Localization and sex steroid regulation of androgen receptor gene expression in rhesus monkey uterus

OBJECTIVE: To characterize the cellular sites and hormonal regulation of uterine androgen receptor gene expression in the monkey. METHODS: Ovariectomized rhesus monkeys (five in each group) were treated with placebo (the control group), estradiol (E2), E2 plus progesterone, or E2 plus testosterone by sustained-release pellets administered subcutaneously. After 3 days of treatment, uteri were removed and uterine sections were analyzed by in situ hybridization for androgen receptor messenger RNA (mRNA). RESULTS: Androgen receptor mRNA was detected in endometrial stromal cells and myometrial smooth muscle cells, with lesser expression in endometrial epithelial cells. Both E2 and E2 plus progesterone treatment doubled androgen receptor mRNA levels in stromal cells (P < .01), whereas E2 plus testosterone treatment increased stromal androgen receptor mRNA levels by about five-fold (P < .001) compared with placebo treatment. In the endometrial epithelium, E2 alone did not increase androgen receptor mRNA levels significantly. However, the E2 plus progesterone and E2 plus testosterone treatments increased epithelial androgen receptor mRNA levels by 4.3 and 5 times, respectively (P = .008 and P < .002, respectively). Androgen receptor mRNA was distributed homogeneously in smooth muscle cells across the myometrium. Estradiol treatment alone did not increase myometrial androgen receptor mRNA levels significantly, but the E2 plus progesterone and E2 plus testosterone treatments increased myometrial androgen receptor mRNA levels by 1.8 and 2 times, respectively (P = .001 and P < .001, respectively). CONCLUSION: Androgen receptor gene expression was detected in all uterine cell compartments where it was subject to significant sex steroid regulation. The fact that androgen receptor mRNA levels were consistently up-regulated by a combined E2 plus testosterone treatment while E2 treatment alone had little or no effect shows that a collaborative action of E2 and testosterone enhances androgen receptor expression in the monkey uterus.     

Cytokine-mediated regulation of 92-kilodalton type IV collagenase, tissue inhibitor or metalloproteinase-1 (TIMP-1), and TIMP-3 messenger ribonucleic acid expression in human endometrial stromal cells

Interleukin-1 (IL-1) is expressed in human endometrium and has been shown to play an integral role in local cellular interactions during implantation. In addition, the matrix metalloproteinase (MMP) and its inhibitor, the tissue inhibitor of metalloproteinase (TIMP), are crucial during implantation, mediating in vitro trophoblast penetration, and are regulated by several cytokines expressed by trophoblast cells. We have investigated the roles of IL-1 beta and transforming growth factor-beta (TGF beta) in regulating TIMP-1, TIMP-3, and 92-kDa type IV collagenase messenger ribonucleic acid (mRNA) expression in human endometrial stromal cells using quantitative competitive PCR. Confluent stromal cell cultures treated with progesterone and estradiol for 9 days were stimulated with IL-1 beta, IL-1 beta plus anti-IL-1 beta antibody, TGF beta, and TGF beta plus anti-TGF beta antibody for an additional 24 h. Competitive complementary DNA fragments were constructed by deletion of a defined fragment from each of the target complementary DNA sequences and coamplified in quantitative competitive PCR as an internal standard. TIMP-1 and TIMP-3, but not 92-kDa type IV collagenase mRNA, were expressed in stromal cells. The 92-kDa type IV collagenase mRNA was only expressed after stimulation with IL-1 beta. IL-1 beta both augmented 92-kDa type IV collagenase mRNA expression and decreased TIMP-1 and TIMP-3 mRNA expression in a dose-dependent manner. Conversely, TGF beta augmented TIMP-1 and TIMP-3 mRNA expression, but did not affect 92-kDa type IV collagenase expression. IL-1 and TGF beta-mediated changes were both neutralized by specific antibodies. These results provide indirect evidence that IL-1 and TGF beta may play crucial roles at the embryo-maternal interface during trophoblast invasion by regulating stromal cell expression of TIMP-1, TIMP-3, and 92-kDa type IV collagenase, all of which are known to be important in trophoblast invasion.     

Regulation of cyclooxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-tau in bovine endometrial cells

Estradiol (E2) and progesterone are responsible for regulating PG synthesis in the endometrium during the estrous cycle and interferon-tau (IFN-tau) alters PG synthesis during early pregnancy in ruminants. In this study, we examined the effects of these steroid hormones and recombinant bovine IFN-tau (rbIFN-tau) on PG production and on cyclooxygenase-2 (COX-2) and PG F (PGF) synthase (PGFS) gene expression in isolated endometrial cells. E2 decreased both PGF2alpha and PG E2 (PGE2) whereas progesterone increased PGF2alpha secretion in epithelial cells. Steroid hormones had no effect on PG production in stromal cells. rbIFN-tau attenuated both PGF2alpha and PGE2 production in epithelial cells and enhanced their production, and the ratio of PGE2 to PGF2alpha, in stromal cells. Northern blot analysis showed that E2 and rbIFN-tau decreased COX-2 messenger RNA (mRNA) levels in epithelial cells. Conversely, rbIFN-tau increased COX-2 mRNA in stromal cells. Furthermore, rbIFN-tau decreased PGFS mRNA in both cell types and this was associated with the increase in PGE2/PGF2alpha ratio. These results show that the regulation of PG synthesis by steroid hormones is different in endometrial epithelial and stromal cells in vitro. The attenuation of PGF2alpha secretion from epithelial cells and increased PGE2 production in stromal cells by rbIFN-tau are modulated by steroid hormones.     

Interleukin-6, hepatocyte growth factor, and their receptors in biliary epithelial cells during a type I ductular reaction in mice: interactions between the periductal inflammatory and stromal cells and the biliary epithelium

The interleukin-6 (IL-6)/gp-80 and hepatocyte growth factor (HGF)/met ligand/receptor systems have been shown to stimulate biliary epithelial cell (BEC) DNA synthesis in vitro. The mRNA and protein production of these two in vitro mitogens were mapped in vivo during the first week after bile duct ligation (BDL) when peak BEC DNA synthesis is seen. Changes around the biliary tree were compared with those seen in the peripheral liver using a combination of Northern blotting and a unique biliary tree isolation technique, in which the bile ducts and the surrounding portal stroma and inflammatory cells are separated from the hepatocytes by perfusion digestion. Further localization was performed with in situ hybridization and immunohistochemistry. In the normal liver, there is low-level expression of HGF mRNA by periportal stellate cells, and HGF protein localizes to these cells and to neutrophils; extracellular HGF protein is present in the bile. There is no detectable IL-6 mRNA by Northern analysis or IL-6 protein expression in the normal liver, but both met and IL-6 receptor (IL-6R) mRNA are detectable; met mRNA is expressed strongly in the biliary tree, and met protein is expressed weakly on hepatocytes and strongly on BEC. IL-6R mRNA is weakly expressed in the biliary tree, and IL-6R protein is detectable on hepatocytes, with a periportal-to-perivenular gradient, but not on BEC. During the first 3 days after BDL, HGF mRNA expression is increased in both the biliary tree and in the peripheral liver, and production is localized to stellate cells, periductal neutrophils, and stromal cells, which typically accompany the proliferating ductules. IL-6 mRNA and protein were detected only near the biliary tree after BDL, and not in the peripheral liver, and the production was localized to periductal hematolymphoid cells, which had the morphological appearance of macrophages and/or dendritic cells. There is also a distinct up-regulation of met and gp-80 mRNA and protein in the biliary tree, which is stronger than that seen in the peripheral liver. Met protein expression is increased, and IL-6R(gp-80) protein is induced on the proliferating BEC, consistent with the participation of both the HGF/met and IL-6/gp-80 systems in the early phases of type I ductular reactions. These observations show that periductal hematolymphoid and stromal cells are the source of BEC growth factors, and receptors for these factors are up-regulated on BEC during active ductular proliferation. Complex interactions between the inflammatory, stromal, and BEC results in a dysmorphogenic repair response that eventually leads to cirrhosis.     

Tissue interleukin 1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats

BACKGROUND/AIMS: Subserosal injection of purified group A streptococcal peptidoglycan-polysaccharide (PG-APS) induces chronic relapsing granulomatous enterocolitis and systemic inflammation in susceptible inbred Lewis rats but only transient intestinal injury in Buffalo and Fischer rats. Cecal interleukin 1 (IL-1) and IL-1 receptor antagonist (IL-1ra) expression was measured in inbred rats displaying differential susceptibility to experimental enterocolitis. METHODS: The ileum and cecum of Lewis, Buffalo, and Fischer rats were subserosally injected with purified PG-APS or albumin. IL-1 and IL-1ra messenger RNA (mRNA) and protein (IL-1 only) were measured 1 or 27 days later. PG-APS-injected Lewis rats were treated with recombinant human IL-1ra. Kinetics of IL-1 and IL-1ra mRNA expression were studied in peritoneal cells. RESULTS: All rats strains developed acute inflammation with increased cecal concentrations of IL-1 beta and IL-1ra mRNA. Lewis rats developed chronic enterocolitis and had higher IL-1 and IL-1ra mRNA tissue levels than Buffalo or Fischer rats, which displayed no chronic inflammation. IL-1 beta and IL-1ra were produced by submucosal granulomas and correlated with inflammation. IL-1 alpha protein levels paralleled IL-1 beta mRNA expression. IL-1ra treatment attenuated acute and chronic enterocolitis, adhesions, and arthritis. PG-APS induced IL-1 and IL-1ra expression in peritoneal cells from Lewis and Fischer rats. CONCLUSIONS: Bacterial cell wall polymers stimulate IL-1 and IL-1ra expression in vivo and in vitro. These counterbalancing cytokines are increased in experimental enterocolitis and have important immunoregulatory roles in intestinal inflammation.     

Immune response against large tumors eradicated by treatment with cyclophosphamide and IL-12

Androgen has an important role in development of the prostate, and the actions of androgen are mediated, in part, by locally produced growth factors. These growth factors are postulated to mediate stromal-epithelial interaction in the prostate to maintain normal tissue physiology. Transforming growth factor-alpha (TGF-alpha) is one of the growth factors that can stimulate prostatic growth. The expression of TGF-alpha is thought to be regulated by androgen. The expression of epidermal growth factor receptor (EGFR), which is the receptor of TGF-alpha and EGF, also may be regulated by androgen. The hormonal and developmental regulation of TGF-alpha and EGFR messenger RNA (mRNA) levels in isolated epithelial and stromal cells from rat ventral prostate was investigated. The expression of mRNA for TGF-alpha and EGFR was analyzed by a quantitative RT-PCR (QRT-PCR) procedure developed. Observations from this assay demonstrated that both epithelial and stromal cells expressed the mRNA for TGF-alpha and EGFR. TGF-alpha mRNA expression was constant during postnatal, pubertal, and adult development of the prostate. EGFR mRNA expression was elevated at the midpubertal period and decreased with age. After castration of 60-day-old adult rats, both TGF-alpha and EGFR mRNA were significantly enhanced. TGF-alpha mRNA expression was stimulated by EGF in stromal cells (4.5-fold increase) but was not changed by any treatment in epithelial cells. EGFR mRNA levels were stimulated by EGF and keratinocyte growth factor treatment and inhibited by testosterone treatment in epithelial cells. Stromal cell EGFR mRNA levels were not affected by any treatment. Both testosterone and EGF stimulated incorporation of 3H-thymidine into prostatic stromal and epithelial cells. Anti-TGF-alpha antibody significantly inhibited testosterone-stimulated 3H-thymidine incorporation into stromal cells and epithelial cells. Immunocytochemical localization of TGF-alpha and EGFR demonstrated expression on the luminal surface of epithelial cells within prostatic ducts, and minimal expression was observed in stromal cells. Results indicate that testosterone does not directly regulate TGF-alpha mRNA levels but does inhibit EGFR mRNA levels. Interestingly, anti TGF-alpha antibody suppressed the effect of testosterone on 3H-thymidine incorporation into prostatic stromal and epithelial cells. This finding suggests that testosterone may act indirectly on prostatic cells to influence TGF-alpha actions. TGF-alpha mRNA levels were influenced by EGF in stromal cells only, and EGFR mRNA levels were influenced by testosterone, EGF, and keratinocyte growth factor in epithelial cells. These observations suggest that regulation of TGF-alpha and EGFR is distinct between the cell types. In conclusion, a network of hormonally controlled growth factor-mediated stromal-epithelial interactions is needed to maintain prostate development and function.     

Tumor necrosis factor-alpha mRNA and protein in endometrial tumors: analysis by in situ hybridization and immunocytochemistry

Abnormal expression of polypeptide growth factors and their receptors is closely associated with tumorigenic transformation. In this study tumor necrosis factor-alpha (TNF-alpha) mRNA and protein were analyzed in polyps and proliferative lesions of endometrium as well as in low and high grade endometrial tumors by using in situ hybridization and immunocytochemistry. All samples contained products of the TNF-alpha gene. Histochemical scores (HS), which reflect the proportion of cells positive for TNF-alpha message or protein and the intensities of the signals, were higher for epithelial than for stromal cells. Benign lesions (endometrial polyps) contained little TNF-alpha mRNA or protein, whereas specific message was abundant in proliferative lesions (hyperplasia, adenofibroma). Although neoplastic cells in both low and high grade endometrial tumors contained TNF-alpha mRNA, two major differences were observed: HS for TNF-alpha mRNA were significantly less in low grade than in high grade neoplasms, and TNF-alpha message was restricted to the nucleus in low grade adenocarcinoma cells but was abundant in the cytoplasm of high grade tumor cells. In contrast to cells in benign and proliferative lesions, TNF-alpha protein scores in endometrial tumor cells were inversely rather than positively correlated with TNF-alpha mRNA scores. Collectively, the findings in this study are consistent with the postulate that TNF-alpha is useful to endometrial tumor cells and suggest that production may increase as cells diverge from normal.     

Minocycline for the treatment of rheumatoid arthritis

This study examines the expression of the multi-functional cytokine, vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) in the rat uterus during early proestrus, proestrus, estrus and diestrus. Groups of ovariectomized or hypophysectomized rats served as endocrine controls. Expression of VPF/VEGF mRNA was 2-fold greater in uteri during proestrus and estrus than in other phases of the estrous cycle. In situ hybridization techniques indicated that VPF/VEGF mRNA expression was confined to the luminal epithelium during proestrus, but shifted to the stromal compartment during estrus. Ovariectomized, hypophysectomized or diestrus rats exhibited scattered localization of VPF/VEGF mRNA among glandular epithelium and endometrial stromal compartments. Although VPF/VEGF mRNA was expressed throughout the estrous cycle, but in different compartments of the endometrium depending on the stage of the estrous cycle, VPF/VEGF protein expression appears to be restricted to the epithelial compartment during proestrus and estrus. Results indicate that circulating levels of gonadal steroids and LH may be associated with the differential expression of VPF/VEGF mRNA and its translation activity in the endometrium during different stages of the estrous cycle.     

Interleukin-8 expression in normal nasal epithelium and its modulation by infection with respiratory syncytial virus and cytokines tumor necrosis factor, interleukin-1, and interleukin-6

Inflammation in nasal and airway tissue caused by allergens, microbial infection, and air pollution are likely to be regulated by inflammatory mediators produced by airway epithelial cells. We have therefore investigated the baseline expression of a number of cytokine genes known to be important inducers and modulators of inflammation, in freshly isolated human nasal epithelium. Cells were obtained by superficial scraping of turbinate tissue, and cDNA for polymerase chain reaction (PCR) amplification was reverse-transcribed directly from lysates of 3 x 10(3) to 5 x 10(3) epithelial cells using random hexamers. Constitutive expression of relatively high levels of interleukin-8 (IL-8) mRNA but undetectable levels (< 1 mRNA copy/cell) of granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-6, IL-1, or tumor necrosis factor (TNF) mRNA were found after PCR amplification of the cDNA. IL-8 protein, but not IL-6, was identified in the nasal epithelial cells by immunocytochemistry. Infection with respiratory syncytial virus (RSV) or stimulation of nasal epithelium for 4 h with TNF or IL-1 in vitro resulted in a 4- to 10-fold increase in IL-8 mRNA expression but not in the expression of detectable levels of mRNA for the other cytokines. IL-8 was secreted by RSV-, IL-1-, and TNF-stimulated as well as unstimulated nasal epithelial cells after 6 to 20 h of culture. Neither IL-6, GM-CSF, nor TNF activity/immunoreactivity was detectable in the culture supernatants. Thus, it appears that IL-8 is a major cytokine of human nasal epithelium, constitutively expressed and readily secreted upon virus infection or stimulation with IL-1 and TNF.     

MaMi, a human endometrial stromal sarcoma cell line that constitutively produces interleukin-6, interleukin-8, and monocyte chemoattractant protein-1

BACKGROUND: Uterine endometrial stromal sarcoma is a rare neoplasm with a morphology that closely resembles that of the proliferative endometrial stroma. To understand its pathologic characteristics, we established a novel cell line, MaMi, from a primary culture of an endometrial stromal sarcoma obtained from a 65-year-old Japanese woman. METHODS: We observed the morphology of MaMi cells and performed immunohistochemical analysis on the primary tumor and transplants in nude mice. Prolactin, insulin-like growth factor-binding protein-1, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1, and fibronectin production in the culture medium of MaMi cells were also examined. RESULTS: MaMi cells were shown to exhibit a fibroblast-like morphology in vitro, and they adopted a more elongated appearance in response to 12-O-tetradecanoyl phorbol-13-acetate (TPA). On injection into nude mice, the cells gave rise to subcutaneous tumors. Immunohistologically, both the primary tumor and MaMi cell-induced tumors stained positively with antibodies to neuron-specific enolase or vimentin. MaMi cells constitutively produced IL-6, IL-8, and monocyte chemoattractant protein-1 in vitro. Interleukin-1beta, (100 pmol/L), tumor necrosis factor-alpha (1 nmol/L), and lipopolysaccharide (1 microg/mL) each increased the release of IL-6, IL-8, and monocyte chemoattractant protein-1 by MaMi cells. TPA (10 nmol/L) also stimulated the production of IL-6 and IL-8 by these cells, but inhibited that of monocyte chemoattractant protein-1. CONCLUSIONS: We demonstrated that MaMi cells closely resemble proliferative endometrial stromal cells not only morphologically, but also functionally. This cell line may prove valuable in understanding the role of cytokines produced by tumor cells in the pathogenesis of endometrial stromal sarcoma and may also be useful as an in vitro model of functioning endometrial stromal cells.