Biomedical subjects

Frank J Michel

Publications and source records attributed to Frank J Michel.

4 recordsLinked to original sources

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

Secretory leukocyte protease inhibitor mediates proliferation of human endometrial epithelial cells by positive and negative regulation of growth-associated genes.

Secretory leukocyte protease inhibitor (SLPI) inhibits chymotrypsin, trypsin, elastase, and cathepsin G. This protein also exhibits proliferative effects, although little is known about the molecular mechanisms underlying this activity. We have generated SLPI-ablated epithelial sublines by stably transfecting the Ishikawa human endometrial cell line with an antisense human SLPI RNA expression vector. We demonstrate a positive correlation between cellular SLPI production and proliferation. We further show that Ishikawa sublines expressing low to undetectable SLPI have correspondingly increased and decreased expression, respectively, of transforming growth factor-beta 1 and cyclin D1 genes, relative to parental cells. SLPI selectively increased cyclin D1 gene expression, with the effect occurring in part at the level of promoter activity. Cellular SLPI levels negatively influenced the anti-proliferative and pro-apoptotic insulin-like growth factor-binding protein-3 expression. We also identified lysyl oxidase, a phenotypic inhibitor of the ras oncogenic pathway and a tumor suppressor, as SLPI-repressed gene, whose expression is up-regulated by transforming growth factor-beta1. Our results suggest that SLPI acts at the node(s) of at least three major interacting growth inhibitory pathways. Because expression of SLPI is generally high in epithelial cells exhibiting abnormal proliferation such as in carcinomas, SLPI may define a novel pathway by which cellular growth is modulated.

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PubMed2002

Molecular markers of endometrial epithelial cell mitogenesis mediated by the Sp/Krüppel-like factor BTEB1.

Basic transcription element binding (BTEB1) protein is one of at least 20 Sp/KLF family members that function as transcriptional activators or repressors by binding to GC/GT-rich sequences within target genes to influence cellular homeostasis in mammals. Previously, we demonstrated that increased expression of BTEB1 in a human endometrial epithelial cell line Hec-1-A resulted in serum dependent-enhanced proliferation, which was accompanied by heightened expression of cell cycle- and growth-associated genes. In the present study, we examined the mechanism underlying the altered proliferative potential associated with BTEB1 by the identification of additional BTEB1 downstream gene targets and by the demonstration of BTEB1 transactivation of promoters for a number of growth-associated genes. Using mRNA differential display in the analysis of RNA populations from Hec-1-A sublines with high (4S, 9S) and low (2As, 3As) BTEB1 cellular content, we identified 10 distinct differentially expressed transcripts, nine of which had higher levels in S than in As sublines. The expression levels of two of these cDNAs, Axl receptor tyrosine kinase and mitosin, whose encoded products are implicated in cellular proliferation, were modestly induced by serum, albeit in a BTEB1-independent manner. Moreover, insulin-like growth factor-I, a mitogen present in serum, had no significant effect on their expression in either subline. In transient reporter assays, the basal activities of the Axl gene promoter and those for two other growth-regulatory genes, namely p21(WAF1) and IGFBP-2, were increased by serum and were significantly higher in 4S than in 2As lines. However, while BTEB1 and its ubiquitous family member Sp1 increased basal p21(WAF1) and IGFBP-2 transcription when added as expression constructs in the parental Hec-1-A cell line, only Sp1 activated Axl transcription, despite the presence in all three gene promoters of GC-enriched regions that presumably can bind BTEB1 and Sp1 with similar affinities. To elucidate intracellular signaling pathways that might involve BTEB1, inhibitors of specific kinase-dependent transducers were used in transient transfection assays involving the IGFBP-2 gene promoter in 4S and 2As sublines. While inhibitors of the MAPK, PI-3K, and PKA pathways elicited similar effects on the IGFBP-2 gene promoter activity, irrespective of cellular BTEB1 content, that for JNK had a more pronounced effect on Hec-1-A sublines exhibiting higher BTEB1 expression levels. Taken together, the results suggest that BTEB1 mediates the expression of growth-associated genes through direct and indirect transactivation mechanisms, one of which may involve the participation of a JNK family member.

Animals↗

PubMed2002

Direct interaction of the Krüppel-like family (KLF) member, BTEB1, and PR mediates progesterone-responsive gene expression in endometrial epithelial cells.

The present study was undertaken to evaluate the underlying mechanism(s) by which PR and a Krüppel-like family member, basic transcription element binding protein (BTEB1), mediate endometrial epithelial expression of pregnancy-associated genes. Human endometrial carcinoma cell lines (Hec-1-A) expressing high and low levels of BTEB1 were transiently transfected with a human PR isoform (PR-B) expression construct and a luciferase reporter gene driven by the uteroferrin gene promoter that is responsive to both BTEB1 and the PR ligand progesterone. Unliganded PR inhibited luciferase activity in low and high BTEB backgrounds, and this effect was reversed by the synthetic progestin R5020 in both lines. Transactivation by PR of uteroferrin promoter activity (approximately 4-fold) was maximal at lower R5020 concentrations (10 nM) in endometrial cells with higher BTEB1 expression, suggesting that nuclear BTEB1content influenced target gene promoter sensitivity to progesterone. BTEB1 and PR-B were found to physically interact in a progesterone-independent manner, using a coimmunoprecipitation assay that employed antibodies specific to either protein. Moreover, the formation of the BTEB1/PR complex, independent of progesterone, occurred within the context of uterine endometrial proteins and was diminished in late-pregnancy endometrium. Mammalian two-hybrid assays using the entire open reading frame of BTEB1 and/or PR-B fused to either the GAL4 DNA-binding domain or VP16 activation domain and a reporter gene (pG5CAT) under the control of GAL4-binding sites were used to evaluate the formation of functional PR-B/BTEB1 dimer in Cos-1 cells. GAL4/PR-B and VP16/PR-B induced ( approximately 3- to 4-fold) chloramphenicol acetyltransferase (CAT) activity in a progesterone-dependent manner, suggesting PR-dimer formation. By contrast, VP16/PR-B and GAL4/BTEB1 had no effect on basal CAT activity. The combination of VP16- and GAL4-PR-B fusion proteins with the BTEB1 expression construct, pCDNA3-BTEB1 enhanced ligand-bound PR-mediated CAT activity by approximately 3-fold. In transient cotransfection assays using the CAT reporter gene driven by the mouse mammary tumor virus-long terminal repeat promoter, which is responsive to ligand-bound PR but not BTEB1, BTEB1 increased PR-B-mediated CAT activity in a progesterone-dependent manner, consistent with a BTEB1/PR-dimer complex occurring independent of BTEB1 binding to DNA. Unliganded PR-B disrupted the DNA-binding activity of BTEB1 in gel retardation assays, and this effect was enhanced by the presence of PR ligand. Together, these findings support the conclusion that BTEB1 and PR-B are coregulatory proteins that mediate progesterone responsiveness of target genes by direct interactions, leading to the formation of a functional BTEB1/PR-dimer complex.

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