ISOLATION AND CHARACTERIZATION OF THE 5'-FLANKING REGULATORY REGION OF THE HUMAN NATRIURETIC PEPTIDE RECEPTOR-C GENE

Phenotypic modulation of vascular smooth muscle cells (SMCs) plays a c entral role in the pathogenesis of atherosclerosis. Natriuretic peptid e receptor-C (NPR-C) is highly expressed in vascular SMCs in the exper imental arteriosclerotic neointimal area as well as in cultured SMCs, suggesting that increased expression of the NPR-C gene is related to t he phenotypic alteration of vascular SMCs. To elucidate the molecular mechanisms and to identify the essential DNA sequences in NPR-C gene e xpression, a genomic clone containing over 8 kilobases of the 5'-flank ing region of the human NPR-C gene has been isolated. Sequence analysi s revealed that a number of putative regulatory elements including unu sual tandem repeated AP-2-like sequences were observed in the 6'-flank ing region. Primer extension and ribonuclease protection analyses reve aled that transcription of the human NPR-C gene starts from two major regions. Promoter analysis using deletion constructs in human cells, h ighly producing NPR-C transcripts, showing that the region (from -33 t o +13 relative to the transcription start point) had a potential promo ter activity suggested that the region from -33 to +13, containing a p yrimidine-rich stretch composed of four CTTTTT-repeated sequences, is sufficient for the proximal promoter activity. Moreover, three distinc t DNA sequences surrounding the transcription start site (Pi, from -60 to -33; P2, from +14 to +40; P3, from +41 to +66) were revealed to be functional as a cis-acting positive enhancer, and a nuclear protein(s ) from the human cells was demonstrated to specifically bind to the se quences, respectively. However, promoter analysis has shown that the P 2 and P3 sequences could not activate the human NPR-C promoter in a sy nergistic manner. On the basis of deoxyribonuclease I footprinting ana lysis showing that a DNA element from +48 to +60 within the P3 sequenc e is preferentially protected, the P3 sequence appears to contain a po tential regulatory element involved in NPR-C gene expression. The pres ent study demonstrated the structure of the 5'-regulatory region of th e human NPR-C gene and multiple cis-acting positive sequences closely located around the transcription start points with an important role i n regulation of human NPR-C gene expression.