Binhai Cong

Differential Regulation of Prostaglandin Production Mediated by Corticotropin-Releasing Hormone Receptor Type 1 and Type 2 in Cultured Human Placental Trophoblasts

Prostaglandin (PG) production by intrauterine tissues plays a key part in the control of pregnancy and parturition. The present study was to investigate the role of placenta-derived CRH and CRH-related peptides in the regulation of PG synthesis and metabolism. We found that placental trophoblasts expressed both CRH-R1 and CRH-R2. Treatment of cultured placental cells with either a CRH or urocortin I (UCNI) antibody resulted in a significant decrease in PGE2 release. Both CRH and UCNI antibodies significantly decreased mRNA and protein expression of synthetic enzymes cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2 and increased mRNA and protein expression of 15-hydroxyprostaglandin dehydrogenase (PGDH), the key enzyme of PG metabolism. CRH-R1/-R2 antagonist astressin and CRH-R1 antagonist antalarmin significantly inhibited PGE2 release, whereas CRH-R2 antagonist astressin-2b had no effect on PGE(2) release. Administration of astressin decreased expression of cPLA2 but had no effect on COX-2 expression. Antalarmin reduced cPLA2 and COX-2 expression, whereas astressin-2b did not alter cPLA2 expression but increased COX-2 expression. PGDH expression was enhanced by these three antagonists. Cells treated with exogenous CRH and UCNI showed an increase in PGE(2) release and expression of cPLA2 and COX-2 but a decrease in PGDH expression. UCNII and UCNIII had no effect on PGE2 release but decreased COX-2 and PGDH expression. Our results suggested CRH and CRH-related peptides act on CRH-R1 and CRH-R2 to exert different effects on PG biosynthetic enzymes cPLA2 and COX-2 and thereby modulate output of PGs from placenta, which would be important for controlling pregnancy and parturition.

Estrogens increase cystathionine-γ-lyase expression and decrease inflammation and oxidative stress in the myocardium of ovariectomized rats.

Objective Hydrogen sulfide (H2S), generated in the myocardium predominantly via cystathionine-&ggr;-lyase (CSE), is cardioprotective. The objectives of the present study were to investigate the effects of estrogens on CSE expression and H2S generation in the myocardium and to examine whether serum 17&bgr;-estradiol (E2) level is associated with CSE activity and H2S generation and whether H2S or E2 level is associated with proinflammatory cytokines and oxidative stress status. Methods Ovariectomized Sprague-Dawley rats received subcutaneous E2 (30 &mgr;g/kg/d) or vehicle for 12 weeks. At the end of the 12-week treatment, CSE expression, H2S generation, reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, catalase (CAT) activity, interleukin (IL)-6 concentration, and tumor necrosis factor-&agr; (TNF-&agr;) concentration in the left ventricle were determined. Results E2 increased CSE expression and H2S generation in the myocardium of ovariectomized rats. H2S production rate and serum E2 were positively correlated. E2 increased GSH/GSSG ratio, T-AOC, CAT, and SOD activity but decreased IL-6 and TNF-&agr; levels. Serum E2 level was positively correlated with GSH/GSSG ratio, T-AOC, CAT, and SOD activity, and inversely correlated with IL-6 and TNF-&agr; levels. H2S generation rate was positively correlated with T-AOC and GSH/GSSG ratio, and inversely correlated with IL-6 and TNF-&agr; levels. Conclusions E2 increases CSE expression and endogenous H2S generation in the myocardium. The effects of E2 are associated with decreased oxidative stress and inflammatory status. Our data suggest that estrogens might exert cardioprotective effects through up-regulation of CSE expression and H2S generation.

Reduced expression of CRH receptor type 1 in upper segment human myometrium during labour

BackgroundCorticotropin-releasing hormone (CRH) and CRH-related peptide are shown to modulate uterine contractility through two CRH receptor subtype, CRH-R1 and CRH-R2 during pregnancy. Through different signaling pathways, CRH-R1 maintains myometrial quiescence whereas CRH-R2 promotes smooth muscle contractility. We hypothesized that the expression of CRH receptors in myometrium might be changed during pregnancy and labour.MethodImmunohistochemistry, Western blot and RT-PCR were used to quantify the cellular localization, the protein levels and the mRNA variants of both CRH-R1 and CRH-R2 in upper segment (US) and lower segment (LS) myometrium from nonpregnant and pregnant women at term before or after labour.ResultsCRH-R1 and CRH-R2 were predominately localized to myometrial smooth muscle cells in US and LS. The protein level of CRH-R1 in US was significantly down-regulated in pregnancy, with a further decrease at the onset of labour. However, the expression of CRH-R1 in LS remained unchanged during pregnancy and labour. No significant changes in CRH-R2 expression were observed in US or LS. Six variants of CRH-R1, CRH-R1alpha,-R1beta,-R1c, -R1e,-R1f and -R1g, were identified in nonpregnant and pregnant myometrium. CRH-R2alpha was identified in pregnant myometrium, whereas CRH-R2beta was identified in nonpregnant myometriumConclusionCRH-R1 and CRH-R2 are expressed in nonpregnant and pregnant US and LS myometrium. Changed expression of CRH receptors during labour may underlie the initiation of uterine contractility during parturition.

Upregulation of microRNA-22 contributes to myocardial ischemia-reperfusion injury by interfering with the mitochondrial function

Mitochondrial oxidative damage is critically involved in cardiac ischemia reperfusion (I/R) injury. MicroRNA-22 (miR-22) has been predicted to potentially target sirtuin-1 (Sirt1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), both of which are known to provide protection against mitochondrial oxidative injury. The present study aims to investigate whether miR-22 is involved in the regulation of cardiac I/R injury by regulation of mitochondrial function. We found that miR-22 level was significantly increased in rat hearts subjected to I/R injury, as compared with the sham group. Intra-myocardial injection of 20 ug miR-22 inhibitor reduced I/R injury as evidenced by significant decreases in cardiac infarct size, serum lactate dehydrogenase (LDH) and creatine kinase (CK) levels and the number of apoptotic cardiomyocytes. H9c2 cardiomyocytes exposed to hypoxia/reoxygenation (H/R) insult exhibited an increase in miR-22 expression, which was blocked by reactive oxygen species (ROS) scavenger and p53 inhibitor. In addition, miR-22 inhibitor attenuated, whereas miR-22 mimic aggravated H/R-induced injury in H9c2 cardiomyocytes. MiR-22 inhibitor per se had no significant effect on cardiac mitochondrial function. Mitochondria from rat receiving miR-22 inhibitor 48h before ischemia were found to have a significantly less mitochondrial superoxide production and greater mitochondrial membrane potential and ATP production as compared with rat receiving miR control. In H9c2 cardiomyocyte, it was found that miR-22 mimic aggravated, whilst miR-22 inhibitor significantly attenuated H/R-induced mitochondrial damage. By using real time PCR, western blot and dual-luciferase reporter gene analyses, we identified Sirt1 and PGC1α as miR-22 targets in cardiomyocytes. It was found that silencing of Sirt1 abolished the protective effect of miR-22 inhibitor against H/R-induced mitochondrial dysfunction and cell injury in cardiomyocytes. Taken together, our findings reveal a novel molecular mechanism for cardiac mitochondrial dysfunction during myocardial I/R injury at the miRNA level and demonstrate the therapeutic potential of miR-22 inhibition for acute myocardial I/R injury by maintaining cardiac mitochondrial function.

Estrogens protect myocardium against ischemia/reperfusion insult by up-regulation of CRH receptor type 2 in female rats

BACKGROUND The mechanism underlying estrogen cardioprotection remains largely unknown. Urocortin (UCN), a member of corticotropin-releasing hormone (CRH) family, is one of endogenous cardioprotective factors. The goal of present study is to investigate whether estrogens regulate UCN and its receptor CRH receptor type 2 (CRHR2) in female rat heart. METHODS 17β-estradiol (E2) was subcutaneously administrated to ovariectomized (OVX) rats for eight weeks. UCN was administrated before simulated myocardial ischemia/reperfusion (I/R). Cell damage was assessed by measurement of infarct size, activity of serum creatine kinase (CK) and lactate dehydrogenase (LDH) and percentage of TUNEL staining in myocardium. The mRNA and protein levels of UCN and CRHR2 were determined in sham operated and OVX rats with or without E2 replacement. DNA methylation frequency of CRHR2 gene promoter was determined by bisulfite-sequencing. RESULTS UCN administration reduced infarct size, LDH and CK level and percentage of TUNEL staining upon I/R injury. The cardioprotective effects of UCN were abrogated in OVX rats and E2 replacement restored UCN-induced cardioprotection.CRHR2 mRNA and protein expression were down-regulated more than 40% in OVX rats, both of which were restored by E2 replacement. UCN mRNA and protein levels were not affected by ovariectomy and E2 replacement. Hypermethylation in CRHR2 promoter was found in OVX rats, and two of the methylated CpG sites were seated at cis-acting elements. Hypermethylation induced by OVX could also be ameliorated by E2 replacement. CONCLUSION Estrogens maintain CRHR2 expression in myocardium, which may through an epigenetic mechanism, and enhance UCN-induced cardioprotective effects against I/R injury.

Cardioprotection of 17β-estradiol against hypoxia/reoxygenation in cardiomyocytes is partly through up-regulation of CRH receptor type 2.

Estrogens have been suggested to exert cardioprotection through maintaining endogenous cardioprotective mechanisms. In the present study, we investigated whether estrogens protect cardiomyocytes against hypoxia/reoxygenation (H/R) via modulating urocortins (UCNs) and their receptor corticotrophin-releasing hormone receptor type 2 (CRHR2). We found that 17β-estradiol (E2) enhanced UCN cardioprotection against H/R and increased CRHR2 expression in neonatal rat cardiomyocytes. E2 protected cardiomyocytes against H/R, which was impaired by CRHR2 antagonist or knockdown of CRHR2. Estrogen receptor α (ERα) antagonist treatment or ERα knockdown could abolish E2-induced CRHR2 up-regulation. Moreover, knockdown of Sp1 also attenuated E2-induced CRHR2 up-regulation. Ovariectomy resulted in down-regulation of CRHR2 and Sp-1 in myocardium of mice, which was restored by E2 or ERα agonist treatment. These results suggest that estrogens act on ERα to up-regulate CRHR2 expression in cardiomyocytes, thereby enhancing cardioprotection of UCNs against H/R.

Estrogenic Action on Arterial Smooth Muscle: Permissive for Maintenance of CRHR2 Expression

Urocortin (Ucn), a member of CRH family, has been implicated to be one of the endogenous regulators in the cardiovascular system and exerts its effects locally via an autocrine/paracrine fashion. Previous studies have shown the gender difference in CRH-induced vasodilation in human skin, which is related to the concentration of estrogens during the menstrual cycle. The aim of this study was to investigate whether estrogens modulate Ucn/CRH receptor type 2 (CRHR2) expression in vascular smooth muscle, thereby leading to vasodilation. We performed sham operation or bilateral ovariectomy (OVX) on female Sprague Dawley rats. OVX rats were sc administered 17β-estradiol (E₂) at a dose of 30 μg/kg·d or with placebo for 12 wk. Primary smooth muscle cells of aorta were used for the in vitro study. It was found that the Ucn-induced vasodilation and CRHR2 expression were decreased in OVX rats and restored by E₂ replacement treatment for 12 wk. E₂ increased the expression of CRHR2 in cultured smooth muscle cells, which was blocked by estrogen receptor-β antagonist. Ucn significantly suppressed the phenylephrine-induced phospholipase Cβ3 activation, inositol 1,4,5-trisphosphate (IP₃) production, and intracellular Ca²⁺ elevation. Ucn stimulated the expression of active GTP-bound Gαs protein and cAMP production. The suppressive effects of Ucn on phenylephrine-induced IP₃ production and intracellular Ca²⁺ elevation were blocked by the inhibitors of adenylate cyclase and protein kinase A. Our results demonstrate that estrogen maintains the expression of CRHR2 in aorta smooth muscle, thereby enhancing vasodilator actions of Ucn. Ucn exerts its vasorelaxant effects via Gαs-cAMP-protein kinase A signaling, leading to down-regulation of the phospholipase Cβ-IP₃-Ca²⁺ signaling pathway.

SGK1 is involved in cardioprotection of urocortin-1 against hypoxia/reoxygenation in cardiomyocytes.

BACKGROUND Urocortin-1 (UCN1) exerts protective effects on hypoxia/reoxygenation injury in the heart. Serum- and glucocorticoid- responsive kinase-1 (SGK1), a serine-threonine kinase, has been shown to be crucial for cardiomyocyte survival. The purpose of the present study was to investigate whether SGK1 is involved in UCN1-induced cardioprotection. METHODS Cardiomyocytes were obtained from neonatal rats and used as a model to investigate UCN1 regulation of SGK1. Specific small interfering RNA targeting SGK1 was used to knock down SGK1 expression. The messenger RNA (mRNA) level of SGK1 was measured using quantitative real time reverse transcription polymerase chain reaction, and the protein levels of SGK1 and phosphorylated SGK1 were determined using Western blot analysis. RESULTS SGK1 knockdown attenuated the protective effects of UCN1 against hypoxia/reoxygenation injury in cardiomyocytes. Treatment of cardiomyocytes with UCN1 stimulated SGK1 mRNA and protein expression and time-dependently increased phosphorylated SGK1 level. These effects were completely reversed with corticotrophin-releasing hormone receptor type 2 antagonist. Adenylate cyclase and protein kinase A inhibitors abolished the stimulatory effect of UCN1 on SGK1 expression. SGK1 phosphorylation induced by UCN1 was blocked by phosphorinositide-3-kinase inhibitor. CONCLUSIONS SGK1 is involved in the cardioprotective effects of UCN1 in cardiomyocytes. UCN1 stimulates SGK1 phosphorylation via the phosphorinositide-3-kinase signalling pathway and it induces SGK1 expression via the adenylate cyclase/protein kinase A pathway.

Estrogen enhancement of SGK1 expression induced by urocortin contributes to its cardioprotection against ischemia/reperfusion insult

☆ This work is supported by the National Natural Sci 31000516 and No. 31371197), Key Laboratory of Exe Ministry of Education, Shanghai University of Sport and I Undergraduates, and Education Commission of Shanghai ⁎ Correspondence to: J. Lu, School of Kinesiology, Sha Changhai Road, Shanghai 200438, China. ⁎⁎ Correspondence to: X. Ni, Department of Physiol University, Shanghai 200433, China. E-mail addresses: lujianqiang@sus.edu.cn (J. Lu), nixin

Reduced expression of CRHR2 and Sp-1 in myocardium of ovariectomized rats is improved by exercise training

Abstract Exercise training has been looked on as a non-pharmacologic approach to treating ovariectomy (OVX)-induced dysfunctions. In this study, we investigated whether chronic exercise impacts on expression of urocortins (UCNs) and corticotropin-releasing hormone receptor type 2 (CRHR2) in myocardium of OVX rats. Bilateral OVX or sham-operation was performed under anesthesia. Both groups were then divided into two subgroups, with or without treadmill training for 8 weeks. It was found that OVX as well as exercise did not affect the mRNA levels of UCN, UCN2 and UCN3 in myocardium. OVX caused down-regulation of CRHR2 in myocardium. Exercise training reversed the OVX-induced reduction of CRHR2, but had no influence on CRHR2 level in sham rats. OVX resulted in a decrease in estrogen receptor α (ERα) expression in myocardium, which was restored by exercise. Moreover, exercise training also reversed OVX-induced down-regulation of specific protein-1 (Sp-1) expression in myocardium. CRHR2 expression level correlated with Sp-1 and ERα level in myocardium. These results indicate that exercise training can restore the CRHR2 level in myocardium of OVX rats, which is associated with ERα and Sp-1 expression. Chinese abstract 运动训练已被视为治疗去卵巢（OVX）引起的功能障碍的一个非药物方法。在这项研究中，我们观察慢性运动是否对去卵巢大鼠心肌中尿皮质激素及促肾上腺皮质激素释放激素受体2型（CRHR2）的表达有影响。在麻醉下进行双侧卵巢切除或假手术。两组又各分成两个亚组，其中一组进行活动平板训练8周，另一组则不进行训练。结果发现：卵巢切除及运动均不影响心肌中UCN、UCN2和UCN3的mRNA水平。卵巢切除引起心肌中CRHR2下调。运动训练逆转卵巢切除导致的CRHR2减少，但对假手术组大鼠CRHR2的水平没有影响。卵巢切除可导致心肌中雌激素受体α(ERα)的表达降低，但可通过锻炼恢复。此外，运动训练能逆转卵巢切除所导致的心肌中特异蛋白-1(Sp-1)的降调节。心肌中CRHR2的表达水平与SP-1和ERα的水平相关。这些结果表明：运动训练可以恢复去卵巢大鼠心肌CRHR2的水平，而CRHR2的表达水平与SP-1和ERα的水平相关。