RENIN GENE PROMOTER ACTIVITY IN GC CELLS IS REGULATED BY CAMP AND THYROID-HORMONE THROUGH PIT-1-DEPENDENT MECHANISMS

Transcriptional activity of human renin gene (hREN) 5'-flanking DNA se quences in pituitary cells is highly dependent on binding of the pitui tary-specific transcription factor Pit-1. Pit-1 has been implicated in cAMP regulation of a number of pituitary genes and has also been show n to interact with thyroid hormone (T-3) receptors in mediating T-3 re sponsiveness of the rat growth hormone gene. In the present study we e xamine the effects of forskolin and T-3 on the expression of luciferas e hybrid genes containing hREN 5'-flanking DNAs (hREN.luc) transiently transfected into the pituitary cell line GC. Basal activities of all hREN.luc constructs transfected into cells grown in media containing s erum stripped of hormones were low. Addition of forskolin stimulated e xpression up to 48 fold, depending on the hREN sequences present. The hREN sequence -148 to +18 was sufficient for both maximal expression a nd maximal stimulation by forskolin. Mutagenesis of the Pit-1 site bet ween -82 and -58 reduced forskolin induction 4-5-fold. In addition to the Pit-1 site, the sequence between -148 and -98 was also required fo r maximal activity and forskolin induction. T-3 on its own had no effe ct on hREN promoter activity in GC cells, but suppressed the effects o f forskolin. Gel mobility shift and Western blot analyses indicated th at forskolin treatment had no effect on Pit-1 DNA binding or Pit-1 lev els. However, T-3 reduced Pit-1 levels which was reflected in lower DN A binding under the conditions employed. Taken together, these finding s emphasize the importance of cAMP-dependent mechanisms in directing r enin gene expression.