Hitoshi Kurose

TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy.

Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II‐induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca2+‐responsive transcriptional factor. It is believed that phospholipase C (PLC)‐mediated production of inositol‐1,4,5‐trisphosphate (IP3) is responsible for Ca2+ increase that is necessary for NFAT activation. However, we demonstrate that PLC‐mediated production of diacylglycerol (DAG) but not IP3 is essential for Ang II‐induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca2+ oscillation triggered by membrane depolarization through activation of DAG‐sensitive TRPC channels, which leads to activation of L‐type Ca2+ channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG‐sensitive TRPC‐like currents. Among DAG‐activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II‐induced NFAT activation and hypertrophic responses. These data suggest that DAG‐induced Ca2+ signaling pathway through TRPC3 and TRPC6 is essential for Ang II‐induced NFAT activation and cardiac hypertrophy.

Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound

Canonical transient receptor potential (TRPC) channels control influxes of Ca2+ and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca2+ influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca2+ response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca2+-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.

G|[alpha]|i and G|[alpha]|o are target proteins ofreactive oxygen species

Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events. In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury and in cytokine-stimulated hypertrophy. Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS; however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the βγ-subunit of G protein (Gβγ) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. The Gβγ-responsive ERK activation induced by H2O2 is independent of ligands binding to Gi-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H 2O2 increases [35S]GTP-γS binding to cardiac membranes and directly activates purified heterotrimeric Gi and Go but not Gs. Analysis using heterotrimeric G o and its individual subunits indicates that H2O2 modifies Gαo but not Gβγ, which leads to subunit dissociation. We conclude that Gαi and Gαo are critical targets of oxidative stress for activation of ERK.

P2Y6 receptor‐Gα12/13 signalling in cardiomyocytes triggers pressure overload‐induced cardiac fibrosis

Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)‐β. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (Gα12/13) in cardiomyocytes suppressed pressure overload‐induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF‐β, connective tissue growth factor, and periostin) and Ang‐converting enzyme (ACE) was suppressed by the functional inhibition of Gα12/13. The expression of these fibrogenic genes through Gα12/13 by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G‐protein‐coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of Gα12/13 in cardiomyocytes by the extracellular nucleotides‐stimulated P2Y6 receptor triggers fibrosis in pressure overload‐induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF‐β.

Inhibition of TRPC6 Channel Activity Contributes to the Antihypertrophic Effects of Natriuretic Peptides-Guanylyl Cyclase-A Signaling in the Heart

Rationale: Atrial and brain natriuretic peptides (ANP and BNP, respectively) exert antihypertrophic effects in the heart via their common receptor, guanylyl cyclase (GC)-A, which catalyzes the synthesis of cGMP, leading to activation of protein kinase (PK)G. Still, much of the network of molecular mediators via which ANP/BNP-GC-A signaling inhibit cardiac hypertrophy remains to be characterized. Objective: We investigated the effect of ANP-GC-A signaling on transient receptor potential subfamily C (TRPC)6, a receptor-operated Ca2+ channel known to positively regulate prohypertrophic calcineurin–nuclear factor of activated T cells (NFAT) signaling. Methods and Results: In cardiac myocytes, ANP induced phosphorylation of TRPC6 at threonine 69, the PKG phosphorylation site, and significantly inhibited agonist-evoked NFAT activation and Ca2+ influx, whereas in HEK293 cells, it dramatically inhibited agonist-evoked TRPC6 channel activity. These inhibitory effects of ANP were abolished in the presence of specific PKG inhibitors or by substituting an alanine for threonine 69 in TRPC6. In model mice lacking GC-A, the calcineurin-NFAT pathway is constitutively activated, and BTP2, a selective TRPC channel blocker, significantly attenuated the cardiac hypertrophy otherwise seen. Conversely, overexpression of TRPC6 in mice lacking GC-A exacerbated cardiac hypertrophy. BTP2 also significantly inhibited angiotensin II–induced cardiac hypertrophy in mice. Conclusions: Collectively, these findings suggest that TRPC6 is a critical target of antihypertrophic effects elicited via the cardiac ANP/BNP-GC-A pathway and suggest TRPC6 blockade could be an effective therapeutic strategy for preventing pathological cardiac remodeling.

Gα12/13-mediated Up-regulation of TRPC6 Negatively Regulates Endothelin-1-induced Cardiac Myofibroblast Formation and Collagen Synthesis through Nuclear Factor of Activated T Cells Activation

Sustained elevation of [Ca2+]i has been implicated in many cellular events. We previously reported that α subunits of G12 family G proteins (Gα12/13) participate in sustained Ca2+ influx required for the activation of nuclear factor of activated T cells (NFAT), a Ca2+-responsive transcriptional factor, in rat neonatal cardiac fibroblasts. Here, we demonstrate that Gα12/13-mediated up-regulation of canonical transient receptor potential 6 (TRPC6) channels participates in sustained Ca2+ influx and NFAT activation by endothelin (ET)-1 treatment. Expression of constitutively active Gα12 or Gα13 increased the expression of TRPC6 proteins and basal Ca2+ influx activity. The treatment with ET-1 increased TRPC6 protein levels through Gα12/13, reactive oxygen species, and c-Jun N-terminal kinase (JNK)-dependent pathways. NFAT is activated by sustained increase in [Ca2+]i through up-regulated TRPC6. A Gα12/13-inhibitory polypeptide derived from the regulator of the G-protein signaling domain of p115-Rho guanine nucleotide exchange factor and a JNK inhibitor, SP600125, suppressed the ET-1-induced increase in expression of marker proteins of myofibroblast formation through a Gα12/13-reactive oxygen species-JNK pathway. The ET-1-induced myofibroblast formation was suppressed by overexpression of TRPC6 and CA NFAT, whereas it was enhanced by TRPC6 small interfering RNAs and cyclosporine A. These results suggest two opposite roles of Gα12/13 in cardiac fibroblasts. First, Gα12/13 mediate ET-1-induced myofibroblast formation. Second, Gα12/13 mediate TRPC6 up-regulation and NFAT activation that negatively regulates ET-1-induced myofibroblast formation. Furthermore, TRPC6 mediates hypertrophic responses in cardiac myocytes but suppresses fibrotic responses in cardiac fibroblasts. Thus, TRPC6 mediates opposite responses in cardiac myocytes and fibroblasts.

Sphingosine 1-Phosphate Induces Membrane Ruffling and Increases Motility of Human Umbilical Vein Endothelial Cells via Vascular Endothelial Growth Factor Receptor and CrkII

Sphingosine 1-phosphate (S1P), a ligand for endothelial differentiation gene family proteins, is one of the most potent signal mediators released from activated platelets. Here, we report that S1P induces membrane ruffling of human umbilical vein endothelial cells (HUVECs) via the vascular endothelial growth factor receptor (VEGFR), Src family tyrosine kinase(s), and the CrkII adaptor protein. S1P induced prominent phosphorylation of CrkII in HUVECs, indicating that CrkII was involved in the S1P-induced signaling pathway. S1P-induced CrkII phosphorylation was blocked by pertussis toxin and overexpression of the carboxyl terminus of β-adrenergic receptor kinase, indicating that the βγ subunit of Gi was required for the phosphorylation. Notably, the S1P-induced CrkII phosphorylation was also abolished by inhibitors of VEGFR or Src family tyrosine kinases. By using Picchu, a real time monitoring protein for CrkII phosphorylation, we found that S1P induced rapid CrkII phosphorylation at membrane ruffles. Finally, we observed that expression of a dominant negative mutant of CrkII inhibited the S1P-induced membrane ruffling and cell migration. These results delineated a novel S1P signaling pathway that involves sequential activation of Gi-coupled receptor(s), VEGFR, Src family tyrosine kinase(s), and the CrkII adaptor protein, and which is responsible for both the induction of membrane ruffling and the increase in cell motility.

Gα12/13 mediates α1-Adrenergic receptor-induced cardiac hypertrophy

In neonatal cardiomyocytes, activation of the G(q)-coupled alpha(1)-adrenergic receptor (alpha(1)AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH(2)-terminal kinase (JNK). Here, we show that JNK activation is essential for alpha(1)AR-induced hypertrophy, in that alpha(1)AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate alpha(1)AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. alpha(1)AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of Galpha(q), Galpha(12), and Galpha(13). JNK activation was also inhibited by the Galpha(q/11)- or Galpha(12/13)-specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2, a polypeptide that sequesters Gbetagamma. alpha(1)AR-induced hypertrophic responses were inhibited by Galpha(q/11)- and Galpha(12/13)-specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein-coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of Galpha(12) and Galpha(13) but not by Galpha(q). Our findings suggest that alpha(1)AR-induced hypertrophic responses are mediated in part by a Galpha(12/13)-Rho-JNK pathway, in part by a G(q/11)-JNK pathway that is Rho independent, and in part by a Gbetagamma pathway that is JNK independent.

Gαi and Gαo are target proteins of reactive oxygen species

Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events. In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury and in cytokine-stimulated hypertrophy. Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS; however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the βγ-subunit of G protein (Gβγ) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. The Gβγ-responsive ERK activation induced by H2O2 is independent of ligands binding to Gi-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H 2O2 increases [35S]GTP-γS binding to cardiac membranes and directly activates purified heterotrimeric Gi and Go but not Gs. Analysis using heterotrimeric G o and its individual subunits indicates that H2O2 modifies Gαo but not Gβγ, which leads to subunit dissociation. We conclude that Gαi and Gαo are critical targets of oxidative stress for activation of ERK.

Gi/o Protein-Dependent and -Independent Actions of Pertussis Toxin (PTX)

Pertussis toxin (PTX) is a typical A-B toxin. The A-protomer (S1 subunit) exhibits ADP-ribosyltransferase activity. The B-oligomer consists of four subunits (S2 to S5) and binds extracellular molecules that allow the toxin to enter the cells. The A-protomer ADP-ribosylates the α subunits of heterotrimeric Gi/o proteins, resulting in the receptors being uncoupled from the Gi/o proteins. The B-oligomer binds proteins expressed on the cell surface, such as Toll-like receptor 4, and activates an intracellular signal transduction cascade. Thus, PTX modifies cellular responses by at least two different signaling pathways; ADP-ribosylation of the Gαi/o proteins by the A-protomer (Gi/o protein-dependent action) and the interaction of the B-oligomer with cell surface proteins (Gi/o protein-independent action).