BTEB2, a Krüppel-Like Transcription Factor, Regulates Expression of the SMemb/Nonmuscle Myosin Heavy Chain B (SMemb/NMHC-B) Gene

Abstract

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 Ϫ105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this cis-regulatory element. We screened a 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 corresponds to the rabbit homologue of basic transcriptional element binding protein-2 (BTEB2), which has previously been identified as one of the Krüppel-like transcription factor. Gel mobility shift assays and antibody supershift analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factorϺSE1 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 activity and BTEB2-induced transcriptional activation were markedly attenuated by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was most highly expressed in intestine, urinary bladder, and uterus. BTEB2 mRNA levels were downregulated in rabbit aorta during normal development. Moreover, immunohistochemical analysis indicated a marked induction of BTEB2 protein in the neointimal SMC after balloon injury in rat aorta. These results suggest that

Positive- and Negative-acting Krüppel-like Transcription Factors Bind a Transforming Growth Factor β Control Element Required for Expression of the Smooth Muscle Cell Differentiation Marker SM22α in Vivo

“…The monoclonal anti-BTEB2 antibody was a generous gift from Yamasa Corp. (Diagnostics Division, Chiba, Japan) and was raised against a carboxyl-terminal peptide antigen described previously (29). The polyclonal anti-GKLF antibody was purchased commercially (M-19X, Santa Cruz Biotechnology).…”

“…The GKLF-related Factor BTEB2 Transactivates SM22␣ Promoter Activity-Recent studies have implicated a possible role for the GKLF-related BTEB (basic transcriptional element-binding protein) factors in the transactivation of gene expression during SMC phenotypic modulation (29,47). To test whether BTEB2 might contribute to TGF-␤-dependent regulation of SM22␣ in vitro, multipotent embryonic 10T1/2 cells were transiently cotransfected with an SM22␣ promoter-lacZ reporter construct and a BTEB2-containing mammalian expression vector (pcDNA3/BTEB2) (Fig.…”

“…9B). It should be noted that the antibody used was raised against a peptide immunogen derived from the carboxyl-terminal DNA-binding domain of BTEB2 (29). Since this region is highly conserved among the KLF subfamily of Krü ppel-like factors, we cannot rule out the possibility that this antibody can cross-react with other KLF proteins in EMSA reactions.…”

BTEB2 knockdown suppresses neointimal hyperplasia in a rat artery balloon injury model

“…Previous experiments have demonstrated that BTeB2 is down-regulated during aortic development and is re-induced after balloon injury (6), suggesting that BTeB2 may be involved in the development of neointimal formation. The present study demonstrated that the knockdown of BTeB2 significantly reduced balloon injury-induced neointimal formation, indicating that BTeB2 may serve as powerful stimulus of neointimal hyperplasia.…”

“…Basic transcription element-binding protein 2 (BTeB2), a transcription factor that binds Gc boxes, is preferentially expressed in dedifferentiated or activated SMcs (6). it has been reported that atherosclerotic lesions containing BTeB2-positive cells are associated with a higher risk of restenosis than BTeB2-negative lesions (7), suggesting that BTeB2 serves as a regulator of vascular SMc proliferation and phenotypic changes.…”