SARS-CoV-2 E protein-induced THP-1 pyroptosis is reversed by Ruscogenin.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an emerging pathogenic coronavirus, has been reported to cause excessive inflammation and dysfunction in multiple cells and organs, but the underlying mechanisms remain largely unknown. Here we showed exogenous addition of SARS-CoV-2 envelop protein (E protein) potently induced cell death in cultured cell lines, including THP-1 monocytic leukemia cells, endothelial cells, and bronchial epithelial cells, in a time- and concentration-dependent manner. SARS-CoV-2 E protein caused pyroptosis-like cell death in THP-1 and led to GSDMD cleavage. In addition, SARS-CoV-2 E protein upregulated the expression of multiple pro-inflammatory cytokines that may be attributed to activation of NF-κB, JNK and p38 signal pathways. Notably, we identified a natural compound, Ruscogenin, effectively reversed E protein-induced THP-1 death via inhibition of NLRP3 activation and GSDMD cleavage. In conclusion, these findings suggested that Ruscogenin may have beneficial effects on preventing SARS-CoV-2 E protein-induced cell death and might be a promising treatment for the complications of COVID-19.

CD38 deficiency promotes skeletal muscle and brown adipose tissue energy expenditure through activating NAD+-Sirt1-PGC1α signaling pathway.

Obesity is a metabolic syndrome characterized by abnormal lipid deposition and energy imbalance. CD38 is a single-chain transmembrane glycoprotein widely expressed in a variety of cell types. The roles of skeletal muscle and brown fat in CD38 deficiency under HFD-induced obesity remain unknown. In this study, we established obesity model with HFD and examined the changes in metabolites with metabonomics. Our results showed that CD38 expression was increased in muscle and brown fat after HFD treatment. Moreover, the results of metabonomics showed that CD38 deficiency significantly altered the metabolites in energy metabolism, cofactor generation, and redox homeostasis. Furthermore, CD38 deficiency reduced the expressions of NADPH oxidase 2 and FASN in mRNA level. We found that the expressions of Sirt1, Sirt3, and PGC1α were upregulated in CD38-deficient muscle tissue. In brown fat, the Sirt1-3, cell death inducing DFFA-like effector A, ELOVL3, and Dio2 expressions were increased in CD38-deficient mice. Our results showed the uncoupling protein 1 expression was upregulated. And NAD+ supplementation increased the expression of Sirt1 and PGC1α after palmitic acid treatment. Taken together, our results demonstrated that the protection of CD38 deficiency on HFD-induced obesity was related to the inhibition of oxidative stress and increasing energy expenditure via activating NAD+/Sirtuins signaling pathways in muscle and brown fat.

CD38 Deficiency Alleviates Diabetic Cardiomyopathy by Coordinately Inhibiting Pyroptosis and Apoptosis.

Diabetic cardiomyopathy is one of the diabetes mellitus-induced cardiovascular complications that can result in heart failure in severe cases, which is characterized by cardiomyocyte apoptosis, local inflammation, oxidative stress, and myocardial fibrosis. CD38, a main hydrolase of NAD+ in mammals, plays an important role in various cardiovascular diseases, according to our previous studies. However, the role of CD38 in diabetes-induced cardiomyopathy is still unknown. Here, we report that global deletion of the CD38 gene significantly prevented diabetic cardiomyopathy induced by high-fat diet plus streptozotocin (STZ) injection in CD38 knockout (CD38-KO) mice. We observed that CD38 expression was up-regulated, whereas the expression of Sirt3 was down-regulated in the hearts of diabetic mice. CD38 deficiency significantly promoted glucose metabolism and improved cardiac functions, exemplified by increased left ventricular ejection fraction and fractional shortening. In addition, we observed that CD38 deficiency markedly decreased diabetes or high glucose and palmitic acid (HG + PA)-induced pyroptosis and apoptosis in CD38 knockout hearts or cardiomyocytes, respectively. Furthermore, we found that the expression levels of Sirt3, mainly located in mitochondria, and its target gene FOXO3a were increased in CD38-deficient hearts and cardiomyocytes with CD38 knockdown under diabetic induction conditions. In conclusion, we demonstrated that CD38 deficiency protected mice from diabetes-induced diabetic cardiomyopathy by reducing pyroptosis and apoptosis via activating NAD+/Sirt3/FOXO3a signaling pathways.

FKBP12.6 protects heart from AngII-induced hypertrophy through inhibiting Ca2+ /calmodulin-mediated signalling pathways in vivo and in vitro.

We previously observed that disruption of FK506-binding protein 12.6 (FKBP12.6) gene resulted in cardiac hypertrophy in male mice. Studies showed that overexpression of FKBP12.6 attenuated thoracic aortic constriction (TAC)-induced cardiac hypertrophy in mice, whereas the adenovirus-mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial. In this study, we aimed to investigate the roles and mechanisms of FKBP12.6 in angiotensin II (AngII)-induced cardiac hypertrophy using various transgenic mouse models in vivo and in vitro. FKBP12.6 knockout (FKBP12.6-/- ) mice and cardiac-specific FKBP12.6 overexpressing (FKBP12.6 TG) mice were infused with AngII (1500 ng/kg/min) for 14 days subcutaneously by implantation of an osmotic mini-pump. The results showed that FKBP12.6 deficiency aggravated AngII-induced cardiac hypertrophy, while cardiac-specific overexpression of FKBP12.6 prevented hearts from the hypertrophic response to AngII stimulation in mice. Consistent with the results in vivo, overexpression of FKBP12.6 in H9c2 cells significantly repressed the AngII-induced cardiomyocyte hypertrophy, seen as reductions in the cell sizes and the expressions of hypertrophic genes. Furthermore, we demonstrated that the protection of FKBP12.6 on AngII-induced cardiac hypertrophy was involved in reducing the concentration of intracellular Ca2+ ([Ca2+ ]i), in which the protein significantly inhibited the key Ca2+ /calmodulin-dependent signalling pathways such as calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4), calmodulin kinaseII (CaMKII)/MEF-2, AKT/Glycogen synthase kinase 3ß (GSK3ß)/NFATc4 and AKT/mTOR signalling pathways. Our study demonstrated that FKBP12.6 protects heart from AngII-induced cardiac hypertrophy through inhibiting Ca2+ /calmodulin-mediated signalling pathways.

CD38 Deficiency Protects Heart from High Fat Diet-Induced Oxidative Stress Via Activating Sirt3/FOXO3 Pathway.

BACKGROUND/AIMS: Previous studies showed that CD38 deficiency protected heart from ischemia/reperfusion injury and high fat diet (HFD)-induced obesity in mice. However, the role of CD38 in HFD-induced heart injury remains unclear. In the present study, we have investigated the effects and mechanisms of CD38 deficiency on HFD-induced heart injury. METHODS: The metabolites in heart from wild type (WT) and CD38 knockout (CD38-/-) mice were examined using metabolomics analysis. Cell viability, lactate hydrogenase (LDH) release, super oxide dismutase (SOD) activity, reactive oxygen species (ROS) production, triglyceride concentration and gene expression were examined by biochemical analysis and QPCR. RESULTS: Our results revealed that CD38 deficiency significantly elevated the intracellular glutathione (GSH) concentration and GSH/GSSG ratio, decreased the contents of free fatty acids and increased intracellular NAD+ level in heart from CD38-/- mice fed with HFD. In addition, in vitro knockdown of CD38 significantly attenuated OA-induced cellular injury, ROS production and lipid synthesis. Furthermore, the expression of mitochondrial deacetylase Sirt3 as well as its target genes FOXO3 and SOD2 were markedly upregulated in the H9C2 cell lines after OA stimulation. In contrast, the expressions of NOX2 and NOX4 were significantly decreased in the cells after OA stimulation. CONCLUSION: Our results demonstrated that CD38 deficiency protected heart from HFD-induced oxidative stress via activating Sirt3/FOXO3-mediated anti-oxidative stress pathway.

CD38 promotes angiotensin II-induced cardiac hypertrophy.

Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and regulated by various signalling pathways. Recently, we observed that mouse embryonic fibroblasts from CD38 knockout mice were significantly resistant to oxidative stress such as H2 O2 -induced injury and hypoxia/reoxygenation-induced injury. In addition, we also found that CD38 knockout mice protected heart from ischaemia reperfusion injury through activating SIRT1/FOXOs-mediated antioxidative stress pathway. However, the role of CD38 in cardiac hypertrophy is not explored. Here, we investigated the roles and mechanisms of CD38 in angiotensin II (Ang-II)-induced cardiac hypertrophy. Following 14 days of Ang-II infusion with osmotic mini-pumps, a comparable hypertension was generated in both of CD38 knockout and wild-type mice. However, the cardiac hypertrophy and fibrosis were much more severe in wild-type mice compared with CD38 knockout mice. Consistently, RNAi-induced knockdown of CD38 decreased the gene expressions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and reactive oxygen species generation in Ang-II-stimulated H9c2 cells. In addition, the expression of SIRT3 was elevated in CD38 knockdown H9c2 cells, in which SIRT3 may further activate the FOXO3 antioxidant pathway. The intracellular Ca2+ release induced by Ang-II markedly decreased in CD38 knockdown H9c2 cells, which might be associated with the decrease of nuclear translocation of NFATc4 and inhibition of ERK/AKT phosphorylation. We concluded that CD38 plays an essential role in cardiac hypertrophy probably via inhibition of SIRT3 expression and activation of Ca2+ -NFAT signalling pathway. Thus, CD38 may be a novel target for treating cardiac hypertrophy.

Inhibition of NAMPT aggravates high fat diet-induced hepatic steatosis in mice through regulating Sirt1/AMPKα/SREBP1 signaling pathway.

BACKGROUND: Nonalcoholic fatty liver disease is one of the most common liver diseases in the world and is a typical hepatic manifestation of metabolic syndrome which is characterized with lipid accumulation in liver. Nicotinamide phosphoribosyltransferase (NAMPT) has been recently identified as an enzyme involved in nicotinamide adenine dinucleotide (NAD+) biosynthesis and plays an important role in cellular metabolism in variety of organs in mammals. The aim of this study was to investigate the effects of NAMPT on high fat diet-induced hepatic steatosis. METHODS: Hepatic steatosis model was induced by high fat diet (HFD) in C57BL/6 mice in vivo. HepG2 and Hep1-6 hepatocytes were transfected with NAMPT vector plasmid or treated with NAMPT inhibitor FK866 and then incubated with oleic acid. Lipids accumulation was examined by HE staining or oil red staining. Quantitative RT-PCR and Western blot were used to measure expressions of the genes involved in lipogenic synthesis. RESULTS: FK866 significantly promoted liver steatosis in the mice fed with HFD and hepatic lipid accumulation in vitro, accompanied by the increases of the expressions of lipogenic genes such as sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FASN). Nicotinamide mononucleotide (NMN) and NAD+ significantly rescued the actions of FK866 in vitro. In contrast, overexpression of NAMPT in HepG2 and Hep1-6 hepatocytes ameliorated hepatic lipid accumulation. In addition, FK866 decreased the protein levels of Sirt1 and phospho-AMPKα in liver of the HFD fed mice. Furthermore, Resveratrol, a Sirt1 activator, significantly reduced lipogenic gene expressions, while EX-527, a Sirt1 specific inhibitor, had the opposite effects. CONCLUSION: Our results demonstrated that inhibition of NAMPT aggravated the HFD- or oleic acid-induced hepatic steatosis through suppressing Sirt1-mediated signaling pathway. On the one hand, the inhibition of NAMPT reduced the production of NAD+ through inhibiting the NAD+ salvage pathway, resulting in the decrease of Sirt1 activity, and then attenuated the deacetylation of SREBP1 in which the inhibition of SREBP1 activity promoted the expressions of FASN and ACC. On the other hand, the reduced Sirt1 activity alleviated the activation of AMPKα to further enhance SREBP1 activities.

Sleep and circadian rhythm disturbance in kidney stone disease: a narrative review.

The circadian rhythm generated by circadian clock genes functions as an internal timing system. Since the circadian rhythm controls abundant physiological processes, the circadian rhythm evolved in organisms is salient for adaptation to environmental change. A disturbed circadian rhythm is a trigger for numerous pathological events. Recently, accumulated data have indicated that kidney stone disease (KSD) is related to circadian rhythm disturbance. However, the mechanism between them has not been fully elucidated. In this narrative review, we summarized existing evidence to illustrate the possible association between circadian rhythm disturbance and KSD based on the epidemiological studies and risk factors that are linked to circadian rhythm disturbance and discuss some chronotherapies for KSD. In summary, KSD is associated with systemic disorders. Metabolic syndrome, inflammatory bowel disease, and microbiome dysbiosis are the major risk factors supported by sufficient data to cause KSD in patients with circadian rhythm disturbance, while others including hypertension, vitamin D deficiency, parathyroid gland dysfunction, and renal tubular damage/dysfunction need further investigation. Then, some chronotherapies for KSD were confirmed to be effective, but the molecular mechanism is still unclear.

Nicaraven protects against endotoxemia-induced inflammation and organ injury through modulation of AMPK/Sirt1 signaling in macrophages.

Endotoxemia is a disease characterized by systemic inflammatory responses and organ injury caused by lipopolysaccharide (LPS) infection, with high mortality. Nicaraven (AVS), a potent hydroxyl radical scavenger, has been proven to regulate the inflammatory response in tumors. To investigate the protective effects and mechanisms of AVS in endotoxemia, mice were injected intraperitoneally with LPS to induce endotoxemia. AVS treatment significantly decreased the levels of pro-inflammatory cytokines in the serum, reduced neutrophil infiltration, attenuated multiple organ injury, and increased the survival rate in LPS-challenged mice. In the LPS-induced inflammatory model of macrophages, AVS inhibited macrophage activation, suppressed nitric oxide (NO) production, and inhibited the expression and secretion of pro-inflammatory cytokines. Mechanistically, AVS treatment up-regulated silence information regulator transcript-1 (Sirt1) expression in a time- and dose-dependent manner. AVS treatment activated the AMP-dependent protein kinase (AMPK)/Sirt1 signaling pathway and suppressed the activation of nuclear factor kappa B (NF-κB) in macrophages exposed to LPS. However, the anti-inflammatory effects of AVS could be reversed by the AMPK, the Sirt1 inhibitor, or the histone deacetylase inhibitor. We confirmed that the AMPK inhibitor inhibited AVS-mediated AMPK/Sirt1 activation and NF-κB p65 acetylation. These results suggested that AVS alleviated endotoxemia by activating the AMPK/Sirt1 signaling pathway in macrophages.

Human amniotic mesenchymal stem cells-conditioned medium protects mice from high-fat diet-induced obesity.

BACKGROUND: Obesity is a metabolic disorder syndrome characterized by excessive fat accumulation that is related to many diseases. Human amniotic mesenchymal stem cells (hAMSCs) have a great potential for cell-based therapy due to their characteristics such as pluripotency, low immunogenicity, no tumorigenicity, potent paracrine effects, and no ethical concern. Recently, we observed that both hAMSCs and their conditioned medium (hAMSCs-CM) efficiently repaired skin injury, inhibited hepatocellular carcinoma, and alleviated high-fat diet (HFD)-induced diabetes. However, the effects and the underlying mechanisms of hAMSCs-CM on high-fat diet (HFD)-induced obesity were not explored. METHODS: The characteristics of hAMSCs were confirmed by flow cytometry, RT-PCR, and immunofluorescence. Obese mice were induced by administrating HFD for 15 weeks and simultaneously, the mice were intraperitoneally injected with hAMSCs-CM weekly to evaluate the effects of hAMSCs-CM on HFD-induced obesity. GTT and ITT assays were used to assess the effects of hAMSCs-CM on HFD-induced glucose tolerance and insulin resistance. The lipid accumulation and adipocytes hypertrophy in mouse adipose tissues were determined by histological staining, in which the alterations of blood lipid, liver, and kidney function were also examined. The role of hAMSCs-CM in energy homeostasis was monitored by examining the oxygen consumption (VO2), carbon dioxide production (VCO2), and food and water intake in mice. Furthermore, the expressions of the genes related to glucose metabolism, fatty acid ß oxidation, thermogenesis, adipogenesis, and inflammation were determined by western blot analysis, RT-PCR, and immunofluorescence staining. The roles of hAMSCs-CM in adipogenesis and M1/M2 macrophage polarization were investigated with 3T3-L1 preadipocytes or RAW264.7 cells in vitro. RESULTS: hAMSCs-CM significantly restrained HFD-induced obesity in mice by inhibiting adipogenesis and lipogenesis, promoting energy expenditure, and reducing inflammation. The underlying mechanisms of the anti-obesity of hAMSCs-CM might be involved in inhibiting PPARγ and C/EBPα-mediated lipid synthesis and adipogenesis, promoting GLUT4-mediated glucose metabolism, elevating UCP1/PPARα/PGC1α-regulated energy expenditure, and enhancing STAT3-ARG1-mediated M2-type macrophage polarization. CONCLUSION: Our studies demonstrated that hAMSCs significantly alleviated HFD-induced obesity through their paracrine effects. Obviously, our results open up an attractive therapeutic modality for the prevention and treatment of obesity and other metabolic disorders clinically. The cytokines, exosomes, or micro-vesicles secreted from hAMSCs significantly inhibited HFD-induced obesity in mice by inhibiting lipid production and adipogenesis, promoting energy consumption, and reducing inflammation.

CD38 Deficiency Protects Mice from High Fat Diet-Induced Nonalcoholic Fatty Liver Disease through Activating NAD+/Sirtuins Signaling Pathways-Mediated Inhibition of Lipid Accumulation and Oxidative Stress in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in hepatocytes. CD38 was initially identified as a lymphocyte surface antigen and then has been found to exist in a variety of cell types. Our previous studies showed that CD38-/- mice were resistant to high-fat diet (HFD)-induced obesity. However, the role and mechanism of CD38 in HFD-induced NAFLD is still unclear. Here, we reported that CD38-/- mice significantly alleviated HFD-induced hepatic steatosis. HFD or oleic acid (OA) remarkably increased the mRNA and protein expressions of CD38 in mouse hepatic tissues and primary hepatocytes or hepatic cell lines in vitro and in vivo, suggesting that CD38 might play a role in HFD-induced hepatic steatosis. We observed that CD38 deficiency markedly decreased HFD- or OA-induced the lipid accumulation and oxidative stress in CD38-/- livers or primary hepatocytes, respectively. In contrast, overexpression of CD38 in Hep1-6 cells aggravated OA-induced lipid accumulation and oxidative stress. Furthermore, CD38 deficiency markedly inhibited HFD- or OA-induced the expressions of NOX4, and increased the expression of PPARα, CPT1, ACOX1 and SOD2 in liver tissue and hepatocytes from CD38-/- mice, indicating that CD38 deficiency-mediated the enhancement of fatty acid oxidation and the inhibition of oxidative stress contributed to protecting NAFLD. More importantly, Ex527 (Sirt1 inhibitor) and 3-TYP (Sirt3 inhibitor) significantly enhanced OA-induced lipid accumulation and oxidative stress in CD38-/- primary hepatocytes, suggesting that the anti-lipid accumulation of CD38 deficiency might be dependent on NAD/Sirtuins-mediated enhancement of FAA ß-oxidation and suppression of oxidative stress in hepatocytes. In conclusion, we demonstrated that CD38 deficiency protected mice from HFD-induced NAFLD by reducing lipid accumulation and suppressing oxidative stress via activating NAD/Sirtuins signaling pathways.

Role of lateral amygdala calstabin2 in regulation of fear memory.

Calstabin2, also named FK506 binding protein 12.6 (FKBP12.6), is a subunit of ryanodine receptor subtype 2 (RyR2) macromolecular complex, an intracellular calcium channel. Studies from our and other's lab have shown that hippocampal calstabin2 regulates spatial memory. Calstabin2 and RyR2 are widely distributed in the brain, including the amygdala, a key brain area involved in the regulation of emotion including fear. Little is known about the role of calstabin2 in fear memory. Here, we found that genetic deletion of calstabin2 impaired long-term memory in cued fear conditioning test. Knockdown calstabin2 in the lateral amygdala (LA) by viral vector also impaired long-term cued fear memory expression. Furthermore, calstabin2 knockout reduced long-term potentiation (LTP) at both cortical and thalamic inputs to the LA. In conclusion, our present data indicate that calstabin2 in the LA plays a crucial role in the regulating of emotional memory.

CD38 Deficiency Alleviates D-Galactose-Induced Myocardial Cell Senescence Through NAD+/Sirt1 Signaling Pathway.

Our previous research showed that CD38 played vital roles in Ang-II induced hypertrophy and high fat diet induced heart injury. However, the role of CD38 in heart aging is still unknown. In the present study, we reported that CD38 knockdown significantly protected cardiomyocytes from D-galactose (D-gal)-induced cellular senescence. Cellular senescence was evaluated by ß-galactosidase staining, the expressions of genes closely related to aging including p16 and p21, and the ROS production, MDA content and the expressions of oxidant stress related genes were examined by biochemical analysis, Western blot and QPCR. Our results showed that the expression of CD38 was increased in H9c2 cells after D-gal treatment and the expressions of NAMPT and Sirt1 were downregulated in heart tissue from old mice. CD38 knockdown significantly reduced the number of SA-ß-gal-positive cells and the expressions of p16 and p21 in H9c2 cells with or without D-gal treatment. The acetylation level of total protein was decreased in CD38 knockdown group, but the expression of Sirt3 was increased in CD38 knockdown group treated with D-gal. In addition, knockdown of CD38 significantly attenuated D-gal induced ROS production, MDA content and NOX4 expression in the cells. Inhibition Sirt1 partially reversed the effects of CD38 knockdown on D-gal induced senescence and oxidative stress. Furthermore, NAD+ supplementation reduced D-gal induced cellular senescence, ROS production and MDA content. The expression of SOD2 was increased and the NOX4 expression was decreased in H9c2 cells after NAD+ supplementation. Taken together, our results demonstrated that CD38 knockdown alleviated D-gal induced cell senescence and oxidative stress via NAD+/Sirt1 signaling pathway.

CD38 Deficiency Protects the Heart from Ischemia/Reperfusion Injury through Activating SIRT1/FOXOs-Mediated Antioxidative Stress Pathway.

Ischemia/reperfusion (I/R) injury induces irreversible oxidative stress damage to the cardiac muscle. We previously observed that CD38 deficiency remarkably protects mouse embryonic fibroblasts (MEFs) from oxidative stress-induced injury. However, whether CD38 deficiency protects from I/R injury in the heart is not explored. Here, we showed that the hearts of CD38 deficient mice or wild type mice supplied with exogenous NAD were significantly protected from ischemia/reperfusion injury, seen as reduction of the myocardial infarct sizes when the mice were subjected to 30 min ischemia followed by 24 hours of reperfusion. Consistently, the protection of CD38 deficiency on hypoxia/reoxygenation (H/R) injury was confirmed with a CD38 knockdown H9c2 stable cell line. Furthermore, we observed that knockdown of CD38 remarkably inhibited ROS generation and intracellular Ca(2+) overloading induced by H/R in H9c2 cells. The FOXO1 and FOXO3 expressions were significantly elevated by H/R injury in CD38 knockdown cells compared with normal H9c2 cells. The cell immunofluorescence assay showed that FOXO1 nuclear translocation was significantly increased in CD38 knockdown H9c2 cells. In addition, we demonstrated that the increase of FOXO1 nuclear translocation was associated with the increased expressions of antioxidant catalase and SOD2 and the attenuated expression of the ROS generation enzyme NOX4. In conclusion, our results provide new evidence that CD38 deficiency protects the heart from I/R injury through activating SIRT1/FOXOs-mediated antioxidative stress pathway.

Histone deacetylases regulate gonadotropin-releasing hormone I gene expression via modulating Otx2-driven transcriptional activity.

BACKGROUND: Precise coordination of the hypothalamic-pituitary-gonadal axis orchestrates the normal reproductive function. As a central regulator, the appropriate synthesis and secretion of gonadotropin-releasing hormone I (GnRH-I) from the hypothalamus is essential for the coordination. Recently, emerging evidence indicates that histone deacetylases (HDACs) play an important role in maintaining normal reproductive function. In this study, we identify the potential effects of HDACs on Gnrh1 gene transcription. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of HDACs activities by trichostatin A (TSA) and valproic acid (VPA) promptly and dramatically repressed transcription of Gnrh1 gene in the mouse immortalized mature GnRH neuronal cells GT1-7. The suppression was connected with a specific region of Gnrh1 gene promoter, which contains two consensus Otx2 binding sites. Otx2 has been known to activate the basal and also enhancer-driven transcription of Gnrh1 gene. The transcriptional activity of Otx2 is negatively modulated by Grg4, a member of the Groucho-related-gene (Grg) family. In the present study, the expression of Otx2 was downregulated by TSA and VPA in GT1-7 cells, accompanied with the opposite changes of Grg4 expression. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the DNA-binding activity of Otx2 to Gnrh1 gene was suppressed by TSA and VPA. Overexpression of Otx2 partly abolished the TSA- and VPA-induced downregulation of Gnrh1 gene expression. CONCLUSIONS/SIGNIFICANCE: Our data indicate that HDAC inhibitors downregulate Gnrh1 gene expression via repressing Otx2-driven transcriptional activity. This study should provide an insight for our understanding on the effects of HDACs in the reproductive system and suggests that HDACs could be potential novel targets for the therapy of GnRH-related diseases.

Disruption of CD38 gene enhances cardiac functions by elevating serum testosterone in the male null mice.

AIMS: Gender-related phenotypes in the cardiovascular system have been observed in various genetically modified mice. Here, we report that cardiac functions are significantly improved only in male CD38-null mice and we explore the potential mechanisms of the sexual dimorphism mediated by CD38 deficiency. MAIN METHODS: Cardiac functions of mice were measured by pressure-volume conductance catheter technique and echocardiography. Serum sex steroids were determined by radioimmunoassay. Relative mRNA levels of myocardial contractile-associated proteins in cardiomyocytes were analyzed by real-time PCR analysis. To clarify the effects of testosterone on the sexual dimorphism, flutamide, an androgen receptor antagonist, was subcutaneously infused into the male null mice for 6 weeks with an osmotic mini-pump. KEY FINDINGS: The myocardial contractility, contraction and relaxation velocities were significantly enhanced only in male CD38-null mice, in which the levels of serum testosterone were markedly elevated. The elevated testosterone levels in the null mice were correlated to an obvious decrease in expression of androgen receptor and dramatic increases in expressions of major genes involved in myocardial contraction, including ryanodine receptor type 2 (RyR2), sarcoplasmic reticular Ca(2+) ATPase (SERCA2) and Na(+)/Ca(2+)-exchanger protein 1 (NCX1), and α myosin heavy chain (α-MHC). More importantly, all of the alternations that were observed in the male null mice were almost completely restored by flutamide administration. SIGNIFICANCE: Elevated serum level of testosterone in the male CD38(-/-) mice enhances cardiac functions through upregulation of major calcium regulatory proteins, which improve our understanding on sex disparities and molecular mechanisms in the incidence and manifestation of heart diseases.