Insulin expression in pancreatic islet cells relies on cooperative interactions between the helix loop helix factor E47 and the homeobox factor STF-1.

146

142

We show that the mitogen-activated protein kinases ERK1/2 are components of the mechanism by which glucose stimulates insulin gene expression. ERK1/2 activity is required for glucose-dependent transcription from both the full-length rat insulin I promoter and the glucose-sensitive isolated E2A3/4 promoter element in intact islets and ␤ cell lines. Dominant negative ERK2 and MEK inhibitors suppress glucose stimulation of the rat insulin I promoter and the E2A3/4 element. Overexpression of ERK2 is sufficient to stimulate transcription from the E2A3/4 element. The glucose-induced response is dependent upon ERK1/2 phosphorylation of a subset of transcription factors that include Beta2 (also known as NeuroD1) and PDX-1. Phosphorylation increases their functional activity and results in a cumulative transactivation of the promoter. Thus, ERK1/2 act at multiple points to transduce a glucose signal to insulin gene transcription.

Section: Resultsmentioning

confidence: 99%

“…The affinity for coactivators such as p300/CBP (58, 59) could be increased, and interactions with repressors could be reduced. Phosphorylation could also enhance cooperative interactions, such as the interaction between PDX-1 and the bHLH heterodimer (55).…”

Section: Fig 6 Erk2 Phosphorylation Sites On E47mentioning

confidence: 99%

Regulation of Insulin Gene Transcription by ERK1 and ERK2 in Pancreatic β Cells

Khoo

Griffen

Xia

et al. 2003

146

142

“…The E2A gene products, E12, E47, and/or E2-5, bind to the Far and Nir boxes and activate the rat insulin I gene promoter synergistically with the homeodomain proteins IDX-1 (IPF-1/STF-1/ PDX-1), HNF-1␣, and Lmx-1 (52,54,60), which bind to the FLAT and P1 elements. Although C/EBP␤ activates the rat insulin I gene promoter in non-␤-cells through binding to the newly identified CEB box, this interaction does not mediate the repression of the rat insulin I promoter by C/EBP␤ in ␤-cells.…”

Section: Discussionmentioning

confidence: 99%

Pancreatic β-Cell-specific Repression of Insulin Gene Transcription by CCAAT/Enhancer-binding Protein β

Lu¹,

Seufert²,

Habener

1997

120

Chronic exposure of ␤-cells to supraphysiologic glucose concentrations results in decreased insulin gene transcription. Here we identify the basic leucine zipper transcription factor, CCAAT/enhancer-binding protein ␤ (C/EBP␤), as a repressor of insulin gene transcription in conditions of supraphysiological glucose levels. C/EBP␤ is expressed in primary rat islets. Moreover, after exposure to high glucose concentrations the ␤-cell lines HIT-T15 and INS-1 express increased levels of C/ EBP␤. The rat insulin I gene promoter contains a consensus binding motif for C/EBP␤ (CEB box) that binds C/EBP␤. In non-␤-cells C/EBP␤ stimulates the activity of the rat insulin I gene promoter through the CEB box. Paradoxically, in ␤-cells C/EBP␤ inhibits transcription, directed by the promoter of the rat insulin I gene by direct protein-protein interaction with a heptad leucine repeat sequence within activation domain 2 of the basic helix-loop-helix transcription factor E47. This interaction leads to the inhibition of both dimerization and DNA binding of E47 to the E-elements of the insulin promoter, thereby reducing functionally the transactivation potential of E47 on insulin gene transcription. We suggest that the induction of C/EBP␤ in pancreatic ␤-cells by chronically elevated glucose levels may contribute to the impaired insulin secretion in severe type II diabetes mellitus.Insulin is a hormone essential for the control of mammalian glucose homeostasis and is produced predominantly in pancreatic ␤-cells of adult animals (1). The expression of the insulin gene occurs to a large extent at the level of transcription. Control elements residing in the 5Ј 350-base pair sequence flanking exon 1 of the rat insulin I gene are sufficient to direct ␤-cell-specific expression (2) (Fig. 1A). Arrays of A and E elements 1 (Far-FLAT, Nir-P1) constitute symmetrical enhansons that cooperatively account for Ͼ90% of the transcriptional activity of the insulin gene promoter (3). The E elements are recognition motifs for transcription factors in the basic helixloop-helix (bHLH) 2 family, such as E12 and E47, which activate the insulin promoter in close synergism with A element binding homeobox transcription factors, such as IDX-1.Chronic hyperglycemia may contribute to the pancreatic ␤-cell dysfunction observed in patients with type II diabetes, a phenomenon attributed to the concept of glucose toxicity (4). Studies using in vivo animal models and in vitro ␤-cell lines have demonstrated that a reduction of insulin gene transcription by glucose toxicity is associated with the loss of transactivator proteins such as IDX-1/IPF-1/STF-1 and RIPE3b1-binding protein (5-10). Because insulin gene transcription is both positively and negatively regulated, we sought to identify repressors that might also mediate the effects of glucose toxicity on insulin gene transcription. In this report we describe CCAAT/enhancer binding protein-␤ (C/EBP␤) as a glucoseinduced repressor of insulin gene transcription.C/EBPs are a family of transcription factors that regulate...

“…2 There is precedence for ubiquitous HLH proteins allowing cell-specific gene expression via functional interactions with other transactivators. For example, the insulin gene has been proposed to be controlled by the combinatorial actions of E47 HLH proteins and cell-specific homeodomain proteins (22,23). The USF HLH proteins have also been shown to functionally interact with other DNA binding proteins to activate transcription, including in a cell-specific manner (32,33,38).…”

Section: Activity Of the Ct/cgrp Enhancer In The Context Of Flanking mentioning

confidence: 99%

“…The ubiquitous E12/E47, ITF-2, and USF proteins have been implicated in combinatorial control with other proteins, including cell-specific factors. The E12/E47 and ITF-2 proteins have been shown to functionally interact with homeodomain proteins to help direct cell-specific gene expression (22)(23)(24). The USF proteins were initially identified as upstream stimulatory factors that control the adenovirus major late promoter (25)(26)(27).…”

mentioning

confidence: 99%

Binding of Upstream Stimulatory Factor and a Cell-specific Activator to the Calcitonin/Calcitonin Gene-related Peptide Enhancer

Lanigan

Russo

1997

The calcitonin/calcitonin gene-related peptide (CT/ CGRP) gene is selectively transcribed in thyroid C cells and neurons. We have previously shown that the rat CT/CGRP cell-specific enhancer is synergistically regulated by a helix-loop-helix (HLH) protein and the OB2 octamer-binding protein. In this report, we show that the HLH-OB2 enhancer is required for full promoter activity, even in the context of other HLH elements.Since this enhancer appears to be a major controlling element, we have characterized the HLH and OB2 DNA binding proteins. We have identified the major HLH complex as a heterodimer of the ubiquitous upstream stimulatory factor (USF)-1 and USF-2 proteins. USF bound the enhancer with a reasonably high affinity (K D 1.6 nM), comparable to other genes. Characterization of a series of mutations revealed that a portion of the HLH motif is also recognized by OB2 and confirmed that HLH activity requires OB2. We have shown that OB2 is a single DNA binding protein based on UV cross-linking studies. The 68-kDa protein-DNA complex was detected only in C cell lines, including a human C cell line that has robust HLH-OB2 enhancer activity. These results suggest that the calcitonin/CGRP gene is controlled by the combinatorial activity of a ubiquitous USF HLH heterodimer and an associated cell-specific activator.