Rhynchosia volubilis Promotes Cell Survival via cAMP-PKA/ERK-CREB Pathway.
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ABSTRACT:
SUBMITTER: Ahn SH
PROVIDER: S-EPMC8778899 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:How the nervous system regulates bone remodeling is an exciting area of emerging research in bone biology. Accumulating evidence suggest that neurotransmitter-mediated inputs from neurons may act directly on osteoclasts. Dopamine is a neurotransmitter that can be released by hypothalamic neurons to regulate bone metabolism through the hypothalamic-pituitary-gonadal axis. Dopamine is also present in sympathetic nerves that penetrate skeletal structures throughout the body. It has been shown that dopamine suppresses osteoclast differentiation via a D2-like receptors (D2R)-dependent manner, but the intracellular secondary signaling pathway has not been elucidated. In this study, we found that cAMP-response element binding protein (CREB) activity responds to dopamine treatment during osteoclastogenesis. Considering the critical role of CREB in osteoclastogenesis, we hypothesize that CREB may be a critical target in dopamine's regulation of osteoclast differentiation. We confirmed that D2R is also present in RAW cells and activated by dopamine. Binding of dopamine to D2R inhibits the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway which ultimately decreases CREB phosphorylation during osteoclastogenesis. This was also associated with diminished expression of osteoclast markers that are downstream of CREB. Pharmacological activation of adenylate cyclase (to increase cAMP production) and PKA reverses the effect of dopamine on CREB activity and osteoclastogenesis. Therefore, we have identified D2R/cAMP/PKA/CREB as a candidate pathway that mediates dopamine's inhibition of osteoclast differentiation. These findings will contribute to our understanding of how the nervous and skeletal systems interact to regulate bone remodeling. This will enable future work toward elucidating the role of the nervous system in bone development, repair, aging, and degenerative disease.
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Project description:BACKGROUND:Genetic causes of premature ovarian insufficiency (POI) account for approximately 20?~?25% of patients. So far, only a few genes have been identified. RESULTS:Here, we first identified the c.1840C?>?A on G-protein signaling modulator 1 (GPSM1) as a susceptibility locus for POI in 10 sporadic POI patients by whole-exome sequencing. The frequency of GPSM1 c.1840C?>?A was then verified as 3/20 in a POI sample of 20 patients (including the above 10 patients) by Sanger sequencing. RT-PCR and western blot analysis showed the expression of GPSM1 in rat ovaries was increased in the large antral follicle stage compared to the primordial follicle stage (P?<?0.01). The cell proliferation assay (CCK8) and flow cytometry suggested that the small-interfering RNA-induced silencing of Gpsm1 significantly increased apoptosis and decreased proliferation of rat ovarian granulosa cells (GCs) (P?<?0.01). Furthermore, suppression of Gpsm1 in GCs reduced levels of cAMP, PKAc, p-CREB as well as the ratio of Bcl-2/Bax, and increased the expression of Caspase-3 and Cleaved Caspase-3 (P?<?0.01). CONCLUSIONS:In summary, this study identified a susceptibility variant GPSM1 c.1840C?>?A of POI for the first time. Gpsm1 was related to rat follicle development, and silencing of Gpsm1 increased apoptosis and decreased proliferation in rat GCs, possibly through inhibition of the cAMP-PKA-CREB pathway.
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Project description:In the cochlea, non-sensory supporting cells are directly connected to adjacent supporting cells via gap junctions that allow the exchange of small molecules. We have previously shown that the pharmacological regulation of gap junctions alleviates cisplatin (CDDP)-induced ototoxicity in animal models. In this study, we aimed to identify specific small molecules that pass through gap junctions in the process of CDDP-induced auditory cell death and suggest new mechanisms to prevent hearing loss. We found that the cyclic adenosine monophosphate (cAMP) inducer forskolin (FSK) significantly attenuated CDDP-induced auditory cell death in vitro and ex vivo. The activation of cAMP/PKA/CREB signaling was observed in organ of Corti primary cells treated with FSK, especially in supporting cells. Co-treatment with gap junction enhancers such as all-trans retinoic acid (ATRA) and quinoline showed potentiating effects with FSK on cell survival via activation of cAMP/PKA/CREB. In vivo, the combination of FSK and ATRA was more effective for preventing ototoxicity compared to either single treatment. Our study provides the new insight that gap junction-mediated intercellular communication of cAMP may prevent CDDP-induced ototoxicity.
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Project description:Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.
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Project description:The Nm23 metastasis suppressor family is involved in physiological and pathological processes including tumorigenesis and metastasis. Although the inverse correlation of Nm23 level with tumor metastasis potential has been widely observed, the mechanisms that regulate the expression of Nm23 remain poorly understood. Our previous studies have revealed that Nm23-H1/2 isoforms are upregulated by RGS19, a regulator of G protein signaling (RGS) protein which accelerates the termination of Gi signals. Here, we examined the ability of RGS19 to stimulate transcriptional regulation of Nm23 by screening a panel of luciferase reporter genes. Transient and stable overexpression of RGS19 upregulated the Nm23-H1/2 protein levels and activated several transcription factors including CREB, AP-1 and SRE in HEK293 cells. Interestingly, agents that increase the intracellular cAMP level and the phosphorylation of CREB (e.g., adrenergic receptor agonist, forskolin, and cAMP analogues) upregulated the expression of Nm23-H1/2 in HEK293 cells and several cancer cell lines including A549, HeLa, MDA-MB-231, and MDA-MB-435s cells. Conversely, inhibition of protein kinase A (PKA) by H-89 suppressed the phosphorylation of CREB and reduced the expression of Nm23-H1/2. Furthermore, activation of PKA attenuated cancer cell migration in wound healing and transwell assays. Collectively, these results revealed a PKA-dependent mechanism for controlling Nm23-H1/2 expression.
| S-EPMC5642529 | biostudies-literature
Project description:Nicotinamide (NAM) is the amide of nicotinic acid and a predominant precursor for NAD(+) biosynthesis via the salvage pathway. Sirt1 is a NAD(+)-dependent deacetylase, playing an important role in regulating cellular functions. Although hepatoprotective effect of NAM has been reported, the underlying mechanism remains elusive. ER stress, induced by saturated fatty acids, in specific palmitate, plays a pathological role in the development of nonalcoholic fatty liver disease. This study aims to determine the effect of NAM on palmitate-induced ER stress in hepatocytes and to elucidate molecular mechanisms behind. Both HepG2 cells and primary mouse hepatocytes were exposed to palmitate (conjugated to BSA at a 2:1 M ratio), NAM, or their combination for different durations. Cellular NAD(+) level, Sirt1 expression/activity, ER stress, as well as cAMP/PKA/CREB pathway activation were determined. NAM increased Sirt1 expression and enzymatic activity, which contributes to the ameliorative effect of NAM on palmitate-triggered ER stress. NAM increased intracellular NAD(+) level in hepatocytes, however, blocking the salvage pathway, a pathway for NAD(+) synthesis from NAM, only partially prevented NAM-induced Sirt1 upregulation while completely prevented NAD+ increase in response to NAM. Further mechanistic investigations revealed that NAM elevated intracellular cAMP level via suppressing PDE activity, leading to downstream PKA and CREB activation. Importantly, cAMP/PKA/CREB pathway blockade abolished not only NAM-induced Sirt1 upregulation, but also its protective effect against ER stress. Our results demonstrate that NAM protects hepatocytes against palmitate-induced ER stress in hepatocytes via upregulating Sirt1. Activation of the cAMP/PKA/CREB pathway plays a key role in NAM-induced Sirt1 upregulation.
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Project description:Oxytocin (OT) and its receptor (OTR) are expressed in the heart and are involved in the physiological cardiovascular functional system. Although it is known that OT/OTR signaling is cardioprotective by reducing the inflammatory response and improving cardiovascular function, the role of OT in the cardiac electrical excitation modulation has not been clarified. This study investigates the molecular mechanism of the action of OT on cardiomyocyte membrane excitation focusing on the L-type Ca2+ channel. Our methodology uses molecular biological methods and a patch-clamp technique on rat cardiomyocytes with OT, combined with several signal inhibitors and/or activators. Our results show that long-term treatment of OT significantly decreases the expression of Cav1.2 mRNA, and reduces the L-type Ca2+ channel current (ICa.L) in cardiomyocytes. OT downregulates the phosphorylated component of a transcription factor adenosine-3',5'-cyclic monophosphate (cAMP) response element binding protein (CREB), whose action is blocked by OTR antagonist and pertussis toxin, a specific inhibitor of the inhibitory GTP-binding regulators of adenylate cyclase, Gi. On the other hand, the upregulation of Cav1.2 mRNA expression by isoproterenol is halted by OT. Furthermore, inhibition of phospholipase C (PLC) was without effect on the OT action to downregulate Cav1.2 mRNA-which suggests a signal pathway of Gi/protein kinase A (PKA)/CREB mediated by OT/OTR. These findings indicate novel signaling pathways of OT contributing to a downregulation of the Cav1.2-L-type Ca2+ channel in cardiomyocytes.
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Project description:Non-alcoholic fatty liver disease (NAFLD), hallmarked by liver steatosis, is becoming a global concern, but effective and safe drugs for NAFLD are still lacking at present. Parathyroid hormone (PTH), the only FDA-approved anabolic treatment for osteoporosis, is important in calcium-phosphate homeostasis. However, little is known about its potential therapeutic effects on other diseases. Here, we report that intermittent administration of PTH ameliorated non-alcoholic liver steatosis in diet-induced obese (DIO) mice and db/db mice, as well as fasting-induced hepatic steatosis. In vitro, PTH inhibits palmitic acid-induced intracellular lipid accumulation in a parathyroid hormone 1 receptor (PTH1R)-dependent manner. Mechanistically, PTH upregulates the expression of genes involved in lipid β-oxidation and suppresses the expression of genes related to lipid uptake and de novo lipogenesis by activating the cAMP/PKA/CREB pathway. Taken together, our current finding proposes a new therapeutic role of PTH on NAFLD.
| S-EPMC9428460 | biostudies-literature
Project description:Malignant gliomas are one of the leading causes of cancer deaths worldwide, but chemoprevention strategies for them are few and poorly investigated. Here, we show that cholera toxin, the traditional biotoxin and well known inducer of accumulation of cellular cAMP, is capable of inducing differentiation on malignant gliomas in vitro with rat C6 and primary cultured human glioma cells. Cholera toxin-induced differentiation was characterized by typical morphological changes, increased expression of glial fibrillary acid protein, decreased expression of Ki-67, inhibition of cellular proliferation, and accumulation of cells in the G(1) phase of the cell cycle. Cholera toxin also triggered a significant reduction in the G(1) cell-cycle regulatory proteins cyclin D1 and Cdk2 along with an overexpression of cell-cycle inhibitory proteins p21(Cip1) and p27(Kip1). Abrogation of cAMP-dependent protein kinase A activity by protein kinase A inhibitor or silencing of cAMP-responsive element binding proteins by RNA interference resulted in suppressed differentiation. These findings imply the attractiveness of cholera toxin as a drug candidate for further development of differentiation therapy. Furthermore, activation of the protein kinase A/cAMP-responsive element binding protein pathway may be a key and requisite factor in glioma differentiation.
| S-EPMC1940034 | biostudies-literature
Project description:BackgroundRestoration of salivary gland function in Sjogren's syndrome (SS) is still a challenge. Dental pulp stem cells (DPSCs) derived exosomes had shown anti-inflammatory, anti-oxidative, immunomodulatory, and tissue function restorative abilities. However, the salivary gland function restoration potential of DPSCs-derived exosomes (DPSC-Exos) during SS has not been investigated yet.MethodsDPSC-Exos was isolated by ultracentrifugation methods and characterized. Salivary gland epithelial cells (SGEC) were treated with interferon-gamma (IFN-γ) to mimic SS in vitro and cultured with or without DPSC-Exos. SGEC survival and aquaporin 5 (AQP5) expression were analyzed. mRNA sequencing and bioinformatics analysis were performed in IFN-γ vs. DPSC-Exos+ IFN-γ treated SGEC. Non-obese diabetic (NOD)/ltj female mice (SS model), were intravenously administered with DPSC-Exos, and salivary gland functions and SS pathogenicity were analyzed. Furthermore, the mRNA sequencing and bioinformatics predicted mechanism of the therapeutic effect of DPSC-Exos was further investigated both in vitro and in vivo using RT-qPCR, Western blot, immunohistochemistry, immunofluorescence, flowcytometry analysis.ResultsDPSC-Exos partially rescued IFN-γ triggered SGEC death. IFN-γ inhibited AQP5 expression in SGEC and DPSC-Exos reversed this effect. Transcriptome analysis showed GPER was the upregulated DEG in DPSC-Exos-treated SGEC with a positive correlation with salivary secretion-related DEGs. Pathway enrichment analysis revealed that DEGs were mainly attributed to estrogen 16 alpha-hydroxylase activity, extracellular exosome function, cAMP signaling, salivary secretion, and estrogen signaling. Intravenous injection of DPSC-Exos in NOD/ltj mice alleviated the SS syndrome as indicated by the increased salivary flow rate, attenuated glandular inflammation, and increased AQP5 expression. GPER was also upregulated in the salivary gland of DPSC-Exos-treated NOD/ltj mice compared with the PBS-treated NOD/ltj mice. IFN-γ+DPSC-Exos-treated SGEC showed higher expression of AQP5, p-PKA, cAMP, and intracellular Ca2+ levels compared with IFN-γ-treated SGEC. These effects were reversed by the inhibition of GPER.ConclusionsOur results showed that DPSC-Exos revitalize salivary gland epithelial cell function during SS via the GPER-mediated cAMP/PKA/CREB pathway suggesting the possible therapeutic potential of DPSC-Exos in SS-treatment.
| S-EPMC10239098 | biostudies-literature