NF1 Negatively Regulates Gadd153 Expression in Vascular Smooth Muscle Cells.
OBJECTIVES: Vascular remodeling is determined by the balance of proliferation and apoptosis in vascular smooth muscle cells (VSMC). Endoplasmic reticulum (ER) stress has been reported as an inducible factor of apoptosis. We have recently reported that growth arrest and DNA damage inducible gene (Gadd)153, which is known to be upregulated by ER stress, plays a pivotal role in the regulation of apoptosis in VSMC. The aim of this study is to characterize a promoter region of the Gadd153 gene for further elucidation of underlying mechanisms of VSMC apoptosis mediated by Gadd153. METHODS: Promoter region was isolated by screening of a rat genomic library. Transcription start sites were determined by primer extension analysis. Progressive 5′-deletions were prepared and used for the construction of luciferase-fusion vectors. For all experiments, VSMC were maintaining in the presence of 10% FCS to make them a proliferative state. Promoter activity was measured by luciferase assay, and DNA-binding studies were performed by electromobility shift and supershift assays. RESULTS: A 1500-bp fragment containing a Gadd153 promoter region was obtained, and its sequence alignment was determined. Primer extension analysis revealed that there were two major transcription start sites. Further, one TATA-like box was found within 30 bp from the transcription start sites. Although the 5′-deletion that was deleted the sequence spanning -1500 through -400 showed the highest promoter activity in VSMC, the presence of sequence spanning -447 and -400 reduced promoter activity drastically to the almost equal level of promoter less control. This domain between -447 and -400 contained a well-known consensus element, NF1 binding site, located at positions between -447 and -417. DNA-binding studies indicated that nuclear extracts from VSMC specifically produced a retarded band that was supershifted by addition of antibodies against NF1. CONCLUSION: In proliferative VSMC, basal transcriptional activity of the Gadd153 gene was regulated mainly by a nuclear factor NF1 via a negative regulatory element located at positions between -447 and -417, suggesting that NF1 may act as an anti-apoptotic factor of VSMC.