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.