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Biomedical subjects

Frank A Simmen

Publications and source records attributed to Frank A Simmen.

At least 19 recordsLinked to original sources
PubMed2006

Chronic ethanol intake impairs insulin signaling in rats by disrupting Akt association with the cell membrane. Role of TRB3 in inhibition of Akt/protein kinase B activation.

Chronic and excessive alcohol consumption is an important and modifiable risk factor for type 2 diabetes. We previously reported elevations in hepatic Class 1 alcohol dehydrogenase (ADH) expression in ethanol-fed rats correspondent with reduced levels of mature, nuclear sterol-regulatory element-binding protein-1 (SREBP-1), an insulin-induced transcriptional repressor of the ADH gene. In this report, we have studied the effects of insulin and ethanol on ADH gene expression in a highly differentiated rat hepatoma cell line (FGC-4), as well as the in vivo effects of chronic intake of an ethanol-containing diet on hepatic insulin signaling. Insulin inhibited ADH gene expression, and this was abolished by LY294002 (a phosphatidylinositol 3-kinase inhibitor) and small interfering RNA knockdown of SREBP-1. Chronic ethanol intake led to decreased phosphorylation of Akt (protein kinase B) at Thr308, increased phosphorylation of Akt at Ser473, and decreased phosphorylation of glycogen synthase kinase-3beta (a downstream effector of Akt). Hepatic membrane-associated Akt content was decreased and cytosolic Akt content was increased in rats fed an ethanol-containing diet. Thus, disruptive effects of ethanol on insulin signaling occurred via impaired phosphorylation of Akt at Thr308. TRB3, a negative regulator of Akt, was induced in liver of ethanol-fed rats. In ethanol-treated FGC-4 cells, small interfering RNA knockdown of TRB3 increased membrane-associated Akt and the phosphorylation of Akt at Thr308. Our results suggest that ethanol induces TRB3, which, through binding to the pleckstrin homology domain of Akt, prevents its plasma membrane association, Akt-Thr308 phosphorylation, and subsequent Akt-mediated signaling. Ethanol inhibition of insulin signaling reduces nuclear SREBP accumulation and results in disinhibition of Class 1 ADH transcription.

Alcohol Dehydrogenase↗
PubMed2006

Enhancer- and silencer-like sequences that mediate insulin-like growth factor-binding protein-2 gene expression in uterine cells of pregnancy.

The regulation of insulin-like growth factor-binding protein-2 (IGFBP-2) gene transcription in specific cell and developmental contexts is not well understood. Here, we identified DNA regions that mediate IGFBP-2 gene transcription in two of the three major cell types of the uterine endometrium of the early pregnant pig. Two clusters of transcriptional start sites at nucleotides -109/-105 and -96/-87 (+1, translational initiation site) in the porcine IGFBP-2 gene were localized in uterine endometrium and in primary cultures of endometrial glandular epithelial (GE) and stromal (ST) fibroblastic cells. Upstream regions of this gene (spanning -1,397/+73) were fused to a luciferase reporter gene, and the constructs were transiently transfected into endometrial GE and ST cells representative of pregnancy days 12 and 18 (day 115 = parturition). A short (110 bp) upstream region (-874/-765) stimulated the IGFBP-2 and heterologous SV40 promoters in the two cell types at both pregnancy days. Two noncontiguous copies of the novel sequence motif TCAGGG within the 110-bp fragment were implicated in transcriptional activity, since block mutation of these sequences led to a repression of SV40 basal promoter activity in endometrial cells. Southwestern blotting identified an endometrial nuclear protein of 34-kDa molecular weight that bound an oligonucleotide containing this motif, and EMSA suggested robust expression of this protein in early pregnancy endometrium and in ovary but at much reduced levels in endometrium at later pregnancy. A pair of E-box elements (CANNTG) within the 110 bp region was stimulatory to IGFBP-2 promoter activity; block mutation of these converted the 110-bp region into a potent transcriptional silencer in all but day 18 ST cells. Results identify novel DNA motifs that regulate the IGFBP-2 gene promoter in uterine endometrium in pregnancy-associated fashion.

Animals↗
PubMed2006

Physiologic and genomic analyses of nutrition-ethanol interactions during gestation: Implications for fetal ethanol toxicity.

Nutrition-ethanol (EtOH) interactions during gestation remain unclear primarily due to the lack of appropriate rodent models. In the present report we utilize total enteral nutrition (TEN) to specifically understand the roles of nutrition and caloric intake in EtOH-induced fetal toxicity. Time-impregnated rats were intragastrically fed either control or diets containing EtOH (8-14 g/kg/day) at a recommended caloric intake for pregnant rats or rats 30% undernourished, from gestation day (GD) 6-20. Decreased fetal weight and litter size (P < 0.05) and increased full litter resorptions (33% vs. 0%), were observed in undernourished dams compared to adequately fed rats given the same dose of EtOH, while undernutrition alone did not produce any fetal toxicity. Undernutrition led to impairment of EtOH metabolism, increased blood EtOH concentrations (160%), and decreased maternal hepatic ADH1 mRNA, protein, and activity. Microarray analyses of maternal hepatic gene expression on GD15 revealed that 369 genes were altered by EtOH in the presence of undernutrition, as compared to only 37 genes by EtOH per se (+/-2-fold, P < 0.05). Hierarchical clustering and gene ontology analysis revealed that stress and external stimulus responses, transcriptional regulation, cellular homeostasis, and protein metabolism were affected uniquely in the EtOH-under-nutrition group, but not by EtOH alone. Microarray data were confirmed using real-time RT-PCR. Undernourished EtOH-fed animals had 2-fold lower IGF-1 mRNA and 10-fold lower serum IGF-1 protein levels compared to undernourished controls (P < 0.0005). Examination of maternal GH signaling via STAT5a and -5b revealed significant reduction in both gene and protein expression produced by both EtOH and undernutrition. However, despite significantly elevated fetal BECs, fetal IGF-1 mRNA and protein were not affected by EtOH or EtOH-undernutrition combinations. Our data suggest that undernutrition potentiates the fetal toxicity of EtOH in part by disrupting maternal GH-IGF-1, signaling thereby decreasing maternal uterine capacity and placental growth.

Alcohol Dehydrogenase↗
PubMed2006

Tumor-protective and tumor-promoting actions of dietary whey proteins in an N-methyl-N-nitrosourea model of rat mammary carcinogenesis.

The mammary tumor-protective effects of dietary factors are considered to be mediated by multiple signaling pathways, consistent with the heterogeneous nature of the disease and the distinct genetic profiles of tumors arising from diverse mammary cell populations. In a 7,12-dimethylbenz(a)anthracene-induced model of carcinogenesis, we showed previously that female Sprague-Dawley rats exposed to AIN-93G diet containing whey protein hydrolysate (WPH) beginning at gestation Day 4 had reduced tumor incidence than those exposed to diet containing casein (CAS), due partly to increased mammary differentiation and reduced activity of phase I metabolic enzymes. Here, we evaluated the tumor-protective effects of these same dietary proteins to the direct-acting carcinogen N-methyl-N-nitrosourea (NMU). We found that lifetime exposure to WPH, relative to CAS, decreased mammary tumor incidence and prolonged the appearance of tumors in NMU-treated female rats, with no corresponding effects on tumor multiplicity. At 115 days post-NMU, histologically normal mammary glands from WPH-fed tumor-bearing rats had increased gene expression for the tumor suppressor BRCA1 and the differentiation marker kappa-casein than those of CAS-fed tumor-bearing rats. Tumor-bearing rats from the WPH group had more advanced tumors, with a greater incidence of invasive ductal carcinoma than ductal carcinoma in situ and higher serum C-peptide levels than corresponding rats fed CAS. WPH-fed tumor-bearing rats were also heavier after NMU administration than CAS tumor-bearing rats, although no correlation was noted between body weight and C-peptide levels for either diet group. Results demonstrate the context-dependent tumor-protective and tumor-promoting effects of WPH; provide support for distinct signaling pathways underlying dietary effects on development of mammary carcinoma; and raise provocative questions on the role of diet in altering the prognosis of existing breast tumors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2005

Progesterone receptor transactivation of the secretory leukocyte protease inhibitor gene in Ishikawa endometrial epithelial cells involves recruitment of Krüppel-like factor 9/basic transcription element binding protein-1.

Progesterone receptor (PR), a ligand-inducible transcription factor, mediates the physiological actions of progesterone (P) through two distinct isoforms, PR-A and PR-B, and numerous nuclear coregulators. We previously demonstrated that basic transcription element binding protein-1 (BTEB1), a transcription factor of the Krüppel-like family, is a functional PR-interacting protein, based on the subfertility phenotype and reduced P sensitivity of uterine PR target genes, on BTEB1 null mutation. Here we examined the role of BTEB1 in PR-mediated signaling in endometrial epithelial cells using Ishikawa human endocarcinoma cells and the P-responsive secretory leukocyte protease inhibitor (SLPI) gene. Treatment of Ishikawa cells with P for 24 h increased SLPI and BTEB1 transcript levels without similar effects on PR expression. P induction was abolished by the PR antagonist RU486, whereas knockdown of BTEB1 with short interfering RNA reduced P-responsive BTEB1 but not SLPI expression to basal levels. Forced expression of BTEB1, either by stable or transient transfections of BTEB1 expression constructs in endometrial carcinoma cells, enhanced SLPI promoter activity. Chromatin immunoprecipitation with anti-BTEB1 antibody demonstrated BTEB1 recruitment to the proximal GC-rich containing SLPI promoter region (-97 to -86) in human endometrial carcinoma (Hec1A) cells overexpressing BTEB1. In Ishikawa cells, recruitment of BTEB1 to the proximal, GC-rich region and the distal, progesterone-responsive element-like containing region (-635 to -514) was P dependent and was accompanied by corecruitment of PR and the PR coactivator cAMP-response element binding protein-binding protein. PR-B, rather than PR-A isoform, preferentially associated with BTEB1 in the GC-rich region, whereas both PR isoforms were recruited to the progesterone-responsive element-like site along with BTEB1. Our findings define a novel pathway for BTEB1/PR cross-talk to facilitate P-dependent gene transcription in endometrial epithelial cells.

Cell Line, Tumor↗
PubMed2005

Dietary whey protein lowers serum C-peptide concentration and duodenal SREBP-1c mRNA abundance, and reduces occurrence of duodenal tumors and colon aberrant crypt foci in azoxymethane-treated male rats.

We evaluated partially hydrolyzed whey protein (WPH) for inhibitory effects on the development of colon aberrant crypt foci (ACF) and intestinal tumors in azoxymethane (AOM)-treated rats. Pregnant Sprague-Dawley rats and their progeny were fed AIN-93G diets containing casein (CAS, control diet) or WPH as the sole protein source. Colons and small intestines from the male progeny were obtained at 6, 12, 20 and 23 weeks after AOM treatment. At 6 and 23 weeks, post-AOM, WPH-fed rats had fewer ACF than did CAS-fed rats. Intestinal tumors were most frequent at 23 weeks, post-AOM. At this time point, differences in colon tumor incidence with diet were not observed; however, WPH-fed rats had fewer tumors in the small intestine (7.6% vs. 26% incidence, P=.004). Partially hydrolized whey protein suppressed circulating C-peptide concentration (a stable indicator of steady-state insulin secretion) at all four time points relative to the corresponding CAS-fed animals. The relative mRNA abundance for the insulin-responsive, transcription factor gene, SREBP-1c, was reduced by WPH in the duodenum but not colon. Results indicate potential physiological linkages of dietary protein type with circulating C-peptide (and by inference insulin), local expression of SREBP-1c gene and propensity for small intestine tumorigenesis.

Animals↗
PubMed2005

Null mutation of Kruppel-like factor9/basic transcription element binding protein-1 alters peri-implantation uterine development in mice.

Female mice null for the basic transcription element binding protein-1 (Bteb1) gene have reduced numbers of implanting embryos. We hypothesized that the implantation defect, resulting in subfertility, is a consequence of developmental asynchrony between the embryo and uterine endometrium at peri-implantation. To address this, endometrium from wild-type (WT) and Bteb1(-/-) females at 0.5 to 5.5 days postcoitum (dpc) were evaluated for proliferation (BrdU labeling), apoptosis (TUNEL), and steroid hormone receptor expression (immunohistochemistry). Loss of BTEB1 did not affect serum estrogen (E) and progesterone (P) levels. In stroma (ST), the numbers of progesterone receptor (PGR) and HomeoboxA10 (HOXA10)-expressing cells were lower (3.5 and 4.5 dpc), while those of estrogen receptor-alpha (ESR1) were higher (3.5 dpc), with Bteb1 ablation. The peak of proliferation in luminal epithelium (LE), glandular epithelium (GE), and ST was delayed, while the apoptotic index in all cell types was increased (2.5 dpc) in Bteb1(-/-) relative to WT mice. The numbers of PGR-positive ST cells was negatively correlated with LE proliferation in WT mice; this correlation was lost in Bteb1(-/-) mice and was not observed before 2.5 dpc for both genotypes. Proliferation and apoptosis in all endometrial compartments, as well as the numbers of PGR-, HOXA10-, and ESR1-expressing ST cells, were lower in Bteb1(-/-) relative to WT mice after ovariectomy and E + P treatment. Results suggest that BTEB1, by regulating ST PGR expression and transactivation, participates in the paracrine control of LE proliferation by PGR and thus is important for establishment of a receptive uterus critical for successful implantation.

Animals↗
PubMed2005

Dietary exposure to soy or whey proteins alters colonic global gene expression profiles during rat colon tumorigenesis.

BACKGROUND: We previously reported that lifetime consumption of soy proteins or whey proteins reduced the incidence of azoxymethane (AOM)-induced colon tumors in rats. To obtain insights into these effects, global gene expression profiles of colons from rats with lifetime ingestion of casein (CAS, control diet), soy protein isolate (SPI), and whey protein hydrolysate (WPH) diets were determined. RESULTS: Male Sprague Dawley rats, fed one of the three purified diets, were studied at 40 weeks after AOM injection and when tumors had developed in some animals of each group. Total RNA, purified from non-tumor tissue within the proximal half of each colon, was used to prepare biotinylated probes, which were hybridized to Affymetrix RG_U34A rat microarrays containing probes sets for 8799 rat genes. Microarray data were analyzed using DMT (Affymetrix), SAM (Stanford) and pair-wise comparisons. Differentially expressed genes (SPI and/or WPH vs. CAS) were found. We identified 31 induced and 49 repressed genes in the proximal colons of the SPI-fed group and 44 induced and 119 repressed genes in the proximal colons of the WPH-fed group, relative to CAS. Hierarchical clustering identified the co-induction or co-repression of multiple genes by SPI and WPH. The differential expression of I-FABP (2.92-, 3.97-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively), cyclin D1 (1.61-, 2.42-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively), and the c-neu proto-oncogene (2.46-, 4.10-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively) mRNAs were confirmed by real-time quantitative RT-PCR. SPI and WPH affected colonic neuro-endocrine gene expression: peptide YY (PYY) and glucagon mRNAs were down-regulated in WPH fed rats, whereas somatostatin mRNA and corresponding circulating protein levels, were enhanced by SPI and WPH. CONCLUSIONS: The identification of transcripts co- or differentially-regulated by SPI and WPH diets suggests common as well as unique anti-tumorigenesis mechanisms of action which may involve growth factor, neuroendocrine and immune system genes. SPI and WPH induction of somatostatin, a known anti-proliferative agent for colon cancer cells, would inhibit tumorigenesis.

Administration, Oral↗
PubMed2005

Uterine phenotype of young adult rats exposed to dietary soy or genistein during development.

Dietary soy intake is associated with protection from breast cancer, but questions persist on the potential risks of the major soy isoflavone genistein (GEN) on female reproductive health. Here, we evaluated intermediate markers of cancer risk in uteri of cycling, young adult Sprague-Dawley rats lifetime exposed to one of three AIN-93G semipurified diets: casein (CAS), soy protein isolate (SPI+ with 276 mg GEN aglycone equivalents/kg) and CAS+GEN (GEN at 250 mg/kg). Postnatal day 50 (PND50) rats lifetime exposed to GEN or SPI+ had similar uterine luminal epithelium height, myometrial thickness, endometrial gland numbers, endometrial immunoreactive proliferating cell nuclear antigen (PCNA), and serum estrogen and progesterone, as CAS-fed rats. GEN-fed rats showed modestly increased apoptosis in uterine glandular epithelium, compared to those of CAS- or SPI+-fed groups. Diet had no effect on the uterine expression of genes for the tumor suppressors PTEN, p53 and p21, and the apoptotic-associated proteins Bcl2, Bax and progesterone receptor. Uterine tissue and serum concentrations of total GEN were higher in rats fed GEN than in those fed SPI+. Human Ishikawa endocarcinoma cells treated with GEN-fed rat serum tended to exhibit increased apoptotic status than those treated with CAS-fed rat serum. Exogenously added GEN (0.2 and 2 microM) increased, while estradiol-17beta (0.1 microM) decreased Ishikawa cell apoptosis, relative to untreated cells. Results suggest that lifetime dietary exposure to soy foods does not alter uterine cell phenotype in young adult rats, while GEN, by enhancing uterine endometrial glandular apoptosis in vivo and in vitro, may confer protection against uterine carcinoma. Given its limited influence on uterine phenotype of young adult females, GEN, when taken as part of soy foods or as supplement, should be favorably considered for other potential health benefits.

Animals↗
PubMed2005

Soy protein isolate and protection against cancer.

OBJECTIVE: Results from epidemiological and animal studies suggest that consuming soy-containing diets reduces the incidence of certain cancers. The purpose of this presentation was to evaluate the potential of soy protein to prevent occurrence of prostate, breast and colon cancer. METHODS: Meta-analyses of published epidemiologic studies associating cancer risk with soy intake were performed. The incidence of chemically-induced mammary or colon tumors was determined for rats fed AIN-93G diets made with either casein or soy protein isolate (SPI). Western and Northern blot and microarray analyses were performed on rat mammary and colon tissues to study mechanisms underlying the effects of soy. RESULTS: Meta-analyses revealed reductions in the mean overall risk estimate for mammary (0.78, p < 0.001), colon (0.70, p < 0.001) and prostate (0.66, p < 0.001) cancer for soy consumers. The incidence of AOM-induced colon tumors and DMBA-induced mammary tumors was reduced (p < 0.05) in rats fed SPI-containing diets. Lower incidence of mammary tumors in SPI-fed rats was associated with: 1) reduced terminal end bud numbers (p < 0.05), 2) lower expression of the phase I enzyme CYP1B1 (p < 0.05) and 3) reduced expression of the Ah Receptor and ARNT (p < 0.05). CONCLUSIONS: SPI may protect against cancer via multiple mechanisms, including: 1) increased mammary gland differentiation, 2) decreased activation of procarcinogens to carcinogens and 3) regulation of genes in signal transduction pathways underlying tumor initiation, promotion and/or progression.

Animals↗
PubMed2004

Feeding of soy protein isolate to rats during pregnancy and lactation suppresses formation of aberrant crypt foci in their progeny's colons: interaction of diet with fetal alcohol exposure.

Soy protein isolate (SPI) in the diet may inhibit colon tumorigenesis. We examined azoxymethane (AOM)-induced aberrant crypt foci (ACF) in male rats in relation to lifetime, pre-weaning, or post-weaning dietary exposure to SPI and also within the context of fetal alcohol exposure. Pregnant Sprague Dawley rats were fed AIN-93G diets containing casein (20%, the control diet) or SPI (20%) as the sole protein source starting on gestation day 4 (GD 4). Progeny were weaned on postnatal day (PND) 21 to the same diet as their dams and were fed this diet until termination of the experiment at PND 138. Rats received AOM on PND 89 and 96. Lifetime (GD 4 to PND 138) feeding of SPI led to reduced frequency of ACF with 4 or more crypts in the distal colon. Progeny of dams fed SPI only during pregnancy and lactation or progeny fed SPI only after weaning exhibited similarly reduced frequency of large ACF in distal colon. Number of epithelial cells, in the distal colon, undergoing apoptosis was unaffected by diet. SPI reduced weight gain and adiposity, but these were not correlated with fewer numbers of large ACF. Lifetime SPI exposure similarly inhibited development of large ACF in Sprague Dawley rats whose dams were exposed to ethanol during pregnancy. In summary, feeding of SPI to rat dams during pregnancy and lactation suppresses numbers of large ACF in their progeny, implying a long-term or permanent change elicited by the maternal diet. Moreover, results support the use of ACF as an intermediate endpoint for elucidating effects of SPI and its biochemical constituents in colon cancer prevention in rats.

Journal Article↗
PubMed2004

The planetary biology of cytochrome P450 aromatases.

BACKGROUND: Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system. RESULTS: Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases-enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene. CONCLUSIONS: This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems.

Amino Acid Sequence↗
PubMed2004

Post-transcriptional regulation of sterol regulatory element-binding protein-1 by ethanol induces class I alcohol dehydrogenase in rat liver.

Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors control the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids. Continuous intragastric infusion of ethanol-containing diets as part of total enteral nutrition generates well defined 6-day cycles (pulses) of urine ethanol concentrations (UECs) in rats. Pulsatile UECs are the result of cyclical expression and activity of the principal alcohol-metabolizing enzyme, hepatic Class I alcohol dehydrogenase (ADH), and this mechanism involves regulated CCAAT/enhancer-binding protein-beta expression and binding to the ADH promoter. In this study, we further explore the molecular mechanism for ethanol-induced ADH expression during the UEC pulse in adult male rats fed ethanol by total enteral nutrition for 21-30 days. In hypophysectomized rats, in which the ADH protein increased by approximately 6-fold, the nuclear form of SREBP-1 decreased by approximately 7-fold. Because the ADH promoter contains two canonical sterol response element (SRE) sites (-63 to -53 and -52 to -40 relative to the transcription start site), electrophoretic mobility shift assays were conducted using an ADH-specific SRE site. Hepatic nuclear protein binding decreased by 2.4-fold on the ascending limbs and by 3.6-fold on the descending limbs of UEC pulses (p < 0.05). The specificity of nuclear protein binding to the ADH-SRE site was confirmed using antibody and UV cross-link assays. The in vivo binding status of SREBP-1 to ADH-SRE sites, as measured by the chromatin immunoprecipitation assay, had a pattern very similar to the electrophoretic mobility shift assay results. Functional analysis of the ADH-SREs demonstrated these sites to be essential for ADH transcription. In vitro transcription assays demonstrated that depletion of the SREBP-1 protein from nuclear extracts increased transcription activity by approximately 5-fold and that the liver X receptor agonist T0901317 (a known activator of SREBP-1c transcription) reduced in vitro expression of ADH mRNA by 2-fold. We conclude that SREBP-1 is a negative regulator of the ADH gene and may work in concert with the CCAAT/enhancer-binding proteins to mediate ethanol induction of ADH in vivo.

Alcohol Dehydrogenase↗
PubMed2004

Subfertility, uterine hypoplasia, and partial progesterone resistance in mice lacking the Kruppel-like factor 9/basic transcription element-binding protein-1 (Bteb1) gene.

Progesterone receptor (PR), a ligand-activated transcription factor, is a key regulator of cellular proliferation and differentiation in reproductive tissues. The transcriptional activity of PR is influenced by co-regulatory proteins typically expressed in a tissue- and cell-specific fashion. We previously demonstrated that basic transcription element-binding protein-1 (BTEB1), a member of the Sp/Krüppel-like family of transcription factors, functionally interacts with the two PR isoforms, PR-A and PR-B, to mediate progestin sensitivity of target genes in endometrial epithelial cells in vitro. Here we report that ablation of the Bteb1 gene in female mice results in uterine hypoplasia, reduced litter size, and increased incidence of neonatal deaths in offspring. The reduced litter size is solely a maternal genotype effect and results from fewer numbers of implantation sites, rather than defects in ovulation. In the early pregnant uterus, Bteb1 expression in stromal cells temporally coincides with PR-A isoform-dependent decidual formation at the time of implantation. Expression of two implantation-specific genes, Hoxa10 and cyclin D3, was decreased in uteri of early pregnant Bteb1-null mutants, whereas that of Bteb3, a related family member, was increased, the latter possibly compensating for the loss of Bteb1. Progesterone responsiveness of several uterine genes was altered with Bteb1-null mutation. These results identify Bteb1 as a functionally relevant PR-interacting protein and suggest its selective modulation of cellular processes that are regulated by PR-A in the uterine stroma.

Animals↗
PubMed2004

Uterine androgen receptors: roles in estrogen-mediated gene expressionand DNA synthesis.

The localization of androgen receptors (AR) and their ligands in the uterine microenvironment at early pregnancy suggest a role for AR in uterine physiology. We have evaluated AR expression in the pig uterine endometrium and examined whether AR ligands modulate peri-implantation uterine gene expression. Northern blot analysis demonstrated the approximately 10.5 kilobase AR transcript in endometrium. Endometrial levels of AR mRNA and protein were greater at early than at mid- or late pregnancy. Estrogen receptor-alpha mRNA levels showed similar maximal expression at early pregnancy. Immunocytochemical analysis of endometrium at early pregnancy localized AR to nuclei of glandular epithelial (GE) and stromal (ST) cells. To evaluate a role for AR in uterine gene regulation, the levels of mRNAs for insulin-like growth factor-I (IGF-I), proliferative cell nuclear antigen (PCNA), and AR itself were assessed in uterine endometrial explant cultures treated with estradiol-17beta (E), testosterone (T), and 19-nortestosterone (N). Induction by E of AR mRNA abundance occurred in endometrium from Day 10 but not from Day 12 pregnant animals and this was partially blocked by coaddition of N or T, although neither androgen alone had any effect. Abundance of IGF-I and PCNA mRNAs was increased by E and inhibited by coaddition of either T or N in Day 10 pregnant pig endometrium. In endometrium from Day 12 pregnant animals, addition of either N or T with E increased IGF-I mRNA levels over that of controls, although E alone was without effect. In contrast, PCNA mRNA abundance was suppressed by all steroid treatments in these explants. DNA synthesis in primary cultures of GE cells from endometrium at Days 10 and 12 of pregnancy was increased by E and was suppressed by T, the latter only at Day 12. E did not affect DNA synthesis in ST cells from endometrium at either pregnancy day, although T inhibited this process in an E-dependent manner in ST cells from pregnancy Day 12. Results identify AR in the pig endometrium during the window of maternal receptivity for implantation and demonstrate the functional, albeit complex, interactions of androgens and estrogens in the regulation of uterine endometrial gene expression and cell growth in vitro. Further elucidation of the role of androgens and their receptor in early pregnancy events may be relevant to an understanding of peri-implantation embryo loss.

Androgens↗
PubMed2003

Selective interactions of Kruppel-like factor 9/basic transcription element-binding protein with progesterone receptor isoforms A and B determine transcriptional activity of progesterone-responsive genes in endometrial epithelial cells.

The Sp/KLF transcription factor basic transcription element-binding protein (BTEB1) regulates gene transcription by binding to GC-rich sequence motifs present in the promoters of numerous tissue-specific as well as housekeeping genes. Similar to other members of this family, BTEB1 can act as a transactivator or transrepressor depending on cell and promoter context, although the molecular mechanism underlying these distinct activities remains unclear. Here we report that BTEB1 can mediate signaling pathways involving the nuclear receptor for the steroid hormone progesterone in endometrial epithelial cells by its selective interaction with the progesterone receptor (PR) isoforms, PR-A and PR-B. Functional interaction with ligand-activated PR-B resulted in superactivation of PR-B transactivity, facilitated the recruitment of the transcriptional integrator CREB-binding protein within the PR-dimer, and was dependent on the structure of the ligand bound by PR-B. By contrast, BTEB1 did not influence agonist-bound PR-A transactivity, although it augmented PR-A inhibition of PR-B-mediated transactivation as well as potentiated ligand-independent PR-A transcriptional activity in the presence of CREB-binding protein. We also demonstrate similar positive modulatory actions of BTEB1-related family members Krüppel-like family (KLF) 13/FKLF2/BTEB3 and Sp1 on PR-B transactivity. Further, we provide support for the potential significance of the selective functional interactions of PR isoforms with BTEB1 in the peri-implantation uterus using mouse and pig models and in the breast cancer cell lines MCF-7 and T47D. Our results suggest a novel mechanism for the divergent physiological consequences of PR-A and PR-B on progesterone-dependent gene transcription in the uterus involving select KLF members.

Animals↗
PubMed2002

Progesterone receptors and Sp/Krüppel-like family members in the uterine endometrium .

Sp/KLF family members, of which at least 20 distinct nuclear proteins are known to date, serve as transcription factors by binding to GC-enriched sites within target gene promoters. These molecules regulate diverse cellular processes such as proliferation, differentiation, and apoptosis, via their interactions with various other nuclear proteins in promoter- and cell-context-dependent pathways to effect transactivation or transrepression. Recent studies indicate that Sp-family members may functionally interact with ligand-activated nuclear receptors, especially those for steroid hormones. This review provides a discussion of the putative mechanisms and important participants involved in the crosstalk between selected members of this family and the receptor for the pregnancy hormone progesterone, and the relevant outcomes to the control of the transcriptional network in the uterine endometrium.

Animals↗