Molecular structure and function of rat platelet-derived growth factor beta-receptor gene promoter.
OBJECTIVE
To understand the regulatory mechanism of platelet-derived growth factor beta-receptor gene expression.


METHODS
A 1.7 kb genomic fragment was obtained from a rat genomic library. After we had determined an entire sequence of this fragment, transcription start sites were determined both by primer extension analysis and by riboprobe mapping. We performed a functional promoter assay by using a dual-luciferase reporter system. Progressive 5'-deletions of the fragment and site-directed mutagenesis for the CCAAT motif located at -67 or -94 were used for the assay, and their promoter activities in vascular smooth muscle cells were assessed. Gel-mobility shift analysis was also performed for the CCAAT motif at -67. Effects of the upstream sequence spanning -310 through -120 on heterologous gene promoters were also investigated.


RESULTS
Multiple transcription start sites were observed in the 5'-flanking region, and the 1.7 kb sequence was actually active as a functional promoter in vascular smooth muscle cells. Two important sequences responsible for the basal transcriptional activity were identified by the functional promoter assay. One was the CCAAT motif at -67 which acts as a promoter itself, and the other was the upstream region spanning -310 through -210 which positively regulates the basal promoter activity.


CONCLUSIONS
The basal promoter activity of the rat platelet-derived growth factor beta-receptor gene is mainly regulated by the interaction or coordination of two sequences, the CCAAT motif and the upstream control element.
Kitami
Y
Y
The Second Department of Internal Medicine, Ehime University School of Medicine, Japan. kitamiyk@m.ehime-u.ac.jp
Fukuoka
T
T
Okura
T
T
Takata
Y
Y
Maguchi
M
M
Igase
M
M
Kohara
K
K
Hiwada
K
K
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D016678:Genome; D008297:Male; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics*; D014158:Transcription, Genetic
D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor