Dual Regulation Of Somatostatin Receptor Subtype 1 Gene Expression By Pit-1 In Anterior Pituitary GH3 Cells

Abstract

Somatostatin represents a major release inhibiting factor for hypophyseal hormones and mediates its action via five receptor subtypes, sst1-sst5, that are all present in the anterior pituitary. The pituitary specific transcription factor Pit-1 is essential for the pituitary development and pituitary-specific gene expression. Here the transcriptional regulation of the sst1 gene, which contains putative Pit-1-binding sites, was studied in anterior pituitary GH3 cells. We found that a fragment of 2 kb suffices to drive the expression of a reporter gene specifically in this cell line. Positive and negative cis-regulatory elements contributed to the promoter activity. Among these elements two functional binding sites for Pit-1 were identified. While the proximal site mediated transcriptional activation, the distal site attenuated transcription of reporter gene constructs. Mutations of the proximal Pit-1 site prevented expression of the reporter gene. Targeting Pit-1 mRNA by antisense oligonucleotides caused inhibition of transcription of reporter gene constructs containing the proximal Pit-1-binding site. Moreover, the expression of the endogenous sst1 gene in GH3 anterior pituitary cells was blocked. This resulted in reduced sst1 levels at the plasma membrane. Reduced sst1 levels were associated with a diminished antisecretory response to the sst1-specific agonist CH-275 and somatostatin. These results demonstrate the importance of Pit-1 for the expression of the sst1 gene, which hence is placed under common genetic control with the genes for hypophysiotropic hormones and the gene for the receptor of GH-releasing hormone.

The POU Domain Transcription Factor Tst-1 Activates Somatostatin Receptor 1 Gene Expression in Pancreatic β-Cells

“…In pituitary GH3 cells the POU domain protein Pit-1 trans-activates the sst1 gene by binding to this site in vitro and in vivo (27). Here, the importance of the fp2b element is emphasized by our findings that it also confers transcriptional activity to the sst1 gene in insulin-producing pancreatic cells.…”

“…Reporter Gene Constructs, Cell Culture, Transient Transfections, Luciferase, and Electrophoretic Mobility Shift Assays-All of the plasmids and experimental techniques have been described in detail recently (27)(28)(29). For luciferase assays it should be noted that the luciferase activities were normalized for efficiency of transfection by determination of ␤-galactosidase activities and protein concentrations (Bradford dye kit, Bio-Rad).…”

“…Members of this family, such as Pit-1, Brn-2, and Brn-4, are well known as regulators of neuroendocrine gene expression and cellular differentiation in the brain, pancreatic ␣-cells, and the pituitary (24 -26). In the pituitary, the POU domain protein Pit-1 has been identified recently as an essential regulator of the sst1 gene (27). In the present paper we investigated whether other POU domain proteins could be responsible for the activation of the sst1 gene in pancreatic ␤-cells.…”

Characterization and Transcriptional Regulation of the Human Somatostatin Receptor Subtype 1 Gene

“…The upstream stimulating factor (USF) has been implicated in the regulation of tissue-specifi c and developmentally regulated genes [40] . The POU domain transcription factor Pit-1 plays a key role in terminal pituitary differentiation [41] and was recently shown to dually regulate the rat sst1 [36,42] . Our studies show that the largest − 2834 sst1 promoter contains elements that support gene expression in pituitary cells, whereas − 1813 bp does not transduce signifi cant activity.…”

Regulation and function of somatostatin receptors

“…Interestingly, the negative and positive cis ‐acting elements both contained a functional Pit‐1 binding site. The two Pit‐1 sites conferred opposing effects to a heterologous promoter, thereby reproducing their activating or silencing effect (Baumeister et al . 2000).…”