIMK1-targeted siRNA could suppress TGF-beta mediated ocular inflammation and fibrosis. METHODS: LIMK1 specific siRNAs designed from the human sequence were transfected into human corneal fibroblasts in culture. Immunofluorescence and immunoblotting were performed to examine the fibronectin assembly. The effects of LIMK1 downregulation on actin cytoskeleton organization and focal adhesion formation were studied. A wound closure assay was used to assess cell migration in in vitro fibroblast cultures. The in vivo effects of LIMK1 genetic deletion or downregulation by mouse siRNA were evaluated in a mouse model of ocular inflammation generated by subconjunctival injection of phosphate buffered saline and latex beads. Cellularity on tissue sections was examined after staining with hematoxylin and eosin. Anti-CD45 antibody was used for the leukocyte detection. RESULTS: Downregulation of LIMK1 in cultured corneal fibroblasts impaired fibronectin secretion and assembly, diminished actin polymerization and focal adhesion formation, and retarded cell migration. In the mouse model of ocular inflammation, both genetic deletion and downregulation of LIMK1 by siRNA significantly reduced inflammatory response. CONCLUSIONS: Downregulation of LIMK1 was efficacious to decrease the ocular inflammation. We disclose a possibility that LIMK1 may mediate TGF-beta-dependent signaling during ocular inflammation. A direct application of siRNA into eyes to downregulate LIMK1 expression may provide a novel therapy for suppression and prevention of ocular inflammation and fibrosis.

Regulation of apoptosis signal-regulating kinase 1 degradation by G alpha13.

Apoptosis signal-regulating kinase (ASK1) is a mitogen-activated protein kinase (MAPK) that transduces apoptotic signals from a variety of stresses. We have shown previously that alpha subunits of heterotrimeric G12 and G13 proteins stimulate ASK1 kinase activity and ASK1-dependent apoptosis. Here, we report a novel mechanism of G-protein-dependent regulation of ASK1. We demonstrated that G alpha13 forms a complex with ASK1 in an activation-independent manner. Both N- and C-terminal regulatory domains of ASK1 were essential for the efficient interaction, while its kinase domain was not required. Formation of the G alpha13-ASK1 complex was enhanced by JNK-interacting leucine zipper protein, JLP. Constitutively activated G alpha13Q226L increased ASK1 expression. Short-term activation of a serotonin 5-HT4 receptor that is coupled to G alpha13 also increased ASK1 expression. Importantly, prolonged activation of 5-HT4 receptor in COS-7 cells or prolonged treatment of human umbilical vein endothelial cells with thrombin concomitantly down-regulated both G alpha13 and ASK1. Data showed that G alpha13Q226L reduced the rate of ASK1 degradation, decreased ASK1 ubiquitination, and reduced association of ASK1 with an E3 ubiquitin ligase CHIP, previously shown to mediate ASK1 degradation. Our findings indicate that ASK1 expression levels can be regulated by G alpha13, at least in part via control of ASK1 ubiquitination and degradation.

Morphogenic signaling in neurons via neurotransmitter receptors and small GTPases.

Several neurotransmitters including serotonin and glutamate have been shown to be involved in many aspects of neural development, such as neurite outgrowth, regulation of neuronal morphology, growth cone motility and dendritic spine shape and density, in addition to their well-established role in neuronal communication. This review focuses on recent advances in our understanding of the molecular mechanisms underlying neurotransmitter-induced changes in neuronal morphology. In the first part of the review, we introduce the roles of small GTPases of the Rho family in morphogenic signaling in neurons and discuss signaling pathways, which may link serotonin, operating as a soluble guidance factor, and the Rho GTPase machinery, controlling neuronal morphology and motility. In the second part of the review, we focus on glutamate-induced neuroplasticity and discuss the evidence on involvement of Rho and Ras GTPases in functional and structural synaptic plasticity triggered by the activation of glutamate receptors.

5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology.

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT7 receptor. Expression of 5-HT7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of 5-HT4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT7R/G12 and 5-HT4R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

G Protein betagamma subunits stimulate p114RhoGEF, a guanine nucleotide exchange factor for RhoA and Rac1: regulation of cell shape and reactive oxygen species production.

Rho GTPases integrate the intracellular signaling in a wide range of cellular processes. Activation of these G proteins is tightly controlled by a number of guanine nucleotide exchange factors (GEFs). In this study, we addressed the functional role of the recently identified p114RhoGEF in in vivo experiments. Activation of endogenous G protein-coupled receptors with lysophosphatidic acid resulted in activation of a transcription factor, serum response element (SRE), that was enhanced by p114RhoGEF. This stimulation was inhibited by the functional scavenger of Gbetagamma subunits, transducin. We have determined that Gbetagamma subunits but not Galpha subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gbetagamma subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gbetagamma subunits stimulated SRE activity induced by full-length and DH/PH domain of p114RhoGEF. Using in vivo pull-down assays and dominant-negative mutants of Rho GTPases, we have determined that p114RhoGEF activated RhoA and Rac1 but not Cdc42 proteins. Functional significance of RhoA activation was established by the ability of p114RhoGEF to induce actin stress fibers and cell rounding. Functional significance of Rac1 activation was established by the ability of p114RhoGEF to induce production of reactive oxygen species (ROS) followed by activation of NADPH oxidase enzyme complex. In summary, our data showed that the novel guanine nucleotide exchange factor p114RhoGEF regulates the activity of RhoA and Rac1, and that Gbetagamma subunits of heterotrimeric G proteins are activators of p114RhoGEF under physiological conditions. The findings help to explain the integrated effects of LPA and other G-protein receptor-coupled agonists on actin stress fiber formation, cell shape change, and ROS production.

Differential expression of adenosine receptors in human endothelial cells: role of A2B receptors in angiogenic factor regulation.

Adenosine has been reported to stimulate or inhibit the release of angiogenic factors depending on the cell type examined. To test the hypothesis that differential expression of adenosine receptor subtypes contributes to endothelial cell heterogeneity, we studied microvascular (HMEC-1) and umbilical vein (HUVEC) human endothelial cells. Based on mRNA level and stimulation of adenylate cyclase, we found that HUVECs preferentially express A2A adenosine receptors and HMEC-1 preferentially express A2B receptors. Neither cells expressed A1 or A3 receptors. The nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased expression of interleukin-8 (IL-8), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in HMEC-1, but had no effect in HUVECs. In contrast, the selective A2A agonist 2-p-(2-carboxyethyl)phenylethylamino-NECA (CGS 21680) had no effect on expression of these angiogenic factors. Cotransfection of each type of adenosine receptors with a luciferase reporter in HMEC-1 showed that A2B receptors, but not A1, A2A, or A3, activated IL-8 and VEGF promoters. These effects were mimicked by constitutively active alphaG(q), alphaG12, and alphaG13, but not alphaG(s) or alphaG(i1-3). Furthermore, stimulation of phospholipase C indicated coupling of A2B receptors to G(q) proteins in HMEC-1. Thus, differential expression of adenosine receptor subtypes contributes to functional heterogeneity of human endothelial cells. A2B receptors, predominantly expressed in human microvascular cells, modulate expression of angiogenic factors via coupling to G(q), and possibly via G12/13.

Galpha(q) and Gbetagamma regulate PAR-1 signaling of thrombin-induced NF-kappaB activation and ICAM-1 transcription in endothelial cells.

As thrombin binding to the G protein-coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, Galpha(q), and the Gbetagamma dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits Galpha(q), or alpha-transducin (Galpha(t)), a scavenger of the Gbetagamma, markedly decreased NF-kappaB activity induced by PAR-1 activation. We determined the downstream signaling targets activated by Galpha(q) and Gbetagamma that mediate NF-kappaB activation. Expression of the kinase-defective protein kinase C (PKC)-delta mutant inhibited NF-kappaB activation induced by the constitutively active Galpha(q) mutant, but had no effect on NF-kappaB activity induced by Gbeta(1)gamma(2). In related experiments, NF-kappaB as well as ICAM-1 promoter activation induced by Gbeta(1)gamma(2) were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated Galpha(q). Cotransfection of the catalytically inactive Akt mutant inhibited the NF-kappaB activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of Galpha(q) and PKC-delta. These results support a model in which ligation of PAR-1 induces NF-kappaB activation and ICAM-1 transcription by the engagement of parallel Galphaq/PKC-delta and Gbetagamma/PI3-kinase pathways that converge at Akt.

Heterotrimeric G-proteins associate with microtubules during differentiation in PC12 pheochromocytoma cells.

Tubulin modifies G-protein signaling and heterotrimeric G-proteins regulate microtubule assembly. Here we report an interplay among G-protein-coupled receptor and receptor tyrosine kinase (such as nerve growth factor-NGF) signaling systems in PC12 pheochromocytoma cells that resulted in a translocation of Galpha(s), Galpha(i1), and Galpha(o) from cell bodies to cellular processes where they appear to localize with tubulin-containing structures. This relocation appeared to depend on the integrity of microtubules, as it was blocked and reversed by nocodazole. Latrunculin, which promotes actin filament depolymerization, had no effect. Both deconvolution microscopy and immunoprecipitation showed a significant increase of Galpha association with microtubules that was coincident with the extension of "neurites." There were distinctions among the Galpha subtypes, with Galpha(s) showing the most profound NGF-induced colocalization with tubulin. Translocation of Galpha was blocked by agents that inhibit the MAP kinases required for neuronal differentiation, suggesting that G-protein relocation is triggered by the intracellular signals for differentiation. Consistent with this, Galpha in Neuro-2A cells, which spontaneously differentiate, showed a similar translocation coincident with differentiation. Thus, diverse signals that promote neuronal differentiation and changes in cell morphology may use specific G-proteins to evoke cytoskeletal rearrangement.

Melatonin mt1 and MT2 receptors stimulate c-Jun N-terminal kinase via pertussis toxin-sensitive and -insensitive G proteins.

Melatonin is a pineal hormone involved in neuroendocrine processes in mammals. It has been shown that melatonin inhibits the enzymatic activities of adenylyl cyclases and the transcriptional activities of CREB. In this report, we demonstrate that 2-iodomelatonin (2IMT) treatment on COS-7 cells transfected with melatonin receptors (mt1 and MT2) induces c-Jun N-terminal kinase (JNK) activation, which is pertussis toxin (PTX)-sensitive, Ras/Rac-dependent and may involve Src-family protein tyrosine kinases. Moreover, PTX-insensitive Gs, Gz and G16 are capable of linking activated melatonin receptors to the stimulation of JNK. Agonist stimulation on PTX-pretreated COS-7 cells overexpressing mt1 receptor, Galpha(s) and adenylyl cyclase VI led to increased cAMP accumulation. Stimulation of endogenous mt1 receptors in MCF-7 cells was associated with the activation of both JNK and extracellular signal-regulated kinase (ERK). This report demonstrates the stimulatory effect of melatonin receptors on JNK, and provides experimental evidence for a functional coupling between the G(i)-coupled melatonin receptor and Gs, in terms of adenylyl cyclase activation.

G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin "outside-in" signaling.

Integrins mediate cell adhesion to the extracellular matrix and transmit signals within the cell that stimulate cell spreading, retraction, migration, and proliferation. The mechanism of integrin outside-in signaling has been unclear. We found that the heterotrimeric guanine nucleotide-binding protein (G protein) Galpha13 directly bound to the integrin beta3 cytoplasmic domain and that Galpha13-integrin interaction was promoted by ligand binding to the integrin alphaIIbbeta3 and by guanosine triphosphate (GTP) loading of Galpha13. Interference of Galpha13 expression or a myristoylated fragment of Galpha13 that inhibited interaction of alphaIIbbeta3 with Galpha13 diminished activation of protein kinase c-Src and stimulated the small guanosine triphosphatase RhoA, consequently inhibiting cell spreading and accelerating cell retraction. We conclude that integrins are noncanonical Galpha13-coupled receptors that provide a mechanism for dynamic regulation of RhoA.