Ko Momotani

The cAMP-responsive Rap1 Guanine Nucleotide Exchange Factor, Epac, Induces Smooth Muscle Relaxation by Down-regulation of RhoA Activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC20) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2′-O-Me-cAMP (“007”), significantly reduced agonist-induced contractile force, RLC20, and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI2 analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca2+ desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

p63RhoGEF Couples Gαq/11-Mediated Signaling to Ca2+ Sensitization of Vascular Smooth Muscle Contractility

Rationale: In normal and diseased vascular smooth muscle (SM), the RhoA pathway, which is activated by multiple agonists through G protein-coupled receptors (GPCRs), plays a central role in regulating basal tone and peripheral resistance. This occurs through inhibition of myosin light chain phosphatase, leading to increased phosphorylation of the myosin regulatory light chain. Although it is thought that specific agonists and GPCRs may couple to distinct RhoA guanine nucleotide exchange factors (GEFs), thus raising the possibility of selective targeting of specific GEFs for therapeutic use, this notion is largely unexplored for SM contraction. Objective: We examine whether p63RhoGEF, known to couple specifically to G&agr;q/11 in vitro, is functional in blood vessels as a mediator of RhoA activation and if it is selectively activated by G&agr;q/11 coupled agonists. Methods and Results: We find that p63RhoGEF is present across SM tissues and demonstrate that silencing of the endogenous p63RhoGEF in mouse portal vein inhibits contractile force induced by endothelin-1 to a greater extent than the predominantly G&agr;12/13-mediated thromboxane analog U46619. This is because endothelin-1 acts on G&agr;q/11 as well as G&agr;12/13. Introduction of the exogenous isolated pleckstrin-homology (PH) domain of p63RhoGEF (residues 331–580) into permeabilized rabbit portal vein inhibited Ca2+ sensitized force and activation of RhoA, when phenylephrine was used as an agonist. This reinforces the results based on endothelin-1, because phenylephrine is thought to act exclusively through G&agr;q/11. Conclusion: We demonstrate that p63RhoGEF selectively couples G&agr;q/11 but not G&agr;12/13, to RhoA activation in blood vessels and cultured cells and thus mediates the physiologically important Ca2+ sensitization of force induced with G&agr;q/11-coupled agonists. Our results suggest that signaling through p63RhoGEF provides a novel mechanism for selective regulation of blood pressure.

Thromboxane A2-induced Bi-directional Regulation of Cerebral Arterial Tone

Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces “buffered” vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated.

Cep57, a multidomain protein with unique microtubule and centrosomal localization domains.

The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to gamma-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.

Neurodevelopment in Children Born to Hypothyroid Mothers Restored to Normal Thyroxine (T4) Concentration by Late Pregnancy in Japan: No Apparent Influence of Maternal T4 Deficiency

CONTEXT The importance of maternal T₄ for brain development prior to the onset of fetal thyroid function has been suggested in basic studies, and a correlation between mild maternal T₄ deficiency in early gestation and disturbance of neurodevelopment in progenies has been shown in large case-control studies. These findings suggest that maternal T₄ deficiency in early pregnancy potentially affects neurointellectual development. On the other hand, no apparent adverse effect in children born to mothers with overt hypothyroidism in Japan has been reported where maternal T₄ had been restored to normal by late pregnancy. OBJECTIVE We report five cases in Japan showing no apparent effect of maternal T₄ deficiency on neurodevelopment in progenies where low T₄ levels had been corrected by late pregnancy. METHODS Five women with overt hypothyroidism detected at 6-16 wk gestation initiated T₄ treatment. Four women restored euthyroidism by the 20th week. One remained in a subclinical hypothyroid state. Developmental scores of their children were evaluated between 25 months and 11 yr of age by either the Tsumori-Inage Infants Developmental Test or the Wechsler Intelligence Scale for Children-Third Edition and compared to those of corresponding siblings with no exposure to maternal hypothyroidism. RESULTS The development scores of all the children turned out to be either normal or advanced. CONCLUSIONS In iodine-sufficient areas, maternal T₄ deficiency in early pregnancy does not necessarily affect neurodevelopment. Therefore, other potential factors altering neurodevelopment, such as iodine deficiency, must be investigated.

Rap1b in smooth muscle and endothelium is required for maintenance of vascular tone and normal blood pressure.

Objective—Small GTPase Ras-related protein 1 (Rap1b) controls several basic cellular phenomena, and its deletion in mice leads to several cardiovascular defects, including impaired adhesion of blood cells and defective angiogenesis. We found that Rap1b−/− mice develop cardiac hypertrophy and hypertension. Therefore, we examined the function of Rap1b in regulation of blood pressure. Approach and Results—Rap1b−/− mice developed cardiac hypertrophy and elevated blood pressure, but maintained a normal heart rate. Correcting elevated blood pressure with losartan, an angiotensin II type 1 receptor antagonist, alleviated cardiac hypertrophy in Rap1b−/− mice, suggesting a possibility that cardiac hypertrophy develops secondary to hypertension. The indices of renal function and plasma renin activity were normal in Rap1b−/− mice. Ex vivo, we examined whether the effect of Rap1b deletion on smooth muscle–mediated vessel contraction and endothelium-dependent vessel dilation, 2 major mechanisms controlling basal vascular tone, was the basis for the hypertension. We found increased contractility on stimulation with a thromboxane analog or angiotensin II or phenylephrine along with increased inhibitory phosphorylation of myosin phosphatase under basal conditions consistent with elevated basal tone and the observed hypertension. Cyclic adenosine monophosphate–dependent relaxation in response to Rap1 activator, Epac, was decreased in vessels from Rap1b−/− mice. Defective endothelial release of dilatory nitric oxide in response to elevated blood flow leads to hypertension. We found that nitric oxide–dependent vasodilation was significantly inhibited in Rap1b-deficient vessels. Conclusions—This is the first report to indicate that Rap1b in both smooth muscle and endothelium plays a key role in maintaining blood pressure by controlling normal vascular tone.

On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF

BackgroundThe Dbl-family of guanine nucleotide exchange factors (GEFs) activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology) domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like) domain, act as effectors of select GPCRs via Gα12/13, although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG).ResultsHere we show that the autoinhibition of PRG is caused largely by an interaction of a short negatively charged sequence motif, immediately upstream of the DH-domain and including residues Asp706, Glu708, Glu710 and Asp712, with a patch on the catalytic surface of the DH-domain including Arg867 and Arg868. In the absence of both PDZ and RGSL domains, the DH-PH tandem with additional 21 residues upstream, is 50% autoinhibited. However, within the full-length protein, the PDZ and/or RGSL domains significantly restore autoinhibition.ConclusionOur results suggest a mechanism for autoinhibition of RGSL family of GEFs, in which the RGSL domain and a unique sequence motif upstream of the DH domain, act cooperatively to reduce the ability of the DH domain to bind the nucleotide free RhoA. The activation mechanism is likely to involve two independent steps, i.e. displacement of the RGSL domain and conformational change involving the autoinhibitory sequence motif containing several negatively charged residues.

Ras-Related Protein 1 in Smooth Muscle and Endothelium Is Required for Maintenance of Vascular Tone and Normal Blood Pressure

Objective—Small GTPase Ras-related protein 1 (Rap1b) controls several basic cellular phenomena, and its deletion in mice leads to several cardiovascular defects, including impaired adhesion of blood cells and defective angiogenesis. We found that Rap1b−/− mice develop cardiac hypertrophy and hypertension. Therefore, we examined the function of Rap1b in regulation of blood pressure. Approach and Results—Rap1b−/− mice developed cardiac hypertrophy and elevated blood pressure, but maintained a normal heart rate. Correcting elevated blood pressure with losartan, an angiotensin II type 1 receptor antagonist, alleviated cardiac hypertrophy in Rap1b−/− mice, suggesting a possibility that cardiac hypertrophy develops secondary to hypertension. The indices of renal function and plasma renin activity were normal in Rap1b−/− mice. Ex vivo, we examined whether the effect of Rap1b deletion on smooth muscle–mediated vessel contraction and endothelium-dependent vessel dilation, 2 major mechanisms controlling basal vascular tone, was the basis for the hypertension. We found increased contractility on stimulation with a thromboxane analog or angiotensin II or phenylephrine along with increased inhibitory phosphorylation of myosin phosphatase under basal conditions consistent with elevated basal tone and the observed hypertension. Cyclic adenosine monophosphate–dependent relaxation in response to Rap1 activator, Epac, was decreased in vessels from Rap1b−/− mice. Defective endothelial release of dilatory nitric oxide in response to elevated blood flow leads to hypertension. We found that nitric oxide–dependent vasodilation was significantly inhibited in Rap1b-deficient vessels. Conclusions—This is the first report to indicate that Rap1b in both smooth muscle and endothelium plays a key role in maintaining blood pressure by controlling normal vascular tone.

Agonist-induced Ca2+ Sensitization in Smooth Muscle REDUNDANCY OF RHO GUANINE NUCLEOTIDE EXCHANGE FACTORS (RhoGEFs) AND RESPONSE KINETICS, A CAGED COMPOUND STUDY

Background: Multiple RhoGEFs regulate agonist-induced Ca2+-sensitized force. Results: PDZRhoGEF and LARG are functionally redundant, translocate to the cell membrane, and form hetero- and homodimers to mediate Gα12/13-dependent RhoA activation. Conclusion: Ca2+-sensitized force is induced by parallel signaling through RhoGEFs, which are rate-limiting due to their slow recruitment and activation. Significance: Signaling through RhoGEFs suggests new therapeutic targets for diseases of smooth muscle. Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca2+]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca2+ sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca2+-sensitized force are not well understood. Using permeabilized blood vessels from PRG(−/−) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca2+-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5′-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca2+ transient and phasic force components and the onset of Ca2+-sensitized force.

p63RhoGEF: A New Switch for Gq-Mediated Activation of Smooth Muscle

In normal and diseased vascular smooth muscle (SM), the RhoA pathway, which is activated by multiple agonists through G protein-coupled receptors (GPCRs), plays a central role in regulating basal tone and peripheral resistance. Multiple RhoA GTP exchange factors (GEFs) are expressed in SM, raising the possibility that specific agonists coupled to specific GPCRs may couple to distinct RhoGEFs and provide novel therapeutic targets. This review focuses on the function and mechanisms of activation of p63RhoGEF (Arhgef 25; GEFT) recently identified in SM and its possible role in selective targeting of RhoA-mediated regulation of basal blood pressure through agonists that couple through G(αq/11).