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The prolonged overstimulation of β-adrenergic receptors (β-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with β-AR antagonists (β-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of β-ARs involved in the pathogenesis of HF. As β-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), β-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of β-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for β-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of β-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of β-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

It is believed that G-protein-coupled receptors are the most promising target proteins for drug research. Theβ2-adrenergic receptor has been analyzed extensively in terms of its structure, regulation and signaling as a prototype G-protein-coupled receptor. Recent development of a recombinantβ2-adrenergic receptor reveals that the receptor exists in different conformations in addition to the resting and active forms. Furthermore, different conformations that are important for coupling with G-proteins are induced in a ligand-specific manner. After the receptor binds an agonist, the receptor is phosphorylated by G-protein-coupled receptor kinase or second messenger-dependent kinase.β-Arrestin is a cytosolic protein that binds to receptors phosphorylated by G-protein-coupled receptor kinase, but not those phosphorylated by second messenger-dependent kinase, and plays important roles in agonist-induced desensitization. However,β-arrestin has recently been recognized as a scaffold protein that helps to achieve efficient transmission of internalization and signaling. Receptor agonists can be divided into two groups: full and partial agonists. Partial agonists have an advantage over full agonists because desensitization is less likely to be induced following stimulation with a partial agonist. However, the effects of partial agonists onβ-arrestin-mediated signaling and internalization have not been fully examined so far. Information regarding the binding sites of partial agonists is still not sufficient to explain why they cannot fully activate the receptor. In the present study, recent progress regarding the structure, regulation and signaling ofβ2 adrenergic receptors and about differences between full and partial agonists will be summarized. [ABSTRACT FROM AUTHOR]

It is generally thought that Gα12 and Gα13-induced responses are exclusively mediated by small G protein Rho. However, Gα12 and Gα13 elicit divergent cellular responses: phospholipase C-ϵ activation, phospholipase D activation, cytoskeletal change, oncogenic response, apoptosis, MAP kinase activation and Na/H-exchange activation. In addition to Rho activation through RhoGEF, it has been recently demonstrated that Gα12 and Gα13 interact with several proteins and regulate their activities. However, physiological importance of the interaction of Gα12 and Gα13 with these proteins has not fully established. I summarize the recent progress of Gα12 and Gα13-mediated signaling cascade. [Copyright &y& Elsevier]

Cardiac fibrosis is the excess deposition of extracellular matrix (ECM), such as collagen. Myofibroblasts are major players in the production of collagen, and are differentiated primarily from resident fibroblasts. Collagen can compensate for the dead cells produced by injury. The appropriate production of collagen is beneficial for preserving the structural integrity of the heart, and protects the heart from cardiac rupture. However, excessive deposition of collagen causes cardiac dysfunction. Recent studies have demonstrated that myofibroblasts can change their phenotypes. In addition, myofibroblasts are found to have functions other than ECM production. Myofibroblasts have macrophage-like functions, in which they engulf dead cells and secrete anti-inflammatory cytokines. Research into fibroblasts has been delayed due to the lack of selective markers for the identification of fibroblasts. In recent years, it has become possible to genetically label fibroblasts and perform sequencing at single-cell levels. Based on new technologies, the origins of fibroblasts and myofibroblasts, time-dependent changes in fibroblast states after injury, and fibroblast heterogeneity have been demonstrated. In this paper, recent advances in fibroblast and myofibroblast research are reviewed. [ABSTRACT FROM AUTHOR]

Myocardial infarction is one of the major causes of death worldwide. Many heart cells die during myocardial infarction through various processes such as necrosis, apoptosis, necroptosis, autophagy-related cell death, pyroptosis and ferroptosis. These dead cells in infarcted hearts expose the so-called 'eat-me' signals, such as phosphatidylserine, on their surfaces, enhancing their removal by professional and non-professional phagocytes. Clearance of dead cells by phagocytes in the diseased hearts plays a crucial role in the pathology of myocardial infarction by inhibiting the inflammatory responses caused by the leakage of contents from dead cells. This review focuses on the rapidly growing understanding of the molecular mechanisms of dead cell phagocytosis, termed efferocytosis, during myocardial infarction, which contributes to the pathophysiology of myocardial infarction. [ABSTRACT FROM AUTHOR]

Current drugs for treating heart failure (HF), for example, angiotensin II receptor blockers and β-blockers, possess specific target molecules involved in the regulation of the cardiac circulatory system. However, most clinically approved drugs are effective in the treatment of HF with reduced ejection fraction (HFrEF). Novel drug classes, including angiotensin receptor blocker/neprilysin inhibitor (ARNI), sodium-glucose co-transporter-2 (SGLT2) inhibitor, hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, soluble guanylyl cyclase (sGC) stimulator/activator, and cardiac myosin activator, have recently been introduced for HF intervention based on their proposed novel mechanisms. SGLT2 inhibitors have been shown to be effective not only for HFrEF but also for HF with preserved ejection fraction (HFpEF). In the myocardium, excess cyclic adenosine monophosphate (cAMP) stimulation has detrimental effects on HFrEF, whereas cyclic guanosine monophosphate (cGMP) signaling inhibits cAMP-mediated responses. Thus, molecules participating in cGMP signaling are promising targets of novel drugs for HF. In this review, we summarize molecular pathways of cGMP signaling and clinical trials of emerging drug classes targeting cGMP signaling in the treatment of HF. [ABSTRACT FROM AUTHOR]

Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y6 receptor (P2Y6R) is a pro-inflammatory Gq/G12 family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver. [ABSTRACT FROM AUTHOR]

Retarded revascularization after progressive occlusion of large conductance arteries is a major cause of bad prognosis for peripheral artery disease (PAD). However, pharmacological treatment for PAD is still limited. We previously reported that suppression of transient receptor potential canonical (TRPC) 6 channel activity in vascular smooth muscle cells (VSMCs) facilitates VSMC differentiation without affecting proliferation and migration. In this study, we found that 1-benzilpiperadine derivative (1-BP), a selective inhibitor for TRPC3 and TRPC6 channel activities, induced VSMC differentiation. 1-BP-treated mice showed increased capillary arterialization and improvement of peripheral circulation and skeletal muscle mass after hind-limb ischemia (HLI) in mice. 1-BP had no additive effect on the facilitation of blood flow recovery after HLI in TRPC6-deficient mice, suggesting that suppression of TRPC6 underlies facilitation of the blood flow recovery by 1-BP. 1-BP also improved vascular nitric oxide bioavailability and blood flow recovery after HLI in hypercholesterolemic mice with endothelial dysfunction, suggesting the retrograde interaction from VSMCs to endothelium. These results suggest that 1-BP becomes a potential seed for PAD treatments that target vascular TRPC6 channels. [ABSTRACT FROM AUTHOR]

Fibrosis is attributed to excess deposition of extracellular matrix (ECM) proteins including collagen and is associated with various organ dysfunction. This excessive ECM is produced by myofibroblasts, which are differentiated from various cells by a variety of stimuli, represented by TGF-β. However, molecular mechanisms for the regulation of ECM production in myofibroblasts remain obscure. In this study, we demonstrate that the expression of drebrin, which binds to and increases the stability of actin filament in neurons, is increased in mouse hearts and lungs upon fibrosis. Drebrin is mainly expressed in myofibroblasts in the fibrotic hearts and lungs and promotes the expression of fibrosis-related genes, such as Acta2 and Col1a1. Taken together, our study identifies drebrin as a molecule that promotes the production of fibrosis-related genes in myofibroblasts. • Dbn1 expression is increased in infarcted mouse hearts. • Dbn1 is predominantly expressed in myofibroblasts in infarcted mouse hearts. • Knockdown of Dbn1 attenuates fibrosis-related gene production in myofibroblasts. • Dbn1 induced during differentiation of lung myofibroblasts promotes lung fibrosis. [ABSTRACT FROM AUTHOR]

Leukotriene B4 receptor 1 (BLT1), a high‐affinity G‐protein‐coupled receptor for leukotriene B4 (LTB4), is expressed on various inflammatory cells and plays critical roles in several inflammatory diseases. In myocardial infarction (MI), various inflammatory cells are known to be recruited to the infarcted area, but the function of BLT1 in MI is poorly understood. Here, we investigated the role of BLT1 in MI and the therapeutic effect of a BLT1 antagonist, ONO‐4057, on MI. Mice with infarcted hearts showed increased BLT1 expression and LTB4 levels. BLT1‐knockout mice with infarcted hearts exhibited attenuated leukocyte infiltration, proinflammatory cytokine production, and cell death, which led to reduced mortality and improved cardiac function after MI. Bone‐marrow transplantation studies showed that BLT1 expressed on bone marrow‐derived cells was responsible for the exacerbation of inflammation in infarcted hearts. Furthermore, ONO‐4057 administration attenuated the inflammatory responses in hearts surgically treated for MI, which resulted in reduced mortality and improved cardiac function after MI. Our study demonstrated that BLT1 contributes to excessive inflammation after MI and could represent a new therapeutic target for MI. [ABSTRACT FROM AUTHOR]

We propose a new approach of an indirect enzyme-linked immunosorbent assay (ELISA) for determination of D-glutamic acid (D-Glu) using a monoclonal antibody against D-glutamic acid (D-Glu-MAb), which recognizes D-Glu-glutaraldehyde (GA) molecule but not D-Glu molecule. Human serum albumin (HSA) was coated on an immunoplate and reacted with D-Glu via GA to produce D-Glu-GA-HSA conjugatesin situin the well to be recognized by D-Glu-MAb, which enabled the development of an indirect ELISA for the determination of free D-Glu. In this indirect ELISA, D-Glu can be specifically detected with limit of detection of 7.81μg/mL. Since anti-conjugate antibodies are often produced, even though anti-hapten antibodies are desired, this new approach could be very useful as an application of anti-conjugate antibodies to the development of quantitative analysis for detecting hapten. [ABSTRACT FROM PUBLISHER]

G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions. [ABSTRACT FROM AUTHOR]

G protein-coupled receptor kinases (GRKs) comprise a family of seven serine/threonine kinases that phosphorylate agonist-activated G protein-coupled receptors (GPCRs). It has recently been reported that GRKs regulate GPCR-independent signaling through the phosphorylation of intracellular proteins. To date, several intracellular substrates for GRK2 and GRK5 have been reported. However, those for GRK6 are poorly understood. Here we identified IκBα, a negative regulator of NF-κB signaling, as a substrate for GRK6. GRK6 directly phosphorylated IκBα at Ser 32 /Ser 36 , and the kinase activity of GRK6 was required for the promotion of NF-κB signaling after TNF-α stimulation. Knockout of GRK6 in peritoneal macrophages remarkably attenuated the transcription of inflammatory genes after TNF-α stimulation. In addition, we developed a bioluminescence resonance energy transfer (BRET) probe to monitor GRK6 activity. Using this probe, we revealed that the conformational change of GRK6 was induced by TNF-α. In summary, our study demonstrates that TNF-α induces GRK6 activation, and GRK6 promotes inflammatory responses through the phosphorylation of IκBα. [ABSTRACT FROM AUTHOR]

Prokineticins are angiogenic hormones that activate two G protein-coupled receptors: PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. Identification of non-peptide PKR1 agonists that contribute to myocardial repair and collateral vessel growth hold promises for treatment of heart diseases. Through a combination of in silico studies, medicinal chemistry, and pharmacological profiling approaches, we designed, synthesized, and characterized the first PKR1 agonists, demonstrating their cardioprotective activity against myocardial infarction (MI) in mice. Based on high throughput docking protocol, 250,000 compounds were computationally screened for putative PKR1 agonistic activity, using a homology model, and 10 virtual hits were pharmacologically evaluated. One hit internalizes PKR1, increases calcium release and activates ERK and Akt kinases. Among the 30 derivatives of the hit compound, the most potent derivative, IS20, was confirmed for its selectivity and specificity through genetic gain- and loss-of-function of PKR1. Importantly, IS20 prevented cardiac lesion formation and improved cardiac function after MI in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis. The preclinical investigation of the first PKR1 agonists provides a novel approach to promote cardiac neovasculogenesis after MI. [ABSTRACT FROM AUTHOR]

Most of the functions of D-amino acids (D-AA) remain unclear because of little analytic methods for specific detection/determination. In this study, a highly specific monoclonal antibody to D-glutamic acid (D-Glu-MAb) was produced using a hybridoma method. Characterization of D-Glu-MAb by indirect enzyme-linked immunosorbent assay (ELISA) revealed that it has high selectivity against D-Glu-glutaraldehyde (GA) conjugates, while no cross-reaction was observed when 38 other kinds of AA-GA conjugates were used. Moreover, subsequent indirect competitive ELISA disclosed that an epitope of D-Glu-MAb is a D-Glu-GA molecule in the conjugates, suggesting that D-Glu-MAb could be a useful tool to investigate the functional analysis of D-Glu in immunostaining. [ABSTRACT FROM AUTHOR]

Hyperactivation of Gq signaling causes cardiac hypertrophy, and β-adrenergic receptor-mediated Gs signaling is attenuated in hypertrophic cardiomyocytes. Here, we found the increase in a global ubiquitination in hypertrophic mouse heart. The activation of Gq signaling resulted in the ubiquitination of Gαs in neonatal rat cardiomyocytes, reduced Gαs expression, and suppressed cAMP response to β-adrenergic receptor stimulation. Ectopic expression of Gαq induced a similar suppression, which is due to the degradation of Gαs by a ubiquitin-proteasome pathway. Co-expression of Ric-8B, a positive regulator of Gαs, effectively canceled the Gαq-induced ubiquitination of Gαs and recovered the cAMP accumulation. In vitro, Gαq competes for the binding of Gαs to Ric-8B. These data show a new role of Ric-8B in the crosstalk of two distinct G protein signaling pathways, which are possibly involved in a part of mechanisms of chronic heart failure. [ABSTRACT FROM AUTHOR]

Beta-arrestins (β-arrestin1 and β-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, β-arrestins have been found to work as adaptor proteins for intracellular signaling pathways. β-arrestin1 and β-arrestin2 are expressed in the heart and are reported to participate in normal cardiac function. However, the physiological and pathological roles of β-arrestin1/2 in myocardial infarction (MI) have not been examined. Here, we demonstrate that β-arrestin2 negatively regulates inflammatory responses of macrophages recruited to the infarct area. β-arrestin2 knockout (KO) mice have higher mortality than wild-type (WT) mice after MI. In infarcted hearts, β-arrestin2 was strongly expressed in infiltrated macrophages. The production of inflammatory cytokines was enhanced in β-arrestin2 KO mice. In addition, p65 phosphorylation in the macrophages from the infarcted hearts of β-arrestin2 KO mice was increased in comparison to that of WT mice. These results suggest that the infiltrated macrophages of β-arrestin2 KO mice induce excessive inflammation at the infarct area. Furthermore, the inflammation in WT mice transplanted with bone marrow cells of β-arrestin2 KO mice is enhanced by MI, which is similar to that in β-arrestin2 KO mice. In contrast, the inflammation after MI in β-arrestin2 KO mice transplanted with bone marrow cells of WT mice is comparable to that in WT mice transplanted with bone marrow cells of WT mice. In summary, our present study demonstrates that β-arrestin2 of infiltrated macrophages negatively regulates inflammation in infarcted hearts, thereby enhancing inflammation when the β-arrestin2 gene is knocked out. β-arrestin2 plays a protective role in MI-induced inflammation. [ABSTRACT FROM AUTHOR]

Background: Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells. Methodology/Principal Findings: In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the antiadipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gas had the opposite effects. Conclusions: 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism. [ABSTRACT FROM AUTHOR]

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to theGprotein-dependent pathways, recent reports have revealed that several ligands called "biased ligands" elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by theβ-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β1-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β1-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β1-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)- knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2- dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestindependent signaling is a reason for the diversity of the effectiveness of β-blockers [ABSTRACT FROM AUTHOR]

An emerging aspect of redox signaling is the pathway mediated by electrophilic byproducts, such as nitrated cyclic nucleotide (for example, 8-nitroguanosine 3?,5?-cyclic monophosphate (8-nitro-cGMP)) and nitro or keto derivatives of unsaturated fatty acids, generated via reactions of inflammation-related enzymes, reactive oxygen species, nitric oxide and secondary products. Here we report that enzymatically generated hydrogen sulfide anion (HS?) regulates the metabolism and signaling actions of various electrophiles. HS? reacts with electrophiles, best represented by 8-nitro-cGMP, via direct sulfhydration and modulates cellular redox signaling. The relevance of this reaction is reinforced by the significant 8-nitro-cGMP formation in mouse cardiac tissue after myocardial infarction that is modulated by alterations in HS? biosynthesis. Cardiac HS?, in turn, suppresses electrophile-mediated H-Ras activation and cardiac cell senescence, contributing to the beneficial effects of HS? on myocardial infarction-associated heart failure. Thus, this study reveals HS?-induced electrophile sulfhydration as a unique mechanism for regulating electrophile-mediated redox signaling. [ABSTRACT FROM AUTHOR]

Abstract: The overexpression of mitochondrial transcription factor A (TFAM) attenuates the decrease in mtDNA copy number after myocardial infarction, ameliorates pathological hypertrophy, and markedly improves survival. However, non-transgenic strategy to increase mtDNA for the treatment of pathological hypertrophy remains unknown. We produced recombinant human TFAM protein (rhTFAM). rhTFAM rapidly entered into mitochondria of cultured cardiac myocytes. rhTFAM increased mtDNA and abolished the activation of nuclear factor of activated T cells (NFAT), which is well known to activate pathological hypertrophy. rhTFAM attenuated subsequent morphological hypertrophy of myocytes as well. rhTFAM would be an attractive molecule in attenuating cardiac pathological hypertrophy. [Copyright &y& Elsevier]

Aims We investigated the mechanisms of action of lysophosphatidic acid (LPA) to regulate vascular endothelial (VE)-cadherin dynamics and cell–cell contact. Methods and results While a low concentration of LPA stimulated VE-cadherin internalization and subsequent cell–cell dissociation, a high concentration of LPA masked the disruptive actions on VE-cadherin and protected the barrier function in human vascular endothelial cells. Knockdown experiments of major LPA receptor subtypes, i.e. LPA1 and p2y5 (also termed LPA6), with their specific small interfering RNAs, showed that LPA1 and LPA6 mediate the LPA-induced disruptive and protective actions on barrier integrity, respectively. LPA6-mediated tube formation, reflecting stabilization of barrier integrity, was confirmed by in vitro angiogenesis assay. The LPA1-mediated disruptive actions were inhibited by pertussis toxin, dominant-negative Rac1, and inhibitors for c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), but not by dominant-negative RhoA. In contrast, the LPA6-mediated protective actions were associated with activation of Src and Rap1 and attenuated by abrogation of their activities. Further characterization showed that Rap1 is located downstream of Src and dependent on C3G, a Rap1 guanine nucleotide exchange factor. Finally, an LPA antagonist significantly inhibited lactic acid-induced limb lesions in vivo, which may be attributed to dysfunction of endothelial cells. Conclusion LPA induced disruption and protection of VE-cadherin integrity through LPA1–Gi protein–Rac1–JNK/p38MAPK and LPA6–G12/13 protein–Src–C3G–Rap1 pathways, respectively. [ABSTRACT FROM PUBLISHER]

Abstract: Angiotensin II (Ang II) is a major vasoactive peptide of the renin-angiotensin system. Ang II is originally found as one of potent vasoconstrictors, but is now attracted attention as an essential mediator of many cardiovascular problems, including endothelial dysfunction, arrhythmia and structural remodeling of cardiovascular systems. Most of the known pathophysiological effects of Ang II are mediated through Ang type1 receptors (AT1Rs), and the up-regulation of AT1Rs is one of important causes by which Ang II can contribute to cardiovascular diseases. A growing body of evidence has suggested that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in the regulation of AT1R signaling. In cardiac fibroblasts, stimulation with cytokines or bacterial toxins induces AT1R up-regulation through NADPH oxidase-dependent ROS production. In contrast, nitric oxide (NO) decreases AT1R density through cysteine modification (S-nitrosylation) of a transcriptional factor, nuclear factor κB (NF-κB). The difference between the effects of ROS and NO on AT1R expression may be caused by the difference between intracellular location of ROS signaling and that of NO signaling, as the agonist-induced S-nitrosylation of NF-κB requires a local interaction between NO synthase (NOS) and NF-κB in the perinuclear region. Thus, the spatial and temporal regulation of cysteine modification by ROS or RNS may underlie the resultant changes of AT1R signaling induced by agonist stimulation. [Copyright &y& Elsevier]

Abstract: Dilated cardiomyopathy (DCM) is a myocardial disorder that is characterized by dilation and dysfunction of the left ventricle (LV). Accumulating evidence has implicated aberrant Ca2+ signaling and oxidative stress in the progression of DCM, but the molecular details are unknown. In the present study, we report that inhibition of the transient receptor potential canonical 3 (TRPC3) channels partially prevents LV dilation and dysfunction in muscle LIM protein-deficient (MLP (−/−)) mice, a murine model of DCM. The expression level of TRPC3 and the activity of Ca2+/calmodulin-dependent kinase II (CaMKII) were increased in MLP (−/−) mouse hearts. Acitivity of Rac1, a small GTP-binding protein that participates in NADPH oxidase (Nox) activation, and the production of reactive oxygen species (ROS) were also increased in MLP (−/−) mouse hearts. Treatment with pyrazole-3, a TRPC3 selective inhibitor, strongly suppressed the increased activities of CaMKII and Rac1, as well as ROS production. In contrast, activation of TRPC3 by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or by mechanical stretch, induced ROS production in rat neonatal cardiomyocytes. These results suggest that up-regulation of TRPC3 is responsible for the increase in CaMKII activity and the Nox-mediated ROS production in MLP (−/−) mouse cardiomyocytes, and that inhibition of TRPC3 is an effective therapeutic strategy to prevent the progression of DCM. [Copyright &y& Elsevier]

Cross-talk between G protein-coupled receptor (GPCR) signaling pathways serves to fine tune cellular responsiveness by neurohumoral factors. Accumulating evidence has implicated nitric oxide (NO)-based signaling downstream of GPCRs, but the molecular details are unknown. Here, we show that adenosine triphosphate (ATP) decreases angiotensin type 1 receptor (AT1R) density through NO-mediated S-nitrosylation of nuclear factor KB (NF-κB) in rat cardiac fibroblasts. Stimulation of purinergic P2Y2 receptor by ATP increased expression of inducible NO synthase (iNOS) through activation of nuclear factor of activated T cells, NFATc1 and NFATc3. The ATP-induced iNOS interacted with p65 subunit of NF-κB in the cytosol through flavin-binding domain, which was indispensable for the locally generated NO-mediated S-nitrosylation of p65 at Cys38. β-Arrestins anchored the formation of p65/ lκBα/β-arrestins/iNOS quaternary complex. The S-nitrosylated p65 resulted in decreases in NF-κB transcriptional activity and ATκR density. In pressure-overloaded mouse hearts, ATP released from cardiomyocytes led to decrease in AT1R density through iNOS-mediated S-nitrosylation of p65. These results show a unique regulatory mechanism of heterologous regulation of GPCRs in which cysteine modification of transcriptional factor rather than protein phosphorylation plays essential roles. [ABSTRACT FROM AUTHOR]

Pertussis toxin (PTX) is recognized as a specific tool that uncouples receptors from Gi and Go through ADP-ribosylation. During the study analyzing the effects of PTX on Ang II type 1 receptor (AT1R) function in cardiac fibroblasts, we found that PTX increases the number of AT1Rs and enhances AT1R-mediated response. Microarray analysis revealed that PTX increases the induction of interleukin (IL)-1β among cytokines. Inhibition of IL-1β suppressed the enhancement of AT1R-mediated response by PTX. PTX increased the expression of IL-1β and AT1R through NF-κB, and a small GTP-binding protein, Rac, mediated PTX-induced NF-κB activation through NADPH oxidase-dependent production of reactive oxygen species. PTX induced biphasic increases in Rac activity, and the Rac activation in a late but not an early phase was suppressed by IL-1β siRNA, suggesting that IL-1β-induced Rac activation contributes to the amplification of Rac-dependent signaling induced by PTX. Furthermore, inhibition of TLR4 (Toll-like receptor 4) abolished PTX-induced Rac activation and enhancement of AT1R function. However, ADP-ribosylation of Gi/Go by PTX was not affected by inhibition of TLR4. Thus, PTX binds to two receptors; one is TLR4, which activates Rac, and another is the binding site that is required for ADP-ribosylation of Gi/Go. [ABSTRACT FROM AUTHOR]

Activation of Ca2+ signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca2+ signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca2+ influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca2+ spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCI or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca2+ influx itself or molecule(s) upstream of Ca2+ influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr69 and prevented TRPC6-mediated Ca2+ influx. Substitution of Ala for Thr69 in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce Gαq-mediated signaling, did not affect the suppression of receptor-activated Ca2+ influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition. [ABSTRACT FROM AUTHOR]

Prokineticins are secreted peptides that activate two G protein-coupled receptors: PKR 1 and PKR2. Prokineticins induce angiogenesis and fenestration, but the cognate receptors involved in these functions are unknown. We hypothesized a role for prokineticin receptor signaling pathways and expression profiles in determining the selective effects of prokineticins on coronary endothelial cells (H5V). Activation of the PKR1/MAPK/Akt signaling pathway stimulates proliferation, migration, and angiogenesis in H5V cells, in which PKRI predominates over PKR2. PKR1 was colocalized with Gα11 and was internalized following the stimulation of these cells with prokineticin-2. Knock down of PKR1 or Gα1 expression in H5V cells effectively inhibited prokineticin-2- induced vessel formation and MAPK/Akt activation, indicating a role for PKR1/Gα11 in this process. However, in conditions in which PKR2 predominated over PKR 1, these cells displayed a fenestrated endothelial cell phenotype. H5V cells overexpressing PKR2 displayed large numbers of multivesicular bodies and caveolar clusters and a disruption of the distribution of zonula occluden-1 tight junction protein. Prokineticin-2 induced the colocalization of PKR2 with Gα12, and activated Gα12, which bound to zonula occluden-1 to trigger the degradation of this protein in these cells. Prokineticin-2 induced the formation of vessel-like structures by human aortic endothelial cells expressing only PKR1, and disorganized the tight junctions in human hepatic sinusoidal endothelial cells expressing only PKR2, confirming the divergent roles of these receptors. Our findings show the functional characteristics of coronary endothelial cells depend on the expression of PKR1 and PKR2 levels and the divergent signaling pathways used by these receptors. [ABSTRACT FROM AUTHOR]

The upstream signaling pathway leading to the activation of AMP-activated protein kinase (AMPK) by high density lipoprotein (HDL) and the role of AMPK in HDL-induced antiatherogenic actions were investigated. Experiments using genetic and pharmacological tools showed that HDL-induced activation of AMPK is dependent on both sphingosine 1-phosphate receptors and scavenger receptor class B type l through calcium/calmodulin dependent protein kinase kinase and, for scavenger receptor class B type I system, additionally serine-threonine kinase LKB1 in human umbilical vein endothelial cells. HDL-induced activation of Akt and endothelial NO synthase, stimulation of migration, and inhibition of monocyte adhesion and adhesion molecule expression were dependent on AMPK activation. The inhibitory role of AMPK in the adhesion molecule expression and monocyte adhesion on endothelium of mouse aorta was confirmed in vivo and ex vivo. On the other hand, stimulation of ERK and proliferation were hardly affected by AMPK knockdown but completely inhibited by an N17Ras, whereas the dominant-negative Ras was ineffective for AMPK activation. In conclusion, dual HDL receptor systems differentially regulate AMPK activity through calcium/calmodulin-dependent protein kinase kinase and/or LKB1. Several HDL-induced antiatherogenic actions are regulated by AMPK, but proliferation-related actions are regulated by Ras rather than AMPK. [ABSTRACT FROM AUTHOR]

Abstract: The physiological role of the thromboxane A2 (TXA2) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA2 analogue. In the present study, we examined the detailed mechanisms of TXA2 receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na+/H+-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [3H]H2O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA2 receptor mediates water influx through aquaporins in astrocytoma cells via TXA2 receptor-mediated activation of Gα12/13, Rho A, Rho kinase and Na+/H+-exchanger. [Copyright &y& Elsevier]

Canonical transient receptor potential (TRPC) channels control influxes of Ca[sup2+] 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 TRPC5 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 Ca[sup2+] influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptorinduced Ca[sup2+] response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca[sup2+]-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 TRPC3selective inhibitor Pyr3 isa 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF-dependent neuritogenesis enhancer potential of a food-derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor-initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic-promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi ( Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6-HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low-concentration of NGF that alone was insufficient to induce neuronal differentiation. 6-HITC also facilitated the sustained-phosphorylation of the extracellular signal-regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr-490 of TrkA and was inactivated by 6-HITC in a redox-dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates. [ABSTRACT FROM AUTHOR]

Abstract: Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation and gene expression. ERK5 is twice the size of ERK1/2, the amino-terminal half contains the kinase domain that shares the homology with ERK1/2 and TEY activation motif, whereas the carboxy-terminal half is unique. In this study, we examined the cross-talk mechanism between G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases, focusing on ERK1/2 and 5. The pretreatment of rat pheochromocytoma cells (PC12) with pertussis toxin (PTX) specifically enhanced epidermal growth factor (EGF)-induced ERK5 phosphorylation. In addition, lysophosphatidic acid (LPA) attenuated the EGF-induced ERK5 phosphorylation in LPA1 receptor- and Gi/o-dependent manners. On the other hand, LPA alone activated ERK1/2 via Gβγ subunits and Ras and potentiated EGF-induced ERK1/2 phosphorylation at late time points. These results suggest Gi/o negatively regulates ERK5, while it positively regulates ERK1/2. LPA did not affect cAMP levels after EGF treatment, and the reagents promoting cAMP production such as forskolin and cholera toxin also attenuated the EGF-induced ERK5 phosphorylation, indicating that the inhibitory effect of LPA on ERK5 inhibition via Gi/o is not due to inhibition of adenylyl cyclase by Gαi/o. However, the inhibitory effect of LPA on ERK5 was abolished in PC12 cells stably overexpressing C-terminus of GPCR kinase2 (GRK2), and overexpression of Gβ1 and γ2 subunits also suppressed ERK5 phosphorylation by EGF. In response to LPA, Gβγ subunits interacted with EGF receptor in a time-dependent manner. These results strongly suggest that LPA negatively regulates the EGF-induced ERK5 phosphorylation through Gβγ subunits. [Copyright &y& Elsevier]

Kelch-like ECH-associated protein 1 (Keap1), a BTB-Kelch substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex, regulates the induction of the phase 2 enzymes, such as glutathione S-transferase (GST), by repressing the transcription factor Nrf2. It is known that, in the human gastrointestinal tract, both GST A1 and P1 are constitutively expressed as the major GST isozymes. In the present study, using the Keap1-overexpressing derivatives of Caco-2 cells, human carcinoma cell line of colonic origin, by stable transfection of wild type Keap1, we investigated the molecular mechanism underlying the constitutive expression of these GST isozymes during differentiation. It was revealed that the overexpression of Keap1 completely repressed the constitutive expression of GST A1, but not GST P1. In Keap1-overexpressed cells, dome formation disappeared, and the formation of the intact actin cytoskeletal organization at cell-cell contact sites and the recruitment of E-cadherin and β-catenin to adherens junctions were inhibited. The constitutive GST A1 expression in Caco-2 cells was repressed by disruption of E-cadherin-mediated cell-cell adhesion, suggesting the correlation between epithelial cell polarization and induction of the basal GST A1 expressions during Caco-2 differentiation, Keap1 overexpression indeed inhibited the activation of the small guanosine triphosphatase Rac1 on the formation of E-cadherin-mediated cell-cell adhesion. The transfection of V12Rac1, the constitutively active Rac1 mutant, into Keap1-overexpressed cells promoted the basal GST A1 expression, suggesting that Keap1 regulated the basal GST A1 expression during Caco-2 differentiation via Rac1 activation on the formation of E-cadherin-mediated cell-cell adhesion. The results of this study suggest the involvement of a novel Keap1-dependent signaling pathway for the induction of the constitutive GST A1 expression during epithelial cell differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P2 receptor, a carboxyl-terminal region of Gα12 or Gα13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P2 receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P2 receptors/G12/13-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P2 receptor-mediated action in glioma cells. [Copyright &y& Elsevier]

Prokineticins are potent angiogenic factors that bind to two G protein-coupled receptors to initiate their biological effects. We hypothesize that prokineticin receptor-1 (PKR1/GPR73) signaling may contribute to cardiomyocyte survival or repair in myocardial infarction. Since we showed that prokineticin-2 and PKR1 are expressed in adult mouse heart and cardiac cells, we investigated the role of prokineticin-2 on capillary endothelial cell and cardiomyocyte function. In cultured cardiac endothelial cells, prokineticin-2 or overexpression of PKR1 induces vessel-like formation without increasing VEGF levels. In cardiomyocytes and H9c2 cells, prokineticin-2 or overexpressing PKR1 activates Akt to protect cardiomyocytes against oxidative stress. The survival and angiogenesis promoting effects of prokineticin-2 in cardiac cells were completely reversed by siRNA-PKR1, indicating PKR1 involvement. We thus, further investigated whether intramyocardial gene transfer of DNA encoding PKR1 may rescue the myocardium against myocardial infarction in mouse model. Transient PKR1 gene transfer after coronary ligation reduces mortality and preserves left ventricular function by promoting neovascularization and protecting cardiomyocytes without altering VEGF levels. In human end-stage failing heart samples, reduced PKR1 and prokineticin-2 transcripts and protein levels implicate a more important role for prokineticin-2/PKR1 signaling in heart. Our results suggest that PKR1 may represent a novel therapeutic target to limit myocardial injury following ischemic events. [ABSTRACT FROM AUTHOR]

Sustained elevation of [Ca2+]i has been implicated in many cellular events. We previously reported that a 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. [ABSTRACT FROM AUTHOR]

Abstract: GPR4 was initially identified as a receptor for sphingosylphosphorylcholine and lysophosphatidylcholine; however, lipid actions have not always been confirmed. Instead, ligand-independent actions have sometimes been observed in GPR4- and other OGR1 family receptor-expressing cells. Here, we examined the possible involvement of extracellular protons, which have recently been proposed as another ligand for GPR4. At pH 7.4, the epidermal growth factor-induced extracellular signal-regulated kinase activity was lower in GPR4-transfected RH7777 cells, in association with increased cAMP accumulation, than in vector-transfected cells. The serum response element (SRE)-driven transcriptional activity was also clearly higher in GPR4-expressing HEK293 cells than in vector-transfected cells at pH 7.4. These apparent ligand-independent actions were very small at alkalinic 7.8. The SRE activity was further increased by extracellular acidification in a manner dependent on the G13 protein/Rho signaling pathway in HEK293 cells expressing GPR4 or other OGR1 receptor family members. GPR4-expressing cells also showed a calcineurin-dependent nuclear factor of activated T cell (NFAT) promoter activation at pH 7.4, and this activity was further increased by pH below 7.2 in association with inositol phosphate production. In contrast to the cAMP and SRE responses, however, alkalinization to pH 7.8 hardly affected the high basal activity. Finally, the expression of GPR4 hardly modulated the sphingosylphosphorylcholine- or lysophosphatidylcholine-induced action. These results suggest that an extracellular proton play a role as a ligand in some of previously postulated ligand-independent actions through GPR4 receptors. Moreover, GPR4 may be a multi-functional receptor coupling to Gs, G13, and Gq/11 proteins in response to extracellular acidification. [Copyright &y& Elsevier]

Abstract: We assessed the characteristics of human osteoblasts, focusing on small GTPase Rho signaling. β1 Integrin were highly expressed on osteoblasts. Engagement of β1 integrins by type I collagen augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor κB ligand (RANKL) on osteoblasts. Rho was activated by β1 stimulation in osteoblasts. β1 Integrin-induced up-regulation of ICAM-1 and RANKL was inhibited by transfection with adenoviruses encoding C3 transferase or pretreated with Y-27632, specific Rho and Rho-kinase inhibitors. Engagement of β1 integrin on osteoblasts induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) in a coculture system of osteoblasts and peripheral monocytes, but this action was completely abrogated by transfection of C3 transferase. Our results indicate the direct involvement of Rho-mediated signaling in β1 integrin-induced up-regulation of ICAM-1 and RANKL and RANKL-dependent osteoclast maturation. Thus, Rho-mediated signaling in osteoblasts seems to introduce major biases to bone resorption. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Clathrin is a major component of clathrin-coated pits and serves as a binding scaffold for endocytic machinery through the binding of a specific sequence known as the clathrin-binding motif. This motif is also found in cellular signaling proteins other than endocytic components, including G protein-coupled receptor kinase 2 (GRK2), which phosphorylates G protein-coupled receptors and promotes uncoupling of receptor-G protein interaction. However, the functions of clathrin in the regulation of GRK2 are unknown. Here we demonstrated that overexpression of GRK2 mutated at the clathrin-binding motif with alanine (GRK2-5A) results in inhibition of phosphorylation and internalization of the β2-adrenergic receptor (β2AR). However, the interaction of β2AR with GRK2-5A is the same as that of wild type GRK2 as determined by bioluminescence resonance energy transfer. Furthermore, GRK2-5A phosphorylates rhodopsin essentially to the same extent as wild type GRK2 in vitro. Depletion of the clathrin heavy chain using small interference RNA inhibits agonist-induced phosphorylation and internalization of β2AR. Thus, clathrin works as a regulator of GRK2 in cells. These results indicate that clathrin is a novel player in cellular functions in addition to being a component of endocytosis. [ABSTRACT FROM AUTHOR]

Abstract: Glial cells express thromboxane A2 receptor, but its physiological role remains unknown. The present study was performed to examine thromboxane A2 receptor-mediated morphological change in 1321N1 human astrocytoma cells. Thromboxane A2 receptor agonists U46619 and STA2 caused a rapid morphological change to spindle shape from stellate form of the cells pretreated with dibutyryl cyclic AMP, but neither carbachol nor histamine caused the change, suggesting that Gq pathway may not mainly contribute to the change. Rho kinase inhibitor Y-27632 inhibited U46619-induced morphological change, and U46619 increased the GTP-bound form of RhoA accompanied with actin stress fiber formation. These responses were reduced by expression of p115-RGS that inhibits G12/13 signaling pathway. U46619 also caused the phosphorylation of extracellular signal-regulated kinase (ERK) and [3H]thymidine incorporation mainly through G12/13-Rho pathway. These results suggest that stimulation of thromboxane A2 receptor causes the morphological change with proliferation mainly through G12/13 activation in glial cells. [Copyright &y& Elsevier]

Abstract: Sphingosine 1-phosphate (S1P) stimulates expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells. S1P-induced actions were associated with nuclear factor kappa-B activation and inhibited by pertussis toxin as well as by antisense oligonucleotides specific to S1P receptors, especially, S1P3. S1P also stimulated endothelial nitric oxide synthase (eNOS) and its activation was markedly inhibited by the antisense oligonucleotide for the S1P1 receptor rather than that for the S1P3 receptor. The dose–response curve of S1P to stimulate adhesion molecule expression was shifted to the left in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin and the NOS inhibitor Nω-nitro-l-arginine methyl ester. NO donor S-nitroso-N-acetylpenicillamine inhibited S1P-induced adhesion molecule expression. Moreover, tumor necrosis factor-α-induced adhesion molecule expression was markedly inhibited by S1P in a manner sensitive to inhibitors for PI3-K and NOS. These results suggest that S1P receptors are coupled to both stimulatory and inhibitory pathways for adhesion molecule expression. The stimulatory pathway involves nuclear factor kappa-B and inhibitory one does phosphatidylinositol 3-kinase and NOS. [Copyright &y& Elsevier]

A potential role for 1-oleoyl-sn-glycero-3-phosphate or lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) in the regulation of malignant diseases has been widely considered. In this study, we found that in transformed astroglial cells, the expression profile of lysophospholipid receptor mRNA and the action modes of LPA and SIP on cell motility were changed: there was a change in the acquisition of the ability of LPA to stimulate cell migration and a change in the migratory response to S1P from stimulation through S1P1 to inhibition through S1P2. LPA-induced cell migration was almost completely inhibited by either pertussis toxin, LPA1 receptor antagonists including Ki16425 (3-(4-[4-([1- (2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-iso- xazolyl] benzylsulfonyl)propanoic acid) or an inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin. The LPA-induced action was also suppressed, although incompletely, by several specific inhibitors for intracellular signaling pathways induding Rac1, Cdc42, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun terminal kinase (JNK), but not extracellular signal-regulated kinase. Nearly complete inhibition of migration response to LPA, however, required simultaneous inhibition of both the p38MAPK and JNK pathways. inhibition of Rac1 suppressed JNK but not p38MAPK, while the activity of p38MAPK was abolished by a dominant-negative form of Cdc42. These findings suggest that, in glioma cells, the PI3K/Cdc42/p38MAPK and PI3K/ Rac1/JNK pathways are equally important for LPA1 receptor-mediated migration. [ABSTRACT FROM AUTHOR]

Abstract: β-Arrestin mediates desensitization and internalization of β-adrenergic receptors (βARs), but also acts as a scaffold protein in extracellular signal-regulated kinase (ERK) cascade. Thus, we have examined the role of β-arrestin2 in the βAR-mediated ERK signaling pathways. Isoproterenol stimulation equally activated cytoplasmic and nuclear ERK in COS-7 cells expressing β1AR or β2AR. However, the activity of nuclear ERK was enhanced by co-expression of β-arrestin2 in β2AR—but not β1AR-expressing cells. Pertussis toxin treatment and blockade of Gβγ action inhibited β-arrestin2-enhanced nuclear activation of ERK, suggesting that β-arrestin2 promotes nuclear ERK localization in a Gβγ dependent mechanism upon receptor stimulation. β2AR containing the carboxyl terminal region of β1AR lost the β-arrestin2-promoted nuclear translocation. As the carboxyl terminal region is important for β-arrestin binding, these results demonstrate that recruitment of β-arrestin2 to carboxyl terminal region of β2AR is important for ERK localization to the nucleus. [Copyright &y& Elsevier]

In the early development of the central nervous system, neural progenitor cells divide in an asymmetric manner and migrate along the radial glia cells. The radial migration is an important process for the proper lamination of the cerebral cortex. Recently, a new mode of the radial migration was found at the intermediate zone where the neural progenitor cells become multipolar and reduce the migration rate. However, the regulatory signals for the radial migration are unknown. Using the migration assay in vitro, we examined how neural progenitor cell migration is regulated Neural progenitor cells derived from embryonic mouse telencephalon migrated on laminin-coated dishes. Endothelin (ET)-1 inhibited the neural progenitor cell migration. This ET-1 effect was blocked by BQ788, a specific inhibitor of the ETB receptor, and by the expression of a carboxyl-terminal peptide of Gαq but not Gαi. The expression of constitutively active mutant of Gαq, GαqR183C, inhibited the migration of neural progenitor cells. Moreover, the inhibitory effect of ET-1 was suppressed by the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the expression of the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of the JNK pathway. Using the slice culture system of embryonic brain, we demonstrated that ET-1 and the constitutively active mutant of Gαq caused the retention of the neural progenitor cells in the intermediate zone and JNK-binding domain of JNK-interacting protein-1 abrogated the effect of ET-1. These results indicated that G protein-coupled receptor signaling negatively regulates neural progenitor cell migration through Gq and JNK. [ABSTRACT FROM AUTHOR]