Antidepression action of BDNF requires and is mimicked by Gαi1/3 expression in the hippocampus.

Stress-related alterations in brain-derived neurotrophic factor (BDNF) expression, a neurotrophin that plays a key role in synaptic plasticity, are believed to contribute to the pathophysiology of depression. Here, we show that in a chronic mild stress (CMS) model of depression the Gαi1 and Gαi3 subunits of heterotrimeric G proteins are down-regulated in the hippocampus, a key limbic structure associated with major depressive disorder. We provide evidence that Gαi1 and Gαi3 (Gαi1/3) are required for the activation of TrkB downstream signaling pathways. In mouse embryonic fibroblasts (MEFs) and CNS neurons, Gαi1/3 knockdown inhibited BDNF-induced tropomyosin-related kinase B (TrkB) endocytosis, adaptor protein activation, and Akt-mTORC1 and Erk-MAPK signaling. Functional studies show that Gαi1 and Gαi3 knockdown decreases the number of dendrites and dendritic spines in hippocampal neurons. In vivo, hippocampal Gαi1/3 knockdown after bilateral microinjection of lentiviral constructs containing Gαi1 and Gαi3 shRNA elicited depressive behaviors. Critically, exogenous expression of Gαi3 in the hippocampus reversed depressive behaviors in CMS mice. Similar results were observed in Gαi1/Gαi3 double-knockout mice, which exhibited severe depressive behaviors. These results demonstrate that heterotrimeric Gαi1 and Gαi3 proteins are essential for TrkB signaling and that disruption of Gαi1 or Gαi3 function could contribute to depressive behaviors.

Gαi2-protein-mediated signal transduction: central nervous system molecular mechanism countering the development of sodium-dependent hypertension.

Excess dietary salt intake is an established cause of hypertension. At present, our understanding of the neuropathophysiology of salt-sensitive hypertension is limited by a lack of identification of the central nervous system mechanisms that modulate sympathetic outflow and blood pressure in response to dietary salt intake. We hypothesized that impairment of brain Gαi2-protein-gated signal transduction pathways would result in increased sympathetically mediated renal sodium retention, thus promoting the development of salt-sensitive hypertension. To test this hypothesis, naive or renal denervated Dahl salt-resistant and Dahl salt-sensitive (DSS) rats were assigned to receive a continuous intracerebroventricular control scrambled or a targeted Gαi2-oligodeoxynucleotide infusion, and naive Brown Norway and 8-congenic DSS rats were fed a 21-day normal or high-salt diet. High salt intake did not alter blood pressure, suppressed plasma norepinephrine, and evoked a site-specific increase in hypothalamic paraventricular nucleus Gαi2-protein levels in naive Brown Norway, Dahl salt-resistant, and scrambled oligodeoxynucleotide-infused Dahl salt-resistant but not DSS rats. In Dahl salt-resistant rats, Gαi2 downregulation evoked rapid renal nerve-dependent hypertension, sodium retention, and sympathoexcitation. In DSS rats, Gαi2 downregulation exacerbated salt-sensitive hypertension via a renal nerve-dependent mechanism. Congenic-8 DSS rats exhibited sodium-evoked paraventricular nucleus-specific Gαi2-protein upregulation and attenuated hypertension, sodium retention, and global sympathoexcitation compared with DSS rats. These data demonstrate that paraventricular nucleus Gαi2-protein-gated pathways represent a conserved central molecular pathway mediating sympathoinhibitory renal nerve-dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Impairment of this mechanism contributes to the development of salt-sensitive hypertension.

Gαi2- and Gαi3-deficient mice display opposite severity of myocardial ischemia reperfusion injury.

G-protein-coupled receptors (GPCRs) are the most abundant receptors in the heart and therefore are common targets for cardiovascular therapeutics. The activated GPCRs transduce their signals via heterotrimeric G-proteins. The four major families of G-proteins identified so far are specified through their α-subunit: Gαi, Gαs, Gαq and G12/13. Gαi-proteins have been reported to protect hearts from ischemia reperfusion injury. However, determining the individual impact of Gαi2 or Gαi3 on myocardial ischemia injury has not been clarified yet. Here, we first investigated expression of Gαi2 and Gαi3 on transcriptional level by quantitative PCR and on protein level by immunoblot analysis as well as by immunofluorescence in cardiac tissues of wild-type, Gαi2-, and Gαi3-deficient mice. Gαi2 was expressed at higher levels than Gαi3 in murine hearts, and irrespective of the isoform being knocked out we observed an up regulation of the remaining Gαi-protein. Myocardial ischemia promptly regulated cardiac mRNA and with a slight delay protein levels of both Gαi2 and Gαi3, indicating important roles for both Gαi isoforms. Furthermore, ischemia reperfusion injury in Gαi2- and Gαi3-deficient mice exhibited opposite outcomes. Whereas the absence of Gαi2 significantly increased the infarct size in the heart, the absence of Gαi3 or the concomitant upregulation of Gαi2 dramatically reduced cardiac infarction. In conclusion, we demonstrate for the first time that the genetic ablation of Gαi proteins has protective or deleterious effects on cardiac ischemia reperfusion injury depending on the isoform being absent.

Differential effects of statins (pravastatin or simvastatin) on ventricular ectopic complexes: Galpha(i2), a possible molecular marker for ventricular irritability.

Retrospective studies suggest that statins might exert an antiarrhythmic effect on the heart. The mechanism of this effect is unclear. Parasympathetic stimulation of the heart has been shown to protect against ventricular arrhythmias. The goal of this study was to determine the effect of statins on ventricular arrhythmias and its correlation with changes in parasympathetic responsiveness and Galpha(i2) expression. Patients were randomized to pravastatin and simvastatin in a double-blind crossover design. Ventricular arrhythmias were determined by analysis of 24-hour Holter recordings. Spectral RR interval analysis of Holter studies determined peak high-frequency power fraction, which reflects parasympathetic modulation of heart rate. Expression of Galpha(i2), a molecular component of the parasympathetic response pathway, was determined by Western blots of patients' lymphocytes. Pravastatin treatment decreased the incidence of ventricular premature complexes by 22.5 + or - 3.4% (n = 20, p <0.05), couplets, and runs of 3 to 6 beats of nonsustained ventricular tachycardia from 9.8 + or - 2.67 to 3.9 + or - 1.25 events/patient/24 hours (n = 12, p <0.05). Pravastatin increased peak high-frequency fraction by 29.8 + or - 4.3% (n = 33, p <0.001), while Galpha(i2) expression increased by 51.3 + or - 22.5% (n = 21, p <0.05). Effects of simvastatin on ventricular premature complexes and nonsustained ventricular tachycardia were not significant. Relative changes in couplets and nonsustained ventricular tachycardia in pravastatin-treated patients correlated negatively with changes in Galpha(i2) and high-frequency fraction (rho = -0.588 and rho = -0.763, respectively, n = 12, p <0.05). In conclusion, these data suggest that pravastatin might decrease cardiac irritability via an increase in parasympathetic responsiveness and that changes in Galpha(i2) expression might serve as a molecular marker for this effect, which might play a role in the molecular mechanism of the antiarrhythmic effect of statins.

Expression profiling identifies genes that predict recurrence of breast cancer after adjuvant CMF-based chemotherapy.

Cyclophosphamide, methotrexate and 5-fluorouracile (CMF)-based chemotherapy for adjuvant treatment of breast cancer reduces the risk of relapse. In this exploratory study, we tested the feasibility of identifying molecular markers of recurrence in CMF-treated patients. Using Affymetrix U133A GeneChips, RNA samples from 19 patients with primary breast cancer who had been uniformly treated with adjuvant CMF chemotherapy were analyzed. Two supervised class prediction approaches were used to identify gene markers that can best discriminate between patients who would experience relapse and patients who would remain disease-free. An additional independent validation set of 51 patients and 21 genes were analyzed by quantitative RT-PCR. Applying different algorithms to evaluate our microarray data, we identified two gene expression signatures of 21 and 12 genes containing eight overlapping genes, that predict recurrence in 19 cases with high accuracy (94%). Quantitative RT-PCR demonstrated that six genes from the combined signatures (CXCL9, ITSN2, GNAI2, H2AFX, INDO, and MGC10986) were significantly differentially expressed in the recurrence versus the non-recurrence group of the 19 cases and the independent breast cancer patient cohort (n = 51) treated with CMF. High expression levels of CXCL9, ITSN2, and GNAI2 were associated with prolonged disease-free survival (DFS) (P = 0.029, 0.018 and 0.032, respectively). When patients were stratified by combined CXCL9/ITSN2 or CXCL9/FLJ22028 tumor levels, they exhibited significantly different disease-free survival curves (P = 0.0073 and P = 0.005, respectively). Finally, the CXCL9/ITSN2 and CXCL9/FLJ22028 ratio was an independent prognostic factor (P = 0.034 and P = 0.003, respectively) for DFS by multivariate Cox analysis in the 70-patient cohort. Our data highlight the feasibility of a prognostic assay that is applicable to therapeutic decision-making for breast cancer. Whether the biomarker profile is chemotherapy-specific or whether it is a more general indicator of bad prognosis of breast cancer patients remains to be explored.

Cryptic peptide derived from the rat neuropeptide FF precursor affects G-proteins linked to opioid receptors in the rat brain.

Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place preference induced by morphine and reversed the antinociceptive activity of morphine (5mg/kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA coding for Galpha(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor.

Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system.

To date, over 100 vomeronasal receptor type 1 (V1R) genes have been identified in rodents. V1R is specifically expressed in the rodent vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. Recently, 21 putatively functional V1R genes were identified in the genome database of the amphibian Xenopus tropicalis. Amphibians are the first vertebrates to possess a VNO. In order to determine at which point during evolution the vertebrate V1R genes began to function in the vomeronasal system, we analyzed the expression of all putatively functional V1R genes in Xenopus olfactory organs. We found that V1R expression was not detected in the VNO but was specifically detected in the main olfactory epithelium (MOE). We also observed that V1R-expressing cells in the MOE coexpressed Gi2, thus suggesting that the V1R-Gi2-mediated signal transduction pathway, which is considered to play an important role in pheromone reception in the rodent VNO, exists in the amphibian MOE. These results suggest that V1R-mediated signal transduction pathway functions in Xenopus main olfactory system.

Specific leukotriene receptors couple to distinct G proteins to effect stimulation of alveolar macrophage host defense functions.

Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB(4) and LTD(4) also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLT1) receptor and cysteinyl LT type 1 (cysLT1) receptor, respectively. Although both Galpha(i) and Galpha(q) proteins have been shown to be coupled to both BLT1 and cysLT1 receptors in transfected cell systems, there is little known about specific G protein subunit coupling to LT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB(4) and LTD(4). LTB(4) but not LTD(4) reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of FcgammaR-mediated phagocytosis and bacterial killing by LTB(4) was also PTX-sensitive, whereas that induced by LTD(4) was not. LTD(4) and LTB(4) induced Ca(2+) and intracellular inositol monophosphate accumulation, respectively, highlighting the role of Galpha(q) protein in mediating PTX-insensitive LTD(4) enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific Galpha(q/11) and Galpha(i3) subunits, but not Galpha(i2) or G(beta)gamma, in LTB(4)-enhanced phagocytosis. The selective importance of Galpha(q/11) protein was also demonstrated in LTD(4)-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.

Impaired trafficking of Gnai2+/- and Gnai2-/- T lymphocytes: implications for T cell movement within lymph nodes.

Signals generated by the engagement of chemoattractants with their cognate receptors orchestrate lymphocyte movements into and out of lymphoid organs and sites of inflammation. Yet, the role of chemokines in organizing lymphocyte movements in lymphoid organs is controversial. Recent evidence suggests that the extensive network of fibroblastic reticular cells within the T cell areas helps guide T cells. The expression of adhesion molecules and chemokines by fibroblastic reticular cells most likely facilitates their influence on T cell movements. Consistent with this hypothesis, CD4 T cells with defective chemokine receptor signaling move very differently within lymph nodes than do normal cells. For the imaging studies, we used CD4 T cells prepared from Gnai2(-/-) mice, which lack G(alphai2) expression. We first demonstrate that CD4 as well as CD8 T cells from these mice are markedly defective in chemokine receptor signaling. Gnai2(-/-) T cells have profound defects in chemokine-induced intracellular calcium mobilization, chemotaxis, and homing, whereas Gnai2(+/-) T cells exhibit modest defects. Intravital imaging revealed that within the inguinal lymph nodes Gnai2(-/-) CD4 T accumulate at the cortical ridge, poorly accessing the lymph node paracortex. They also lack the customary amoeboid-like cell movements and active membrane projections observed with normal CD4 T cells. These results demonstrate the importance of G(alphai2) for T lymphocyte chemokine receptor signaling and argue that local chemoattractants regulate the movement of CD4 T cells in lymph nodes.

Intracerebroventricular antisense knockdown of G alpha i2 results in ciliary stasis and ventricular dilatation in the rat.

BACKGROUND: In the CNS, the heterotrimeric G protein Galphai2 is a minor Galpha subunit with restricted localization in the ventricular regions including the ependymal cilia. The localization of Galphai2 is conserved in cilia of different tissues, suggesting a particular role in ciliary function. Although studies with Galphai2-knockout mice have provided information on the role of this Galpha subunit in peripheral tissues, its role in the CNS is largely unknown. We used intracerebroventricular (icv) antisense administration to clarify the physiological role of Galphai2 in the ventricular system. RESULTS: High resolution MRI studies revealed that continuous icv-infusion of Galphai2-specific antisense oligonucleotide caused unilateral ventricular dilatation that was restricted to the antisense-receiving ventricle. Microscopic analysis demonstrated ependymal cell damage and loss of ependymal cilia. Attenuation of Galphai2 in ependymal cells was confirmed by immunohistochemistry. Ciliary beat frequency measurements on cultured ependymal cells indicated that antisense administration resulted in ciliary stasis. CONCLUSION: Our results establish that Galphai2 has an essential regulatory role in ciliary function and CSF homeostasis.

Localization and variable expression of G alpha(i2) in human endometrium and Fallopian tubes.

BACKGROUND: Heterotrimeric G proteins take part in membrane-mediated cell signalling and have a role in hormonal regulation. This study clarifies the expression and localization of the G protein subunit G alpha(i2) in the human endometrium and Fallopian tube and changes in G alpha(i2) expression in human endometrium during the menstrual cycle. METHODS: The expression of G alpha(i2) was identified by Polymerase chain reaction (PCR), and localization confirmed by immunostaining. Cyclic changes in G alpha(i2) expression during the menstrual cycle were evaluated by quantitative real-time PCR. RESULTS: We found G alpha(i2) to be expressed in human endometrium, Fallopian tube tissue and in primary cultures of Fallopian tube epithelial cells. Our studies revealed enriched localization of G alpha(i2) in Fallopian tube cilia and in endometrial glands. We showed that G alpha(i2) expression in human endometrium changes significantly during the menstrual cycle, with a higher level in the secretory versus proliferative and menstrual phases (P < 0.05). CONCLUSIONS: G alpha(i2) is specifically localized in human Fallopian tube epithelial cells, particularly in the cilia, and is likely to have a cilia-specific role in reproduction. Significantly variable expression of G alpha(i2) during the menstrual cycle suggests G alpha(i2) might be under hormonal regulation in the female reproductive tract in vivo.

Valproic acid-induced gene expression through production of reactive oxygen species.

Valproic acid (VPA) is a widely used anticonvulsive agent that has profound antiproliferative effects in many cell types, as well as inductive effects on a number of genes. The mechanism of its gene-inducing effect has been reported to involve transcription factors, Sp1 and activator protein-1. Using two well-characterized antioxidant response element (ARE)-driven gene promoters, i.e., mouse heme oxygenase-1 and human NAD(P)H:quinone oxidoreductase 1 genes as tools to monitor the transcriptional response to VPA, we show here that VPA-induced gene transcription was abrogated by antioxidants. With the human Galpha(i2) gene promoter, which was previously used to establish the involvement of Sp1 in the transcriptional action of VPA, we found that VPA-induced gene transcription was also blocked by antioxidants. Mutation of the ARE (5'-TGACtggGC-3') in this promoter abrogated the transcriptional response to VPA. With such mutants, the NADPH oxidase inhibitor, diphenyleneiodonium, had no effect on VPA-induced transcription. In gel mobility shift assays, VPA-induced binding of nuclear proteins to a DNA probe containing the relevant ARE sequence in the Galpha(i2) gene promoter was decreased in nuclear extracts from cells pretreated with antioxidants. Chromatin immunoprecipitation assays showed that the prototype redox-sensitive transcription factors, Nrf2, small Maf protein(s), and c-Fos, were recruited to this promoter in VPA-treated cells. Overall, this study reveals that the mechanism of the transcriptional response to VPA includes VPA-induced production of reactive oxygen species which induce the activation of redox-sensitive transcription factors that interact with the ARE.