Regulation of gene activation by the estrogen receptor (ER) is complex and involves co-regulatory proteins, oncoproteins (such as Fos and Jun), and phosphorylation signaling pathways. Here we report the cloning and initial characterization of a novel protein, Brx, that contains a region of identity to the oncogenic Rhoguanine nucleotide exchange (Rho-GEF) protein Lbc, and a unique region capable of binding to nuclear hormone receptors, including the ER. Western and immunohistochemistry studies showed Brx to be expressed in estrogen-responsive reproductive tissues, including breast ductal epithelium. Brx bound speci®cally to the ER via an interaction that required distinct regions of ER and Brx. Furthermore, overexpression of Brx in transfection experiments using an estrogenresponsive reporter revealed that Brx augmented gene activation by the ER in an element-speci®c and liganddependent manner. Moreover, activation of ER by Brx could be speci®cally inhibited by a dominant-negative mutant of Cdc42Hs, but not by dominant negative mutants of RhoA or Rac1. Taken together, these data suggest that Brx represents a novel modular protein that may integrate cytoplasmic signaling pathways involving Rho family GTPases and nuclear hormone receptors.

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BackgroundEarly molecular changes of nutritionally-induced insulin resistance are still enigmatic. It is also unclear if acute overnutrition alone can alter insulin signaling in humans or if the macronutrient composition of the diet can modulate such effects.MethodsTo investigate the molecular correlates of metabolic adaptation to either high-carbohydrate (HC) or high-fat (HF) overfeeding, we conducted overfeeding studies in 21 healthy lean (BMI < 25) individuals (10 women, 11 men), age 20-45, with normal glucose metabolism and no family history of diabetes. Subjects were studied first following a 5-day eucaloric (EC) diet (30% fat, 50% CHO, 20% protein) and then in a counter balanced manner after 5 days of 40% overfeeding of both a HC (20% fat, 60% CHO) diet and a HF (50% fat, 30% CHO) diet. At the end of each diet phase, in vivo insulin sensitivity was assessed using the hyperinsulinemic-euglycemic clamp technique. Ex vivo insulin action was measured from skeletal muscle tissue samples obtained 15 minutes after insulin infusion was initiated.ResultsOverall there was no change in whole-body insulin sensitivity as measured by glucose disposal rate (GDR, EC: 12.1 ± 4.7; HC: 10.9 ± 2.7; HF: 10.8 ± 3.4). Assessment of skeletal muscle insulin signaling demonstrated increased tyrosine phosphorylation of IRS-1 (p < 0.001) and increased IRS-1-associated phosphatidylinositol 3 (PI 3)-kinase activity (p < 0.001) following HC overfeeding. In contrast, HF overfeeding increased skeletal muscle serine phosophorylation of IRS-1 (p < 0.001) and increased total expression of p85α (P < 0.001).ConclusionWe conclude that acute bouts of overnutrition lead to changes at the cellular level before whole-body insulin sensitivity is altered. On a signaling level, HC overfeeding resulted in changes compatible with increased insulin sensitivity. In contrast, molecular changes in HF overfeeding were compatible with a reduced insulin sensitivity.

The effects of estrogen on the reproductive tract involve cell proliferation, migration, and differentiation, which need to be well coordinated. Polypeptide growth factors are believed to play a vital role in a number of these cellular processes. Among the growth factors now documented to be associated with estrogen action are epidermal growth factor, transforming growth factor-alpha (TGF alpha), transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, TGF beta 3, and insulin-like growth factor-I (IGF-I). Platelet-derived growth factors (PDGFs) are also potent mitogens, which consist of two peptide chains, denoted A and B, that dimerize to isoforms (PDGF-AA, -AB, and -BB) which differ in their functional properties, secretory behavior, receptor binding, and physiological effects. To study the role of the PDGF-A and -B chains and the PDGF receptor subunits, alpha and beta, during estrogen action in the mouse reproductive tract, time-dependent changes in the expression of these genes were examined by Northern and in situ RNA analyses and by immunohistochemistry after a single treatment of immature CD-1 (17- to 19-day-old) mice with the synthetic estrogen, diethylstilbestrol (DES). Our results demonstrate estrogen modulation of the expression of messenger RNA (mRNA) and protein for the PDGF ligands and receptors in both the uterus and vagina of the mouse. Northern and in situ RNA analyses demonstrate time-dependent estrogen induction of the mRNA levels for these genes in both tissues within 3 h after treatment. However, distinctive mRNA expression profiles for the PDGF ligand and receptor genes are exhibited by the uterus and vagina in response to DES, especially in that the induction of transcripts for PDGF-A and both receptor subunits is more transient in the vagina than in the uterus. Steroid specificity studies demonstrate predominant estrogen-specific regulation of mRNA induction for these genes. Analysis of cell-specific RNA expression by in situ hybridization reveals prominent induction of transcripts for the PDGF chains and receptor subunits in the uterine and vaginal epithelium after estrogen treatment, although enhanced expression of mRNA is also noted in the stroma, particularly for the PDGF receptor subunit genes. Cellular localization of the PDGF ligand and receptor protein molecules by immunohistochemistry detected significant immunostaining for all of these proteins in both the uterus and vagina of control animals. After DES treatment, the uterus exhibits a significant decrease in the level of PDGF ligand and receptor proteins immunostained within 6 h, whereas less dramatic effects ar observed in the vagina.(ABSTRACT TRUNCATED AT 400 WORDS)

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The incidence of cervical adenocarcinomas in young women over the last two decades has increased. Even with increasing knowledge of the role of human papillomavirus in the etiology of adenocarcinoma of the cervix, there is a paucity of data concerning the genetic and epigenetic factors that contribute to the histologic features and biologic behaviors of these tumors. Lactoferrin is a basic glycoprotein found in human milk, secondary granules of neutrophils, and many body secretions, and it has been associated with carcinogenesis of the endometrium, breast, and lymphoid systems. In this study, we examined the expression of lactoferrin in normal human endocervical epithelium and in cervical adenocarcinomas in relation to proliferative index, steroid receptor status, p53 protein expression, and apoptosis. Immunohistochemical and in situ studies demonstrated that lactoferrin protein and mRNA were strikingly downregulated upon neoplastic transformation of the endocervix as early as in adenocarcinoma in situ when compared with the prominent expression exhibited by the normal cervical epithelium. Furthermore, neoplastic transformation of endocervical epithelial cells was accompanied by a pronounced stimulation of proliferation and a substantial reduction in the expression of the estrogen and progesterone receptors and p53 but little or no change in the number of apoptotic cells. In conclusion, we identified lactoferrin as a novel cancer-specific marker of endocervical adenocarcinomas that may be useful in the early detection of the disease, prediction of prognosis, and the development of new therapeutic modalities.

These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer.

The effects of estrogen on the reproductive tract involve cell proliferation, migration, and differentiation, which need to be well coordinated. Polypeptide growth factors are believed to play a vital role in a number of these cellular processes. Among the growth factors now documented to be associated with estrogen action are epidermal growth factor, transforming growth factor-alpha (TGF alpha), transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, TGF beta 3, and insulin-like growth factor-I (IGF-I). Platelet-derived growth factors (PDGFs) are also potent mitogens, which consist of two peptide chains, denoted A and B, that dimerize to isoforms (PDGF-AA, -AB, and -BB) which differ in their functional properties, secretory behavior, receptor binding, and physiological effects. To study the role of the PDGF-A and -B chains and the PDGF receptor subunits, alpha and beta, during estrogen action in the mouse reproductive tract, time-dependent changes in the expression of these genes were examined by Northern and in situ RNA analyses and by immunohistochemistry after a single treatment of immature CD-1 (17- to 19-day-old) mice with the synthetic estrogen, diethylstilbestrol (DES). Our results demonstrate estrogen modulation of the expression of messenger RNA (mRNA) and protein for the PDGF ligands and receptors in both the uterus and vagina of the mouse. Northern and in situ RNA analyses demonstrate time-dependent estrogen induction of the mRNA levels for these genes in both tissues within 3 h after treatment. However, distinctive mRNA expression profiles for the PDGF ligand and receptor genes are exhibited by the uterus and vagina in response to DES, especially in that the induction of transcripts for PDGF-A and both receptor subunits is more transient in the vagina than in the uterus. Steroid specificity studies demonstrate predominant estrogen-specific regulation of mRNA induction for these genes. Analysis of cell-specific RNA expression by in situ hybridization reveals prominent induction of transcripts for the PDGF chains and receptor subunits in the uterine and vaginal epithelium after estrogen treatment, although enhanced expression of mRNA is also noted in the stroma, particularly for the PDGF receptor subunit genes. Cellular localization of the PDGF ligand and receptor protein molecules by immunohistochemistry detected significant immunostaining for all of these proteins in both the uterus and vagina of control animals. After DES treatment, the uterus exhibits a significant decrease in the level of PDGF ligand and receptor proteins immunostained within 6 h, whereas less dramatic effects ar observed in the vagina.(ABSTRACT TRUNCATED AT 400 WORDS)