BTEB2, a Kruppel-like transcription factor, regulates expression of the SMemb/nonmuscle myosin heavy chain B (SMemb/NMHC-B) gene

We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15- bp sequence, designated SE1, located at -105 from the transcriptional start site as an important regulatory element for its transcriptional activity i n a smooth muscle cell (SMC) line, In this study, we attempted to isolate c DNA clones encoding for the transcription factors that control the expressi on of the SMemb gene through binding to this cis-regulatory element. We scr eened a lambda gt11 cDNA library prepared from C2/2 cells, a rabbit-derived SMC line, by using a radiolabeled concatenated oligonucleotide containing SE1 as a probe. Sequence analysis revealed that one of the cDNA clones corr esponds to the rabbit homologue of basic transcriptional element binding pr otein-2 (BTEB2), which has previously been identified as one of the Kruppel -like transcription factor. Gel mobility shift assays and antibody supershi ft analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factor:SE1 complexes. Furthermore, a glutathione S-transferase-BTEB2 fusion protein binds to the SE1 in a sequence-specific manner. In support of the functionality of BTEB2 binding, basal promoter a ctivity and BTEB2-induced transcriptional activation were markedly attenuat ed by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was mo st highly expressed in intestine, urinary bladder, and uterus, BTEB2 mRNA l evels were downregulated in rabbit aorta during normal development. Moreove r, immunohistochemical analysis indicated a marked induction of BTEB2 prote in in the neointimal SMC after balloon injury in rat aorta. These results s uggest that BTEB2 mediates the transcriptional regulation of the SMemb/NMHC -B gene and possibly plays a role in regulating gene expression during phen otypic modulation of vascular SMC.