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The sst2 somatostatin receptor mediates the inhibitory effects of somatostatin on secretive and proliferative processes. We previously showed that sst2 is one of the major subtypes expressed in the rat pituitary, and its messenger RNA level is up-regulated by chronic treatment with estrogen. To investigate the molecular mechanisms regulating sst2 gene expression, we cloned the upstream region (9.5 kb) from the translation initiation codon of the rat sst2 gene. It contained a single intron (5.0 kb) at the 5'-untranslated region, lacked TATA and CCAAT boxes, and had multiple transcriptional start sites. Transient transfection analysis with deleted mutants of a luciferase reporter construct showed that the promoter activity was regulated negatively and positively in the distal and proximal promoter regions, respectively. The promoter activity of each construct was more efficient in GH(3) pituitary cells than in nonpituitary cells. The construct (-77/+172/luc) containing a cAMP response element (CRE; -54/-47) provided maximum promoter activity, but a further 5'-deleted construct dramatically reduced the activity. Competitive gel shift and supershift assays indicated that Sp2 and Sp3 were bound to an Sp1 site (-40/-31), and activating transcription factor-2 and c-Jun were bound to a CRE site. Both Sp1 and CRE sites were essential for the full promoter activity. Overexpression of the pituitary homeoprotein Pitx1 activated the promoter activity of the -4066/+172/luc construct, and mapping analysis indicated the existence of two Pitx1 response sites, including the CRE site. Estrogen also increased the promoter activity of -77/+172/luc in GH(3) cells or in HeLa cells overexpressing both the estrogen receptor and c-Jun. These studies demonstrated the nature of the rat sst2 gene and the functional importance of both Sp1 and CRE sites in regulating sst2 gene expression and suggest that the CRE site mediates, at least partly, the promoter activity activated by Pitx1 or estrogen.

endocrinology

Characterization of 5′-Flanking Region of Rat Somatostatin Receptor sst2 Gene: Transcriptional Regulatory Elements and Activation by Pitx1 and Estrogen<sup>1</sup>

Characterization of 5′-Flanking Region of Rat Somatostatin Receptor sst2 Gene: Transcriptional Regulatory Elements and Activation by Pitx1 and Estrogen1

Characterization of 5′-Flanking Region of Rat Somatostatin Receptor sst2 Gene: Transcriptional Regulatory Elements and Activation by Pitx1 and Estrogen¹