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.
Simmen
Rosalia C M
RC
Department of Animal Sciences and Interdisciplinary Concentration in Animal Molecular & Cell Biology, University of Florida, Gainesville, Florida 32611-0910, USA. simmen@animal.ufl.edu
Zhang
Xue-Lian
XL
Michel
Frank J
FJ
Min
Seok Hong
SH
Zhao
Ge
G
Simmen
Frank A
FA
D000818:Animals; D015415:Biomarkers; D002277:Carcinoma / Q000473:pathology; D002455:Cell Division / Q000235:genetics; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D016889:Endometrial Neoplasms / Q000473:pathology; D004717:Endometrium / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000096:biosynthesis / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D051741:Kruppel-Like Transcription Factors; D008840:Microfilament Proteins; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D005810:Multigene Family; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured / Q000166:cytology; D000094963:Axl Receptor Tyrosine Kinase
D015415:Biomarkers; C493956:CDKN1A protein, human; C493958:Cdkn1a protein, rat; D002868:Chromosomal Proteins, Non-Histone; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D018971:Insulin-Like Growth Factor Binding Protein 2; C492214:KLF5 protein, human; C489676:KLF9 protein, human; C492215:Klf5 protein, rat; D051741:Kruppel-Like Transcription Factors; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C094772:centromere protein F; C077017:Klf9 protein, rat; D007334:Insulin-Like Growth Factor I; D020794:Receptor Protein-Tyrosine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D000094963:Axl Receptor Tyrosine Kinase