GATA-4 upregulates glucose-dependent insulinotropic polypeptide expression in cells of pancreatic and intestinal lineage
Introduction
Glucose-dependent insulinotropic polypeptide (GIP), like glucagon-like-peptide-1 (GLP-1), is an incretin, a mediator of the enteroinsular axis that helps to maintain glucose homeostasis under physiological conditions (Murphy et al., 1995). In the presence of elevated blood glucose, incretins stimulate insulin secretion from pancreatic β-cells (O’Harte et al., 1998, Usdin et al., 1993, Yip et al., 1998). Although GLP-1 appears to be the more potent pharmacological stimulator of insulin release (Gutniak et al., 1992, Nauck et al., 1997), using GIP and GLP-1 specific receptor antagonists, it has been demonstrated that GIP is the major physiological incretin, accounting for ∼80% of nutrient induced enteroinsular pancreatic β-cell stimulation (Gault et al., 2003, Tseng et al., 1996). GIP is a 42-amino acid peptide that is produced in specific enteroendocrine cells, termed K-cells, dispersed primarily within the small intestinal mucosa (Buchan et al., 1978, Cheung et al., 2000, Yeung et al., 1999) and is released into circulation in response to the ingestion of nutrients (Cataland et al., 1974, Pederson et al., 1975).
We have begun to elucidate the mechanisms by which GIP expression is regulated. Characterization of the rat GIP gene has demonstrated that transcription initiates from a promoter located in the 5′-flanking region of the gene (Boylan et al., 1997). Furthermore, deletional analysis of the GIP promoter revealed that the first 193 bp upstream of the transcription initiation site are sufficient to direct specific expression of the GIP gene in the neuroendocrine cell line STC-1 (Boylan et al., 1997). A subsequent mutation analysis of this region demonstrated that GIP expression requires the binding of three transcription factors (Boylan et al., 1997, Jepeal et al., 2003, Jepeal et al., 2005).
The region located between base pairs −193 and −182 with respect to the transcription initiation site (+1) of the GIP promoter contains a consensus binding motif for the GATA family of DNA binding proteins, WGATAR (Jepeal et al., 2003, Orkin, 1992). Previous studies in our laboratory have shown that binding of GATA-4 to this site is essential for high GIP promoter activity in STC-1 cells (Jepeal et al., 2003). A second cis-regulatory region located between base pairs −156 and −151 of the GIP promoter also has been shown to be essential for transcription of the GIP gene in STC-1 cells (Jepeal et al., 2003) The transcription factors ISL-1 and PDX-1 were found to bind to this region and appear to function in conjunction with GATA-4 to regulate GIP gene expression (Jepeal et al., 2003, Jepeal et al., 2005).
Unfortunately, the molecular analysis of GIP expression and K-cell function has been hampered by the inability to adequately purify populations of GIP-producing K-cells, and consequently has required the identification and use of surrogate cell lines as models for such in vitro studies. Most of what is known about the regulation of GIP expression has been determined using a single cell line, STC-1 (Boylan et al., 1997, Jepeal et al., 2003, Jepeal et al., 2005, Kieffer et al., 1995). STC-1 cells were derived from an intestinal tumor isolated from a transgenic mouse expressing a viral oncogene under control of the insulin promoter (Rindi et al., 1990). These cells express multiple peptides, including GIP, glucagon, somatostatin, CCK, chromatogranin, and amylin, but not insulin (Boylan et al., 1997, Jepeal et al., 2003, Kieffer et al., 1995, Rindi et al., 1990). This plurihormonal pattern of gene expression is consistent with previous studies demonstrating the expression of multiple hormones in cells of endocrine neoplasms (Brubaker et al., 1998, Philippe et al., 1987, Philippe et al., 1988, Sidhu et al., 2000).
In addition to intestinal tumors, the transgenic mouse line that gave rise to STC-1 cells also developed pancreatic neoplasms. The cell line βTC-3 was derived from one such pancreatic β-cell tumor. Because βTC-3 cells were found to possess many of the characteristics of native differentiated β-cells, including the synthesis and secretion of considerable amounts of insulin, they have been used extensively to study insulin regulation and β-cell function (D’Ambra et al., 1990, Matsuoka et al., 2003). However, βTC-3 cells do not behave entirely like native islet β-cells by virtue of their capacity to synthesize proglucagon-derived peptides in addition to insulin (Efrat et al., 1988).
In the present study, we have demonstrated the expression and secretion of biologically active GIP from βTC-3 cells. In addition, we have examined the transcriptional regulation of GIP expression in these cells and have specifically evaluated the role of the transcription factors GATA-4, ISL-1 and PDX-1.
Section snippets
Cell culture
STC-1 cells (mouse neuroendocrine tumor derived) (Boylan et al., 1997), GTC-1 cells (a high GIP-expressing subpopulation of STC-1 cells) IEC-6 cells (normal rat small intestine derived immature intestinal stem cells [ATCC, Manassas, VA]), βTC-3 cells (mouse pancreatic β-cell tumor [ATCC]) and LGIPR2 cells (provided by Dr. Ted B. Usdin, Bethesda, MD) (Usdin et al., 1993) were grown in Dulbecco's minimal essential medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) at 37 °C in an
GIP is secreted by the pancreatic cell line βTC-3
Biologically active GIP peptide was measured reliably using a sensitive and specific in vitro bioassay. The LGIPR2 cells used in this assay are mouse L-cells cells that express both the human GIP receptor and a β-galactosidase (β-gal) reporter coupled to the cyclic adenosine monophosphate (cAMP) responsive vasoactive intestinal polypeptide (VIP) promoter (Usdin et al., 1993). When LGIPR2 cells are incubated with samples containing active GIP, the peptide present binds to and activates the GIP
Discussion
GIP expression in vitro had previously been detected only in the intestinal-like cell line STC-1 (Boylan et al., 1997, Jepeal et al., 2005, Rindi et al., 1990) and cell lines derived from STC-1 cells such as GTC-1 (Cheung et al., 2000), STC6-14 (Kieffer et al., 1995), and GIP-INS (Ramshur et al., 2002). Although numerous other intestinal cell lines have been screened by northern blot analysis, GIP bioassay and RT-PCR (Boylan et al., 1997), none have been found to express GIP. However, because
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