Homeobox protein Hex induces SMemb/nonmuscle myosin heavy chain-B gene expression through the cAMP-responsive element.
Recent studies have shown that the homeobox gene Hex plays an important role in inducing differentiation of vascular endothelial cells. In this study, we examined the expression of Hex in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Immunohistochemistry showed a marked induction of Hex protein in neointimal VSMCs after balloon injury in rat aorta. Western and reverse transcriptase-polymerase chain reaction analyses demonstrated that Hex was abundantly expressed in cultured VSMCs, whereas it was undetectable in other cell types or in normal aorta. The expression pattern of Hex was similar to that of SMemb/NMHC-B, a nonmuscle isoform of myosin heavy chain that we have previously reported to be a molecular marker of dedifferentiated VSMCs. We next examined the role of Hex in SMemb gene transcription. Promoter analysis demonstrated that the sequence identical to consensus cAMP-responsive element (CRE) located at -481 of the SMemb promoter was critical for Hex responsiveness. Mutant Hex expression vector, which lacks the homeodomain, failed to stimulate SMemb gene transcription, suggesting the requirement of the homeodomain for its transactivation. Elecrophoretic mobility shift assay showed that Hex binds to a consensus binding sequence for homeobox proteins, but not to CRE. Cotransfection of protein kinase A expression vector increased the ability of Hex to stimulate SMemb promoter activity in a CRE-dependent manner. Overexpression of CRE binding protein (CREB), but not Mut-CREB which contains mutation at Ser133, strongly activated Hex-induced SMemb promoter activity. These results suggest that Hex mediates transcriptional induction of the SMemb/NMHC-B gene via its homeodomain, and Hex can function as a transcriptional modulator of CRE-dependent transcription in VSMCs.
Sekiguchi
K
K
Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan.
Kurabayashi
M
M
Oyama
Y
Y
Aihara
Y
Y
Tanaka
T
T
Sakamoto
H
H
Hoshino
Y
Y
Kanda
T
T
Yokoyama
T
T
Shimomura
Y
Y
Iijima
H
H
Ohyama
Y
Y
Nagai
R
R
D016475:3T3 Cells; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002404:Catheterization; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D009154:Mutation; D018995:Myosin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D024501:Nonmuscle Myosin Type IIB; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014157:Transcription Factors; D015533:Transcriptional Activation; D017539:Tunica Intima / Q000378:metabolism / Q000473:pathology
D017362:Cyclic AMP Response Element-Binding Protein; C513080:Hhex protein, mouse; C513079:Hhex protein, rat; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000242:Cyclic AMP; D008156:Luciferases; D024501:Nonmuscle Myosin Type IIB; C103815:nonmuscle myosin type IIB heavy chain; D018995:Myosin Heavy Chains