Sex specific response of cultured human bone cells to ERα and ERβ specific agonists by modulation of cell proliferation and creatine kinase specific activity

We have previously reported that human cultured bone cells (hObs) respond to estradiol-17β (E2) by stimulating DNA synthesis, creatine kinase BB specific activity (CK) and other parameters sex-specifically. We now investigate the sex specificity of the response of these hObs to estrogen receptor (ER) α and ERβ specific agonists. Real time PCR revealed that all cells express mRNA for both ERs. ERα mRNA but not ERβ mRNA was stimulated by all estrogenic compounds in both pre- and post-menopausal hObs with no effect in male hObs. Cells treated with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα specific agonist) showed increased DNA synthesis and CK in all female but not male hObs. Raloxifene (Ral), a specific ERα antagonist, inhibited the stimulation of DNA synthesis and CK by E2 or PPT, but not by DPN. DPN and PPT like E2 modulated the expression of both 12 and 15 lipooxygenase (LO) mRNA in both female but not male hObs. 12 and 15 HETE production was modulated only by DPN and PPT in these cells. The LO inhibitor baicaleine inhibited only E2 and PPT but not DPN effects in both female hObs. In conclusion, we provide herein evidence for the separation of age- and sex-dependent mediation via both ERα and ERβ pathways in the effects of estrogens on hObs, with a yet unknown mechanism.     

DT56a (Femarelle), contrary to estradiol-17β, is effective in human derived female osteoblasts in hyperglycemic condition

We have reported previously, that female-derived cultured osteoblasts (hObs) responded to DT56a (Femarelle) measured by the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness and (3)[H] thymidine incorporation into DNA (DNA synthesis). Since the skeletal protective effects of estrogens are not discernable in hyperglycemic diabetic women, we sought to analyze the effect of estrogenic compounds on CK and DNA synthesis in hObs when grown in high glucose concentration (HG). Cells were grown either in normal glucose (NG) (4.5g/L; 22mM) or HG (9.0g/L; 44mM) for 7 days. HG increased constitutive CK but, the response of CK activity and DNA synthesis to estradiol-17β (E(2)) treatment was reduced. In contrary, DT56a was found to be active (as measured by CK activity and DNA synthesis) in both NG and HG. HG decreases the hormonal responsiveness and might block important effects of estrogenic compounds, most likely contributing to their decreased skeletal preserving properties in hyperglycemic women. In hObs from post-menopausal women grown in HG, ERs mRNA expressions were unchanged. On the other hand, in hObs from pre-menopausal women HG increased ERs mRNA expressions. Since DT56a unlike E(2) is active in HG environment as well as in normal glucose, it may be an effective bone restoring agent in diabetic post-menopausal women.     

Modulation of response to estrogens in cultured human female bone cells by a non-calcemic Vitamin D analog: changes in nuclear and membranal binding

Estradiol17beta (E2) and the phytoestrogens genistein (G), and daidzein (D) increase creatine kinase (CK) specific activity in primary cell cultures of human female to a greater extent in cells from pre-menopausal than post-menopausal women. Pretreatment with the non-calcemic analog of Vitamin D, JK 1624 F2-2 (JKF), upregulated this estrogenic response at all ages. In contrast, biochainin A (BA) and quercertin (Qu) increased CK with no age dependence or modulation by JKF pretreatment. Both ERalpha and ERbeta present in the cells were upregulated by pretreatment with JKF, as measured by Western blot analysis. Real time PCR showed no significant change in ERalpha mRNA but a marked decrease in ERbeta mRNA in both age groups after JKF treatment. Cells from both age groups had surface binding sites for E2, shown by assays using cell impermeable Europium labeled ovalbumin-E2 conjugate (Eu-Ov-E2). Binding of [3H]-E2 to intracellular E2 receptors (ERs) was similar in both age groups with differences in phytoestrogenic competition. JKF pretreatment increased nuclear but decreased membranal binding in both age groups. These results provide evidence for membranal, in addition to nuclear estrogen receptors which are differentially modulated by a Vitamin D analog.     

The effects of estrogen receptors α- and β-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.     

A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.     

Responsiveness to phytoestrogens in primary human osteoblasts is modulated differentially by a "less-calcemic" analog of 1,25 dihydroxyvitamin D(3): JK 1624F(2)-2 (JKF)

We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and raloxifene (Ral), and male-derived cells responded only to dihydrotestosterone (DHT) when the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness, was measured. We also found that pre-treatment with the less-calcemic analog of Vitamin D, JK 1624 F(2)-2 (JKF), upregulated the response of CK to E(2) and Ral. In this study, we analyzed the response of human bone cells from pre- and post-menopausal females and males, to phytoestrogens. Bone cells derived from pre-menopausal women showed greater stimulation of CK than cells from post-menopausal women, after treatment with E(2) (30 nM), daidzein (D, 3000 nM), genistein (G, 3000 nM) and Ral (3000 nM); whereas the responses to biochainin A (BA 3000 nM), quecertin (Qu 3000 nM) or the carboxy derivative of G (cG 300 nM) were not age-dependent. Male-derived cells did not respond to phytoestrogens. When cells derived from female bones at both age groups were pre-treated with JKF, by daily addition of 1nM, for 3 days, there was an upregulation of the response to E(2), Ral, G and D but not to BA or Qu. Nuclear binding of (3)[H] E(2) was characteristic of the different phytoestrogens, with increase of the specific binding after pre-treatment with JKF. In contrast, the membranal binding of E(2)-Ov-Eu, which was specific for the estrogenic compounds except Ral, was inhibited by pre-treatment with JKF except for ICI 161480 (ICI). Male bone cells did not bind E(2)-Ov-Eu but bound T-BSA-Eu; this binding was abolished by pre-treatment with JKF. Pre-treatment with JKF increased mRNA for ERalpha and decreased mRNA for ERbeta in bone cells from both age groups of females and from males, all of which expressed both ERs, with a ratio of ERalpha to ERbeta of 121:1 in pre- and 78:1 in post-menopausal and 105:1 in male bone cells. This study raises the possibility of combined Vitamin D analog and phytoestrogen for hormonal replacement therapy to prevent post-menopausal osteoporosis, which is a subject of ongoing research in animal models.     

Vitamin D metabolites and analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cell line

Vitamin D metabolites and its less-calcemic analogs (vitamin D compounds) are beneficial for bone and modulate cell growth and energy metabolism. We now analyze whether 25(OH)D(3) (25D), 1,25(OH)(2)D(3) (1,25D), 24,25(OH)(2)D(3) (24,25D), JKF1624F(2)-2 (JKF) or QW1624F(2)-2 (QW) regulate lipooxygenase (LO) mRNA expression and its products; hydroxyl-eicosatetraenoic acid (12 and 15HETE) formation, as well as reactive oxygen species (ROS) production in human bone cell line (SaOS2) and their interplay with modulation of cell proliferation and energy metabolism. All compounds except 25D increased 12LO mRNA expression and modulated 12 and 15HETE production whereas ROS production was increased by all compounds, and inhibited by NADPH oxidase inhibitors diphenyleneiodonium (DPI) and N-acetylcysteine (NAc). Baicaleine (baic) the inhibitor of 12 and 15LO activity blocked only slightly the stimulation of DNA synthesis by all compounds, whereas DPI inhibited almost completely the stimulation of DNA and CK by all compounds. Treatments of cells with 12 or 15HETE increased DNA synthesis and CK that were only slightly inhibited by DPI. These results indicate that vitamin D compounds increased oxidative stress in osteoblasts in part via induction of LO expression and activity. The increased ROS production mediates partially elevated cell proliferation and energy metabolism, whereas the LO mediation is not essential. This new feature of vitamin D compounds is mediated by intracellular and/or membranal binding sites and its potential hazard could lead to damage due to increased lipid oxidation, although the transient mediation of ROS in cell proliferation is beneficial to bone growth in a yet unknown mechanism.     

Progesterone inhibits estrogen-mediated neuroprotection against excitotoxicity by down-regulating estrogen receptor-β

While both 17β-estradiol (E2) and progesterone (P4) are neuroprotective in several experimental paradigms, P4 also counteracts E2 neuroprotective effects. We recently reported that a 4-h treatment of cultured hippocampal slices with P4 following a prolonged (20?h) treatment with E2 eliminated estrogenic neuroprotection against NMDA toxicity and induction of brain-derived neurotrophic factor (BDNF) expression. In the present study, we evaluated the effects of the same treatment on levels of estrogen receptors, ERα and ERβ, and BDNF using a similar paradigm. E2 treatment resulted in elevated ERβ mRNA and protein levels, did not modify ERα mRNA, but increased ERα protein levels, and increased BDNF mRNA levels. P4 reversed E2-elicited increases in ERβ mRNA and protein levels, in ERα protein levels, and in BDNF mRNA levels. Experiments with an ERβ-specific antagonist, PHTPP, and specific agonists of ERα and ERβ, propylpyrazoletriol and diarylpropionitrile, respectively, indicated that E2-mediated neuroprotection against NMDA toxicity was, at least in part, mediated via ERβ receptor. In support of this conclusion, E2 did not protect against NMDA toxicity in cultured hippocampal slices from ERβ-/- mice. Thus, E2-mediated neuroprotection against NMDA toxicity may be because of estrogenic induction of BDNF via its ERβ receptor, and P4-mediated inhibition of E2 neuroprotective effects treatment to P4-induced down-regulation of ERβ and BDNF.     

Leptin regulates estrogen receptor gene expression in ATDC5 cells through the extracellular signal regulated kinase signaling pathway

Both estrogen and leptin play an important role in the regulation of physiological processes of endochondral bone formation in linear growth. Estrogen receptors (ERα and ERβ) are known as members of the superfamily of nuclear steroid hormone receptors and are detected in all zones of growth plate chondrocytes. They can be regulated in a ligand-independent manner. Whether leptin regulates ERs in the growth plate is still not clear. To explore this issue, chondrogenic ATDC5 cells were used in the present study. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting. We found that both ERα and ERβ were dynamically expressed during the ATDC5 cell differentiation for 21 days. Leptin (50 ng/ml) significantly upregulated ERα and ERβ mRNA and protein levels 48 h after leptin stimulation (P<0.05) at day 14. The up-regulation of ERα and ERβ mRNA by leptin was shown in a dose-dependent manner, but the most effective dose of leptin was different (100 and 1,000 ng/ml, respectively). Furthermore, we confirmed that leptin augmented the phosphorylation of ERK1/2 in a time-dependent manner. A maximum eightfold change was observed at 15 min. Finally, a specific ERK1/2 inhibitor, UO126, blocked leptin-induced ERs regulation in ATDC5 cells, indicating that ERK1/2 mediates, partly, the effects of leptin on ERs. These data demonstrate, for the first time, that leptin regulates the expression of ERs in growth plate chondrocytes via ERK signaling pathway, thereby suggesting a crosstalk between leptin and estrogen receptors in the regulation of bone formation.     

Fatty acids derived from royal jelly are modulators of estrogen receptor functions

Royal jelly (RJ) excreted by honeybees and used as a nutritional and medicinal agent has estrogen-like effects, yet the compounds mediating these effects remain unidentified. The possible effects of three RJ fatty acids (FAs) (10-hydroxy-2-decenoic-10H2DA, 3,10-dihydroxydecanoic-3,10DDA, sebacic acid-SA) on estrogen signaling was investigated in various cellular systems. In MCF-7 cells, FAs, in absence of estradiol (E(2)), modulated the estrogen receptor (ER) recruitment to the pS2 promoter and pS2 mRNA levels via only ERβ but not ERα, while in presence of E(2) FAs modulated both ERβ and ERα. Moreover, in presence of FAs, the E(2)-induced recruitment of the EAB1 co-activator peptide to ERα is masked and the E(2)-induced estrogen response element (ERE)-mediated transactivation is inhibited. In HeLa cells, in absence of E(2), FAs inhibited the ERE-mediated transactivation by ERβ but not ERα, while in presence of E(2), FAs inhibited ERE-activity by both ERβ and ERα. Molecular modeling revealed favorable binding of FAs to ERα at the co-activator-binding site, while binding assays showed that FAs did not bind to the ligand-binding pocket of ERα or ERβ. In KS483 osteoblasts, FAs, like E(2), induced mineralization via an ER-dependent way. Our data propose a possible molecular mechanism for the estrogenic activities of RJ's components which, although structurally entirely different from E(2), mediate estrogen signaling, at least in part, by modulating the recruitment of ERα, ERβ and co-activators to target genes.     

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.     

Effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils (Meriones unguiculatus)

Quinestrol, a synthetic estrogen with marked estrogenic effects and prolonged activity, has potential as a contraceptive for Mongolian gerbils. The objective of this study was to describe the effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils. Serum and pituitary concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were decreased, whereas serum concentrations of estradiol (E2) and progesterone (P4) were increased after quinestrol treatment; the effects were both time- and dose-dependent. Furthermore, quinestrol downregulated expression of FSHβ and LHβ mRNA in the pituitary gland, as well as FSH receptor (FSHR) and estrogen receptor (ER) β in the ovary. However, it up-regulated mRNA expression levels of ERα and progesterone receptor (PR) in the pituitary gland and uterus, as well as mRNA for LH receptor (LHR) and PR in the ovary (these effects were time- and dose-dependent). In contrast, quinestrol had no significant effects on the mRNA expression levels of ERα in the ovary, or the gonadotropin α (GtHα) subunit in the pituitary gland. We inferred that quinestrol impaired synthesis and secretion of FSH and LH and that the predominant ER subtype in the pituitary gland of Mongolian gerbils may be ERα. Overall, quinestrol disrupted reproductive hormone receptor expression at the mRNA level in the pituitary-gonadal axis of the Mongolian gerbil.     

Modulation of the response to estradiol-17beta of rat vascular tissues by a non calcemic vitamin D analog

Estradiol-17beta (E(2)) increases creatine kinase (CK) specific activity in aorta (Ao) and left ventricle of the heart (Lv) from rat females. In the present study, we analyzed the effects of pretreatment with the non calcemic analog of vitamin D, JK 1624 F2-2 (JKF) on the response to E(2) (either 0.5 or 5 microg/rat) of Ao and Lv from prepubertal female rats. JKF did not affect CK in either organ. However, pretreatment with JKF (0.1 ng/g body weight for 1 or 2 weeks) increased the CK response to E(2) (0.5 microg/rat) by 50 +/- 10% in Ao and by 150 +/- 12% in Lv. The CK response to 5 microg/rat of E(2) in intact female rats, was increased by 118 +/- 15% and 99 +/- 11% in the Ao and by 89 +/- 6% and 112 +/- 13% in the Lv, in animals treated daily with JKF for 1 or 2 weeks, respectively, before administration of E(2). JKF also increased the response to 500 microg/rat raloxifene (Ral) by 47 +/- 8% in Ao and by 56 +/- 12% in Lv. Preliminary experiments showed that JKF treatment induced a approximately 50% increase in estradiol receptor ERalpha in both organs. The results indicate that the vitamin D analog JKF upregulates the response and sensitivity of vascular tissues to E(2), in association with increased expression of their ERalpha. These results should prompt examination of the possibility that the effects estrogen therapy in postmenopausal women can be augmented by vitamin D or its analogs.     

SIRT1‐mediated ERβ suppression in the endothelium contributes to vascular aging

SIRT1 has many important molecular functions in aging, and the estrogen receptors (ERs) have a vasculoprotective effect, although the detailed mechanism for the roles of SIRT1 and ERs in vascular aging remains unclear. We found that ERβ expression in the endothelium was reduced in aging mice, and the expression of ERα and SIRT1 did not change, while SIRT1 activity declined. Further investigation showed that the ERβ expression was regulated by SIRT1 through complexes of SIRT1‐PPARγ/RXR‐p300 that bind to a PPRE (PPAR response element) site on the ERβ promoter, and the declined SIRT1 function in aging mice was due to compromised phosphorylation at S154. A single‐mutant SIRT1‐C152(D) restored the reduced ERβ expression in the endothelium with minimized reactive oxygen species generation and DNA damage and increased mitochondrial function and fatty acid metabolism. In high‐fat diet aging mice, the endothelium‐specific delivery of ERβ or SIRT1‐C152(D) on the vascular wall reduced the circulating lipids with ameliorated vascular damage, including the restored vessel tension and blood pressure. We conclude that SIRT1‐mediated ERβ suppression in the endothelium contributes to vascular aging, and the modulation of SIRT1 phosphorylation through a single‐mutant SIRT1‐C152(D) restores this effect.     

Human sperm physiology: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) influence sperm metabolism and may be involved in the pathophysiology of varicocele-associated male infertility

The mechanisms by which varicocele affects fertility remain undetermined. Estrogens play a key role in the human male reproduction and human sperm expresses the estrogen receptors (ERs) and aromatase. In this study, by Western blotting we evidenced the ERs content concomitantly in healthy sperm and in oligoastenoteratozoospermic (OAT) samples without and with varicocele. In varicocele a strong reduction of the ERβ was observed, while the ERα was almost absent. Besides, transmission electron microscopy (TEM) confirmed the reduction of ERs expression in "varicocele" sperm, indicating that varicocele has a detrimental effect on sperm structure at molecular level. To further define the estrogen significance in male gamete and the pathophysiology of varicocele we investigated both the expression of ERα and ERβ in normal and pathologic sperm samples as well as we evaluated estradiol (E2) action on lipid and glucose sperm metabolism. Responses to E2 treatments on cholesterol efflux, protein tyrosine phosphorylations, motility, and acrosin activity in varicocele sperm were reduced or absent. The evaluation of the triglycerides content, lipase and acyl-CoA dehydrogenase activities, suggest that E2 exerts a lipolytic effect on human sperm metabolism. Concerning glucose metabolism, it appears that E2 induces G6PDH activity concomitantly to the insulin secretion. In "varicocele" sperm, the E2 did not induce energy expenditure. OAT sperm had E2-responsiveness but in a lesser extent with respect healthy sperm. This study discovered a novel role for E2/ERs in human sperm physiology, since they modulate sperm metabolism and new detrimental effects related to the pathophysiology of the varicocele condition.     

The hepatitis B virus-associated estrogen receptor alpha (ERalpha) was regulated by microRNA-130a in HepG2.2.15 human hepatocellular carcinoma cells

The estrogen receptors (ERs) play important roles in hepatocarcinogenesis, which is always fostered by persistent hepatitis B virus (HBV) infection. Recent studies have linked microRNAs (miRNAs) to viral pathogenesis or oncogenesis. ERα could lead to cell cycle progression or inhibition of apoptosis, while ERβ had opposite effects. Here we proposed that HBV affected ERs expression in viral oncogenesis, which might be triggered by miRNAs. The protein expression of ERα in HepG2.2.15 cells was much stronger than that in HepG2 cells, which was not consistent with its mRNA expression in these cells. MicroRNA-130a (miR-130a) was predicted to be a regulator of ERα by targeting its 3' untranslated region (3'-UTR). The enhanced green fluorescence protein (EGFP) reporter experiment confirmed the direct interaction of miR-130a and ERα. Moreover, ERα protein level was inversely correlated with the miR-130a level. Taken together, our studies supported that HBV infection might attenuate miR-130a expression and ERα was a direct target of miR-130a. Difference in miR-130a levels between HepG2 and HepG2.2.15 cells resulted in the difference in ERα expression, implying host-virus crosstalk in viral pathogenesis mediated by miRNAs.