Deletion of Gαq/11 or Gαs Proteins in Gonadotropes Differentially Affects Gonadotropin Production and Secretion in Mice.

Gonadotropin-releasing hormone (GnRH) regulates gonadal function via its stimulatory effects on gonadotropin production by pituitary gonadotrope cells. GnRH is released from the hypothalamus in pulses and GnRH pulse frequency differentially regulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis and secretion. The GnRH receptor (GnRHR) is a G protein-coupled receptor that canonically activates Gα q/11-dependent signaling on ligand binding. However, the receptor can also couple to Gα s and in vitro data suggest that toggling between different G proteins may contribute to GnRH pulse frequency decoding. For example, as we show here, knockdown of Gα s impairs GnRH-stimulated FSH synthesis at low- but not high-pulse frequency in a model gonadotrope-derived cell line. We next used a Cre-lox conditional knockout approach to interrogate the relative roles of Gα q/11 and Gα s proteins in gonadotrope function in mice. Gonadotrope-specific Gα q/11 knockouts exhibit hypogonadotropic hypogonadism and infertility, akin to the phenotypes seen in GnRH- or GnRHR-deficient mice. In contrast, under standard conditions, gonadotrope-specific Gα s knockouts produce gonadotropins at normal levels and are fertile. However, the LH surge amplitude is blunted in Gα s knockout females and postgonadectomy increases in FSH and LH are reduced both in males and females. These data suggest that GnRH may signal principally via Gα q/11 to stimulate gonadotropin production, but that Gα s plays important roles in gonadotrope function in vivo when GnRH secretion is enhanced.

Side-by-side comparison of the effects of Gq- and Gi-DREADD-mediated astrocyte modulation on intracellular calcium dynamics and synaptic plasticity in the hippocampal CA1.

Astrocytes express a plethora of G protein-coupled receptors (GPCRs) that are crucial for shaping synaptic activity. Upon GPCR activation, astrocytes can respond with transient variations in intracellular Ca2+. In addition, Ca2+-dependent and/or Ca2+-independent release of gliotransmitters can occur, allowing them to engage in bidirectional neuron-astrocyte communication. The development of designer receptors exclusively activated by designer drugs (DREADDs) has facilitated many new discoveries on the roles of astrocytes in both physiological and pathological conditions. They are an excellent tool, as they can target endogenous GPCR-mediated intracellular signal transduction pathways specifically in astrocytes. With increasing interest and accumulating research on this topic, several discrepancies on astrocytic Ca2+ signalling and astrocyte-mediated effects on synaptic plasticity have emerged, preventing a clear-cut consensus about the downstream effects of DREADDs in astrocytes. In the present study, we performed a side-by-side evaluation of the effects of bath application of the DREADD agonist, clozapine-N-oxide (10 µM), on Gq- and Gi-DREADD activation in mouse CA1 hippocampal astrocytes. In doing so, we aimed to avoid confounding factors, such as differences in experimental procedures, and to directly compare the actions of both DREADDs on astrocytic intracellular Ca2+ dynamics and synaptic plasticity in acute hippocampal slices. We used an adeno-associated viral vector approach to transduce dorsal hippocampi of male, 8-week-old C57BL6/J mice, to drive expression of either the Gq-DREADD or Gi-DREADD in CA1 astrocytes. A viral vector lacking the DREADD construct was used to generate controls. Here, we show that agonism of Gq-DREADDs, but not Gi-DREADDs, induced consistent increases in spontaneous astrocytic Ca2+ events. Moreover, we demonstrate that both Gq-DREADD as well as Gi-DREADD-mediated activation of CA1 astrocytes induces long-lasting synaptic potentiation in the hippocampal CA1 Schaffer collateral pathway in the absence of a high frequency stimulus. Moreover, we report for the first time that astrocytic Gi-DREADD activation is sufficient to elicit de novo potentiation. Our data demonstrate that activation of either Gq or Gi pathways drives synaptic potentiation through Ca2+-dependent and Ca2+-independent mechanisms, respectively.

Astrocytes in the Ventromedial Hypothalamus Involve Chronic Stress-Induced Anxiety and Bone Loss in Mice.

Chronic stress is one of the main risk factors of bone loss. While the neurons and neural circuits of the ventromedial hypothalamus (VMH) mediate bone loss induced by chronic stress, the detailed intrinsic mechanisms within the VMH nucleus still need to be explored. Astrocytes in brain regions play important roles in the regulation of metabolism and anxiety-like behavior through interactions with surrounding neurons. However, whether astrocytes in the VMH affect neuronal activity and therefore regulate chronic stress-induced anxiety and bone loss remain elusive. In this study, we found that VMH astrocytes were activated during chronic stress-induced anxiety and bone loss. Pharmacogenetic activation of the Gi and Gq pathways in VMH astrocytes reduced and increased the levels of anxiety and bone loss, respectively. Furthermore, activation of VMH astrocytes by optogenetics induced depolarization in neighboring steroidogenic factor-1 (SF-1) neurons, which was diminished by administration of N-methyl-D-aspartic acid (NMDA) receptor blocker but not by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker. These results suggest that there may be a functional "glial-neuron microcircuit" in VMH nuclei that mediates anxiety and bone loss induced by chronic stress. This study not only advances our understanding of glial cell function but also provides a potential intervention target for chronic stress-induced anxiety and bone loss therapy.

Physiological Temperature Changes Fine-Tune ß 2- Adrenergic Receptor-Induced Cytosolic cAMP Accumulation.

Norepinephrine (NE) controls many vital body functions by activating adrenergic receptors (ARs). Average core body temperature (CBT) in mice is 37°C. Of note, CBT fluctuates between 36 and 38°C within 24 hours, but little is known about the effects of CBT changes on the pharmacodynamics of NE. Here, we used Peltier element-controlled incubators and challenged murine hypothalamic mHypoA -2/10 cells with temperature changes of ±1°C. We observed enhanced NE-induced activation of a cAMP-dependent luciferase reporter at 36 compared with 38°C. mRNA analysis and subtype specific antagonists revealed that NE activates ß 2- and ß 3-AR in mHypoA-2/10 cells. Agonist binding to the ß 2-AR was temperature insensitive, but measurements of cytosolic cAMP accumulation revealed an increase in efficacy of 45% ± 27% for NE and of 62% ± 33% for the ß 2-AR-selective agonist salmeterol at 36°C. When monitoring NE-promoted cAMP efflux, we observed an increase in the absolute efflux at 36°C. However, the ratio of exported to cytosolic accumulated cAMP is higher at 38°C. We also stimulated cells with NE at 37°C and measured cAMP degradation at 36 and 38°C afterward. We observed increased cAMP degradation at 38°C, indicating enhanced phosphodiesterase activity at higher temperatures. In line with these data, NE-induced activation of the thyreoliberin promoter was found to be enhanced at 36°C. Overall, we show that physiologic temperature changes fine-tune NE-induced cAMP signaling in hypothalamic cells via ß 2-AR by modulating cAMP degradation and the ratio of intra- and extracellular cAMP. SIGNIFICANCE STATEMENT: Increasing cytosolic cAMP levels by activation of G protein-coupled receptors (GPCR) such as the ß 2-adrenergic receptor (AR) is essential for many body functions. Changes in core body temperature are fundamental and universal factors of mammalian life. This study provides the first data linking physiologically relevant temperature fluctuations to ß 2-AR-induced cAMP signaling, highlighting a so far unappreciated role of body temperature as a modulator of the prototypic class A GPCR.

Functional characterization of uveal melanoma oncogenes.

Uveal melanoma (UM) is a currently untreatable form of melanoma with a 50% mortality rate. Characterization of the essential signaling pathways driving this cancer is critical to develop target therapies. Activating mutations in the Gαq signaling pathway at the level of GNAQ, GNA11, or rarely CYSLTR2 or PLCß4 are considered alterations driving proliferation in UM and several other neoplastic disorders. Here, we systematically examined the oncogenic signaling output of various mutations recurrently identified in human tumors. We demonstrate that CYSLTR2 → GNAQ/11 → PLCß act in a linear signaling cascade that, via protein kinase C (PKC), activates in parallel the MAP-kinase and FAK/Yes-associated protein pathways. Using genetic ablation and pharmacological inhibition, we show that the PKC/RasGRP3/MAPK signaling branch is the essential component that drives the proliferation of UM. Only inhibition of the MAPK branch but not the FAK branch synergizes with inhibition of the proximal cascade, providing a blueprint for combination therapy. All oncogenic signaling could be extinguished by the novel GNAQ/11 inhibitor YM-254890, in all UM cells with driver mutation in the Gαq subunit or the upstream receptor. Our findings highlight the GNAQ/11 → PLCß â†’ PKC → MAPK pathway as the central signaling axis to be suppressed pharmacologically to treat for neoplastic disorders with Gαq pathway mutations.

Sensory satellite glial Gq-GPCR activation alleviates inflammatory pain via peripheral adenosine 1 receptor activation.

Glial fibrillary acidic protein expressing (GFAP+) glia modulate nociceptive neuronal activity in both the peripheral nervous system (PNS) and the central nervous system (CNS). Resident GFAP+ glia in dorsal root ganglia (DRG) known as satellite glial cells (SGCs) potentiate neuronal activity by releasing pro-inflammatory cytokines and neuroactive compounds. In this study, we tested the hypothesis that SGC Gq-coupled receptor (Gq-GPCR) signaling modulates pain sensitivity in vivo using Gfap-hM3Dq mice. Complete Freund's adjuvant (CFA) was used to induce inflammatory pain, and mechanical sensitivity and thermal sensitivity were used to assess the neuromodulatory effect of glial Gq-GPCR activation in awake mice. Pharmacogenetic activation of Gq-GPCR signaling in sensory SGCs decreased heat-induced nociceptive responses and reversed inflammation-induced mechanical allodynia via peripheral adenosine A1 receptor activation. These data reveal a previously unexplored role of sensory SGCs in decreasing afferent excitability. The identified molecular mechanism underlying the analgesic role of SGCs offers new approaches for reversing peripheral nociceptive sensitization.

PAK Membrane Translocation and Phosphorylation Regulate Platelet Aggregation Downstream of Gi and G12/13 Pathways.

Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G12/13 depends on agonist concentrations; coactivation of Gi and G12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G12/13, and PAK may represent a potential antiplatelet and antithrombotic target.

Gnaq Protects PC12 Cells from Oxidative Damage by Activation of Nrf2 and Inhibition of NF-kB.

Reactive oxygen species (ROS) are continuously produced as byproducts of aerobic metabolism. Oxidative stress (OS) plays an important role in the occurrence of several neurodegenerative diseases as well as aging because of the accumulation of ROS. Gnaq is a member of G protein α subunits. It has been reported that the expression level of Gnaq in the mouse forebrain cortex was significantly decreased with age in our previous study; therefore, we supposed that Gnaq contributes to attenuate the OS. In this study, we generated a Gnaq-overexpression cell using gene recombinant technique and lentivirus transfection technique in a neuron-like PC12 cell, and investigated whether Gnaq had antioxidant effects in PC12 cells treated with H2O2. The viability of cells, concentration of ROS, Nrf2 nuclear translocation, expression of antioxidant enzymes, activation of NF-κB and apoptosis were compared between Gnaq-PC12 cells and Vector-PC12 cells. Results showed that, compared with Vector-PC12 cells, the antioxidative ability of Gnaq-PC12 cells was significantly improved, while the ROS level in Gnaq-PC12 cells was significantly decreased. Nrf2 nuclear translocation was up-regulated and NF-κB nuclear translocation was down-regulated in Gnaq-PC12 cells after H2O2 treatment. The results suggest that Gnaq plays a crucial role in neuroprotection in PC12 cells. A possible mechanism for this would be that the overexpressed Gnaq enhances the antioxidative effect mediated by Nrf2 signal pathway and inhibits the cellular damaging effect through NF-κB signal pathway.

WNT16 Requires Gα Subunits as Intracellular Partners for Both Its Canonical and Non-Canonical WNT Signalling Activity in Osteoblasts.

In the past years, WNT16 became an interesting target in the field of skeletal research, as it was identified as an essential regulator of the cortical bone compartment, with the ability to increase both cortical and trabecular bone mass and strength in vivo. Even though there are indications that these advantageous effects are coming from canonical and non-canonical WNT-signalling activity, a clear model of WNT signalling by WNT16 is not yet depicted. We, therefore, investigated the modulation of canonical (WNT/ß-catenin) and non-canonical [WNT/calcium, WNT/planar cell polarity (PCP)] signalling in human embryonic kidney (HEK) 293 T and SaOS2 cells. Here, we demonstrated that WNT16 activates all WNT-signalling pathways in osteoblasts, whereas only WNT/calcium signalling was activated in HEK293T cells. In osteoblasts, we therefore, additionally investigated the role of Gα subunits as intracellular partners in WNT16's mechanism of action by performing knockdown of Gα12, Gα13 and Gαq. These studies point out that the above-mentioned Gα subunits might be involved in the WNT/ß-catenin and WNT/calcium-signalling activity by WNT16 in osteoblasts, and for Gα12 in its WNT/PCP-signalling activity, illustrating a novel possible mechanism of interplay between the different WNT-signalling pathways in osteoblasts. Additional studies are needed to demonstrate whether this mechanism is specific for WNT16 signalling or relevant for all other WNT ligands as well. Altogether, we further defined WNT16's mechanism of action in osteoblasts that might underlie the well-known beneficial effects of WNT16 on skeletal homeostasis. These findings on WNT16 and the activity of specific Gα subunits in osteoblasts could definitely contribute to the development of novel therapeutic approaches for fragility fractures in the future.

Gq-activated fibroblasts induce cardiomyocyte action potential prolongation and automaticity in a three-dimensional microtissue environment.

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, Gq-mediated signaling was selectively enhanced in CFs by Gαq adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca2+-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMPCF). Enhanced Gq signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gαq in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca2+ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive Gq activation in CFs also depolarized RMPCF from -33 to -20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca2+ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias.NEW & NOTEWORTHY In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced Gq signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.

Decreased Gαq expression in T cells correlates with enhanced cytokine production and disease activity in systemic lupus erythematosus.

Aberrant T cell immune responses appear central to the development of systemic lupus erythematosus (SLE). We previously reported that Gαq, the alpha subunit of Gq, regulates T and B cell immune responses, promoting autoimmunity. To address whether Gαq contributes to the pathogenesis of SLE, Gαq mRNA expression was studied using real time-PCR in PBMCs and T cells from SLE patients as well as age- and sex-matched healthy controls. Our results showed that Gαq mRNA expression was decreased in PBMCs and T cells from SLE patients compared to healthy individuals. Correlation analyses showed that Gαq expression in T cells from SLE patients was associated with disease severity (as per SLE Disease Activity Index), the presence of lupus nephritis, and expression of Th1, Th2 and Th17 cytokines. In keeping with clinical results, T-helper cell subsets (Th1, Th2 and Th17) were over-represented in Gαq knockout mice. In addition, Gαq expression in SLE T cells was negatively correlated with the expression of Bcl-2, an anti-apoptotic gene, and positively correlated with the expression of Bax, a pro-apoptotic gene. These data suggest that reduced Gαq levels in T cells may promote enhanced and prolonged T cell activation, contributing to the clinical manifestations of SLE.

Timosaponin AIII induces antiplatelet and antithrombotic activity via Gq-mediated signaling by the thromboxane A2 receptor.

The thromboxane (Tx) A2 pathway is a major contributor to the amplification of initial platelet activation and is therefore a key drug target. To identify potent small-molecule inhibitors of the thromboxane prostaglandin (TP) receptor, we screened a small steroidal saponin library using U46619-induced rat platelet aggregation assays. Timosaponin AIII (TAIII) was identified as a potent inhibitor of U46619-induced rat platelet aggregation and exhibited superior selectivity for the TP receptor versus other G protein-coupled receptors and a PKC activator. TAIII inhibited U46619-induced rat platelet aggregation independent of increases in cAMP and cGMP and the inhibition of TxA2 production. Both PKC and PLC activators restored TAIII-inhibited platelet aggregation, whereas TAIII did not inhibit platelet aggregation induced by co-activation of the G12/13 and Gz pathways. Furthermore, TAIII did not affect the platelet shape change or ROCK2 phosphorylation evoked by low-dose U46619. In vivo, TAIII prolonged tail bleeding time, reduced the mortality of animals with acute pulmonary thromboembolism and significantly reduced venous thrombus weight. Our study suggests that TAIII, by preferentially targeting Gq-mediated PLC/PKC signaling from the TP receptor, induces stronger in vitro antiplatelet activity and in vivo antithrombotic effects and may be an excellent candidate for the treatment of thrombotic disorders.

Activation of the orphan receptor GPR55 by lysophosphatidylinositol promotes metastasis in triple-negative breast cancer.

The orphan G protein-coupled receptor GPR55 has been directly or indirectly related to basic alterations that drive malignant growth: uncontrolled cancer cell proliferation, sustained angiogenesis, and cancer cell adhesion and migration. However, little is known about the involvement of this receptor in metastasis. Here, we show that elevated GPR55 expression in human tumors is associated with the aggressive basal/triple-negative breast cancer population, higher probability to develop metastases, and therefore poor patient prognosis. Activation of GPR55 by its proposed endogenous ligand lysophosphatidylinositol confers pro-invasive features on breast cancer cells both in vitro and in vivo. Specifically, this effect is elicited by coupling to Gq/11 heterotrimeric proteins and the subsequent activation, through ERK, of the transcription factor ETV4/PEA3. Together, these data show that GPR55 promotes breast cancer metastasis, and supports the notion that this orphan receptor may constitute a new therapeutic target and potential biomarker in the highly aggressive triple-negative subtype.

Mechanosensitive Gq/11 Protein-Coupled Receptors Mediate Myogenic Vasoconstriction.

Myogenic vasoconstriction (Bayliss effect) is mediated by vascular smooth muscle cells (VSMCs) of small resistance arteries sensing mechanical forces. During the last three decades, several proteins have been proposed as VSMC mechanosensors. Our previous studies highlighted agonist-independent mechanical activation of Gq/11 protein-coupled receptors (Gq/11 PCRs) in VSMCs of resistance arteries. In particular, angiotensin II AT1 receptors (AT1 Rs) emerged as mechanosensors mediating myogenic tone. Moreover, we found that the AT1B receptor isoform was more mechanosensitive than the AT1A receptor. Interestingly, cysteinyl leukotriene 1 receptors (CysLT1 Rs) were up-regulated in AT1 R-deficient arteries as an essential backup strategy to compensate for the loss of vasoconstrictor receptors. Up-regulation of CysLT1 Rs resulted in increased myogenic tone at low intraluminal pressures resulting in hyperactivity of AT1 R-deficient arteries. Only at high intraluminal pressures myogenic tone was reduced, thus reflecting the loss of AT1 Rs. Further, CysLT1 Rs were involved in myogenic vasoconstriction of wild-type arteries. Simultaneous blockade of AT1 Rs and CysLT1 Rs in wild-type arteries caused reduction in myogenic tone of more than 60% comparable to the application of the selective Gq/11 -protein inhibitor YM-254890. Our findings suggest that AT1 Rs and CysLT1 Rs are crucial mechanosensors in resistance arteries mediating 60% of myogenic vasoconstriction via the Gq/11 -protein pathway without involvement of endogenous agonists.

Endothelial Gαq/11 is required for VEGF-induced vascular permeability and angiogenesis.

AIMS: VEGF A (VEGF-A) is a central regulator of pre- and postnatal vascular development. In vitro studies suggested that heterotrimeric G-proteins of the Gq/11 family contribute to VEGF receptor 2 (VEGFR2) signalling, but the mechanism and physiological relevance of this finding is unknown. The aim of this study is to understand the role of endothelial Gαq/11 in VEGF-dependent regulation of vascular permeability and angiogenesis. METHODS AND RESULTS: We show here that VEGF-A-induced signalling events, such as VEGFR2 autophosphorylation, calcium mobilization, or phosphorylation of Src and Cdh5, were reduced in Gαq/11-deficient endothelial cells (ECs), resulting in impaired VEGF-dependent barrier opening, tube formation, and proliferation. Agonists at Gq/11-coupled receptors facilitated VEGF-A-induced VEGFR2 autophosphorylation in a Gαq/11-dependent manner, thereby enhancing downstream VEGFR2 signalling. In vivo, EC-specific Gαq/11- and Gαq-deficient mice showed reduced VEGF-induced fluid extravasation, and retinal angiogenesis was significantly impaired. Gαq-deficient ECs showed reduced proliferation, Cdh5 phosphorylation, and fluid extravasation, whereas apoptosis was increased. CONCLUSION: Gαq/11 critically contributes to VEGF-A-dependent permeability control and angiogenic behaviour in vitro and in vivo.

Estrogen regulates Hippo signaling via GPER in breast cancer.

The G protein-coupled estrogen receptor (GPER) mediates both the genomic and nongenomic effects of estrogen and has been implicated in breast cancer development. Here, we compared GPER expression in cancerous tissue and adjacent normal tissue in patients with invasive ductal carcinoma (IDC) of the breast and determined that GPER is highly upregulated in cancerous cells. Additionally, our studies revealed that GPER stimulation activates yes-associated protein 1 (YAP) and transcriptional coactivator with a PDZ-binding domain (TAZ), 2 homologous transcription coactivators and key effectors of the Hippo tumor suppressor pathway, via the Gαq-11, PLCß/PKC, and Rho/ROCK signaling pathways. TAZ was required for GPER-induced gene transcription, breast cancer cell proliferation and migration, and tumor growth. Moreover, TAZ expression positively correlated with GPER expression in human IDC specimens. Together, our results suggest that the Hippo/YAP/TAZ pathway is a key downstream signaling branch of GPER and plays a critical role in breast tumorigenesis.

Mitochondrial reprogramming induced by CaMKIIδ mediates hypertrophy decompensation.

RATIONALE: Sustained activation of Gαq transgenic (Gq) signaling during pressure overload causes cardiac hypertrophy that ultimately progresses to dilated cardiomyopathy. The molecular events that drive hypertrophy decompensation are incompletely understood. Ca(2+)/calmodulin-dependent protein kinase II δ (CaMKIIδ) is activated downstream of Gq, and overexpression of Gq and CaMKIIδ recapitulates hypertrophy decompensation. OBJECTIVE: To determine whether CaMKIIδ contributes to hypertrophy decompensation provoked by Gq. METHODS AND RESULTS: Compared with Gq mice, compound Gq/CaMKIIδ knockout mice developed a similar degree of cardiac hypertrophy but exhibited significantly improved left ventricular function, less cardiac fibrosis and cardiomyocyte apoptosis, and fewer ventricular arrhythmias. Markers of oxidative stress were elevated in mitochondria from Gq versus wild-type mice and respiratory rates were lower; these changes in mitochondrial function were restored by CaMKIIδ deletion. Gq-mediated increases in mitochondrial oxidative stress, compromised membrane potential, and cell death were recapitulated in neonatal rat ventricular myocytes infected with constitutively active Gq and attenuated by CaMKII inhibition. Deep RNA sequencing revealed altered expression of 41 mitochondrial genes in Gq hearts, with normalization of ≈40% of these genes by CaMKIIδ deletion. Uncoupling protein 3 was markedly downregulated in Gq or by Gq expression in neonatal rat ventricular myocytes and reversed by CaMKIIδ deletion or inhibition, as was peroxisome proliferator-activated receptor α. The protective effects of CaMKIIδ inhibition on reactive oxygen species generation and cell death were abrogated by knock down of uncoupling protein 3. Conversely, restoration of uncoupling protein 3 expression attenuated reactive oxygen species generation and cell death induced by CaMKIIδ. Our in vivo studies further demonstrated that pressure overload induced decreases in peroxisome proliferator-activated receptor α and uncoupling protein 3, increases in mitochondrial protein oxidation, and hypertrophy decompensation, which were attenuated by CaMKIIδ deletion. CONCLUSIONS: Mitochondrial gene reprogramming induced by CaMKIIδ emerges as an important mechanism contributing to mitotoxicity in decompensating hypertrophy.

Cross talk between the bombesin neuropeptide receptor and Sonic hedgehog pathways in small cell lung carcinoma.

Small cell lung carcinoma (SCLC) often features the upregulation of the Sonic hedgehog (Shh) pathway leading to activation of Gli transcription factors. SCLC cells secrete bombesin (BBS)-like neuropeptides that act as autocrine growth factors. Here, we show that SCLC tumor samples feature co-expression of Shh and BBS-cognate receptor (gastrin-releasing peptide receptor (GRPR)). We also demonstrate that BBS activates Gli in SCLC cells, which is crucial for BBS-mediated SCLC proliferation, because cyclopamine, an inhibitor of the Shh pathway, hampered the BBS-mediated effects. BBS binding to GRPR stimulated Gli through its downstream Gαq and Gα12/13 GTPases, and consistently, other Gαq and Gα13 coupled receptors (such as muscarinic receptor, m1, and thrombin receptor, PAR-1) and constitutively active GαqQL and Gα12/13QL mutants stimulated Gli. By using cells null for Gαq and Gα12/13, we demonstrate that these G proteins are strictly necessary for Gli activation by BBS. Moreover, by using constitutively active Rho small G-protein (Rho QL) as well as its inhibitor, C3 toxin, we show that Rho mediates G-protein-coupled receptor (GPCR)-, Gαq- and Gα12/13-dependent Gli stimulation. At the molecular level, BBS caused a significant increase in Shh gene transcription and protein secretion that was dependent on BBS-induced GPCR/Gαq-12/13/Rho mediated activation of nuclear factor κB (NFκB), which can stimulate a NF-κB response element in the Shh gene promoter. Our data identify a novel molecular network acting in SCLC linking autocrine BBS and Shh circuitries and suggest Shh inhibitors as novel therapeutic strategies against this aggressive cancer type.

Parathyroid hormone (PTH) and PTH-related peptide domains contributing to activation of different PTH receptor-mediated signaling pathways.

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), acting through the osteoblast PTH1 receptor (PTH1R), play important roles in bone remodeling. Intermittent administration of PTH(1-34) (teriparatide) leads to bone formation, whereas continuous administration paradoxically leads to bone resorption. Activation of PTH1R promotes regulation of multiple signaling pathways, including G(s)/cAMP/protein kinase A, G(q)/calcium/protein kinase C, ß-arrestin recruitment, and extracellular signal-related kinase (ERK)1/2 phosphorylation, as well as receptor internalization, but their role in promoting anabolic and catabolic actions of PTH(1-34) are unclear. In the present investigation, a collection of PTH(1-34) and PTHrP(1-34) peptide analogs were evaluated in orthogonal human PTH1R (hPTH1R) functional assays capturing G(s)- and G(q)-signaling, ß-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization to further define the patterns of PTH1R signaling that they stimulate and further establish peptide domains contributing to agonist activity. Results indicate that both N- and C-terminal domains of PTH and PTHrP are critical for activation of signaling pathways. However, modifications of both regions lead to more substantial decreases in agonist potency and efficacy to stimulate G(q)-signaling, ß-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization than to stimulate G(s)-signaling. The substantial contribution of the peptide C-terminal domain in activation of hPTH1R signaling suggests a role in positioning of the peptide N-terminal region into the receptor J-domain. Several PTH and PTHrP peptides evaluated in this study promote different patterns of biased agonist signaling and may serve as useful tools to further elucidate therapeutically relevant PTH1R signaling in osteoblasts. With a better understanding of therapeutically relevant signaling, novel biased peptides with desired signaling could be designed for safer and more effective treatment of osteoporosis.

Gαq G proteins modulate MMP-9 gelatinase during remodeling of the murine femoral artery.

BACKGROUND: Vessels heal after injury and G protein-coupled receptors are involved in the vascular smooth muscle cell proliferation required to form intimal hyperplasia. We have previously identified the role of Gαq in vascular smooth muscle cell proliferation in vitro. This study now examines the role of Gαq in the developing intimal hyperplasia in a murine model and the impact of disruption of Gαq signaling on intimal hyperplasia development. METHODS: We employed a murine femoral wire injury model in which a micro-wire is passed through a branch of the femoral artery and used to denude the common femoral artery. We perfusion-fixed specimens and stained sections with hematoxylin-eosin and Movat's stains such that morphometric analysis could be performed using an Image-Pro system. We also harvested additional specimens of femoral artery and snap-froze them for Western blotting or zymography, to allow for the study of G protein expression and both protease expression and activity. We used contralateral vessels as controls. We immersed additional vessels in pluronic gel containing the chemical Gαq G protein inhibitors GP-2A, siRNA to Gαq or adenovirus containing mutant inactive Gαq. RESULTS: Gαq expression increased in a time-dependent manner after femoral artery injury. Sham-operated vessels did not produce such a response. Inhibition of Gαq reduced cell proliferation without affecting cell migration. Interruption of Gαq signaling also inhibited the development of intimal hyperplasia. Inhibition of Gαq did not alter peak urinary-type plasminogen activator activity and expression, but did increase early plasminogen activator inhibitor-1 activity and expression. Inhibition of Gαq reduced peak metalloproteinase (MMP)-9 activity at Day 3 but did not influence peak MMP-2 activity at Day 7. Protein expression for MMP-9 was also decreased, but that of MMP-2 was not affected. There were no changes in the expression or the activity of the respective inhibitors for MMP-9 and MMP-2, and tissue inhibitor of metalloproteinases-1 and -2. CONCLUSIONS: These data demonstrate that femoral wire injury in the mouse is associated with a time-dependent increase in Gαq expression. Inhibition of Gαq alters cell proliferation and is associated with decreased MMP-9 expression and activity.