Identification of estrogen receptor beta-positive intraepithelial lymphocytes and their possible roles in normal and tubal pregnancy oviducts.

BACKGROUND: Although intraepithelial lymphocytes (IELs) in human oviductal epithelium have been implicated in the regulation of local immunity, the precise kinetics and mechanism of steroid regulation of IEL are largely unknown. METHODS: We examined the localization of estrogen receptors (ERs) and progesterone receptors (PRs) in 41 human oviducts by immunohistochemistry. These tissues were obtained from various menstrual cycles, also from both post-menopausal women and tubal pregnancies. The expressions of ERbeta mRNA and membrane (m)PR mRNA were examined by in situ hybridization and RT-PCR, respectively. RESULTS: Most of the IEL expressed ERbeta at both mRNA and protein levels. The number of ERbeta-positive IEL, which were identified as CD8-positive T lymphocytes and also were mPR positive, was increased in the late proliferative, the mid-secretory and late secretory phases in normally cycling women (P < 0.05). Interestingly, in tubal pregnancy, ERbeta-positive IELs were consistently abundant. In addition, we found a high Ki-67-labelling index for IEL, although ERalpha was entirely absent in the tubal pregnancy oviducts. CONCLUSIONS: These results suggest that the number of IEL fluctuated because of estrogen and progesterone levels probably through ERbeta and mPR, respectively. ERbeta-positive IEL may be involved in regulating immune tolerance in tubal pregnancy oviducts.

Diethylstilbestrol increases the density of prolactin cells in male mouse pituitary by inducing proliferation of prolactin cells and transdifferentiation of gonadotropic cells.

Diethylstilbestrol (DES) has been implicated in mammalian abnormalities. We examined the effects of DES on follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) cells in the pituitaries of male mice treated with various doses of DES for 20 days. DES reduced the density of FSH and LH cells in a dose-dependent manner, but increased that of PRL cells. When the expression of estrogen receptor (ER) alpha and beta was assessed, an induction of ERbeta by DES was found predominantly in PRL cells. However, since these effects were abolished in ERalpha knockout mice, DES appears to act primarily through ERalpha. When the expression of Ki-67 and Pit-1 in PRL cells was examined at various time-points after DES treatment, some PRL cells became Ki-67 positive at 10-15 days, and Pit-1-positive cells were increased at 5-15 days. Furthermore, some FSH and LH cells became Pit-1 positive, and co-localized with PRL at 5-10 days. Our results indicate that DES increases PRL cells by inducing proliferation of PRL cells and transdifferentiation of FSH/LH cells to PRL cells.

Reduction of nitric oxide synthase 2 expression by distamycin A improves survival from endotoxemia.

NO synthase 2 (NOS2) plays an important role in endotoxemia through overproduction of NO. Distamycin A (Dist A) belongs to a class of drugs termed minor-groove DNA binders, which can inhibit transcription factor binding to AT-rich regions of DNA. We and others have previously shown that AT-rich regions of DNA surrounding transcription factor binding sites in the NOS2 promoter are critical for NOS2 induction by inflammatory stimuli in vitro. Therefore, we hypothesized that Dist A would attenuate NOS2 up-regulation in vivo during endotoxemia and improve animal survival. C57BL/6 wild-type (WT) mice treated with Dist A and LPS (endotoxin) showed significantly improved survival compared with animals treated with LPS alone. In contrast, LPS-treated C57BL/6 NOS2-deficient (NOS2-/-) mice did not benefit from the protective effect of Dist A on mortality from endotoxemia. Treatment with Dist A resulted in protection from hypotension in LPS-treated WT mice, but not in NOS2-/- mice. Furthermore, LPS-induced NOS2 expression was attenuated in vivo (WT murine tissues) and in vitro (primary peritoneal and RAW 264.7 murine macrophages) with addition of Dist A. Dist A selectively decreased IFN regulatory factor-1 DNA binding in the enhancer region of the NOS2 promoter, and this IFN regulatory factor-1 site is critical for the effect of Dist A in attenuating LPS induction of NOS2. Our data point to a novel approach in modulating NOS2 expression in vivo during endotoxemia and suggest the potential for alternative treatment approaches for critical illness.

Alteration in heme oxygenase-1 and nitric oxide synthase-2 gene expression during endotoxemia in cyclooxygenase-2-deficient mice.

Sepsis is a systemic inflammatory response to a blood-borne infection that is associated with an extremely high rate of morbidity and mortality. The present article reviews our recent studies involving the role of cyclooxygenase (COX)-2 in host responses to bacterial endotoxemia and its role in the regulation of nitric oxide synthase (NOS)2 and heme oxygenase (HO)-1. COX-2-deficient (-/-) mice display a blunted and delayed induction of the cytokine-inducible genes NOS2 and HO-1 after administration of Escherichia coli lipopolysaccharide (LPS or endotoxin). Translocation and activation of transcription factors important for signaling events during an inflammatory response, such as nuclear factor-kappaB and activating protein-1, are also reduced. In addition, COX-2(-/-) mice have reduced leukocyte infiltration into critical organs (kidneys and lungs) after LPS administration. Interestingly, the absence of COX-2 does not alter the LPS induction of several proinflammatory cytokines in tissue macrophages, but induction of the antiinflammatory cytokine interleukin-10 is exaggerated. After LPS administration, 50% of wild-type (+/+) mice die; however, COX-2(-/-) mice display a dramatic improvement in survival during endotoxemia. Taken together, our findings suggest that COX-2(-/-) mice are resistant to many of the detrimental consequences of endotoxemia.

Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer.

Although estrogen is known to play a crucial role in the pathogenesis of breast cancer, the molecular mechanisms underlying the action of estrogen remain elusive. In the present study, we focused on keratinocyte growth factor (KGF) and its receptor (KGFR) in the pathogenesis of breast cancer, as a growth factor mediating estrogen action, since significant roles of KGF were demonstrated in various steroid hormone-dependent tissues. First, using paraffin-embedded specimens from 42 breast cancer patients, we examined expression patterns of KGF and KGFR by both immunohistochemistry using newly generated antibodies and nonradioactive in situ hybridization with T-T dimerized synthetic oligonucleotide probes. We next compared the results with the expression of estrogen receptor (ER) alpha and beta, proliferative activity and apoptotic frequency (TUNEL staining). Also, the similar approaches were taken to analyze the expression and role of KGF in ER-positive (MCF7, ZR-75-1) and ER-negative (SK-BR-3, MDA-MB-231) human breast cancer cell lines in vitro. In the surgical specimens, KGF was expressed in cancer cells as well as stromal cells in 19/42 cases (45%), while KGFR was found in cancer cells in 24/42 cases (57%). The distribution of protein and mRNA in the analysis of both KGF and KGFR expression generally coincided. Moreover, KGF expression was closely associated with the expression of ER alpha, and the coexpression of KGF and KGFR significantly correlated with lower TUNEL index, but not with proliferative activity. In accordance with the in vivo findings, KGF expression was detected only in ER alpha-positive MCF7 and ZR-75-1 cells in vitro. And more importantly, we found the inhibitory effect of KGF upon the induction of apoptosis by anticancer drugs in MCF7 cells. Collectively, our results indicate that ER alpha may be involved in KGF expression, and that KGF may play antiapoptotic roles, rather than mitogenic, in human breast cancer.

Expression of keratinocyte growth factor and its receptor in human breast cancer: its inhibitory role in the induction of apoptosis possibly through the overexpression of Bcl-2.

Keratinocyte growth factor (KGF), a mesenchymal cell derived paracrine growth factor that regulates normal epithelial cell proliferation, appears to be an essential mediator of steroids in various reproductive organs. The present study was designed to determine the expression and role of KGF and its receptor (KGFR) in human breast carcinoma tissues by immunohistochemistry. We also compared the results with the expression of estrogen receptor alpha(ERalpha), ERbeta, the proliferative activity assessed by the labeling index (LI) for the Ki-67 antigen, apoptotic frequency assessed by terminal dUTP nick end-labeling (TUNEL) index, and the expression of Bcl-2. All of KGF-positive cases were ERalpha- positive (p<0.05), but not that of ERbeta, while all of KGFR-positive cases were ERbeta-positive (p<0.05), but not that of ERalpha. The specimens with the coexpression of KGF and KGFR significantly correlated with a lower TUNEL index (p<0.05), but not with Ki-67 LI in breast cancer tissues. Further analysis at the cellular level revealed that Bcl-2 was colocalized in KGFR-positive cells, and these cells were almost negative for TUNEL staining. Bcl-2-positive cells were also associated with ERbeta, as expected. Therefore, the results indicate that ERalpha may be involved in KGF expression, and that the coexpression of KGF and KGFR may play an inhibitory role in the induction of apoptosis possibly through the up-regulation of Bcl-2 expression in human breast cancer.

Elk-3 is a transcriptional repressor of nitric-oxide synthase 2.

The inducible isoform of nitric-oxide synthase (NOS2), a key enzyme catalyzing the dramatic increase in nitric oxide by lipopolysaccharide (LPS), plays an important role in the pathophysiology of endotoxemia and sepsis. Recent evidence suggests that Ets transcription factors may contribute to NOS2 induction by inflammatory stimuli. In this study, we investigated the role of Ets transcription factors in the regulation of NOS2 by LPS and transforming growth factor (TGF)-beta 1. Transient transfection assays in macrophages showed that Ets-2 produced an increase in NOS2 promoter activity, whereas the induction by Ets-1 was modest and NERF2 had no effect. Elk-3 (Net/Erp/Sap-2a) markedly repressed NOS2 promoter activity in a dose-dependent fashion, and overexpression of Elk-3 blunted the induction of endogenous NOS2 message. Mutation of the Net inhibitory domain of Elk-3, but not the C-terminal-binding protein interaction domain, partially alleviated this repressive effect. We also found that deletion of the Ets domain of Elk-3 completely abolished its repressive effect on the NOS2 promoter. LPS administration to macrophages led to a dose-dependent decrease in endogenous Elk-3 mRNA levels, and this decrease in Elk-3 preceded the induction of NOS2 mRNA. In a mouse model of endotoxemia, the expression of Elk-3 in kidney, lung, and heart was significantly down-regulated after systemic administration of LPS, and this down-regulation also preceded NOS2 induction. Moreover, TGF-beta 1 significantly increased endogenous Elk-3 mRNA levels that had been down-regulated by LPS in macrophages. This increase in Elk-3 correlated with a TGF-beta 1-induced down-regulation of NOS2. Taken together, our data suggest that Elk-3 is a strong repressor of NOS2 promoter activity and mRNA levels and that endogenous expression of Elk-3 inversely correlates with NOS2. Thus, Elk-3 may serve as an important mediator of NOS2 gene expression.

Cyclooxygenase-2-deficient mice are resistant to endotoxin-induced inflammation and death.

Sepsis is a systemic inflammatory response to a blood-borne infection that is associated with an extremely high rate of morbidity and mortality. The present study investigates the role of cyclooxygenase (COX)-2 in host responses to bacterial endotoxemia. After administration of Escherichia coli lipopolysaccharide, 50% of wild-type mice die within 96 h. COX-2 deficient mice displayed a dramatic improvement in survival with reduced leukocyte infiltration into critical organs (kidneys and lungs) and a blunted and delayed induction of the cytokine inducible genes nitric oxide synthase 2 and heme oxygenase-1. Translocation and activation of transcription factors important for signaling events during an inflammatory response, such as nuclear factor (NF)-kappaB, were also markedly reduced. While the absence of COX-2 did not alter the induction of several pro-inflammatory cytokines in tissue macrophages, induction of the anti-inflammatory cytokine IL-10 was exaggerated. Administration of IL-10 to wild-type mice reduced NF-kappaB activation. Taken together, our data suggest that COX-2 deficient mice are resistant to many of the detrimental consequences of endotoxemia. These beneficial effects occur, in part, by a compensatory increase in IL-10 that counterbalances the pro-inflammatory host response to endotoxemia.

Modulation of the thioredoxin system during inflammatory responses and its effect on heme oxygenase-1 expression.

Heme oxygenase (HO) enzymes catalyze the initial reaction in heme catabolism. HO-1 is an inducible isoform that is up-regulated by diverse stimuli, including inflammatory cytokines and factors that promote oxidative stress. HO-1 is a cytoprotective enzyme that degrades heme, a potent oxidant, to generate carbon monoxide, biliverdin (subsequently reduced to bilirubin), and iron. Recently, we found that thioredoxin (TRX), a disulfide reductase enzyme known to be important for the binding of transcription factors to DNA, contributes to the induction of HO-1 by inflammatory mediators. In the present study, we extended this observation and determined that, similar to HO-1, TRX and TRX reductase (TR) are induced by bacterial lipopolysaccharide in macrophages at the level of mRNA and protein. However, maximal induction of TRX and TR precedes that of HO-1. Increased expression of HO-1 in the cytoplasm of inflammatory cells corresponds to a translocation of TRX into the nucleus of these cells. Finally, transfection of TRX into macrophages promoted an increase in HO-1 protein. Taken together, these data support the concept that the TRX system contributes to the up-regulation of HO-1 under conditions associated with increased oxidative stress.