Differential regulation of Rho and Rac through heterotrimeric G-proteins and cyclic nucleotides

Platelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A(2) (TXA(2)) mimetic rapidly activated both small GTPases independently of integrin alpha(IIb)beta(3) activation., which leads to the activation of G(12)/G(13) and G(q) did not induce Rac activation in G alpha(q)-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol 4,5-bisphosphate formation, and to induce shape change. Rac activation by in wild-type platelets could be blocked by chelation of intracellular Ca(2+) and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the G(i)-coupled ADP receptor. Cyclic AMP, which completely blocks platelet function, inhibited the -induced activation of G(q) and G(12)/G(13) as well as of Rac and Rho. In contrast, cGMP, which has no effect on platelet shape change blocked only activation of G(q) and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G(12)/G(13)-mediated Rho activation is involved in the shape change response, whereas Rac is activated through G(q) and is not required for shape change. Cyclic AMP and cGMP differentially interfere with -induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA(2) receptor.     

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.     

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A(2) (TXA(2)) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G(q), G(i) and G(z) (refs. 4-6). However, the role and relative importance of G(12) and G(13), which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha(13), but not Galpha(12), severely reduced the potency of thrombin, TXA(2) and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha(13) deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G(13)-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.     

Lysophosphatidic acid-induced platelet shape change proceeds via Rho/Rho kinase-mediated myosin light-chain and moesin phosphorylation

Platelet activation plays an important role in arterial thrombotic disorders. Here we show that the serum-borne phospholipid lysophosphatidic acid (LPA) activates the GTPase Rho and its target Rho-kinase to induce myosin light-chain (MLC) and moesin phosphorylation, leading to platelet shape change. MLC phosphorylation, moesin phosphorylation, and shape change were blocked by preincubating platelets with C3 transferase from Clostridium botulinum and Y-27632-specific inhibitors of Rho and Rho kinase, respectively. LPA did not increase the cytosolic Ca(2+) concentration during shape change. Our results suggest that LPA via Rho-Rho kinase induces MLC and moesin phosphorylation leading to shape change in the absence of an increase in the cytosolic Ca(2+) concentration. Rho/Rho kinase inhibition could be a therapeutic strategy to prevent pathologic platelet activation during arterial thrombotic disorders.     

Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets

Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.     

Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase.

A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein alpha subunits of the Galpha(12) family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Galpha(12) and Galpha(13) to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. Here, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, we explored whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs. We obtained evidence that FAK can be activated by thrombin, Galpha(12), Galpha(13), and Galpha(q) through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through the tyrosine phosphorylation of RGL-containing RhoGEFs.     

Akt activation in platelets depends on Gi signaling pathways

The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonist-induced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr308 and Ser473 phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y12 receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental Gi or Gz signaling. Unlike wild-type mouse platelets, platelets from Galphaq-deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y1 receptor. However, ADP caused Akt phosphorylation in Galphaq- and P2Y1-deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that Gi, but not Gq or G12/13, signaling pathways are required for activation of Akt in platelets, and Gi signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets.     

Convulxin induces platelet shape change through myosin light chain kinase and Rho kinase.

Once platelets are activated, the first event to occur is a rapid change in shape, associated with Ca2+/calmodulin-dependent myosin light chain (MLC) phosphorylation and with Rho kinase activation. The purpose of this study was to investigate which is the biochemical pathway that leads to platelet shape change in response to convulxin, a selective GpVI activator, and to verify whether MLC phosphorylation is essential for this process. The inhibition of the Ca2+-dependent pathway by means of the Ca2+ chelator BAPTA, the Ca2+/calmodulin inhibitor W-7 or the cAMP enhancing drug iloprost reduced about 50% of platelet shape change in response to convulxin. The treatment with either the Rho kinase inhibitors Y27632 or HA 1077 had no effect on platelet shape change induced by convulxin. When both Ca2+/calmodulin-dependent and Rho kinase-dependent pathways were concomitantly inhibited by the combined use of Y27632 plus BAPTA, W-7 or iloprost, platelet shape change was completely abolished. Our findings suggest that convulxin-induced platelet shape change occurs via both pathways, the Ca2+/calmodulin-dependent, which appears to be more important, and the Rho kinase-dependent one. The pattern of MLC phosphorylation was not modified by Rho kinase inhibitors. Conversely, the inhibition of the Ca2+-dependent pathway caused a strong reduction of MLC phosphorylation in BAPTA-treated platelets, and a total inhibition in W-7 or iloprost-treated platelets. Our results demonstrate that following Rho kinase-dependent pathway platelet shape change can occur without the involvement of MLC phosphorylation.     

G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension

The tone of vascular smooth muscle cells is a primary determinant of the total peripheral vascular resistance and hence the arterial blood pressure. Most forms of hypertension ultimately result from an increased vascular tone that leads to an elevated total peripheral resistance. Regulation of vascular resistance under normotensive and hypertensive conditions involves multiple mediators, many of which act through G protein-coupled receptors on vascular smooth muscle cells. Receptors that mediate vasoconstriction couple with the G-proteins G(q)-G11 and G12-G13 to stimulate phosphorylation of myosin light chain (MLC) via the Ca2+/MLC kinase- and Rho/Rho kinase-mediated signaling pathways, respectively. Using genetically altered mouse models that allow for the acute abrogation of both signaling pathways by inducible Cre/loxP-mediated mutagenesis in smooth muscle cells, we show that G(q)-G11-mediated signaling in smooth muscle cells is required for maintenance of basal blood pressure and for the development of salt-induced hypertension. In contrast, lack of G12-G13, as well as of their major effector, the leukemia-associated Rho guanine nucleotide exchange factor (LARG), did not alter normal blood pressure regulation but did block the development of salt-induced hypertension. This identifies the G12-G13-LARG-mediated signaling pathway as a new target for antihypertensive therapies that would be expected to leave normal blood pressure regulation unaffected.     

Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase

Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways.     

Vav family proteins are required for optimal regulation of PLCgamma2 by integrin alphaIIbbeta3

Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCgamma (phospholipase Cgamma) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin alphaIIbbeta3, which has recently been shown to regulate PLCgamma2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced alphaIIbbeta3-mediated PLCgamma2 tyrosine phosphorylation and reduced Ca(2+) mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCgamma2 by integrin alphaIIbbeta3, but that their requirement is by-passed upon G-protein receptor activation.     

Signal transduction in collagen-stimulated rat platelets is composed of three stages

A stable thromboxane A2 (TXA2) mimetic, U46619, induced a shape change of rat platelets, but did not induce phosphoinositide breakdown, aggregation or secretion. However, when U46619 was added to platelets which had been previously stimulated with collagen in the presence of indomethacin, all biological responses were induced about 1 min after the occurrence of shape change. Furthermore, two phases of phosphorylation of myosin light chain (MLC) were observed under the same conditions, one coinciding with shape change and the other with aggregation. Similar two-phase Ca2+ mobilization has been observed using aequorin (Nakano, T., Terawaki, A., & Arita, H. (1986) J. Biochem. 99, 1285-1288). From these results, collagen-induced signal transduction is considered to be composed of three stages. The first stage is the initial TXA2 generation. The second stage involves inositol trisphosphate-independent first-phase Ca2+ mobilization and the first-phase MLC phosphorylation by the action of TXA2 alone, leading to the shape change of platelets. The third stage is initiated by an abrupt phosphoinositide breakdown via the synergistic action of TXA2 and occupation of the collagen receptor, and the resulting inositol trisphosphate may induce the second-phase Ca2+ mobilization to produce the second-phase MLC phosphorylation together with aggregation and secretion.     

The role of heterotrimeric G proteins in platelet activation

Activation of platelets plays a central role in hemostasis as well as in various thromboembolic diseases like myocardial infarction or stroke. Most platelet activating stimuli function through receptors which couple to heterotrimeric G proteins of the Gi, Gq and G12 families. Recent studies have elucidated the roles of individual G proteins in the regulation of platelet functions like shape change, aggregation and granule secretion. The signaling pathways mediated by heterotrimeric G proteins operate synergistically to induce a full activation of platelets. This review summarizes recent progress in the understanding of upstream regulation of platelet activation through G protein-coupled receptors.     

Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors

The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC(50) 0.7 +/- 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated G(q) and G(13), stimulated G alpha(q)-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release, and increased cytosolic free Ca(2+). PI hydrolysis was blocked by expression of G alpha(q) minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC(50) = 1.0 +/- 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca(2+)-dependent contraction and myosin light-chain (MLC)(20) phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC(20) phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of G alpha(q) or G alpha(13) minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC(20) phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a G alpha(q)-mediated cascade involving Ca(2+)/calmodulin activation of MLC kinase and transient MLC(20) phosphorylation and contraction as well as a sustained G alpha(q)- and G alpha(13)-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC(20) phosphorylation and contraction.     

Coordinated signaling through both G12/13 and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets

Activation of GPIIb/IIIa is known to require agonist-induced inside-out signaling through G(q), G(i), and G(z). Although activated by several platelet agonists, including thrombin and thromboxane A(2), the contribution of the G(12/13) signaling pathway to GPIIb/IIIa activation has not been investigated. In this study, we used selective stimulation of G protein pathways to investigate the contribution of G(12/13) activation to platelet fibrinogen receptor activation. YFLLRNP is a PAR-1-specific partial agonist that, at low concentrations (60 microm), selectively activates the G(12/13) signaling cascade resulting in platelet shape change without stimulating the G(q) or G(i) signaling pathways. YFLLRNP-mediated shape change was completely inhibited by the p160(ROCK) inhibitor, Y-27632. At this low concentration, YFLLRNP-mediated G(12/13) signaling caused platelet aggregation and enhanced PAC-1 binding when combined with selective G(i) or G(z) signaling, via selective stimulation of the P2Y(12) receptor or alpha(2A)-adrenergic receptor, respectively. Similar data were obtained when using low dose (10 nm), a thromboxane A(2) mimetic, to activate G(12/13) in the presence of G(i) signaling. These results suggest that selective activation of G(12/13) causes platelet GPIIb/IIIa activation when combined with G(i) signaling. Unlike either G(12/13) or G(i) activation alone, co-activation of both G(12/13) and G(i) resulted in a small increase in intracellular calcium. Chelation of intracellular calcium with dimethyl BAPTA dramatically blocked G(12/13) and G(i)-mediated platelet aggregation. No significant effect on aggregation was seen when using selective inhibitors for p160(ROCK), PKC, or MEKK1. PI 3-kinase inhibition lead to near abolishment of platelet aggregation induced by co-stimulation of G(q) and G(i) pathways, but not by G(12/13) and G(i) pathways. These data demonstrate that co-stimulation of G(12/13) and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets in a mechanism that involves intracellular calcium, and that PI 3-kinase is an important signaling molecule downstream of G(q) but not downstream of G(12/13) pathway.     

Role of Rho in Ca(2+)-insensitive contraction and paxillin tyrosine phosphorylation in smooth muscle

We investigatedwhether Rho activation is required for Ca²⁺-insensitivepaxillin phosphorylation, myosin light chain (MLC) phosphorylation, andcontraction in tracheal muscle. Tyrosine-phosphorylated proteins havebeen implicated in the Ca²⁺-insensitive contractileactivation of smooth muscle tissues. The contractile activation oftracheal smooth muscle increases tyrosine phosphorylation of thecytoskeletal proteins paxillin and focal adhesion kinase. Paxillin isimplicated in integrin-mediated signal transduction pathways thatregulate cytoskeletal organization and cell motility. In fibroblastsand other nonmuscle cells, paxillin tyrosine phosphorylation depends onthe activation of Rho and is inhibited by cytochalasin, an inhibitor ofactin polymerization. In permeabilized muscle strips, we found that AChinduced Ca²⁺-insensitive contraction, MLC phosphorylation,and paxillin tyrosine phosphorylation. Ca²⁺-insensitivecontraction and MLC phosphorylation induced by ACh were inhibited by C3transferase, an inhibitor of Rho activation; however, C3 transferasedid not inhibit paxillin tyrosine phosphorylation. Ca²⁺-insensitive paxillin tyrosine phosphorylation was alsonot inhibited by the Rho kinase inhibitor Y-27632, by cytochalasin D,or by the inhibition of MLC phosphorylation. We conclude that, intracheal smooth muscle, Rho mediates Ca²⁺-insensitivecontraction and MLC phosphorylation but that Rho is not required forCa²⁺-insensitive paxillin tyrosine phosphorylation.Paxillin phosphorylation also does not require actomyosin activation,nor is it inhibited by the actin filament capping agent cytochalasin D.

     

Protein kinase Calpha-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement

Heterotrimeric G-proteins of the Galpha12/13 family activate Rho GTPase through the guanine nucleotide exchange factor p115RhoGEF. Because Rho activation is also dependent on protein kinase Calpha (PKCalpha), we addressed the possibility that PKCalpha can also induce Rho activation secondary to the phosphorylation of p115RhoGEF. Studies were made using human umbilical vein endothelial cells in which we addressed the mechanisms of PKCalpha-induced Rho activation and its consequences on actin cytoskeletal changes. We observed that PKCalpha associated with p115RhoGEF within 1 min of thrombin stimulation and p115RhoGEF phosphorylation was dependent on PKCalpha. Inhibition of PKCalpha-dependent p115RhoGEF phosphorylation prevented the thrombin-induced Rho activation, indicating that the response occurred downstream of PKCalpha phosphorylation of p115RhoGEF. The regulator of G-protein signaling domain of p115RhoGEF, a GTPase activating protein for G12/13, also prevented thrombin-induced Rho activation, indicating the parallel requirement of G12/13 in signaling Rho activation via p115RhoGEF. These data demonstrate a pathway of Rho activation involving PKCalpha-dependent phosphorylation of p115RhoGEF. Thus, Rho activation in endothelial cells and the subsequent actin cytoskeletal re-arrangement require the cooperative interaction of both G12/13 and PKCalpha pathways that converge at p115RhoGEF.     

Inhibitory and stimulatory regulation of Rac and cell motility by the G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform

The G protein-coupled receptors S1P2/Edg5 and S1P3/Edg3 both mediate sphingosine-1-phosphate (S1P) stimulation of Rho, yet S1P2 but not S1P3 mediates downregulation of Rac activation, membrane ruffling, and cell migration in response to chemoattractants. Specific inhibition of endogenous Galpha12 and Galpha13, but not of Galphaq, by expression of respective C-terminal peptides abolished S1P2-mediated inhibition of Rac, membrane ruffling, and migration, as well as stimulation of Rho and stress fiber formation. Fusion receptors comprising S1P2 and either Galpha12 or Galpha13, but not Galphaq, mediated S1P stimulation of Rho and also inhibition of Rac and migration. Overexpression of Galphai, by contrast, specifically antagonized S1P2-mediated inhibition of Rac and migration. The S1P2 actions were mimicked by expression of V14Rho and were abolished by C3 toxin and N19Rho, but not Rho kinase inhibitors. In contrast to S1P2, S1P3 mediated S1P-directed, pertussis toxin-sensitive chemotaxis and Rac activation despite concurrent stimulation of Rho via G12/13. Upon inactivation of Gi by pertussis toxin, S1P3 mediated inhibition of Rac and migration just like S1P2. These results indicate that integration of counteracting signals from the Gi- and the G12/13-Rho pathways directs either positive or negative regulation of Rac, and thus cell migration, upon activation of a single S1P receptor isoform.     

Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK‐mediated MLC phosphorylation

The retinal pigment epithelium (RPE) forms the outer blood–retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial–mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR‐1 induces the time‐ and dose‐dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho‐MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y‐27632, whereas MLCK inhibitor ML‐7 and PLC‐β inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC‐β‐mediated calcium increase. Additionally, thrombin‐induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP₃/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin‐induced RPE cell transformation subsequent to BRB dysfunction. J. Cell. Physiol. 226: 414–423, 2011. © 2010 Wiley‐Liss, Inc.