Role of Cdx-2 in insulin and proglucagon gene expression: a study using the RIN-1056A cell line with an inducible gene expression system

Abstract

Although the homeobox gene Cdx-2 was initially isolated from the pancreatic cell line HIT-T15, no examination of its role in regulating endogenous insulin gene expression has been reported. To explore further the role of Cdx-2 in regulating both insulin and proglucagon gene expression, we established an ecdysone-inducible Cdx-2 expression system. This report describes a study using the rat insulinoma cell line RIN-1056A, which abundantly expresses both insulin and proglucagon (glu), and relatively high amounts of endogenous Cdx-2. Following the introduction of the inducible Cdx-2 expression system into this cell line and the antibiotic selection procedure, we obtained novel cell lines that displayed dramatically reduced expression of endogenous Cdx-2, in the absence of the inducer. These novel cell lines did not express detectable amounts of glu mRNA or the glucagon hormone, while their insulin expression was not substantially affected. In the presence of the inducer, however, transfected Cdx-2 expression was dramatically increased, accompanied by stimulation of endogenous Cdx-2 expression. More importantly, activated Cdx-2 expression was accompanied by elevated insulin mRNA expression, and insulin synthesis. Cdx-2 bound to the insulin gene promoter enhancer elements, and stimulated the expression of a luciferase reporter gene driven by these enhancer elements. Furthermore, Cdx-2 and insulin gene expressions in the wild-type RIN-1056A cells were stimulated by forskolin treatment, and forskolin-mediated activation on insulin gene expression was attenuated in the absence of Cdx-2. We suggest that Cdx-2 may mediate the second messenger cAMP in regulating insulin gene transcription.

ParaHox genes in pancreatic cell cultures: effects on the insulin promoter regulation

“…CDX2/3, instead, responded differently depending on the glucose concentration. While having no statitistically significant effect at low glucose levels, it was able to transactivate the insulin promoter at high glucose concentration, in accordance to previous reports 22, 23. This effect was particularly observed when high amounts of the transcription factor construct were transfected (4.4-fold increase).…”

“…CDX2/3 is expressed in α-cells, where it binds to the glucagon promoter and activates its transcription 21. In addition, and although this factor is not β cell-specific, CDX2/3 can recognise enhacer elements of the rat insulin I gene promoter usually bound by PDX1 (German et al, 1992) and transactivate endogenous and recombinant rat insulin I and II promoters 22, 23. Moreover, most studies on Cdx and Gsh transcription factors focus on their roles during embryonic development and tumorigenesis, and very scarce data is available on their biological function in adult tissues.…”

Presence of functional hyperpolarisation-activated cyclic nucleotide-gated channels in clonal alpha cell lines and rat islet alpha cells

“…Immunostaining Alpha-TC6 cells and rat islets were dualstained for glucagon and HCN channels using a protocol similar to one used in a previous report [27]. In brief, cells or islets grown on coverslips were fixed with paraformaldehyde (3.7% [wt/vol.]…”

“…Glucagon secretion Glucagon secretion in the clonal alpha cells was measured using a glucagon RIA kit (Linco Research, St Charles, MO, USA) as described previously [27]. For islet assay, Krebs-Ringer bicarbonate buffer (KRB) was used containing (mmol/l) 115 NaCl, 5 KCl, 24 NaHCO 3 , 2.5 CaCl 2 , 1 MgCl 2 , 10 HEPES and 2% (wt/vol.)…”

Multifunctional Magnetic Nanocarriers for Image-Tagged SiRNA Delivery to Intact Pancreatic Islets

“…One very promising and widely explored approach towards this goal involves modification of the gene expression profile of the beta cell. To this end, numerous reports in the literature describe the application of gene transfer in beta-cell derived cell-lines for the study of beta-cell differentiation (1-3), beta-cell function (2, 4-7), immunorecognition in the pathogenesis of type 1 diabetes (8), and the mechanisms behind the pathogenesis of type 2 diabetes (9), to name a few. In the context of gene therapy for diabetes, several ideas have shown promise, including stimulation of beta-cell growth, induction of beta-cell differentiation and regeneration, genetic engineering of non-beta cells to produce insulin, and transplantation of engineered beta cells (10).…”