PubMed Health⌕ Search

Biomedical subjects

S Marshak

Publications and source records attributed to S Marshak.

8 recordsLinked to original sources
PubMed2002

Regulation of insulin gene transcription.

The mammalian insulin gene is exclusively expressed in the beta cells of the endocrine pancreas. Two decades of intensive physiological and biochemical studies have led to the identification of regulatory sequence motifs along the insulin promoter and to the isolation of transcription factors which interact to activate gene transcription. The majority of the islet-restricted (BETA2, PDX-1, RIP3b1-Act/C1) and ubiquitous (E2A, HEB) insulin-binding proteins have been characterized. Transcriptional regulation results not only from specific combinations of these activators through DNA-protein and protein-protein interactions, but also from their relative nuclear concentrations, generating a cooperativity and transcriptional synergism unique to the insulin gene. Their DNA binding activity and their transactivating potency can be modified in response to nutrients (glucose, NEFA) or hormonal stimuli (insulin, leptin, glucagon like peptide-1, growth hormone, prolactin) through kinase-dependent signalling pathways (PI3-K, p38MAPK, PKA, CaMK) modulating their affinities for DNA and/or for each other. From the overview of the research presented, it is clear that much more study is required to fully comprehend the mechanisms involved in the regulated-expression of the insulin gene in the beta cell to prevent its impairment in diabetes.

Animals↗
PubMed2001

A pancreatic beta -cell-specific enhancer in the human PDX-1 gene is regulated by hepatocyte nuclear factor 3beta (HNF-3beta ), HNF-1alpha, and SPs transcription factors.

The PDX-1 transcription factor plays a key role in pancreas development. Although expressed in all cells at the early stages, in the adult it is mainly restricted to the beta-cell. To characterize the regulatory elements and potential transcription factors necessary for human PDX-1 gene expression in beta-cells, we constructed a series of 5' and 3' deletion fragments of the 5'-flanking region of the gene, fused to the luciferase reporter gene. In this report, we identify by transient transfections in beta- and non-beta-cells a novel beta-cell-specific distal enhancer element located between -3.7 and -3.45 kilobases. DNase I footprinting analysis revealed two protected regions, one binding the transcription factors SP1 and SP3 and the other hepatocyte nuclear factor 3beta (HNF-3beta) and HNF-1alpha. Cotransfection experiments suggest that HNF-3beta, HNF-1alpha, and SP1 are positive regulators of the herein-described human PDX-1 enhancer element. Furthermore, mutations within each motif abolished the binding of the corresponding factor(s) and dramatically impaired the enhancer activity, therefore suggesting cooperativity between these factors.

3T3 Cells↗
PubMed2001

Regulatory elements involved in human pdx-1 gene expression.

PDX-1 was shown to be expressed early during development in cells of both exocrine and endocrine origin; later it becomes restricted primarily to beta-cells where it regulates the expression of beta-cell-specific genes and mediates the glucose effect on insulin gene transcription. Therefore, it was important to identify the molecular mechanisms that specifically govern the expression of pdx-1 in the mature beta-cell. To address this question, we analyzed 7 kb of the 5' flanking region of the human pdx-1 gene. By transient transfections of beta- and non-beta-cell lines with different 5' and 3' deletions of that region, a strong beta-cell-specific enhancer element located between -3.71 and 3.46 kb was revealed. We also sequenced about 4.5 kb of the human 5' flanking region and compared it with that of the mouse pdx-1 gene. This comparison revealed three short conserved regions, designated PH1, PH2, and PH3. We showed that HNF-3beta can bind and stimulate the activity of the human PH1 and PH2 elements in non-beta-cells. Results reported by Wu et al. (7) and Sharma et al. (6) also indicate that expression of the mouse pdx-1 is controlled by an HNF-3-like element. Thus, it can be stated that at least some aspects of pdx-1 expression rely on the transcription factor HNF-3beta. Because HNF-3beta is not restricted to beta-cells, the selective transcription of pdx-1 is likely to rely on additional factors. Our findings that the PH1 enhancer element binds both HNF-3beta and PDX-1 and that mutations in each individual site dramatically impair its transcriptional activity suggest that these factors cooperate with one another. We therefore propose that a possible feedback mechanism might control the expression of pdx-1 at different stages during development.

Animals↗
PubMed2000

Functional conservation of regulatory elements in the pdx-1 gene: PDX-1 and hepatocyte nuclear factor 3beta transcription factors mediate beta-cell-specific expression.

The PDX-1 transcription factor plays a key role in pancreatic development and in the regulation of the insulin gene in the adult beta cell. As its functions appear to be similar in humans and mice, we analyzed the functional conservation of homologous sequences important for the maintenance and the cell-specific regulation of the pdx-1 gene. Apart from the proximal promoter region, three highly homologous (PH1 to PH3) sequences were apparent in the human and mouse 5' flanking regions of the gene. By transient transfections in beta and non-beta cells, we show that mainly PH1 and PH2 preferentially confer beta-cell-specific activation on a heterologous promoter. DNase I footprinting and binding analyses revealed that both bind to and are transactivated by hepatocyte nuclear factor 3beta (HNF-3beta). Furthermore, the PH1 enhancer element also binds the PDX-1 transcription factor itself, which acts cooperatively with adjacent HNF-3beta to regulate its transcriptional potency. This finding suggests a possible autoregulatory loop as a mechanism for PDX-1 to control its own expression.

Animals↗
PubMed1999

Impaired beta-cell functions induced by chronic exposure of cultured human pancreatic islets to high glucose.

In type 2 diabetes, chronic hyperglycemia has been suggested to be detrimental to beta-cell function, causing reduced glucose-stimulated insulin secretion and disproportionately elevated proinsulin. In the present study, we investigated the effect on several beta-cell functions of prolonged in vitro exposure of human pancreatic islet cultures to high glucose concentrations. Islets exposed to high glucose levels (33 mmol/l) for 4 and 9 days showed dramatic decreases in glucose-induced insulin release and in islet insulin content, with increased proportion of proinsulin-like peptides relative to insulin. The depletion in insulin stores correlated with the reduction in insulin mRNA levels and human insulin promoter transcriptional activity. We also demonstrated that high glucose dramatically lowered the binding activity of pancreatic duodenal homeobox 1 (the glucose-sensitive transcription factor), whereas the transcription factor rat insulin promoter element 3b1 activator was less influenced and insulin enhancer factor 1 remained unaffected. Most of these beta-cell impairments were partially reversible when islets first incubated for 6 days in high glucose were transferred to normal glucose (5.5 mmol/l) concentrations for 3 days. We conclude that cultured human islets are sensitive to the deleterious effect of high glucose concentrations at multiple functional levels, and that such mechanisms may play an important role in the decreased insulin production and secretion of type 2 diabetic patients.

Adult↗
PubMed1996

Purification of the beta-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells.

The beta cell-specific glucose-sensitive factor (GSF), which binds the A3 motif of the rat I and human insulin promoters, is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose only in normal islets, supporting the suggested physiological role of GSF in the glucose-regulated expression of the insulin gene. GSF binding activity was observed in all insulin-producing cells. We have therefore purified this activity from the rat insulinoma RIN and found that a single polypeptide of 45 kDa was responsible for DNA binding. Its amino acid sequence, determined by microsequencing, provided direct evidence that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as PDX-1) and that, in addition to its essential roles in development and differentiation of pancreatic islets and in beta cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated beta cells. The human cDNA coding for GSF/IPF-1 has been cloned, its cell and tissue distribution is described. Its expression in the glucagon-producing cell line alpha TC1 transactivates the wild-type human insulin promoter more efficiently than the mutated construct. It is demonstrated that high levels of ectopic GSF/IPF-1 inhibit the expression of the human insulin gene in normal islets, but not in transformed beta TC1 cells. These results suggest the existence of a control mechanism, such as requirement for a coactivator of GSF/IPF-1, which may be present in limiting amounts in normal as opposed to transformed beta cells.

Amino Acid Sequence↗