Rat Glucagon Gene Research Articles

Pax6 Controls the Expression of Critical Genes Involved in Pancreatic α Cell Differentiation and Function*

The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased β and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.

Transcriptional control of pancreatic islet hormones gene expression.

Initiation of transcription requires transcription factors that recognize specific DNA sequences present in promoters and enhancers of the respective genes. Cell-specific expression of the pancreatic islet hormone genes is conferred by a cell-specific combination of transcription factors. The DNA binding sites and cDNAs of some of them have been characterized. An islet-specific enhancer-binding protein of the rat glucagon gene recognizes also cis-acting elements of the rat insulin I and rat somatostatin genes. Thus identical or related factors may be involved in the expression of these pancreatic genes, reflecting that the different islet cell types arise from a common progenitor cell. The characterization of islet-specific transcription factors may lead to regulatory genes whose products specify islet cell phenotypes during development. Basal transcriptional activity of islet hormone genes can be enhanced by metabolic, humoral or nerve signals via various second messenger systems. Cyclic AMP acts through distinct response elements. The islet cells are electrically excitable and known secretagogues increase electrical activity and elevate cytosolic calcium concentration. Depolarization-induced activation of voltage-dependent calcium channels stimulates rat glucagon gene transcription suggesting that calcium is an intracellular signal for pancreatic islet hormone gene transcription.

Inhibition of human insulin gene transcription by peroxisome proliferator‐activated receptor γ and thiazolidinedione oral antidiabetic drugs

The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for glucose homeostasis. PPARgamma ligands reducing insulin levels in vivo are used as drugs to treat type 2 diabetes mellitus. Genes regulated by PPARgamma have been found in several tissues including insulin-producing pancreatic islet beta-cells. However, the role of PPARgamma at the insulin gene was unknown. Therefore, the effect of PPARgamma and PPARgamma ligands like rosiglitazone on insulin gene transcription was investigated. Reporter gene assays were used in the beta-cell line HIT and in primary mature pancreatic islets of transgenic mice. Mapping studies and internal mutations were carried out to locate PPARgamma-responsive promoter regions. Rosiglitazone caused a PPARgamma-dependent inhibition of insulin gene transcription in a beta-cell line. This inhibition was concentration-dependent and had an EC(50) similar to that for the activation of a reporter gene under the control of multimerized PPAR binding sites. Also in normal primary pancreatic islets of transgenic mice, known to express high levels of PPARgamma, rosiglitazone inhibited glucose-stimulated insulin gene transcription. Transactivation and mapping experiments suggest that, in contrast to the rat glucagon gene, the inhibition of the human insulin gene promoter by PPARgamma/rosiglitazone does not depend on promoter-bound Pax6 and is attributable to the proximal insulin gene promoter region around the transcription start site from -56 to +18. The human insulin gene represents a novel PPARgamma target that may contribute to the action of thiazolidinediones in type 2 diabetes mellitus.

The homeodomain of PAX6 is essential for PAX6-dependent activation of the rat glucagon gene promoter: Evidence for a PH0-like binding that induces an active conformation

The transcription factor PAX6 plays an important role in transcriptional regulation of the peptide hormone glucagon from pancreatic α-cells. PAX6 contains two DNA binding domains, the paired domain (PD) and the homeodomain (HD). While the interaction of the PD with the PAX6 responsive elements G1 and G3 in the rat glucagon gene promoter is well understood, the role of the PAX6 HD for PAX6 binding and function on G1 and G3 remains unclear. In EMSA studies the PAX6 HD was found to be mandatory for PAX6 binding to G1 but not to G3. Transient transfections with luciferase reporter gene constructs revealed the HD to be critical for proper function of PAX6 on both, G1 and G3. Transfection data with variant promoter constructs and limited proteolysis assays demonstrated that the DNA sequence located 5′ to the PD binding site plays an important role for PAX6 function and its conformation on the elements G1 and G3. Taken together, our data indicate a PH0-like binding of PAX6 to the glucagon promoter elements G1 and G3 where the HD binding site is abutted directly to the PD binding motif. The data suggest that the PH0-like binding induces a transcriptionally active conformation of PAX6.

Heterogeneous Nuclear Ribonucleoprotein K Modulates Angiotensinogen Gene Expression in Kidney Cells

The present studies aimed to identify the 70-kDa nuclear protein that binds to an insulin-responsive element in the rat angiotensinogen gene promoter and to define its action on angiotensinogen gene expression. Nuclear proteins were isolated from rat kidney proximal tubular cells and subjected to two-dimensional electrophoresis. The 70-kDa nuclear protein was detected by Southwestern blotting and subsequently identified by mass spectrometry, which revealed that it was identical to 65-kDa heterogeneous nuclear ribonucleoprotein K (hnRNP K). hnRNP K bound to the insulin-responsive element of the rat angiotensinogen gene was revealed by a gel mobility shift assay and chromatin immunoprecipitation assay. hnRNP K inhibited angiotensinogen mRNA expression and promoter activity. In contrast, hnRNP K down-expression by small interference RNA enhanced angiotensinogen mRNA expression. Moreover, hnRNP K interacted with hnRNP F in pulldown and co-immunoprecipitation assays. Co-transfection of hnRNP K and hnRNP F further suppressed angiotensinogen mRNA expression. Finally, in vitro and in vivo studies demonstrated that high glucose increases and insulin inhibits hnRNP K expression in rat kidney proximal tubular cells. In conclusion, our experiments revealed that hnRNP K is a nuclear protein that binds to the insulin-responsive element of the rat angiotensinogen gene promoter and modulates angiotensinogen gene transcription in the kidney.

Characterization of a novel Foxa (hepatocyte nuclear factor-3) site in the glucagon promoter that is conserved between rodents and humans

The pancreatic islet hormone glucagon stimulates hepatic glucose production and thus maintains blood glucose levels in the fasting state. Transcription factors of the Foxa [Fox (forkhead box) subclass A; also known as HNF-3 (hepatocyte nuclear factor-3)] family are required for cell-specific activation of the glucagon gene in pancreatic islet alpha-cells. However, their action on the glucagon gene is poorly understood. In the present study, comparative sequence analysis and molecular characterization using protein-DNA binding and transient transfection assays revealed that the well-characterized Foxa-binding site in the G2 enhancer element of the rat glucagon gene is not conserved in humans and that the human G2 sequence lacks basal enhancer activity. A novel Foxa site was identified that is conserved in rats, mice and humans. It mediates activation of the glucagon gene by Foxa proteins and confers cell-specific promoter activity in glucagon-producing pancreatic islet alpha-cell lines. In contrast with previously identified Foxa-binding sites in the glucagon promoter, which bind nuclear Foxa2, the novel Foxa site was found to bind preferentially Foxa1 in nuclear extracts of a glucagon-producing pancreatic islet alpha-cell line, offering a mechanism that explains the decrease in glucagon gene expression in Foxa1-deficient mice. This site is located just upstream of the TATA box (between -30 and -50), suggesting a role for Foxa proteins in addition to direct transcriptional activation, such as a role in opening the chromatin at the start site of transcription of the glucagon gene.

Binding of HNF-3α in nuclear extracts of pancreatic islet α-cell line to the novel HNF-3 site-A in the glucagon gene

The pancreatic islet hormone glucagon stimulates hepatic glucose production and thus maintains blood glucose levels in the fasting state. Transcription factors of the hepatocyte nuclear factor-3 (HNF-3, also called Foxa) family are expressed in endoderm-derived tissues such as liver and pancreatic islets and, as part of their metabolic control, are required for cell-specific activation of the glucagon gene in pancreatic islet α-cells. However, their action at the glucagon gene is poorly understood. We have shown previously that the well characterized HNF-3 binding site in the G2 enhancer element of the rat glucagon gene is not conserved in humans and that the human G2 sequence lacks basal enhancer activity. However, a novel HNF-3 site (called site-A) has been identified that is conserved in rat, mouse, and humans and that mediates an activation of the glucagon gene by HNF-3 proteins expressed in an heterologous cell line. In the present study, a molecular characterization using protein-DNA binding and transient transfection assays revealed that the novel HNF-3 site-A confers cell-specific promoter activity in glucagon-producing pancreatic islet α-cell lines (InR1G9, αTC2). In contrast to other HNF-3 binding sites in the glucagon promoter that bind nuclear HNF-3β, the novel HNF-3 site-A was found to bind preferentially HNF-3α in nuclear extracts of a glucagon-producing pancreatic islet α-cell line. Mice with a targeted disruption of the HNF-3α gene have been shown to develop normally but exhibit neonatal growth retardation and die postnatally prior to 4 weeks of age, exhibiting hypoglycemia associated with inappropriately low levels of circulating glucagon. The results of the present study offer a mechanism that explains the decrease in glucagon gene expression in HNF-3α-deficient mice. The novel HNF-3 site-A is located just upstream of the TATA box (between -30 and -50) suggesting a role for HNF-3 proteins in addition to direct transcriptional activation

Pax6 and Cdx2/3 form a functional complex on the rat glucagon gene promoter G1-element

α-cell specific transcription of the glucagon gene is mainly conferred by the glucagon promoter G1-element, while additional elements G2, G3, and G4 have broad islet cell specificity. Transcription of the glucagon gene has been shown to be stimulated by Pax6 through binding to the glucagon gene promoter G3-element. In this report, we show that Pax6 additionally binds the glucagon gene promoter G1-element and forms a transcriptionally active complex with another homeodomain protein, Cdx2/3. Two distinct mutations in the G1-element, that both reduce promoter activity by 85–90%, is shown to eliminate binding of either Pax6 or Cdx2/3. Additionally, Pax6 enhanced Cdx2/3 mediated activation of a glucagon reporter in heterologous cells. We discuss how Pax6 may contribute to cell-type specific transcription in the pancreatic islets by complex formation with different transcription factors.

Study of the promoter of the rat glucagon gene: 1/ Tissue specific polymorphism of the 5′ end of glucagon receptor mRNA

Study of the promoter of the rat glucagon gene: 1/ Tissue specific polymorphism of the 5′ end of glucagon receptor mRNA

The Glucagon Gene Is Transcribed in β-like Pancreatic Cells

In this report we demonstrate that approximately 1.1 kb of the rat glucagon gene promoter upstream of the transcriptional start site specifically directs the transcription of the reporter gene chloramphenicol acetyl transferase (CAT) (p[-1.1]GLU-CAT) in insulinoma β-TC1 cells. On the contrary, the 350 bp closest to the transcription start site (p[-0.35]GLU-CAT) are ineffective in β-TC1 cells. Both constructs are transcriptionally active in InR1-G9 glucagonoma cells. While protein kinase A and protein kinase C activators, acting through independent pathways, strongly increase both the transcription of p[-1.1]GLU-CAT and the accumulation of glucagon transcript in β-TC1 cells, they are weaker activators in InR1-G9 cells. Our experiments suggest that some positive transcription control elements, necessary for the glucagon gene transcription in insulinoma β-TC1 cells, are localized in the -350/-1100 region of the glucagon gene. Furthermore, our data indicate that glucagon gene transcription can be strongly activated through the protein kinase A pathway in some specific cellular contexts.

Hepatocyte-nuclear factor 3 beta gene transcripts generate protein isoforms with different transactivation properties on the glucagon gene.

Hepatocyte-nuclear factor 3 beta (HNF-3 beta), a member of the HNF-3 gene family, is expressed in glucagon-producing islet cells and represses glucagon gene expression. We show here that at least three different HNF-3 beta transcripts that encode HNF-3 beta protein variants are present in glucagon-producing cells, HNF-3 beta 1, HNF-3 beta 2, and HNF-3 beta 3. Compared with the HNF-3 beta 1 cDNA, HNF-3 beta 2 cDNA lacks sequences of exon 1 while exons 1 and 4 are absent from the HNF-3 beta 3 cDNA. The deduced amino-acid (aa) sequence of HNF-3 beta 2 and HNF-3 beta 3 proteins differs from HNF-3 beta 1 by a 6-aa amino-terminal extension and by the absence of the first 30 aa, respectively. HNF-3 beta 1, HNF-3 beta 2, and HNF-3 beta 3 bind to the major enhancer of the rat glucagon gene G2 with similar affinity. By contrast to HNF-3 beta 1, which represses glucagon gene expression when overexpressed in the glucagon-producing cell line InR1G9, HNF-3 beta 2 and HNF-3 beta 3 do not affect transcriptional activity. Furthermore, cotransfection of HNF-3 beta 2 or HNF-3 beta 3 along with HNF-3 beta 1 decreases the negative effects of HNF-3 beta 1. We conclude that glucagon gene expression may be regulated by the relative abundance of the three different HNF-3 beta variants in alpha-cells.

Islet-specific Proteins Interact with the Insulin-response Element of the Glucagon Gene

Glucagon gene expression is negatively regulated by insulin at the transcriptional level. G3, a DNA control element located in the 5'-flanking sequence of the rat glucagon gene mediates the inhibition of transcription, which occurs in response to insulin. We show here that two islet-specific protein complexes C1A and C1B, bind to the A domain of G3, which is critical for the insulin response. These two complexes bind to overlapping sequences of the A domain and display very similar binding specificities. Point mutations in the A domain that affect binding of C1A and C1B result in both decreased G3 enhancer activity and insulin-mediated inhibitory effects with a close correlation between diminution of binding and function. One of the two complexes, C1A, is similar or identical to B1, a protein complexes interacting with the upstream promoter element of the glucagon gene, G1, implicated in the A cell-specific expression of the glucagon gene. Our data indicate that islet-specific proteins are involved in glucagon gene regulation by insulin.

Distinct properties of the cAMP-responsive element of the rat insulin I gene.

The cAMP response element (CRE)-binding transcription factor CREB can mediate induction of gene transcription in response to calcium as well as to cAMP. Since the rat insulin I gene 5'-flanking region contains a CRE with an octamer-like motif (TGACGTCC), CREB binding and cAMP/calcium responsiveness of the insulin CRE were investigated. In an electrophoretic mobility shift assay and in Southwestern blot experiments, bacterially expressed recombinant CREB bound to the insulin CRE as it did to the rat glucagon and rat somatostatin gene CREs. However, in nuclear extracts of the pancreatic islet cell line HIT, protein complexes binding to the insulin CRE did not contain proteins with CREB-like immunoreactivity, although these bound to the glucagon and somatostatin CREs. When reporter fusion genes were transfected into HIT cells, the isolated insulin CRE increased basal activity and mediated transcriptional activation by cAMP. However, cAMP stimulation of transcription through the insulin CRE was weak when compared with the response through the glucagon and somatostatin CREs. Furthermore, the insulin CRE did not confer responsiveness to membrane depolarization and calcium influx, in contrast to the glucagon and somatostatin CREs. These results demonstrate that the functional properties of the rat insulin I gene CRE are different from those of the rat glucagon and somatostatin CREs which may be explained by a distinct pattern of nuclear protein binding and suggest the existence of post-translational mechanisms that decrease the binding of cellular CREB to the insulin CRE.

Glucagon gene expression is negatively regulated by hepatocyte nuclear factor 3 beta.

Pancreatic expression of the glucagon gene depends on multiple transcription factors interacting with at least three DNA control elements: G1, the upstream promoter element, and G2 and G3, two enhancer-like sequences. We report here that the major enhancer of the rat glucagon gene, G2, interacts with three protein complexes, A1, A2, and A3. A2 is detected only in islet cells, and impairment of its binding to mutant G2 causes a marked decrease in transcriptional activity. We identify A1 as hepatocyte nuclear factor 3 beta (HNF-3 beta), a member of the HNF-3 DNA-binding protein family found in abundance in the liver which has been proposed to play a role in the formation of gut-related organs. HNF-3 beta binds G2 on a site which overlaps A2 and acts as a repressor of glucagon gene expression, as demonstrated by mutational analyses of G2 and by cotransfection of HNF-3 beta cDNA along with reporter genes containing G2 into glucagon-producing cells. Our data implicate HNF-3 beta in the control of glucagon gene expression and strengthen the idea of endodermal origin of the islet cells.

Glucagon gene expression is negatively regulated by hepatocyte nuclear factor 3 beta.

Pancreatic expression of the glucagon gene depends on multiple transcription factors interacting with at least three DNA control elements: G1, the upstream promoter element, and G2 and G3, two enhancer-like sequences. We report here that the major enhancer of the rat glucagon gene, G2, interacts with three protein complexes, A1, A2, and A3. A2 is detected only in islet cells, and impairment of its binding to mutant G2 causes a marked decrease in transcriptional activity. We identify A1 as hepatocyte nuclear factor 3 beta (HNF-3 beta), a member of the HNF-3 DNA-binding protein family found in abundance in the liver which has been proposed to play a role in the formation of gut-related organs. HNF-3 beta binds G2 on a site which overlaps A2 and acts as a repressor of glucagon gene expression, as demonstrated by mutational analyses of G2 and by cotransfection of HNF-3 beta cDNA along with reporter genes containing G2 into glucagon-producing cells. Our data implicate HNF-3 beta in the control of glucagon gene expression and strengthen the idea of endodermal origin of the islet cells.

Transcription of the Rat Glucagon Gene by the Cyclic AMP Response Element-Binding Protein CREB Is Modulated by Adjacent CREB-Associated Proteins

The cyclic AMP (cAMP) response element (CRE) of the rat glucagon gene (Glu-CRE, 5'-TGACGTCA-3') mediates transcriptional responses to 8-bromo-cAMP and protein kinase A (PKA) in a glucagon-producing hamster islet cell line (InR1G9). By several different DNA-protein binding assays, we show that the transcription factor CREB binds to the CRE octamer and that additional nuclear proteins bind to sequences adjacent to the CRE. Mutation of the Glu-CRE octamer attenuates both the binding of CREB and cAMP-dependent PKA-stimulated transcriptional activity in transient transfection experiments but does not affect the binding of adjacent CREB-associated proteins. Progressive deletions and clustered point mutations of the sequences flanking the Glu-CRE identify sequences (5'-TCATT-3') located both 5' and 3' to the core CRE octamer that bind several proteins. Two proteins with molecular masses of 80 and 100 kDa bind to each of the 5' and 3' TCATT sites. Formation of additional protein-DNA complexes containing 45- and 20-kDa proteins depends upon the integrity of both TCATT sequences. Deletion or point mutation of the TCATT motif located on the 3' side of the CRE octamer results in enhanced transcriptional responses to PKA, suggesting that the CREB-associated proteins decrease the ability of CREB to mediate PKA-stimulated transcription. Results from these studies demonstrate that nucleotides flanking the core CRE octamer can influence the activity of the CRE by serving as binding sites for proteins that modulate the function of CREB and suggest a mechanism to explain why some consensus palindromic CREs are less responsive to cAMP stimulation than others.

The immunosuppressive drugs cyclosporin A and FK506 inhibit calcineurin phosphatase activity and gene transcription mediated through the cAMP-responsive element in a nonimmune cell line

Cyclosporin A and the macrolide tacrolimus (FK506) are powerful immunosuppressive drugs that in T cells inhibit the calcium/calmodulin-dependent phosphatase calcineurin thereby preventing the activation of T-cell-specific transcription factors, such as NF-AT, involved in lymphokine gene expression. While this may explain, at least in part, the mechanism of cyclosporin A/FK506 immunosuppression, additional mechanisms have to be invoked in order to explain the pharmacological properties and toxic effects of these drugs, such as nephrotoxicity and neurotoxicity. We have studied the effects of cyclosporin A and FK506 on calcineurin phosphatase activity and gene transcription mediated by the cAMP-responsive element (CRE), a binding site of the ubiquitous transcription factor CREB. A reporter gene was placed under the transcriptional control of the CRE of the rat glucagon gene and transiently transfected into the glucagon-expressing cell line alpha TC2. Cyclosporin A and FK506 inhibited depolarization-induced gene transcription in a concentration-dependent manner (IC50 of about 1 nM and 30 nM for FK506 and cyclosporin A, respectively). Both cyclosporin A and FK506 inhibited calcineurin phosphatase activity at drug concentrations that inhibited gene transcription. The FK506 analogue rapamycin had no effect on calcineurin activity and gene transcription, but excess concentrations of rapamycin prevented the effects of FK506 on both calcineurin activity and gene transcription. These results support the notion that the interaction of drug-immunophilin complexes with calcineurin may be the molecular basis of cyclosporin A/FK506-induced inhibition of CREB/CRE-mediated gene transcription. The ability to interfere with CREB/CRE-mediated gene transcription represents a novel mechanism of cyclosporin A/FK506 action which may underlie pharmacological effects and toxic manifestations of these potent immunuosuppressive drugs.

Transcription of the rat glucagon gene by the cyclic AMP response element-binding protein CREB is modulated by adjacent CREB-associated proteins.

The cyclic AMP (cAMP) response element (CRE) of the rat glucagon gene (Glu-CRE, 5'-TGACGTCA-3') mediates transcriptional responses to 8-bromo-cAMP and protein kinase A (PKA) in a glucagon-producing hamster islet cell line (InR1G9). By several different DNA-protein binding assays, we show that the transcription factor CREB binds to the CRE octamer and that additional nuclear proteins bind to sequences adjacent to the CRE. Mutation of the Glu-CRE octamer attenuates both the binding of CREB and cAMP-dependent PKA-stimulated transcriptional activity in transient transfection experiments but does not affect the binding of adjacent CREB-associated proteins. Progressive deletions and clustered point mutations of the sequences flanking the Glu-CRE identify sequences (5'-TCATT-3') located both 5' and 3' to the core CRE octamer that bind several proteins. Two proteins with molecular masses of 80 and 100 kDa bind to each of the 5' and 3' TCATT sites. Formation of additional protein-DNA complexes containing 45- and 20-kDa proteins depends upon the integrity of both TCATT sequences. Deletion or point mutation of the TCATT motif located on the 3' side of the CRE octamer results in enhanced transcriptional responses to PKA, suggesting that the CREB-associated proteins decrease the ability of CREB to mediate PKA-stimulated transcription. Results from these studies demonstrate that nucleotides flanking the core CRE octamer can influence the activity of the CRE by serving as binding sites for proteins that modulate the function of CREB and suggest a mechanism to explain why some consensus palindromic CREs are less responsive to cAMP stimulation than others.

Multiple cis-acting domains mediate basal and adenosine 3',5'-monophosphate-dependent glucagon gene transcription in a mouse neuroendocrine cell line.

A number of cell and tissue-specific differences have been described in studies of the regulation of glucagon gene expression. DNA sequences important for islet cell-specific transcription are not sufficient for expression of the glucagon gene in the intestine, and the posttranslational processing of proglucagon results in the liberation of different peptides in pancreas and intestine. We have studied the control of glucagon gene expression in STC-1 cells, a mouse intestinal neuroendocrine cell line. STC-1 cells are plurihormonal and contain glucagon, somatostatin, amylin, and cholecystokinin, but not insulin mRNA transcripts. Glucagon gene expression is regulated by a cAMP-dependent pathway in STC-1 cells, with an increase in glucagon mRNA transcripts detected 2 h after forskolin stimulation. The levels of glucagon mRNA transcripts remained elevated for 36-48 h after forskolin stimulation, but cycloheximide inhibited the forskolin induction of glucagon gene expression. Although sequences up-stream of -1300 are necessary for intestine-specific glucagon gene transcription in transgenic mice, glucagon-chloramphenicol acetyltransferase (CAT) plasmids containing less than 1300 basepairs of 5'-flanking sequences were transcriptionally active in STC-1 cells. The transcriptional properties of specific DNA elements important for glucagon gene transcription in islet cells differed in STC-1 cells. Deletion of the islet cell-specific enhancer G3 element resulted in an increase in the transcriptional activity of transfected glucagon-CAT plasmids, suggesting that G3 may function as a negative element in STC-1 cells. Deletion of the cAMP response element sequence from -291 to -298 did not eliminate the forskolin induction of glucagon-CAT activity in STC-1 cells, and forskolin responsiveness was maintained with deletions containing only 60 basepairs of rat glucagon gene 5'-flanking sequences. The results of these experiments define novel functional properties for previously characterized domains within the rat glucagon gene 5'-flanking region, suggesting that mouse STC-1 cells may be a useful cell line for studies of the molecular control of glucagon gene expression.

Membrane depolarization and calcium influx induce glucagon gene transcription in pancreatic islet cells through the cyclic AMP-responsive element.

Glucagon-producing pancreatic islet cells generate calcium-dependent action potentials. By the control of calcium influx through voltage-gated calcium channels, calcium is a tightly regulated second messenger in these cells. It is unknown whether calcium is a signal for glucagon gene transcription. Therefore, rat glucagon reporter fusion genes were transiently transfected into pancreatic islet cell lines. High potassium-induced membrane depolarization activated glucagon gene transcription. The effects of a calcium chelator, calcium channel blockers, calmodulin antagonists, and an inhibitor of calcium/calmodulin-dependent protein kinase II (CaM kinase II) indicate that depolarization-induced glucagon gene transcription depends on calcium influx and CaM kinase II. The depolarization-responsive element was mapped to the glucagon cAMP-responsive element (CRE). The CRE-binding protein CREB was shown, by using GAL4-CREB fusion proteins, to function as a depolarization-regulated transcription factor in pancreatic islet cells. Membrane depolarization and cAMP had synergistic effects on glucagon gene transcription. These results suggest that rat glucagon gene transcription is regulated by membrane electrical activity and calcium influx in pancreatic islet cells. This signal may be transmitted via CaM kinase II and CREB to the glucagon CRE.