Sp1/Sp3 binding is associated with cell-specific expression of the glucose-dependent insulinotropic polypeptide receptor gene

Abstract

-The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor (GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between Ϫ1 and Ϫ100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5Ј-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between Ϫ85 and Ϫ40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions Ϫ77, Ϫ60, and Ϫ50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at Ϫ50 and Ϫ60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at Ϫ70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between Ϫ100 and Ϫ40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cellspecific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter. gastric inhibitory polypeptide; transcription factors; RIN38 cells; insulinoma; rat-2 cells; chromatin immunoprecipitation assay GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE (GIP) is a member of the secretin

Adrenal GIPR expression and chromosome 19q13 microduplications in GIP-dependent Cushing’s syndrome

“…We performed pyrosequencing of the GIPR promoter region (chr19: 46,171,165-46,171,452), containing 10 CpG islands. Of note, 3 CpGs were located close to functional Sp1/Sp3 binding motifs as described previously (12). Pyrosequencing showed absence of methylation in the proximal GIPR promoter in normal adrenal tissue, adenoma, and hyperplasia samples (Supplemental Figure 3).…”

The Role of Incretins in Glucose Homeostasis and Diabetes Treatment

“…The 5′-flanking region of the gipr gene is sufficient to direct transcription in a rat insulinoma cell line 2 (RIN38), and this region contains negative regulatory sequences distal to the transcription start site controlling the cell-specific expression of the gipr gene (Boylan et al, 1999). In addition, the 5′-flanking region of gipr gene contains the binding sites for Oct-1, Sp1, and Sp3 transcription factors; binding of these transcription factors is critical not only for high levels of gipr transcription but also for cell-specific expression of the GIPR (Boylan et al, 2006). …”

The GIP/GIPR axis is functionally linked to GH-secretion increase in a significant proportion of gsp− somatotropinomas

“…Somatotroph adenomas were screened for somatic gsp mutations as reported previously (3). In nine GIPR overexpressing cases -included in this and previous cohort (3) -of which we had both somatic and matched germline DNA, the presence of somatic mutations in GIPR promoter (15) and transcribed regions has been evaluated. Lymphocytes DNA was extracted with a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions.…”