p38α MAPK mediates 17β-estradiol inhibition of MMP-2 and -9 expression and cell migration in human lovo colon cancer cells

Epidemiological studies demonstrate that the incidence and mortality rates of colorectal cancer in women are lower than in men. However, it is unknown if 17β‐estradiol (E₂) treatment is sufficient to inhibit cell proliferation and cell migration in human colon cancer cells. Up‐regulation of urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and matrix metallopeptidases (MMPs) is reported to associate with the development of cancer cell mobility, metastasis, and subsequent malignant tumor. In the present study, we treated human LoVo colon cancer cells with E₂ to explore whether E₂ down‐regulates cell proliferation and migration, and to identify the precise molecular and cellular mechanisms behind the down‐regulatory responses. Here, we found that E₂ treatment decreased cell proliferation and cell cycle‐regulating factors such as cyclin A, cyclin D1 and cyclin E. At the same time, E₂ significantly inhibited cell migration and migration‐related factors such as uPA, tPA, MMP‐2, and MMP‐9. However, E₂ treatment showed no effects on upregulating expression of plasminogen activator inhibitor‐1 (PAI‐1), tissue inhibitor of metalloproteinase‐1, ‐2, ‐3, and ‐4 (TIMP‐1, ‐2, ‐3, and ‐4). After administration of inhibitors including QNZ (NFκB inhibitor), LY294002 (Akt activation inhibitor), U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor) or SP600125 (JNK1/2 inhibitor), E₂‐downregulated cell migration and expression of MMP‐2 and MMP‐9 in LoVo cells is markedly inhibited only by p38 MAPK inhibitors, SB203580. Application of specific target gene siRNA (ERα, ERβ, p38α, and p38β) to LoVo cells further confirmed that p38 MAPK mediates E₂/ERs inhibition of MMP‐2 and ‐9 expression and cell motility in LoVo cells. Collectively, these results suggest that E₂ treatment down‐regulates cell proliferation by modulating the expression of cyclin A, cyclin D1 and cyclin E. E₂ treatment simultaneously impaired cell migration by inhibiting the expression of uPA, tPA, MMP‐2, and MMP‐9 through E₂/ERs ? p38α MAPK signaling pathway in human LoVo colon cancer cells. J. Cell. Physiol. 227: 3648–3660, 2012. © 2012 Wiley Periodicals, Inc.     

Modulation of human β-defensin-2 expression by 17β-estradiol and progesterone in vaginal epithelial cells

We investigated the expression of HBD-1 and -2 in vaginal epithelial cells treated with lipopolysaccharide (LPS) and the effects on HBD-2 expressions by 17β-estradiol and progesterone. Primary vaginal epithelial cells were isolated from a segment of normal anterior vaginal wall obtained during vaginoplasty and were cultured in keratinocyte growth medium and were allowed to undergo their 3rd passage. Expression of HBD-1 and -2 by different stimuli using LPS 0.5 μg/ml, 17β-estradiol 2 nM and progesterone 1 μM was measured by RT-PCR, ELISA and real-time RT-PCR, respectively. HBD-1 was produced constitutively in vaginal epithelial cells and the production of HBD-1 was not influenced by LPS, 17β-estradiol and progesterone, but the production of HBD-2 was increased inducibly by LPS. 17β-Estradiol and progesterone did not change the production of HBD-2 in normal state, but 17β-estradiol increased the production of HBD-2 and progesterone suppressed the production of HBD-2 under the circumstances with infection. The HBD-2 plays an important role at innate host defense on genitourinary tract. The lacks of estrogen during menopause or uses of a progesterone-based oral contraceptive in sexually active women may influence production of HBD-2 in vaginal epithelium and may increase susceptibility to bacterial vaginitis or recurrent UTI.     

Characterization of caveolins from human knee joint cartilage: expression of caveolin-1, -2, and -3 in chondrocytes and association with integrin β1

Interactions between the extracellular matrix (ECM) and chondrocytes are of great importance for structure and function of cartilage. The present study was undertaken to answer the question whether caveolins take part in integrin-mediated cell–ECM interactions in the human cartilage. In samples of human knee joint cartilage, we detected the caveolin subtypes -1, -2, and -3 by immunohistochemical methods. Double-label experiments revealed a colocalization of caveolin with β1-integrin. Results of immunoprecipitation and immunoblotting assays show that β1-integrins associate with all three caveolin subtypes in human chondrocytes and indicate that they are part of the same complexes. Furthermore, immunoelectron microscopy shows the localization of β1-integrin in caveolae-like structures of the cell membrane. The data stimulate further investigations on the role of the caveolin–integrin complex for integrin-mediated signaling pathways in chondrocytes. Accepted: 17 December 1999     

Purification and characterization of the human protein tyrosine phosphatase,PTPμ, from a baculovirus expression system

The receptor like PTPase, PTP, displays structural similarity in its extracellular segment to members of the immunoglobulin superfamily of cell adhesion molecules. The full length form of PTP (200 kD) and a construct expressing only the intracellular PTPase domain-containing segment *80 kD) were expressed in the baculovirus/Sf9 cell system, purified and characterized. Full length PTP was membrane associated while the truncated form was recovered in the soluble fraction. PTP preferentially dephosphorylated a reduced carboxamidomethylated and maleylated derivative of lysozyme (RCML) over other tyrosine phosphorylated substrates such as myelin basic protein (MBP) or the synthetic peptide EDNDYINASL. The enzymatic properties of the soluble, truncated form of the enzyme were examined in detail. The pH optimum was 7.5. It dephosphorylated RCML with a K_m of 400 nM and a V_max of 725 nmol/min/mg. This form of the enzyme was 2 fold more active than full length PTP. Trypsinization of the full length form inhibited activity. Vanadate and molybdate, potent tyrosine phosphatase inhibitors, abolished activity of the enzyme. Zn⁺⁺ and Mn⁺⁺ ions, polylysine, poly-glu/tyr, and spermine were also inhibitory.     

Assay and properties of alginate lyase and 1, 3-β-glucanase in intertidal sands

Summary Alginate lyase and 1, 3--glucanase activity were detected in intertidal sands below decomposing seaweeds (Fucus sp. andLaminaria sp). Linear relationships between activity and sand weight; length of incubation and substrate concentration, were established for both enzymes. Other properties of these enzymes in intertidal sands are reported.     

Pharmacology and immune modulating properties of 5-androstene-3β,7β,17β-triol, a DHEA metabolite in the human metabolome

Androst-5-ene-3β,7β,17β-triol (βAET) is an anti-inflammatory metabolite of DHEA that is found naturally in humans, but in rodents only after exogenous DHEA administration. Unlike DHEA, C-7-oxidized DHEA metabolites cannot be metabolized into potent androgens or estrogens, and are not peroxisome proliferators in rodents. The objective of our current studies was to characterize the pharmacology of βAET to enable clinical trials in humans. The pharmacology of βAET was characterized by pharmacokinetics, drug metabolism, nuclear hormone receptor interactions, androgenicity, estrogenicity, and systemic toxicity studies. βAET's acute anti-inflammatory activity and immune modulating characteristics were measured in vitro in RAW264.7 cells and in vivo in murine models with parenteral administration. βAET was rapidly metabolized and cleared from circulation in mice and monkeys. βAET was weakly androgenic and estrogenic in immature rodents, but not bound by androgen, estrogen, progesterone, or glucocorticoid nuclear hormone receptors. βAET did not induce peroxisome proliferation, nor was it systemically toxic or trophic for sex hormone responsive tissues in mature rats and monkeys. βAET significantly attenuated acute inflammation both in vitro and in vivo, augmented immune responses in adult mice, and reversed immune senescence in aged mice. βAET may contribute to the anti-inflammatory activity in rodents attributed to DHEA. Unlike DHEA, βAET's anti-inflammatory activity cannot be ascribed to activation of PPARs, androgen, or estrogen nuclear hormone receptors. Exogenous βAET is unlikely to produce untoward toxicity or hormonal perturbations in humans.     

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.     

LC–MS/MS coupled with immunoaffinity extraction for determination of estrone, 17β-estradiol and estrone 3-sulfate in human plasma

Determination of estrogens in plasma is important in evaluation of effects of some anticancer drugs, such as aromatase inhibitors. However, as reported previously, high performance liquid chromatography–radio immunoassay (HPLC–RIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) with chemical derivatization require complicated sample preparation. In this study, a highly sensitive and simple method for determination of estrone (E1), 17β-estradiol (E2) and estrone 3-sulfate (E1S) in human plasma has been developed. Following diethylether extraction from plasma, analytes were purified by immunosorbents and then determined by LC–MS/MS using electrospray ionization (ESI). Immunosorbents were prepared by immobilization of specific antibodies raised against each analyte onto solid support. Use of selective immunosorbents in sample preparation removed interference in plasma samples that would cause ionization suppression, and markedly improved the sensitivity of LC–MS/MS for these analytes, without derivatization. Calibration curves of each analyte showed good linearity and reproducibility over the range of 0.05–50 pg/injection for E1, 0.2–50 pg/injection for E2 and 0.05–300 pg/injection for E1S, respectively. The mean values of lower limits of quantification (LLOQ) in human plasma corrected by recovery of deuterated estrogens (internal standard, I.S.) were 0.1892 pg/mL for E1, 0.7064 pg/mL for E2 and 0.3333 pg/mL for E1S, respectively. These LLOQ values were comparable to those previous reported using HPLC–RIA and LC–MS/MS. Using this method, the normal levels of three estrogens in healthy female plasma (n = 5) were determined. The mean values of E1, E2 and E1S were 38.0 pg/mL (range 24.8–53.0), 34.3 pg/mL (22.6–46.6) and 786 pg/mL (163–2080), respectively. The immunoaffinity LC–MS/MS described here allows sensitive and accurate quantification of E1, E2 and E1S without laborious sample preparation.     

Effects of 17 β-estradiol on the expression of interstitial collagenases-8 and −13 (MMP-8 and MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in ovariectomized rat osteoblastic cells

Estrogen plays an important role in maintaining normal bone metabolism via the direct or indirect regulation of bone cells. Osteoblastic cells, as the target cells of estrogen, can secrete multiple matrix metalloproteinases (MMPs) that participate in bone remodeling. It has been demonstrated that bone loss induced by estrogen deficiency is closely related to the abnormal expression of multiple MMPs in osteoblastic cells. However, the regulating action of estrogen on the expression of interstitial collagenases MMP-8 and MMP-13 in osteoblastic cells in vivo remains unclear. We used an ovariectomized osteoporotic rat model to analyze the changes in the histomorphometric parameters of bone after and without treatment with 17-estradiol (E₂); We also used immunohistochemistry and in situ hybridization to observe changes in the expression of mRNA and the proteins MMP-8, MMP-13 and TIMP-1 in osteoblastic cells in rat proximal tibia. In this study, we found that in the ovariectomized rat the expression of MMP-13 mRNA and protein increased markedly, whereas the expression of MMP-8 and TIMP-1 mRNA and protein did not change significantly. Our analysis showed that the expression of MMP-13 protein was correlated positively to bone trabecular separation, osteoid surface area, and negatively to trabecular numbers and the percentage of trabecula bone volume/total tissue volume. Our results suggest that MMP-13 plays an important role in estrogen deficiency-induced bone loss, while estrogen can inhibit bone resorption and reduce bone turnover rate by down-regulating the expression of MMP-13 in osteoblastic cells.     

5α-androstane-3β,17β-diol in feedback between gonads and hypothalamo-pituitary system of male rats

Serum level of LH and levels of LH-RH in the arcuate nucleus and in the median eminence of hypothalamus were measured by radioimmunoassay in castrated male rats 1, 3, 6, 12, 24 h after single administration of 25 μg/100 g b.w. testosterone, 5α-androstane-3α,17β-diol (3α-diol) or 5α-androstane-3β, 17β-diol (3β-diol).The levels were then measured 6 h after single administration of 3α-diol and 3β-diol in different doses: 5 μg; 25 μg; 100 μg; 250 μg; 2500 μg/100 g b.w.The results suggest basic differences between the influence of testosterone and of its 5α-reduced metabolites upon the hypothalamo-pituitary system as to speed and strength of effect. Both 3α-diol and 3β-diol are active in the negative feedback between the gonads and the hypothalamo-pituitary system, but their effect depends on the dose. 3β-Diol is active in the low doses we have studied as opposed to its 3α-epimer.     

Peripartal plasma concentrations of 15-ketodihydro-PGF2α, cortisol, progesterone and 17-β-estradiol in Martina Franca jennies

The transition from intra- to extrauterine environment represents a very delicate phase, in which the successful coordination of maturation is strictly connected with several hormonal changes during the last weeks of gestation and at parturition. While the peripartal endocrinology in the mare has been deeply investigated, the peripartal hormonal changes in the jenny need further evaluation. The aim of this study is to evaluate the mean 15-ketodihydro-PGF_2α (PGFM), cortisol (C), progesterone (P4), and 17β-estradiol (E2) levels during the peripartal period in this species. Ten Martina Franca jennies, with normal gestational length and parturition, were enrolled. From each jenny, blood was collected twice a day from 10 d before to 7 d after parturition and from the plasma obtained PGFM, C, P4 and E2 were analyzed by RIA. Higher, constant PGFM concentrations were observed in the pre-foaling days compared to the decreasing levels detected the days after delivery, as previously observed in the mare. During the whole period of observation no significant differences in plasma C levels were detected. In contrast to the mare, P4 has always been detectable and the highest level found at −2.5 days was significantly different compared to samples obtained between −10 and −4.5 days and between 1.5 and 7 days after foaling. Finally, E2 showed higher concentrations before foaling, with the highest values between −3 and −1.5 days, decreasing only one day before foaling. A positive correlation was found between PGFM and P4, during the last 4 days of gestation, while a positive correlation between PGFM and E2 was observed during the prepartum. Despite some similarities with the mare exist, differences have been found in P4 and E2 profiles, underlining once more the differences in the physiology of this two species.     

Characterization and constitutive expression of a novel endo-1,4-β-d-xylanohydrolase from Aspergillus niger in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl10 from Aspergillus niger, encoding a 308-residue mature xylanase belonging to glycosyl hydrolase family 10, was constitutively expressed in Pichia pastoris. The recombinant Xyl10 exhibited optimal activity at pH 5.0 and 60 °C with more than 50 % of the maximum activity from 40 to 70 °C. It retained more than 90 % of the original activity after incubation at 60 °C (pH 5.0) for 30 min and more than 74 % after incubation at pH 3.0–13.0 for 2 h (25 °C). The specific activity, K _m and V _max values for purified Xyl10 were, respectively, 3.2 × 10³ U mg^?1, 3.6 mg ml^?1 and 5.4 × 10³ μmol min^?1 mg^?1 towards beechwood xylan. The enzyme degraded xylan to a series of xylooligosaccharides and xylose. The recombinant enzyme with these properties has the potential for various industrial applications.     

Protein carbonyl, 3β-, and 17β-hydroxysteroid dehydrogenases in testes and serum FSH, LH, and testosterone levels in zinc deficient Wistar rats

The present study evaluated protein oxidation, alteration in hydroxysteroid dehydrogenases (3β- and 17β HSD) in testes and serum hormonal profiles of dietary zinc deficient Wistar rats. Pre-pubertal rats were divided into three groups: zinc control (ZC), pairfed (PF), and zinc deficient (ZD) and fed 100 ppm (ZC and PF groups) and 1.0 ppm (ZD group) zinc diet for 2- and 4-weeks. The testes from zinc deficient groups exhibited significant increase in total protein (2 weeks) and protein carbonyl (2- and 4-weeks) concentration as well as 3β- and 17β-hydroxysteroid dehydrogenase activities (4 weeks), whereas a significant decrease was recorded in total protein (testes 4 weeks; serum 2- and 4-weeks), total zinc (testes and serum 2- and 4-weeks), 3β- and 17β-hydroxysteroid dehydrogenase activities (testes 2 weeks), and serum hormonal profiles (FSH and testosterone 2- and 4-weeks). However, LH was below the detectable limits. These results reflect that zinc deficiency during pre-pubertal period affected total protein and zinc status, elevates protein oxidation, and causes dysregulation of the hydroxysteroid dehydrogenases. Low level of zinc attenuated the gonadal physiology which indicates that the metabolic regulation of testes is mediated by combined effects of a specific response (caused by decreased zinc concentration) and a nonspecific response (inhibition of gonadotrophin secretion). All these contribute to testicular dysfunction.     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Effect of 17β-estradiol and flavonoids on the regulation of expression of newly identified oestrogen responsive genes in a rat raphe nuclei-derived cell line

Due to the health risks attributed to perimenopausal hormone therapy, phytoestrogens such as flavonoids are receiving widespread attention to help alleviate menopausal symptoms, including hormone-driven mood disorders. Based on our previous reporter gene study regarding their transactivational activity in raphe nuclei cells from a brain region involved in regulation of mood disturbances, we herein study their effects on the regulation of expression of 17β-estradiol (E2)-regulated genes. DNA microarray was used to globally assess E2-induced gene expression in RNDA cells, a rat raphe nuclei-derived cellular model expressing oestrogen receptor β. Out of 212 regulated genes, six were selected for verification and as endpoints for the effect of flavonoids on the regulation of mRNA expression in proliferating as well as differentiating RNDA cells. Under proliferative conditions, E2 up-regulated mRNA expression of Cml-5, Sox-18 and Krt-19. Similar effects were observed in response to 8-prenylnaringenin (8-PN), genistein (GEN), daidzein (DAI) and equol (EQ). In line with E2, mRNA expression of Nefm and Zdhhc-2 was down-regulated following 8-PN, GEN, DAI, EQ and naringenin treatment. No regulation was observed on Slc6a4 mRNA expression in response to E2 or the flavonoids in proliferating RNDA cells. When cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards E2 and the tested flavonoids were noticed. These expression studies additionally highlighted some of the genes as markers for RNDA cellular differentiation. RNDA cells should prove useful to elucidate molecular and cellular mechanisms of exogenous oestrogen receptor ligands with neural cell populations.     

Antisense oligonucleotides to crabp I and II alter the expression of TGF-β3, RAR-β, and tenascin in primary cultures of embryonic palate cells

Summary The cellular retinoic acid-binding proteins (CRABPs) are thought to modulate the responsiveness of cells to retinoic acid (RA). We have previously shown that primary cultures of murine embryonic palate mesenchymal (MEPM) cells express both CRABP-I and CRABP-II genes and that this expression is regulated by RA and transforming growth factor β (TGF-β). These cells also express high levels of TGF-β3, which is also regulated by RA and TGF-β. We have used an antisense strategy to investigate the role of the CRABPs in retinoid-induced gene expression. Subconfluent cultures of MEPM cells were treated for several days with phosphorothioate modified 18-mer oligonucleotides antisense to CRABP-I or CRABP-II and then with all-trans-retinoic acid at a concentration of 3.3 μM or 0.33 μM for 5 or 22 h. Total RNA was then extracted and the expression of TGF-β3, retinoic acid receptor β (RAR-β), and tenascin was assessed by northern blot analysis. Antisense oligonucleotides to CRABP-I partially inhibited the RA-induced TGF-β3, RAR-β, and tenascin mRNA expression. The corresponding mis-sense oligonucleotides were without effect. Antisense oligonucleotides to CRABP-II also partially inhibited RA-induced expression of these genes. As with the CRABP-I antisense, mis-sense oligonucleotides to CRABP-II had no effect. These data suggest that both CRABPs modulate the responsiveness of MEPM cells to retinoic acid. Inhibition of endogenous CRABP expression renders MEPM cells less responsive to RA with respect to induction of TGF-β3, RAR-β, and tenascin gene expression. These results have important implications for our understanding of the role of the CRABPs in retinoid teratology.