Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells.     

S1P₂ receptor regulation of sphingosine-1-phosphate effects on conventional outflow physiology

Elevated intraocular pressure is the main risk factor in primary open-angle glaucoma, involving an increased resistance to aqueous humor outflow in the juxtacanalicular region of the conventional outflow pathway which includes the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC). Previously, sphingosine-1-phosphate (S1P) was shown to decrease outflow facility in porcine and human eyes, thus increasing outflow resistance and intraocular pressure. Owing to S1P's known effect of increasing barrier function in endothelial cells and the robust expression of the S1P? receptor on the inner wall of SC, we hypothesized that S1P? receptor activation promotes junction formation and decreases outflow facility. The effects of subtype-specific S1P receptor compounds were tested in human and porcine whole-eye perfusions and human primary cultures of SC and TM cells to determine the receptor responsible for S1P effects on outflow resistance. The S1P?-specific agonist SEW2871 failed to both mimic S1P effects in paired human eye perfusions, as well as increase myosin light chain (MLC) phosphorylation in cell culture, a prominent outcome in S1P-treated SC and TM cells. In contrast, the S1P? antagonist JTE-013, but not the S1P? or S1P?,? antagonists, blocked the S1P-promoted increase in MLC phosphorylation. Moreover, JTE-013 prevented S1P-induced decrease in outflow facility in perfused human eyes (P < 0.05, n = 6 pairs). Similarly, porcine eyes perfused with JTE-013 + S1P did not differ from eyes with JTE-013 alone (P = 0.53, n = 3). These results demonstrate that S1P? , and not S1P? or S1P?, receptor activation increases conventional outflow resistance and is a potential target to regulate intraocular pressure.     

The role of calcium-independent phospholipase A2γ in modulation of aqueous humor drainage and Ca2+ sensitization of trabecular meshwork contraction

The contractile and relaxation characteristics of trabecular meshwork (TM) are presumed to influence aqueous humor (AH) drainage and intraocular pressure. The mechanisms underlying regulation of TM cell contractile properties, however, are not well understood. This study investigates the role of calcium-independent phospholipase A(2) (iPLA(2)), which controls eicosanoid synthesis, in regulation of TM cell contraction and AH outflow using mechanism-based isoform specific inhibitors (R)-bromoenol lactone (R-BEL, iPLA(2)γ specific) and (S)-bromoenol lactone (S-BEL, iPLA(2)β specific). Immunohistochemical analysis revealed intense staining for both iPLA(2)β and γ isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA(2)γ by R-BEL or small interfering RNA-mediated silencing of iPLA(2)γ expression induced dramatic changes in TM cell morphology, and decreased actin stress fibers, focal adhesions, and myosin light-chain (MLC) phosphorylation. AH outflow facility increased progressively and significantly in enucleated porcine eyes perfused with R-BEL. This response was associated with a significant decrease in TM tissue MLC phosphorylation and alterations in the morphology of aqueous plexi in R-BEL-perfused eyes. In contrast, S-BEL did not affect either of these parameters. Additionally, R-BEL-induced cellular relaxation of the TM was associated with a significant decrease in the levels of active Rho GTPase, phospho-MLC phosphatase, phospho-CPI-17, and arachidonic acid. Taken together, these observations demonstrate that iPLA(2)γ plays a significant and isoform-specific role in regulation of AH outflow facility by altering the contractile characteristics of the TM. The effects of iPLA(2)γ on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways.     

Rho-mediated cytoskeletal rearrangement in response to LPA is functionally antagonized by Rac1 and PIP2

G-protein-coupled receptors signal through Rho to induce actin cytoskeletal rearrangement. We previously demonstrated that thrombin stimulates Rho-dependent process retraction and rounding of 1321N1 astrocytoma cells. Surprisingly, while lysophosphatidic acid (LPA) activated RhoA in 1321N1 cells, it failed to produce cell rounding. Thrombin, unlike LPA, decreased Rac1 activity, and activated (GTPase-deficient) Rac1 inhibited thrombin-stimulated cell rounding, while expression of dominant-negative Rac1 promoted LPA-induced rounding. LPA and thrombin receptors appear to differ in coupling to Gi, as LPA but not thrombin-stimulated 1321N1 cell proliferation was pertussis toxin-sensitive. Blocking Gi with pertussis toxin enabled LPA to induce cell rounding and to decrease activated Rac1. These data support the hypothesis that Rac1 and Gi activation antagonize cell rounding. Thrombin and LPA receptors also differentially activated Gq pathways as thrombin but not LPA increased InsP3 formation and reduced phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Microinjection of the plekstrin homology domain of phospholipase C (PLC)delta1, which binds PIP2, enabled LPA to elicit cell rounding, consistent with a requirement for PIP2 reduction. We suggest that Rho-mediated cytoskeletal responses are enhanced by concomitant reductions in cellular levels of PIP2 and Rac1 activation and thus effected only by G-protein-coupled receptors with appropriate subsets of G protein activation.     

Aqueous humor dynamics in the enucleated deer eye

Facility of outflow of aqueous humor was determined in ten deer (Odocoileus virginianus) eyes enucleated within 1 hr post-mortem. Although both outflow facility and anterior chamber volume were much greater than values reported in the literature for a variety of other species, calculated values for aqueous humor turnover were within the reported range for other animals as well as for man. These results strengthen the hypothesis that the rate of turnover of aqueous humor is quite similar in eyes of vastly differing dimensions.     

Adenovirus conducted connective tissue growth factor on extracellular matrix in trabecular meshwork and its role on aqueous humor outflow facility

Deposition of extracellular matrix (ECM) in trabecular meshwork, such as fibronectin, collagen IV, elastin. leads to increased resistance of trabecular meshwork in primary open angle glaucoma (POAG). Connective tissue growth factor (CTGF) is known to regulate the ECM deposits. In this study, we detect the effect of adenovirus conducted CTGF (Adv-CTGF) transfection on either the expression of ECM components or aqueous humor outflow facility. Adv-CTGF was used to transfect rat trabecular meshwork cells in vivo and in vitro. Aqueous humor outflow facility was test by microbeads perfusion. Protein expression of CTGF, fibronectin, and collagen IV was determined using Western blot. In the Adv-CTGF group, the outflow facility displayed a significant decrease from baseline. It appears as though the transfection with Adv-CTGF significantly affects the aqueous humor outflow pattern. A negative correlation between IOP and PEFL indicated that a decrease in the area of bead deposition corresponded to an overall decrease of outflow, leading to an elevated IOP. Adv-CTGF can enhance the expression of CTGF, fibronectin and collagen IV. CTGF is the novel target for treatment of POAG. It is necessary to further study to test inhibition of CTGF expression for treatment of POAG.     

Antithrombin III, a serpin family protease inhibitor, is a major heparin binding protein in porcine aqueous humor

Our hypothesis is that the proteins in aqueous humor may be involved in the regulation of outflow facility through the trabecular meshwork and uveoscleral meshwork. In this study, we analyzed the profile of heparin-binding proteins present in porcine aqueous humor to identify and characterize secretory proteins with a binding affinity for heparin. A single step involving heparin-sepharose affinity chromatography of porcine aqueous humor yielded a approximately 60 kDa protein as the major heparin-binding species. This protein was specifically eluted from the column by heparin. The N-terminal sequence and immunological cross reactivity of this protein confirmed its identity as antithrombin III. Aqueous humor from different species, as well as cells from human trabecular meshwork, Schlemm's canal, and lens epithelium, contained detectable amounts of antithrombin III. Based on its known anticoagulative function in endothelial cells and effects on the production of prostacyclin, it is reasonable to speculate that antithrombin III present in aqueous humor might influence the physiology of the trabecular and uveoscleral meshwork and thereby regulate intraocular pressure.     

Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue

Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.     

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance

Transforming growth factor-beta 2 (TGF-β2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-β2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-β2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-β2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-β2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm’s canal (MSC) cell monolayers was decreased by TGF-β2 treatment. SAHA inhibited the effects of TGF-β2 on the permeability of these cells. TGF-β2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-β2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-β2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-β signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-β. These results imply that SAHA prevents TGF-β2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-β signaling in TM and MSC cells.     

Essential role of type I(alpha) phosphatidylinositol 4-phosphate 5-kinase in neurite remodeling

Rapid neurite remodeling is fundamental to nervous system development and plasticity and is regulated by Rho family GTPases that signal f-actin reorganization in response to various receptor ligands. Neuronal N1E-115 cells show dramatic neurite retraction and cell rounding in response to serum factors such as lysophosphatidic acid (LPA), sphingosine-1 phosphate (S1P), and thrombin, due to activation of the RhoA-Rho kinase pathway. Type I phosphatidylinositol 4-phosphate 5-kinases (PIPkinase), which regulate cellular levels of PtdIns(4,5)P(2), have been suggested as targets of the RhoA-Rho kinase pathway able to modulate cytoskeletal dynamics. Here, we show that the introduction of Type Ialpha PIPkinase into N1E-115 cells leads to cell rounding and complete inhibition of neurite outgrowth, perhaps through the dissociation of vinculin and the destabilization of focal adhesions. This occurs independently of RhoA, Rho kinase, and the activation of actomyosin contraction. Strikingly, expression of kinase-dead PIPkinase promotes the outgrowth of neurites, which fail to retract in response to LPA, S1P, thrombin, or active RhoA. Moreover, neurite retraction in response to an endogenous neuronal guidance cue, Semaphorin3A, was also dependent on Type Ialpha PIPkinase. Our results suggest an essential role for a Type I PIPkinase during neurite retraction in response to a number of diverse stimuli.     

Role of the autotaxin-lysophosphatidic acid axis in glaucoma,aqueous humor drainage and fibrogenic activity

Ocular hypertension due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM) is a major risk factor for glaucoma, a leading cause of irreversible blindness. However, the etiology of ocular hypertension remains unclear. Although autotaxin, a secreted lysophospholipase D and its catalytic product lysophosphatidic acid (LPA) have been shown to modulate AH drainage through TM, we do not have a complete understanding of their role and regulation in glaucoma patients, TM and AH outflow. This study reports a significant increase in the levels of autotaxin, lysophosphatidylcholine (LPC), LPA and connective tissue growth factor (CTGF) in the AH of Caucasian and African American open angle glaucoma patients relative to age-matched non-glaucoma patients. Treatment of human TM cells with dexamethasone, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) increased the levels of autotaxin protein, a response that was mitigated by inhibitors of glucocorticoid receptor, NF-kB and SMAD3. Dexamethasone, TNF-α, IL-1β and LPC treatment of TM cells also led to an increase in the levels of CTGF, fibronectin and collagen type 1 in an autotaxin dependent manner. Additionally, in perfused enucleated mouse eyes, autotaxin and LPC were noted to decrease, while inhibition of autotaxin was increased aqueous outflow through the TM. Taken together, these results provide additional evidence for dysregulation of the autotaxin-LPA axis in the AH of glaucoma patients, reveal molecular insights into the regulation of autotaxin expression in TM cells and the consequences of autotaxin inhibitors in suppressing the fibrogenic response and resistance to AH outflow through the TM.     

Rho/ROCK and Cdk5 effects on phosphorylation of a beta-thymosin repeat protein in Hermissenda

Rho GTPases acting through effector proteins regulate actin dynamics and cytoskeletal structure. In Hermissenda Csp24 is a cytoskeletal-related protein that contributes to the development of intermediate-term memory, and is homologous to other beta-thymosin-like repeat proteins containing multiple actin-binding domains. We have examined the role of Rho GTPase activity and its downstream target ROCK, and cyclin-dependent kinase 5 (Cdk5) on the phosphorylation of Csp24 using 32PO4 labeling of proteins separated with 2-D PAGE. The ROCK inhibitor Y-27632 significantly increased Csp24 phosphorylation, and the Rho activator lysophosphatidic acid (LPA) or the Cdk5 inhibitor butyrolactone significantly decreased Csp24 phosphorylation. Pretreatment with Y-27632 before LPA application significantly reduced the decreased phosphorylation of Csp24 normally detected in nervous systems exposed to LPA. Using a pull-down assay we found that LPA treatments activated Rho and exposure to 5-HT decreased Rho activity. Our results indicate that the Rho/ROCK and Cdk5 signaling pathways contribute to the regulation of Csp24 phosphorylation.     

Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms

Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr(696) and Thr(850) resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly.     

Central involvement of Rho family GTPases in TNF-alpha-mediated bovine pulmonary endothelial cell apoptosis

In our recent studies, we defined a critical role for increased levels of myosin light chain (MLC) phosphorylation, a regulatory event in the interaction between actin and myosin in TNF-alpha-induced pulmonary endothelial cell actomyosin rearrangement and apoptosis. The Rho GTPase effector, Rho kinase is an important signaling effector governing levels of MLC phosphorylation which contributes to plasma membrane blebbing in several models of apoptosis. In this study, we directly assessed the role of Rho kinase in TNF-alpha-induced endothelial cell microfilament rearrangement and apoptosis. Inhibition of RhoA GTPase activity by the overexpression of dominant negative RhoA attenuates TNF-alpha-triggered stress fiber formation, consistent with Rho activation as a key event in TNF-alpha-induced cytoskeletal rearrangement. Furthermore, pharmacologic inhibition of Rho kinase as well as dominant negative RhoA overexpression dramatically reduced TNF-alpha-induced bovine endothelial apoptosis reflected by nucleosomal fragmentation as well as caspase 7, 3, and 8 activation. These results indicate that Rho kinase-dependent cytoskeletal rearrangement is critical for early apoptotic events, possibly in the assembly of the death-inducing signaling complex leading to initiator and effector caspase activation, and suggest a novel role for Rho GTPases in endothelial cell apoptosis.     

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.     

Ligand density and integrin repertoire regulate cellular response to LPA

Engagement of integrin receptors by the extracellular matrix (ECM) protein fibronectin (FN) activates intracellular signaling, cytoskeletal reorganization and cellular tension. The soluble factor lysophosphatidic acid (LPA) acts through Rho GTPase and its effector Rho kinase (ROCK) to enhance α5β1 integrin-mediated cell spreading on the Arg-Gly-Asp (RGD) cell-binding domain of FN. A second cell-binding site for α4 integrins resides in the CS1 segment of the alternatively spliced V region of FN. We show here that LPA treatment of α4β1-expressing CHOα4 cells on FN induced a significant decrease in spread cell area. LPA also decreased apoptosis induced by serum-deprivation in CHOα4 and human A375 melanoma cells in an α4β1-dependent manner. Improvement in cell viability and changes in cell morphology were dependent on ROCK and on the number of substrate binding sites for α4β1. LPA signaling combined with α4β1-mediated adhesion appears to sustain cell viability in situations where FN matrix is limiting. Such cooperation may impact dynamic cellular events such as wound healing, fibrosis, and metastasis.     

Differential P1-purinergic modulation of human Schlemm's canal inner-wall cells

Intraocular pressure is directly dependent on aqueous humor flow into, and resistance to flow out of, the eye. Adenosine has complex effects on intraocular pressure. Stimulation of A₁ and A_2A adenosine receptors changes intraocular pressure oppositely, likely through opposing actions on the outflow of aqueous humor. While the cellular sites regulating outflow resistance are unknown, the cells lining the inner wall of Schlemm's canal (SC) are a likely regulatory site. We applied selective adenosine receptor agonists to SC cells in vitro to compare the responses to A₁ and A_2A stimulation. Parallel studies were conducted with human inner-wall SC cells isolated by a novel enzyme-assisted technique and with cannula-derived mixed inner- and outer-wall SC cells. A₁ agonists increased whole cell currents of both inner-wall and cannula-derived SC cells. An A_2A agonist reduced currents most consistently in specifically inner-wall SC cells. Those currents were also increased by A_2B, but not consistently affected by A₃, stimulation. A₁, A_2A, and A₃ agonists all increased SC-cell intracellular Ca²⁺. The electrophysiological results are consistent with the possibility that inner-wall SC cells may mediate the previously reported modulatory effects of adenosine on outflow resistance. The results are also consistent with the presence of functional A_2B, as well as A₁, A_2A, and A₃ adenosine receptors in SC cells. intraocular pressure; aqueous humor outflow; ion transport; adenosine agonists     

Rho-family GTPases modulate Ca(2+) -dependent ATP release from astrocytes

Previously, we reported that activation of G protein-coupled receptors (GPCR) in 1321N1 human astrocytoma cells elicits a rapid release of ATP that is partially dependent on a G(q)/phophospholipase C (PLC)/Ca(2+) mobilization signaling cascade. In this study we assessed the role of Rho-family GTPase signaling as an additional pathway for the regulation of ATP release in response to activation of protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca(2+) mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme, which inhibit Rho-GTPases, attenuated the thrombin- and LPA-stimulated ATP release but did not decrease carbachol-stimulated release. Thus the ability of certain G(q)-coupled receptors to additionally stimulate Rho-GTPases acts to strongly potentiate a Ca(2+)-activated ATP release pathway. However, pharmacological inhibition of Rho kinase I/II or myosin light chain kinase did not attenuate ATP release. PAR1-induced ATP release was also reduced twofold by brefeldin treatment suggesting the possible mobilization of Golgi-derived, ATP-containing secretory vesicles. ATP release was also markedly repressed by the gap junction channel inhibitor carbenoxolone in the absence of any obvious thrombin-induced change in membrane permeability indicative of hemichannel gating.     

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.