Tanshinone-induced ERs suppresses IGFII activation to alleviate Ang II-mediated cardiac hypertrophy

Cardiomyopathy involves changes in myocardial ultrastructure and cardiac hypertrophy. Angiotensin II (AngII) has previously been shown to stimulate the expression of IGF-2 and IGF-2R in H9c2 cardiomyoblasts and increase of blood pressure, and cardiac hypertrophy. Estrogen receptors (ERs) exert protective effects, such as anti-hypertrophy in cadiomyocytes. Tanshinone IIA (TSN), a main active ingredient from a Chinese medical herb, Salvia miltiorrhiza Bunge (Danshen), was shown to protect cardiomyocytes hypertrophy by different stress signals. We aimed to investigate whether TSN protected H9c2 cardiomyocytes from AngII-induced activation of IGF-2R pathway and hypertrophy by mediating through ERs. AngII resulted in H9c2 cardiomyoblast hypertrophy and increased inflammatory molecular markers. These were down-regulated by TSN via estrogen receptors. AngII resulted in elevation in MAPKs, IGF-2R and hypertrophic protein markers. These, again, were reduced by addition of the phytoestrogen with activation of ERs. Finally, AngII induced phosphorylation of heat shock factor-1 (HSF1) and decreased sirtuin-1 (SIRT1). In addition, AngII also caused an increase in distribution of IGF-2R molecules on cell membrane. In contrast, TSN reduced HSF1 phosphorylation and cell surface IGF-2R while elevating SIRT1 via ERs. TSN was capable of attenuating AngII-induced IGF-2R pathway and hypertrophy through ERs in H9c2 cardiomyoblast cells.     

G0S2 represses PI3K/mTOR signaling and increases sensitivity to PI3K/mTOR pathway inhibitors in breast cancer

G0/G1 switch gene 2 (G0S2) is a direct retinoic acid target implicated in cancer biology and therapy based on frequent methylation-mediated silencing in diverse solid tumors. We recently reported that low G0S2 expression in breast cancer, particularly estrogen receptor-positive (ER+) breast cancer, correlates with increased rates of recurrence, indicating that G0S2 plays a role in breast cancer progression. However, the function(s) and mechanism(s) of G0S2 tumor suppression remain unclear. In order to determine potential mechanisms of G0S2 anti-oncogenic activity, we performed genome-wide expression analysis that revealed an enrichment of gene signatures related to PI3K/mTOR pathway activation in G0S2 null cells as compared to G0S2 wild-type cells. G0S2 null cells also exhibited a dramatic decreased sensitivity to PI3K/mTOR pathway inhibitors. Conversely, restoring G0S2 expression in human ER+ breast cancer cells decreased basal mTOR signaling and sensitized the cells to pharmacologic mTOR pathway inhibitors. Notably, we provide evidence here that the increase in recurrence seen with low G0S2 expression is especially prominent in patients who have undergone antiestrogen therapy. Further, ER+ breast cancer cells with restored G0S2 expression had a relative increased sensitivity to tamoxifen. These findings reveal that in breast cancer G0S2 functions as a tumor suppressor in part by repressing PI3K/mTOR activity, and that G0S2 enhances therapeutic responses to PI3K/mTOR inhibitors. Recent studies implicate hyperactivation of PI3K/mTOR signaling as promoting resistance to antiestrogen therapies in ER+ breast cancer. Our data establishes G0S2 as opposing this form of antiestrogen resistance. This promotes further investigation of the role of G0S2 as an antineoplastic breast cancer target and a biomarker for recurrence and therapy response.     

Reduced dosing and liability in methadone maintenance treatment by targeting oestrogen signal for morphine addiction

Methadone maintenance treatment (MMT) is the major tapering therapy for morphine addictive patients. There have gender differences reported in response to MMT. This study discovered that the estrogen‐response element single nucleotide polymorphism (ERE‐SNP; rs16974799, C/T) of cytochrome 2B6 gene (cyp2b6; methadone catabolic enzyme) responded differently to MMT dosing. Oestradiol was associated with high MMT dosing, high enantiomer (R‐ or S‐) of 2‐ethylidene‐1,5‐dimethyl‐3,3‐dipheny‐pyrrolidine (EDDP; methadone metabolite) to methadone ratio and increased drug‐seeking behaviour, implicating oestradiol‐CYP‐EDDP/methadone axis decreasing MMT efficacy. In mouse model, oestrogen mitigates methadone antinociceptive response, facilitates methadone catabolism and up‐regulates methadone‐associated metabolizing enzymes. Oestrogen also ablates chronic methadone administration‐induced rewarding response. Mechanism dissection revealed the CC genotype of CYP2B6‐ERE‐SNP exerts higher ERE sequence alignment score, higher estrogenic response as compared to TT genotype. At last, preclinical study via targeting estrogen signal that tamoxifen (TMX; selective estrogen receptor modulator, SERM) could facilitate the tolerance phase rewarding response of methadone. Strikingly, TMX also reduces tapering/abstinence phases methadone liability in mice. In conclusion, this study demonstrates altering methadone metabolism through targeting estrogen signals might be able to free morphine addictive patients from the addiction of opioid replacement therapy. Therefore, the add‐on therapy clinical trial introducing SERM in MMT regimen is suggested.     

不同培养时间对鸡卵泡颗粒细胞孕酮、雌激素分泌水平及FSHR、LHR基因表达的影响

目的：研究培养不同时间鸡卵泡颗粒细胞孕酮和雌激素的分泌水平,促卵泡素受体（FSHR）和促黄体素受体（LHR）的基因表达水平,推断体外培养时间对颗粒细胞激素分泌及相关受体基因表达的影响。方法：通过细胞体外培养的方法,分别于0 h、24 h、48 h、72 h、96 h收集鸡卵泡颗粒细胞上清液,采用ELISA法测定细胞上清液内的孕酮及雌激素分泌水平,并采用荧光定量PCR技术检测颗粒细胞内FSHR和LHR基因表达情况。结果：在培养初期0 h~48 h孕酮和雌激素分泌量显著降低（P < 0.05）,随着培养时间增加到72 h两种激素的分泌量又开始增加,并达到培养初期水平,培养至96 h细胞内孕酮和雌激素分泌量再次降低;颗粒细胞FSHR和LHR mRNA的表达水平则随着培养时间的增加而降低（P < 0.05）。结论：体外培养的卵泡颗粒细胞内孕酮和雌激素的分泌量随体外培养时间的延长呈先降低后升高的趋势,可能与体外培养细胞的生长状态相关,从整体上看随着培养时间的延长,细胞内孕酮和雌激素的分泌量均降低,可能与两种促性腺激素受体FSHR和LHR基因表达量下降相关。     

Sex differences in lifespan extension with acarbose and 17‐α estradiol: gonadal hormones underlie male‐specific improvements in glucose tolerance and mTORC2 signaling

Interventions that extend lifespan in mice can show substantial sexual dimorphism. Here, we show that male‐specific lifespan extension with two pharmacological treatments, acarbose (ACA) and 17‐α estradiol (17aE2), is associated, in males only, with increased insulin sensitivity and improved glucose tolerance. Females, which show either smaller (ACA) or no lifespan extension (17aE2), do not derive these metabolic benefits from drug treatment. We find that these male‐specific metabolic improvements are associated with enhanced hepatic mTORC2 signaling, increased Akt activity, and phosphorylation of FOXO1a – changes that might promote metabolic health and survival in males. By manipulating sex hormone levels through gonadectomy, we show that sex‐specific changes in these metabolic pathways are modulated, in opposite directions, by both male and female gonadal hormones: Castrated males show fewer metabolic responses to drug treatment than intact males, and only those that are also observed in intact females, while ovariectomized females show some responses similar to those seen in intact males. Our results demonstrate that sex‐specific metabolic benefits occur concordantly with sexual dimorphism in lifespan extension. These sex‐specific effects can be influenced by the presence of both male and female gonadal hormones, suggesting that gonadally derived hormones from both sexes may contribute to sexual dimorphism in responses to interventions that extend mouse lifespan.     

右归丸与液体钙片对老年骨质疏松症患者血清雌激素和骨钙素水平的影响

目的:研究右归丸联合液体钙片对老年骨质疏松症患者血清雌激素水平、骨钙素(BGP)水平及临床疗效的影响。方法:选取2014年10月至2015年9月本院收治的82例老年骨质疏松症患者,根据入院顺序分为观察组和对照组,每组41例。对照组使用骨化三醇软胶囊和液体钙软胶囊进行治疗,观察组在此基础上加以右归丸进行治疗。比较两组患者治疗前和治疗1个月后血清雌二醇(E2)水平、BGP水平、骨密度变化,分析两组患者的临床疗效。结果:治疗后,观察组的血清E2水平显著高于对照组(P0.05),BGP水平显著低于对照组(P0.05),腰椎L1-4正位、右侧股骨颈、左侧股骨颈骨密度显著高于对照组(p0.05),总临床疗效率显著高于对照组(P0.05)。结论:右归丸联合液体钙片治疗老年骨质疏松症患者能明显改善患者血清雌激素、BGP水平,增加骨密度,临床疗效良好。     

Expression and regulation of Angiopoietins and their receptor Tie-2 in sika deer antler

The cartilage vascularization and chondrocyte survival are essential for endochondral ossification which occurs in the process of antler growth. Angiopoietins (Ang) is a family of major angiogenic growth factors and involved in regulating the vascularization. However, the expression and regulation of Angs in the antler are still unknown. The aim of this study is to localize the expression of Ang-1, Ang-2 and their receptor Tie-2 in sika deer antler using in situ hybridization and focused on analyzing the regulation of testosterone, estrogen, all-trans-retinoic acid (ATRA) and 9cRA on their expression in antler chondrocytes. The results showed that Ang-1, Ang-2 and Tie-2 were highly expressed in antler chondrocytes. Administration of testosterone to antler chondrocytes led to a notable increase in the expression of Ang-1 and Tie-2, and a reduction in the expression of Ang-2. The similar result was also observed after estrogen treatment. In contrast, ATRA and 9cRA could inhibit the expression of Ang-1 in antler chondrocytes and heighten the expression of Ang-2. Simultaneously, ATRA could downregulate the expression of Tie-2 in antler chondrocytes at 12 and 24?h, while 9cRA upregulate the expression of Tie-2 at 3 and 6?h. Collectively, Ang-1, Ang-2 and Tie-2 are expressed in antler chondrocytes and their expression can be affected by testosterone, estrogen, ATRA and 9cRA.     

Effect of overexpression of estrogen receptors in osteoblasts

Summary Our study focused on investigating the mechanism of action of estrogen in regulating p53 levels within osteoblasts. In the studies reported here, we attempted to understand the role of estrogen receptors, ER-alpha and ER-beta, in the regulation of p53 and osteoblast differentiation. We stably expressed ER-alpha and ER-beta in ROS 17/2.8 cells and isolated several single cell clones. These clones were initially characterized for expression of the exogenous receptors, and representative clones from each type were chosen for further analyses. Cell proliferation, alkaline phosphatase activity, and the viability of these clones in culture were tested. The cells expressing exogenous ER-alpha exhibited more differentiated characteristics than cells expressing ER-beta. Morphologically, ER-beta-overexpressing cells were more rounded than the ER-alpha-overexpressing cells, which were more elongated and fibroblastic in appearance. The ER-beta-expressing cells had a higher survival and growth rate when compared with ER-alpha cells. The ER-alpha clones were not as viable as ER-beta clones, and some of the ER-alpha cell lines showed signs of senescence, with an increase in senescence-associated (SA) galactosidase activity. The basal levels of p53 functional activity were higher in cells expressing ER-alpha as was protein expression of the p53-regulated gene p21. The significance of these receptors to osteoblast differentiation and p53 regulation is discussed.     

Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture

The soybean phytoestrogen, genistein (Gen), has anabolic effects on bone through mechanisms that remain to be elucidated. We examined the role of nitric oxide (NO) and its downstream effector guanylyl cyclase (GC) in mediating the effects of Gen on the proliferation and osteoblastic maturation of primary mouse bone marrow-derived mesenchymal stem cells (BMSCs). Gen (10(-8) approximately 10(-6) M) resulted in a dose-dependent increase in cell proliferation as measured by increased [3H]thymidine incorporation, and stimulated osteoblastic maturation as assessed by culture duration-dependent increments in alkaline phosphatase (ALP) activity, calcium deposition into extracellular matrix and Runx2/Cbfa1 gene expression in BMSCs cultures. Gen also resulted in a dose-dependent increase in NO synthase (NOS) activity, NO formation, and cGMP production in BMSCs cultures. The effects of Gen were mimicked by 17beta-estradiol (E2, 10(-8) M). Concurrent treatment with the estrogen receptor (ER) antagonist ICI182,780 (10(-7) M) or the NOS inhibitor L-NAME (3 x 10(-3) M) diminished the Gen (10(-6) M)-mediated increase in NOS activity, NO production, and cGMP content. In contrast, a soluble GC inhibitor 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ, 10(-6) M) selectively blocked the Gen (10(-6) M)-mediated increase in cGMP content but not in NO production and NOS activity. Moreover, inhibition of ER, NOS activity or cGMP blocked Gen-induced proliferation and osteoblastic differentiation of BMSCs and Runx2/Cbfa1 gene expression in culture. Gen has estrogen-like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs at least in part through NO/cGMP pathway.     

Estrogen (17β-Estradiol) Enhances Glutamine Synthetase Activity in C6-Glioma Cells

Glutamine synthetase (GS) is the major glutamine-forming enzyme of vertebrates and is accepted to be a marker of astroglial cells. Maturation of astroglial cells is characterized by an increase of GS activity, and the regulation of this enzyme is the topic of many publications. Because of the fundamental role of the GS in controlling brain glutamate and glutamine level, it is essential to understand the mechanism of expression of this enzyme. To our knowledge, the effect of estrogen (17β-estradiol) on GS activity in glial cells has not been reported. We examined the effect of treatment with estrogen on glutamine synthetase enzyme activity in glial cells. C6-glioma cells in later passage have many astrocytic characteristics and provided a convenient and well-established model system. We adapted a colorimetric method to measure GS-catalyzed γ-glutamyltransferase (GT) activity in C6-glioma cells. The assay monitors GT activity of glutamine synthetase by following the absorbance of the product γ-glutamyl hydroxamate at 540 nm. We observed that, the absorbance of γ-glutamyl hydroxamate significantly increased in estrogen treated cells (0.13±0.03), as compared to untreated cells (0.058±0.015). Estrogen also significantly increased concentration of glutamine in C6-glioma cells as measured by fluorometric assay. In addition, western blot analysis showed that estrogen significantly increased the amount of glutamine synthetase compared to control. This estrogen effect could have important physiological implications on cerebral glutamate and glutamine metabolism.