Transcriptional activation of the zinc finger transcription factor BTEB2 gene by Egr-1 through mitogen-activated protein kinase pathways in vascular smooth muscle cells.

We have recently demonstrated that a developmentally regulated zinc finger protein, basic transcription regulatory element binding protein 2 (BTEB2), is induced in neointimal smooth muscle in response to vascular injury. In this study, we investigated the molecular mechanisms regulating BTEB2 expression in vascular smooth muscle cells (SMCs) in vitro. BTEB2 mRNA expression is rapidly and persistently induced in SMCs by phorbol 12-myristate 13-acetate (PMA) and basic fibroblast growth factor. We have isolated and characterized the promoter region of the human BTEB2 gene to determine the regulatory network controlling expression of this gene in vascular SMCs. Functional studies on the BTEB2 promoter coupled to a luciferase reporter gene demonstrated activation of the promoter by PMA and basic fibroblast growth factor. Both characterization of DNA-protein complexes in vitro and site-specific mutation analysis of the BTEB2 promoter have defined a 9-bp sequence, 5'-CGCCCGCGC-3', located at -25, as the Egr-1 binding site mediating an induction of the BTEB2 promoter activity by PMA. In addition, we show that this site mediates inducible expression through the mitogen-activated protein kinase pathways. These results indicate that BTEB2 is a target of the early-response gene Egr-1, and mitogen-activated protein kinase pathways directly or indirectly activate BTEB2 expression. Given a rapid induction of Egr-1 on stimulation with growth factors or injury, these findings may represent at least one of the molecular mechanisms underlying phenotypic modulation of smooth muscles after vascular injury.

Inducible expression of basic transcription factor-binding protein 2 (BTEB2), a member of zinc finger family of transcription factors, in cardiac allograft vascular disease.

BACKGROUND: We have recently identified basic transcription factor-binding protein 2 (BTEB2), which is involved in phenotypic modulation of vascular vascular smooth muscle cells. The aim of this study was to investigate the expression of BTEB2 in cardiac allograft vascular disease. METHODS: Heterotopic cardiac transplantation was performed in rats. All grafts were stained with antibodies against for BTEB2 and cyclin-dependent kinase 4 for immunohistochemical study. The intensity of BTEB2 expression was also calculated. RESULTS: In the allografts at 4 and 8 weeks after transplantation, smooth muscle cells were positive for BTEB2 in the diffusely thickened coronary arteries and the perivascular space. BTEB2 expression was closely associated with cyclin-dependent kinase 4 expression. The BTEB2 expression score was significantly higher in the allografts compared with the isografts. CONCLUSIONS: The induced expression of BTEB2 may play a potential role in the development of the cardiac allograft vascular disease.

Phenotypic modulation of vascular smooth muscle cells: dissection of transcriptional regulatory mechanisms.

The smooth muscle myosin heavy chain (MHC) gene and its isoforms are excellent molecular markers that reflect smooth muscle phenotypes. The SMemb/Nonmuscle Myosin Heavy Chain B (NMHC-B) is a distinct MHC gene expressed predominantly in phenotypically modulated SMCs (synthetic-type SMC). To dissect the molecular mechanisms governing phenotypic modulation of SMCs, we analyzed the transcriptional regulatory mechanisms underlying expression of the SMemb gene. We previously reported two transcription factors, BTEB2/IKLF and Hex, which transactivate the SMemb gene promoter based on the transient reporter transfection assays. BTEB2/IKLF is a zinc finger transcription factor, whereas Hex is a homeobox protein. BTEB2/IKLF expression in SMCs is downregulated with vascular development in vivo but upregulated in cultured SMCs and in neointima in response to vascular injury after balloon angioplasty. BTEB2/IKLF and Hex activate not only the SMemb gene but also other genes activated in synthetic SMCs including plasminogen activator inhibitor-1 (PAI-1), iNOS, PDGF-A, Egr-1, and VEGF receptors. Mitogenic stimulation activates BTEB2/IKLF gene expression through MEK1 and Egr-1. Elevation of intracellular cAMP is also important in phenotypic modulation of SMCs, because the SMemb promoter is activated under cooperatively by cAMP-response element binding protein (CREB) and Hex.

Quantitative analysis of mast cells in benign and malignant colonic lesions: immunohistochemical study on formalin-fixed, paraffin-embedded tissues.

BACKGROUND: Comparison of the number of mast cells in the active stage and that in remission in the same patients with ulcerative colitis with immunohistochemical staining remains to be elucidated, and analysis of the number of mast cells in benign and malignant colonic lesions is insufficient. METHODS: Using immunohistochemical methods, morphological examinations of mast cells were undertaken in colonic tissues from 8 patients with ulcerative colitis and 10 patients with colonic primary cancer, which were formalin-fixed and paraffin-embedded. Changes in the number of mast cells in the active stage and in remission in the same patients with ulcerative colitis were investigated. Then, the number of mast cells in malignant tissues and adjacent healthy tissues obtained from the same patients with colonic primary cancer were compared, and finally the number of mast cells was compared among the samples from benign and malignant colonic lesions. RESULTS: Accumulation of mast cells was found to be significant in the active stage of ulcerative colitis compared with remission in the same patients. The number of mast cells in colonic primary cancer was significantly increased compared with that in adjacent healthy tissues. The number of mast cells in ulcerative colitis was significantly greater than that in adjacent healthy tissues from patients with colonic primary cancer, irrespective of the stages of ulcerative colitis. CONCLUSIONS: We were the first to analyse mast cells in the active stage and in remission in the same patients with ulcerative colitis using immunohisto-chemical methods, and compared the number of mast cells between benign and malignant colonic lesions.

BTEB2, a Krüppel-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 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 lambdagt11 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 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 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 BTEB2 mediates the transcriptional regulation of the SMemb/NMHC-B gene and possibly plays a role in regulating gene expression during phenotypic modulation of vascular SMC.

Quantitative analysis of mast cells in benign and malignant colonic lesions: immunohistochemical study on formalin-fixed, paraffin-embedded tissues

Background: Comparison of the number of mast cells in the active stage and that in remission in the same patients with ulcerative colitis with immunohistochemical staining remains to be elucidated, and analysis of the number of mast cells in benign and malignant colonic lesions is insufficient. Methods: Using immunohistochemical methods, morphological examinations of mast cells were undertaken in colonic tissues from 8 patients with ulcerative colitis and 10 patients with colonic primary cancer, which were formal in-fixed and paraffin-embedded. Changes in the number of mast cells in the active stage and in remission in the same patients with ulcerative colitis were investigated. Then, the number of mast cells in malignant tissues and adjacent healthy tissues obtained from the same patients with colonic primary cancer were compared, and finally the number of mast cells was compared among the samples from benign and malignant colonic lesions. Results: Accumulation of mast cells was found to be significant in the active stage of ulcerative colitis compared with remission in the same patients. The number of mast cells in colonic primary cancer was significantly increased compared with that in adjacent healthy tissues. The number of mast cells in ulcerative colitis was significantly greater than that inadjacent healthy tissues from patients with colonic primary cancer, irrespective of the stages of ulcerative colitis. Conclusions: We were the first to analyse mast cells in the active stage and in remission in the same patients with ulcerative colitis using immunohistochemical methods, and compared the number of mast cells between benign and malignant colonic lesions

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