Shuhui Zheng

17β-Estradiol enhances breast cancer cell motility and invasion via extra-nuclear activation of actin-binding protein ezrin.

Estrogen promotes breast cancer metastasis. However, the detailed mechanism remains largely unknown. The actin binding protein ezrin is a key component in tumor metastasis and its over-expression is positively correlated to the poor outcome of breast cancer. In this study, we investigate the effects of 17β-estradiol (E2) on the activation of ezrin and its role in estrogen-dependent breast cancer cell movement. In T47-D breast cancer cells, E2 rapidly enhances ezrin phosphorylation at Thr567 in a time- and concentration-dependent manner. The signalling cascade implicated in this action involves estrogen receptor (ER) interaction with the non-receptor tyrosine kinase c-Src, which activates the phosphatidylinositol-3 kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase (ROCK-2) complex. E2 enhances the horizontal cell migration and invasion of T47-D breast cancer cells in three-dimensional matrices, which is reversed by transfection of cells with specific ezrin siRNAs. In conclusion, E2 promotes breast cancer cell movement and invasion by the activation of ezrin. These results provide novel insights into the effects of estrogen on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.

Estrogen improved metabolic syndrome through down-regulation of VEGF and HIF-1α to inhibit hypoxia of periaortic and intra-abdominal fat in ovariectomized female rats

Metabolic syndrome (MBS), a cluster of metabolic abnormalities and visceral fat accumulation, increases cardiovascular risks in postmenopausal women. In addition to visceral fat, perivascular adipose tissue has been recently found to play an important role in vascular pathophysiology. Hence, the present study investigates the effects of estrogen on both intra-abdominal fat (visceral fat) and periaortic fat (perivascular fat) accumulation as well as hypoxia in ovariectomized female rats. Female rats were divided into sham operation, ovariectomy and ovariectomy with 17β-estradiol supplementation groups. Twelve weeks later, we found that estrogen improved MBS via reducing body weight gain, the weight of periaortic and intra-abdominal fat, hepatic triglyceride, and total serum cholesterol levels. Estrogen also increased insulin sensitivity through restoring glucose and serum leptin levels. For periaortic fat, western blot showed estrogen inhibited hypoxia by reducing the levels of VEGF and HIF-1α, which is consistent with the results from immunohistochemical staining. The correlation analysis indicated that perivascular fat had a positive correlation with body weight, intra-abdominal fat or serum total cholesterol, but a negative correlation with insulin sensitivity index. For intra-abdominal fat, real-time fluorescent RT-PCR showed estrogen improved fat dysfunction via reducing the levels of relative leptin, MCP-1 but increasing adiponectin mRNA. Estrogen reduced the levels of VEGF and HIF-1α to inhibit hypoxia but restored the levels of PPARγ and Srebp-1c, which are important for lipid capacity function of intra-abdominal fat. These results demonstrated estrogen improved MBS through down-regulating VEGF and HIF-1α to inhibit hypoxia of periaortic and intra-abdominal fat in ovariectomized female rats.

17β-estradiol down-regulates lipopolysaccharide-induced MCP-1 production and cell migration in vascular smooth muscle cells

Atherosclerosis is an inflammatory disease where lipopolysaccharide (LPS) triggers the release of inflammatory cytokines that accelerate its initiation and progression. Estrogen has been proven to be vasoprotective against atherosclerosis; however, the anti-inflammatory function of estrogen in the vascular system remains obscure. In this study, we investigated the effect of estrogen on LPS-induced monocyte chemoattractant protein-1 (MCP-1; listed as CCL2 in the MGI database) production in vascular smooth muscle cells (VSMCs). LPS significantly enhances MCP-1 production and this is dependent on nuclear factor kappa B (NFkappaB) signaling, since the use of NFkappaB inhibitor pyrrolidine dithiocarbamate or the silencing of NFkappaB subunit p65 expression with specific siRNA largely impairs LPS-enhanced MCP-1 production. On the contrary, 17beta-estradiol (E(2)) inhibits LPS-induced MCP-1 production in a time- and dose-dependent manner, which is related to the suppression of p65 translocation to nucleus. Furthermore, p38 MAPK is rapidly activated in response to LPS, while E(2) markedly inhibits p38 MAPK activation. Transfection with p38 MAPK siRNA or the use of p38 MAPK inhibitor SB203580 markedly attenuates LPS-stimulated p65 translocation to nucleus and MCP-1 production, suggesting that E(2) suppresses NFkappaB signaling by the inactivation of p38 MAPK signaling. LPS promotes VSMCs migration and this is abrogated by MCP-1 antibody, implying that MCP-1 may play a major role as an autocrine factor in atherosclerosis. In addition, E(2) inhibits LPS-promoted cell migration by downregulation of MCP-1 production. Overall, our results demonstrate that E(2) exerts anti-inflammatory property antagonistic to LPS in VSMCs by reducing MCP-1 production, and this effect is related to the inhibition of p38 MAPK/NFkappaB cascade.

Progesterone enhances vascular endothelial cell migration via activation of focal adhesion kinase.

The mechanisms of progesterone on endothelial cell motility are poorly investigated. Previously we showed that progesterone stimulated endothelial cell migration via the activation of actin‐binding protein moesin, leading to actin cytoskeleton remodelling and the formation of cell membrane structures required for cell movement. In this study, we investigated the effects of progesterone on the formation of focal adhesion complexes, which provide anchoring sites for cell movement. In cultured human umbilical endothelial cells, progesterone enhanced focal adhesion kinase (FAK) phosphorylation at Tyr397 in a dose‐ and time‐dependent manner. Several signalling inhibitors interfered with progesterone‐induced FAK activation, including progesterone receptor (PR) antagonist ORG 31710, specific c‐Src kinase inhibitor PP2, phosphatidylinosital‐3 kinase (PI3K) inhibitor wortmannin as well as ρ‐associated kinase (ROCK‐2) inhibitor Y27632. It suggested that PR, c‐Src, PI3K and ROCK‐2 are implicated in this action. In line with this, we found that progesterone rapidly promoted c‐Src/PI3K/Akt activity, which activated the small GTPase RhoA/ρ‐associated kinase (ROCK‐2) complex, resulting in FAK phosphorylation. In the presence of progesterone, endothelial cells displayed enhanced horizontal migration, which was reversed by small interfering RNAs abrogating FAK expression. In conclusion, progesterone promotes endothelial cell movement via the rapid regulation of FAK. These findings provide new information on the biological actions of progesterone on human endothelial cells that are relevant for vascular function.

Protective Effects of Scutellarin on Type II Diabetes Mellitus-Induced Testicular Damages Related to Reactive Oxygen Species/Bcl-2/Bax and Reactive Oxygen Species/Microcirculation/Staving Pathway in Diabetic Rat

The goal of our study is to evaluate the effect of Scutellarin on type II diabetes-induced testicular disorder and show the mechanism of Scutellarins action. We used streptozotocin and high-fat diet to establish type II diabetic rat model. TUNEL and haematoxylin and eosin staining were used to evaluate the testicular apoptotic cells and morphologic changes. Immunohistochemical staining was used to measure the expression level of vascular endothelial growth factor and blood vessel density in testes. Oxidative stress in testes and epididymis was tested by fluorescence spectrophotometer and ELISA. The expression of Bcl-2/Bax and blood flow rate in testicular vessels were measured by western blot and Doppler. Our results for the first time showed that hyperglycemia induced apoptotic cells and morphologic impairments in testes of rats, while administration of Scutellarin can significantly inhibit these damages. This effect of Scutellarin is controlled by two apoptotic triggers: ROS/Bcl-2/Bax and ROS/microcirculation/starving pathway.

2-Methoxyestradiol Induces Vasodilation by Stimulating NO Release via PPARγ/PI3K/Akt Pathway

The endogenous estradiol metabolite 2-methoxyestradiol (2-ME) reduces atherosclerotic lesion formation, while the underlying mechanisms remain obscure. In this work, we investigated the vasodilatory effect of 2-ME and the role of nitric oxide (NO) involved. In vivo studies using noninvasive tail-cuff methods showed that 2-ME decreased blood pressure in Sprague Dawley rats. Furthermore, in vitro studies showed that cumulative addition of 2-ME to the aorta caused a dose- and endothelium-dependent vasodilation. This effect was unaffected by the pretreatment with the pure estrogen receptor antagonist ICI 182,780, but was largely impaired by endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or by phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (WM). Moreover, 2-ME（10−7 ∼10−5 M）enhanced phosphorylation of Akt and eNOS and promoted NO release from cultured human umbilical endothelial cells (HUVECs). These effects were blocked by PI3K inhibitor WM, or by the transfection with Akt specific siRNA, indicating that endothelial Akt/eNOS/NO cascade plays a crucial role in 2-ME-induced vasodilation. The peroxisome proliferator-activated receptor γ (PPARγ) mRNA and protein expression were detected in HUVECs and the antagonist GW9662 or the transfection with specific PPARγ siRNA inhibited 2-ME-induced eNOS and Akt phosphorylation, leading to the impairment of NO production and vasodilation. In conclusion, 2-ME induces vasodilation by stimulating NO release. These actions may be mediated by PPARγ and the subsequent activation of Akt/eNOS cascade in vascular endothelial cells.

17β-Estradiol inhibits vascular smooth muscle cell migration via up-regulation of striatin protein.

Abstract Striatin, an estrogen receptor (ER)-interacting protein, plays an important role in estrogen’s nongenomic actions in vascular endothelial cells. However, the role of striatin in VSMCs is unknown. Here, we investigated the role of striatin in estrogen-regulated VSMCs migration. 17β-Estradiol (E2) at 10 nM largely inhibited VSMCs migration, which was reversed by the silencing of striatin expression. E2 increased striatin protein expression in a dose- and time-dependent manner. ERα agonist PPT, but not ERβ agonist DPN, mimicked the regulatory effect of E2. The regulatory effect of E2 on striatin protein expression was blocked by the pure ER antagonist ICI 182,780 or the mitogen-activated protein kinase inhibitor PD98059, but not by the phosphatidylinositol-3 kinase inhibitor wortmannin or Src inhibitor PP2, suggesting that E2 increased striatin protein expression via extracellular-signal regulated kinase 1/2 (ERK1/2). E2 resulted in phosphorylation of ERK1/2 in a time-dependent manner. The silencing of ERK1/2 largely abolished E2-enhanced striatin expression. Finally, the inhibitory effect of E2 on VSMC migration was reversed by ICI 182,780 or PD98059. Taken together, our results indicate that E2 inhibits VSMC migration by increasing striatin expression via ERα to ERK1/2 pathway, which maybe helpful to understand estrogen’s anti-atherogenic effect in VSMCs. Chinese abstract Striatin，一种雌激素受体相互作用蛋白，在雌激素对血管内皮细胞的非基因作用中发挥非常重要的作用。然而，Striatin对血管平滑肌细胞的作用是未知的。在这里，我们研究Striatin在雌激素调节的血管平滑肌细胞迁移中的作用。17β-雌二醇（E2）水平达到10 nM时很大程度上抑制VSMCs迁移，这是由于Striatin表达沉默的逆转。雌二醇增加striatin蛋白的表达是呈剂量和时间依赖的。ERα激动剂PPT，而不是ERβDPN，模仿雌激素的调节作用。E2对striatin蛋白表达的调节作用是通过纯雌激素受体拮抗剂ICI 182，780或丝裂原活化蛋白激酶抑制剂PD98059所阻断，而不是磷脂酰肌醇-3激酶抑制剂曼青霉素或酪氨酸激酶抑制剂，表明E2增加striatin蛋白表达是通过细胞外信号调节激酶1/2（ERK1 / 2）。E2使ERK1 / 2磷酸化是时间依赖性的。ERK1 / 2的沉默很大程度上抑制了E2 Striatin表达增强。最后，E2对血管平滑肌细胞迁移的抑制作用通过ICI 182780或PD98059所逆转。总之，我们的研究结果表明，E2通过ERα或激酶ERK1／2通路增强Striatin表达而抑制血管平滑肌迁移，这或许有助于理解雌激素作用于血管平滑肌细胞的抗动脉粥样硬化作用。

The role of caveolin1 and sprouty1 in genistein's regulation of vascular smooth muscle cell and endothelial cell proliferation.

Genistein prevents atherosclerosis by exerting protective effects on blood vessels. The aim of this study is to investigate the role of caveolin1 and sprouty1 in the regulation of proliferation of vascular smooth muscle cell (VSMC) and endothelial cell by genistein. Using thiazolyl blue tetrazolium bromide(MTT) and [3H]-TdR assay, we found genistein inhibited angiotensin II-induced proliferation in primary cultured VSMC while it stimulated proliferation of quiescent endothelial cells. The effects were attenuated by caveolin1 or sprouty1 siRNA. Western blot analysis indicated that genistein attenuated the phosphorylation of extracellular regulated kinase1/2(ERK1/2) in angiotensin II-induced proliferated VSMC but stimulated the phosphorylation of ERK1/2 in quiescent endothelial cell. Double staining immunofluorescence identified caveolin1 and sprouty1 coexpressed in the cytoplasm of both VSMC and endothelial cell. Genistein increased the expression of caveolin1, p-caveolin1 and sprouty1 in VSMC, while it had opposite effects in quiescent endothelial cell. Co-immunoprecipitation suggested that genistein exerted its effects through interaction of caveolin1 and sprouty1. Our results demonstrate that the inhibition of angiotensin II-induced proliferation of VSMC and stimulation of quiescent endothelial cell by genistein are regulated by caveolin1 and sprouty1, which are implemented through Ras/MAPK pathway.

Oestrogen exerts anti-inflammation via p38 MAPK/NF-κB cascade in adipocytes

BACKGROUND Oestrogen has anti-inflammatory property in obesity. However, the mechanism is still not defined. OBJECTIVE To investigate the effect of oestrogen on LPS-induced monocyte chemoattractant protein-1 (MCP-1) production in adipocytes. METHODS Lipopolysaccharides (LPS) was used to imitate inflammatory responses and monocyte chemotactic protein-1 (MCP-1) was selected as an inflammatory marker to observe. 17β-Estradiol (E2), SB203580 (SB), pyrrolidine dithiocarbamate (PDTC), pertussis toxin (PTX), wortmannin (WM), p65 siRNA and p38 MAPK siRNA were pre-treated respectively or together in LPS-induced MCP-1. Then p38 MAPK and NF-κB cascade were silenced successively to observe the change of each other. Lastly, oestrogen receptor (ER) α agonist, ERβ agonist and ER antagonist were utilised. RESULTS LPS-induced MCP-1 largely impaired by pre-treatment with E2, SB, PDTC or silencing NF-κB subunit. E2 inhibited LPS-induced MCP-1 in a time- and dose-dependent manner, which was related to the suppression of p65 translocation to nucleus. Furthermore, LPS rapidly activated p38 MAPK, while E2 markedly inhibited this activation. It markedly attenuated LPS-stimulated p65 translocation to nucleus and MCP-1 production by transfecting with p38 MAPK siRNA or using p38 MAPK inhibitor. The oestrogens inhibitory effect was mimicked by the ERα agonist, but not by the ERβ agonist. The inhibition of E2 on p38 MAPK phosphorylation was prevented by ER antagonist. CONCLUSIONS E2 inhibits LPS-stimulated MCP-1 in adipocytes. This effect is related to the inhibition of p38 MAPK/NF-κB cascade, and ERα appears to be the dominant ER subtype in these events.

Hyperglycemia induced testicular damage in type 2 diabetes mellitus rats exhibiting microcirculation impairments associated with vascular endothelial growth factor decreased via PI3K/Akt pathway

As an endocrine disease, type 2 diabetes mellitus (T2DM) can cause testicular damage which induces male infertility. However, the underlying mechanism is still not clear. We prove that T2DM induced testicular microcirculation impairment involves the decrease of VEGF and these actions are regulated by PI3K/Akt pathway. In our study, rats were divided into three groups (n=8): control group, diabetes group and diabetes + VEGF group. Intraperitoneal injection of streptozotocin (STZ, 65mg/Kg, at 9th week) and daily high-fat diet were used to establish T2DM rat model. Serum glucose in diabetes group and diabetes + VEGF group obviously exceeded 13mmol/L after STZ injection. Immunohistochemical studies indicated that VEGF level in diabetes group significantly decreased. In diabetes group, testicular blood velocity and vascular area reduced evaluated by Doppler and FITC. Furthermore, atrophic testicular morphology and increasing apoptosis cells were evaluated by haematoxylin and eosin staining and TUNEL assay. In diabetes + VEGF group, the administration of VEGF (intraperitoneally, 10mg/kg) can significantly alleviated hyperglycemia-induced impairment of testes in above aspects. Finally, we used Western blot to analyze the mechanism of hyperglycemia-induced testicular VEGF decrease. The results indicated that hyperglycemia-induced VEGF decreased is regulated by PI3K/Akt pathway in Rats testicular sertoli cells (RTSCs). Together, we demonstrate that T2DM can reduce testicular VEGF expression, which results in testicular microcirculation impairment, and then induces testicular morphological disarrangement and functional disorder. These actions are triggered by PI3K/Akt pathway. Our findings provide solid evidence for VEGF becoming a therapeutic target in T2DM related male infertility.