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.     

Progesterone and levonorgestrel regulate expression of 17βHSD-enzymes in progesterone receptor positive breast cancer cell line T47D

The use of combined hormone replacement therapy (HRT) with oestrogens and progestins in postmenopausal women has been associated with an increased risk for developing breast cancer. The reasons are not fully understood, but influence of HRT on endogenous conversion of female sex hormones may be involved. The expression of 17β hydroxysteroid dehydrogenases (17βHSD), which are enzymes catalysing the conversion between more or less potent oestrogens, may partly be regulated by progestins. The breast cancer cell lines T47D, MCF7 and ZR75-1 were treated with progesterone, medroxyprogesterone acetate (MPA) or levonorgestrel for 48 and 72 h at 10(-7) and 10(-9)M to investigate influence on 17βHSD1, 17βHSD2 and 17βHSD5 mRNA expression measured by real time PCR. The expression of 17βHSD1 increased in progesterone and levonorgestrel treated T47D cells (48 h 10(-7)M P=0.002; P<0.001) and 17βHSD5 increased after progesterone treatment (48 h 10(-7)M P=0.003), whereas the expression of 17βHSD2 decreased after the (48 h 10(-7)M P=0.003; P<0.001). Similar, but less prominent effects were seen in MCF7 and ZR75-1. The progestin effects on 17βHSD-expression were lost when T47D cells were co-treated with progestins and the progesterone receptor (PgR) inhibitor mifprestone. We show that both reductive (17βHSD1 and 17βHSD5) and oxidative (17βHSD2) members of the 17βHSD-family are under control of progesterone and progestins in breast cancer cell lines. This is most clear in T47D cells which have high PgR expression. 17βHSD-enzymes are important players in the regulation of sex steroids locally in breast tumours and tumoural expression of various 17βHSD-enzymes have prognostic and treatment predictive relevance. We propose a mechanism for increased breast cancer risk after HRT in which hormone replacement affects the expression of 17βHSD-enzymes, favouring the expression of reductive enzymes, which in turn could increase levels of bioactive and mitogenic estrogens in local tissue, e.g. breast tissue.     

Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17beta given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [(14)C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17beta has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [(14)C]lysine into stromal nuclear proteins, but changes after oestradiol-17beta treatment were similar to those seen in epithelium with oestradiol-17beta alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17beta. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17beta alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-(3)H]oestradiol-17beta by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.     

Secretion of gonadotrophins change during the luteal phase of the bovine oestrous cycle in the absence of corresponding changes in progesterone or 17β-oestradiol

The hypothesis in the present study was that changes in circulating luteinizing hormone (LH) and follicle stimulating hormone (FSH) would occur during the luteal phase of the oestrous cycle (Days 4–19; Day 0, day of behaviourial oestrus) that were not related to corresponding changes in concentrations of progesterone and 17β-oestradiol. The stage of the oestrous cycle of cows (n = 18) was synchronised to obtain cows that were on alternate days of the cycle. Blood samples were collected every other day at 15 min intervals for 12 h from all cows: Days 4, 6, 8, 10, 12, 14, 16, 18 (n = 9) and Days 5, 7, 9, 11, 13, 15, 17, 19 (n = 9). Concentrations of LH, FSH, 17β-oestradiol and progesterone were determined in these samples. Data were compared across days to determine when significant changes occurred in concentrations or patterns of secretion of the gonadotrophins and ovarian steroid hormones during the oestrous cycle. There were significant changes in mean concentrations of FSH in circulation between Days 6 and 12. The most striking changes in secretion of gonadotrophins that could not be explained by changes in gonadal steroids were the fluctuations in amplitude of LH pulses between Days 7 and 12. Amplitude of LH pulses increased between Days 7 and 11 and subsequently decreased between Days 11 and 12 of the oestrous cycle. Some changes in gonadotrophin secretion that occurred in the present study can be explained by fluctuations in concentrations of progesterone and 17β-oestradiol in circulation. Other changes cannot be explained by fluctuations in circulating concentrations of these steroids. We accept our hypothesis because the concomitant changes in mean concentration of FSH between Days 6 and 11 and amplitude of LH pulses between Days 7 and 12 of the bovine oestrous cycle cannot be explained by changes in circulating concentrations of progesterone and 17β-oestradiol.     

The enhanced expression of estrogen-related receptor α in human bladder cancer tissues and the effects of estrogen-related receptor α knockdown on bladder cancer cells

Estrogen-related receptor α (ERRα) belongs to the superfamily of nuclear orphan receptors. However, the role of ERRα in bladder cancer remains unknown. This study examined the expression of ERRα in bladder cancer tissues and explored the molecular mechanisms of ERRα in bladder cancer progression. The expression of ERRα in bladder cancer tissues from 61 patients was determined by immunohistochemistry. We performed quantitative real-time polymerase chain reaction assay to detect the gene expression levels and carried out Western blot assay to measure protein levels. In vitro functional assays, including colony formation, Cell Counting Kit-8, Transwell invasion, and migration assays, were performed to detect bladder cancer cell growth, proliferation, invasion, and migration, respectively. Flow cytometry was used to determine the cell apoptotic rate of bladder cancer cells. Among the 61 detected bladder cancer tissues, 39 bladder cancer tissues showed positive ERRα immunoreactivity. Higher ERRα immunoreactivity score was significantly associated with TNM stage, tumor grade, distant metastasis, and poor survival in patients with bladder cancer. Univariate and multivariate analyses showed that ERRα immunoreactivity was an independent prognostic factor for overall survival in patients with bladder cancer. ERRα was found to be upregulated in bladder cancer cell lines, and knockdown of ERRα suppressed bladder cancer cell growth, proliferation, invasion, and migration; promoted bladder cancer cell apoptosis; and inhibited the epithelial-mesenchymal transition of bladder cancer cells. On the other hand, bladder cancer cell proliferation, invasion, and migration were significantly enhanced after cells were transfected with an ERRα-overexpressing vector. In vivo tumor growth and metastasis assays showed that ERRα knockdown resulted in remarkable inhibition of tumor growth and tumor metastasis in nude mice. Collectively, our results suggest that the enhanced expression of ERRα may play a key role in the development and progression of bladder cancer and ERRα may serve as an important prognostic factor for bladder cancer.     

Determination of 17β-oestradiol by fluorescence immunoassay with streptavidin-conjugated quantum dots as label

A sensitive and selective method for the determination of 17β-oestradiol by fluorescence immunoassay (FIA) was established on the basis of quantum dots (QDs) as label. The complex of biotin-labelled anti-rabbit IgG and strepavidin conjugated by quantum dots (QD-SA) was regarded as a probe in this system and the strepavidin-biotin system as signal amplification system. After optimising the conditions of the immunoreaction, such as the concentration of the reagent and the pH of the buffer solution, the linear range and the limit of detection of 17β-oestradiol were 0.01-10,000 ng ml⁻¹ and 0.00542 ng ml⁻¹, respectively. This method was applied to determine oestradiol in water samples, with the percent recoveries in the range of 86-113%.     

Synchronization of estrus in post-partum mares with progesterone and estradiol 17β

Thirty-six mares which foaled over a 10-day period were given 1 to 10 daily intramuscular injections of a combination of 150 mg. progesterone and 10 mg. estradiol 17β. The first injection was given within 18 hours after parturition. Because individual mares foaled on different dates during the 10 day period, commencement of treatment varied, but treatment for all mares ceased on the same day. Teasing and breeding began seven days after the final treatment. The mares were teased daily for 10 days and artifically inseminated every second day until ovulation occurred. The mean interval from the end of treatment to beginning of estrus was 9.4 days (range 7 to 14) and 33 of 26 mares (94.7%) ovulated 10 to 16 days after the final treatment. Both estrus and ovulation were effectively synchronized, resulting in a first estrus pregnancy rate of 80.6% (29 of 36).     

β−Adrenergic receptor subtype expression in myocyte and non-myocyte cells in human female bladder

β(3)-Adrenergic receptor agonists are currently under clinical development for the treatment of overactive bladder, a condition that is prevalent in postmenopausal women. These agents purportedly relax bladder smooth muscle through a direct action at the myocyte β(3)-receptor. The aim of this study was to examine the expression of the individual beta-adrenergic receptors in full thickness sections from ageing human female bladder. We obtained a series of rabbit polyclonal antibodies generated against each of the three β-adrenergic receptors, and validated their receptor specificity in CHOK1 cells expressing each of the individual receptors. Immunostaining for β(1), β(2), and β(3) were each more prominent in the urothelium than in the detrusor, with all receptors expressed in the same cell types, indicating co-expression of all three receptors throughout the urothelium in addition to the detrusor. Staining of all receptors was also observed in suburothelial myofibroblast-like cells, intramural ganglion cells, and in Schwann cells of intramural nerves. The β(3)-receptor in the human urothelium appears to be functional, as two different selective β(3)-receptor agonists, TAK677 and BRL37344, stimulate cAMP formation in URO tsa cells. Densitometry analysis indicates a persistent expression of all receptors throughout the bladder with increasing age, with the exception of the β(2)-receptor in the urothelium of the trigone, which appears to decrease slightly in older women. These data indicate that β(3)-receptor expression is maintained with age, but may function in concert with other β-receptors. Activation of the myocyte receptor may be influenced by action on non-myocyte structures including the intramural ganglion cells and myofibroblasts.     

Localization of type 5 17β-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization

The mouse enzyme type 5 17-hydroxysteroid dehydrogenase (17-HSD) catalyzes the conversion of androstenedione to testosterone and, to a lesser degree, the conversion of estrone to estradiol. In order to determine the exact sites of action of type 5 17-HSD, we studied the cellular localization of the mRNA of the enzyme in mouse tissues by using in situ hybridization. Specific hybridization signal was found in the liver, ovary, adrenal cortex, and kidney. In the liver of mice of both sexes, a strong signal was observed in all hepatocytes. In the ovary, specific labeling was detected in the granulosa and theca interna cells in growing follicles and in luteal cells. In the female adrenal cortex, intense labeling was restricted to the zona reticularis, whereas no type 5 17-HSD mRNA expression could be found in the male adrenal cortex. In the kidney of mice of both sexes, type 5 17-HSD mRNA was expressed in epithelial cells in both the proximal and distal convoluted tubules. The data indicate that androgens and estrogens are formed via the action of type 5 17-HSD in specific cell types in the liver, ovary, adrenal cortex, and kidney.This work was supported by Genome Canada and Genome Québec.     

Comparative analysis of receptor binding by chicken and human interleukin-1β

Interleukin-1β (IL-1β) is an important cytokine in the immune system. Mammalian and avian IL-1βs share only 31-35% sequence identity, and the function of avian IL-1βs is less well understood by comparison. Although chicken and mammalian IL-1βs have similar tertiary structures, these ILs differ significantly with respect to receptor activation. Analysis of the structures and sequences of IL-1βs reveals that the major differences lie in loops. Modeling docking of chicken IL-1β to its receptor reveals that these variable loops are critical for receptor binding. Molecular dynamics simulations of the IL-1βs reveal significant changes in the dynamic range of motion upon receptor binding. Loops 3 and 9 of the unbound chicken IL-1β had greater fluctuations compared with the other loops. Upon binding, the flexibility of these loops, which directly contact the receptor, markedly decreases. Taken together, these results suggest that receptor binding leads to not only favorable enthalpy but also lower conformational entropy.     

Estrogen and progesterone receptors in mammary tumors induced in rats by simultaneous administration of 17ß-estradiol and progesterone

Mammary tumors were promoted in male rats of the Wistar WAG strain by continuous and simultaneous administration of 17ß-estradiol and progesterone. Tumor induction and growth were dependent on estradiol and on progesterone. Their histological features were comparable with those of human breast cancers. Hormone receptors were present in tumor cells. Estradiol receptor was found in 95% of them, at a higher level in nuclei than in cytosol. Progesterone receptor was present in 75% of tumors. In all cases, the level of androgen receptor was low.     

Estradiol and progesterone receptor concentrations and 17β-hydroxysteroid-dehydrogenase activity in estrogenprogestin stimulated endometrium of women with gonadal dysgenesis

Four hypergonadotrophic women between 25 and 37 years of age with gonadal dysgenesis were treated sequentially with estrogens and a progestin. The hormonal environment induced by this therapy was similar to that of ovulating women, as demonstrated by serum levels of estradiol, endometrial histology and pituitary gonadotropin secretion before and after LH-RH double stimulation.The concentrations of estradiol and progesterone receptors (ER and PR) and the activity of the 17β-hydroxysteroid-dehydrogenase (17β-HSD) were determined in endometrial curettings obtained from the above patients at 5 different days of their 28-day artificial cycles. The results were correlated to the histology of the endometrium and the serum concentrations of estradiol at the corresponding days of the cycle.The cytoplasmic ER and PR concentrations in the endometrium were 3–6 times higher during the estrogen than progestin phase of the induced endometrial cycle. For the activity of the 17β-HSD, the contrary was the case, being 6–10 fold higher during the progestin phase. A 22-day administration of estrogens only did not lead to a rise in enzyme activity or decrease in receptor content as observed under progestin influence after day 14. Since ER and PR concentrations and 17β-HSD activities were similar to those in the endometrium of normally ovulating women, these results confirm experimentally the present concept concerning the dependence of the cytoplasmic ER and PR content and 17β-HSD activity on female sex hormone action.     

Potentiation of paclitaxel effect by resveratrol in human breast cancer cells by counteracting the 17β-estradiol/estrogen receptor α/neuroglobin pathway

Neuroglobin (NGB), an antiapoptotic protein upregulated by 17β-estradiol (E2), is part of E2/estrogen receptor α (ERα) pathway pointed to preserve cancer cell survival in presence of microenvironmental stressors including chemotherapeutic drugs. Here, the possibility that resveratrol (Res), an anticancer plant polyphenol, could increase the susceptibility of breast cancer cells to paclitaxel (Pacl) by affecting E2/ERα/NGB pathway has been evaluated. In MCF-7 and T47D (ERα-positive), but not in MDA-MB 231 (ERα-negative) nor in SK-N-BE (ERα and ERβ positive), Res decreases NGB levels interfering with E2/ERα-induced NGB upregulation and with E2-induced ERα and protein kinase B phosphorylation. Although Res treatment does not reduce cell viability by itself, this compound potentiates Pacl proapoptotic effects. Notably, the increase of NGB levels by NGB expression vector transfection prevents Pacl or Res/Pacl effects. Taken together, these findings indicate a new Res-based mechanism that acts on tumor cells impairing the E2/ERα/NGB signaling pathways and increasing cancer cell susceptibility to chemotherapeutic agent.     

Induction of cardiac angiogenesis requires killer cell lectin-like receptor 1 and α4β7 integrin expression by NK cells

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice, myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6, accompanied by substantial expression of IL-2, TNF-α, and CCL2. In contrast, NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo, IL-2-activated NK cells promoted TNF-α-stimulated endothelial cell proliferation, enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2-activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4β7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin-specific binding. In this study, we show that IL-2-activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases.     

Distribution of GDNF family receptor α3 and RET in rat and human non-neural tissues

The neurotrophic growth factor artemin binds selectively to GDNF family receptor α3 (GFRα3), forming a molecular complex with the co-receptor RET which mediates downstream signaling. This signaling pathway has been demonstrated to play an important role in the survival and maintenance of nociceptive sensory neurons and in the development of sympathetic neurons. However, the presence and potential role of this artemin-responsive pathway in non-neural tissues has not been fully explored to-date. To study the distribution of GFRα3 and RET in adult rat and human non-neural tissues, we carried out a comprehensive immunohistochemical study. We stained major organs from the digestive, urinary, reproductive, immune, respiratory and endocrine systems, and from other systems (cardiovascular, skeletal muscle), as well as regions of the nervous system for comparison. In both rat and human, the majority of non-neural cells did not exhibit detectable GFRα3-like immunoreactivity. In the rat, GFRα3- and RET-like staining were found in the same non-neural cell type only in kidney. In the human digestive and reproductive systems, a subset of epithelial cells exhibited GFRα3- and RET-like staining, suggesting co-localization. In other tissues, sub-populations of cells expressed either GFRα3- or RET-like immunoreactivity. The functional consequences of GFRα3 expression in non-neural cells remain to be determined.     

STAT5 signaling in expression of the α-subunit of interleukin-2 receptor in human blood lymphocytes

A comparative study of STAT3 and STAT5 activity (assessed by tyrosine phosphorylation level) and the expression of an α-subunit of the interleukin-2 receptor (examined by cytophotometric evaluation of CD25 cell number) during phytohemaglutinin (PHA)-induced proliferation of human blood lymphocytes (HBLs) has been carried out. It was found that the level of STAT3 phosphorylation was high both in resting and competent HBLs and remained unchanged in the presence of PHA or interleukin-2 (IL-2). In contrast to STAT3, phosphorylation of STAT5 was not seen either in resting or competent HBL. In the presence of PHA, STAT5 phosphorylation was observed no earlier than in 2–5 h; maximal phosphorylation was detected after 24 h. In competent HBLs, exogenous IL-2 induced high phosphorylation of STAT5 in 30 min that was retained for the next 24–48 h. Alterations in the level of tyrosine phosphorylation of STAT5 correlated with CD25 expression. WHI-P131, a JAK3 kinase inhibitor, prevents STAT5 activation, CD25 surface expression, and lymphocyte proliferation. It is concluded that JAK3/STAT5 signaling via an IL-2 receptor is necessary to support the long-term expression of a high-affinity αβγ_c-receptor of IL-2 and HBL optimal proliferation.     

Transient expression of the β-glucuronidase gene in tissues ofArabidopsis thaliana by bombardment-mediated transformation

Particle bombardment has proved to be useful for the transformation of plants. We have previously reported successful transient expression of the beta-glucuronidase (GUS) gene in cultured plant cells and tissues and the stable transformation of various plants using a pneumatic particle gun. In this chapter, we describe transient expression of the GUS gene in Arabidopsis thaliana leaves and roots using the pneumatic particle gun.     

Distribution of the α2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues

Summary The hepatic ₁-macroglobulin receptor (₂MR)/low density lipoprotein receptor-related protein (LRP) binds and endocytoses ₂-macroglobulin-proteinase complexes in plasma. In addition, it binds lipoproteins, a novel 40 kDa protein, and complexes between plasminogen activators and plasminogen activator inhibitor type-1. This study shows, for the first time, the tissue distribution of ₂MR/LRP as determined by immunohistochemistry with specific monoclonal antibodies. The analysis revealed ₂MR/LRP-expression in a restricted spectrum of cell types, including neurons and astrocytes in the central nervous system, epithelial cells of the gastrointestinal tract, smooth muscle cells, fibroblasts, Leydig cells in testis, granulosa cells in ovary, and dendritic interstitial cells of kidney. Monocytederived cells displayed marked ₂MR/LRP expression in the phagocytes of liver, lung and lymphoid tissues, but no or low expression in antigen-presenting cells including Langerhans' cells of the skin. The high abundance of ₂MR/LRP in certain cell types of most organs suggests two main routes for ₂MR/LRP ligand clearance: (1) systemic removal in liver of circulating ligands, and (2) non-hepatic interstitial removal in different organs, including the brain.