Cyclic AMP response element binding protein CREB and modulator protein CREM are products of distinct genes.
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Amino Acid Sequence↗
Biomedical subjects
J F Habener
Publications and source records attributed to J F Habener.
Somatostatin gene transcription regulated by a bipartite pancreatic islet D-cell-specific enhancer coupled synergetically to a cAMP response element.
The insulin-, glucagon-, and somatostatin-producing cells in the pancreatic islets derive from a common precursor stem cell and differentiate sequentially during embryonic development, thereby providing an informative model for the study of the transcriptional mechanisms involved in the control of cell-specific gene expression. Relative to the early expression of the glucagon and insulin genes on embryonic days 10 and 12, respectively, the expression of the somatostatin gene is delayed (day 17). The relatively late expression of the somatostatin gene indicates the involvement of both negative and positive transcriptional control mechanisms. We show that the expression of the somatostatin gene in pancreatic islet cells is accomplished by the interplay of both positive and negative cis-regulatory DNA elements. We have characterized the functional properties of one of these positive control elements, the somatostatin gene upstream enhancer element (SMS-UE). The SMS-UE is a pancreatic islet D-cell-specific transcriptional regulator that acts synergistically with the cyclic AMP response element. Mutation-expression and cell-free transcription analyses show that the SMS-UE is a bipartite element with two interdependent functional domains. Our results indicate that the SMS-UE is part of a functional unit that includes other transcriptional control elements of the somatostatin gene proximal promoter, and that they act together to regulate the D-cell-specific transcription of the somatostatin gene in the islet cells of the pancreas.
Animals↗
Somatostatin gene upstream enhancer element activated by a protein complex consisting of CREB, Isl-1-like, and alpha-CBF-like transcription factors.
The expression of the genes encoding the hormones glucagon, insulin, somatostatin, and pancreatic polypeptide in the endocrine islets of the pancreas is regulated in a cell-specific manner, defining four distinct cellular phenotypes (A-, B-, D-, and F-cells, respectively). Binding of nuclear proteins to cognate DNA sequences within cis-acting regulatory elements mediates the transcriptional events that result in the cell-specific activation or repression of gene expression. In a parallel study, we describe the functional properties of the SMS-UE, a pancreatic islet D-cell specific enhancer element that regulates the expression of the somatostatin gene and contains two interdependent domains, A and B. In the studies described herein, we have characterized the nuclear proteins that recognize the SMS-UE. Domain A of the SMS-UE is a DNA enhancer sequence that is identical to that bound by the ubiquitously distributed CCAAT box-binding protein alpha-CBF, a transcription factor that regulates the expression of the human chorionic gonadotrophin alpha-subunit gene. The B-domain, on the other hand, binds an islet cell-specific protein with characteristics similar to those of Isl-1, a transcriptional activator protein that binds to the E2 enhancer of the rat insulin-1 gene. In addition, the SMS-UE binds transcription factor CREB but not CREM, the close homolog of CREB, on a site adjacent to, or overlapping, the 3' end of domain B. We show that the carboxyl-terminal bZIP domain of CREB binds to the cAMP response element of the somatostatin gene but is not sufficient for binding to the SMS-UE, and we present evidence suggesting that CREB.SMS-UE binding requires stabilization by a region of the protein located within the transactivation domain.
Animals↗
Signal transduction crosstalk in the endocrine system: pancreatic beta-cells and the glucose competence concept.
Crosstalk between intracellular signalling systems is recognized as the principal means by which a cell orchestrates coordinate responses to stimulation by neurotransmitters, hormones or growth factors. The functional consequences of crosstalk are evident at multiple levels within a given signalling cascade, including the regulation of receptor-ligand interactions, guanine nucleotide-binding proteins, enzyme activities, ion channel function and gene expression. Here we focus on the pancreatic beta-cells of the islets of Langerhans to illustrate the important role crosstalk plays in the regulation of glucose-induced insulin secretion. Recent studies indicating a synergistic interaction in beta-cells between the glucose-regulated ATP-dependent signalling system and the hormonally regulated cAMP-dependent signalling system are emphasized. This interaction gives beta-cells the ability to match the ambient concentration of glucose to an appropriate insulin secretory response, a process we refer to as the induction of glucose competence. The glucose competence concept may provide new insights into the etiology and treatment of non-insulin-dependent diabetes mellitus (Type II diabetes).
Animals↗
CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription.
We report on the identification of a nuclear protein that serves as a dominant-negative inhibitor of the transcription factors C/EBP and LAP. A 32P-labeled LAP DNA-binding and dimerization domain "zipper probe" was used to isolate a clone that encodes a new C/EBP-homologous protein: CHOP-10. CHOP-10 has strong sequence similarity to C/EBP-like proteins within the bZIP region corresponding to the DNA-binding domain consisting of a leucine zipper and a basic region. Notably, however, CHOP-10 contains 2 prolines substituting for 2 residues in the basic region, critical for binding to DNA. Thus, heterodimers of CHOP-10 and C/EBP-like proteins are unable to bind their cognate DNA enhancer element. CHOP-10 mRNA is expressed in many different rat tissues. Antisera raised against CHOP-10 recognize a nuclear protein with an apparent molecular mass of 29 kD. CHOP-10 is induced upon differentiation of 3T3-L1 fibroblasts to adipocytes, and cytokine-induced dedifferentiation of adipocytes is preceded by the loss of nuclear CHOP-10. Coimmunoprecipitation of CHOP-10 and LAP from transfected COS-1 cells demonstrated a direct interaction between the two proteins, in vivo. Consistent with the structure of its defective basic region, bacterially expressed CHOP-10 inhibits the DNA-binding activity of C/EBP and LAP by forming heterodimers that cannot bind DNA. In transfected HepG2 cells, expression of CHOP-10 attenuates activation of C/EBP- and LAP-driven promoters. We suggest that CHOP-10 is a negative modulator of the activity of C/EBP-like proteins in certain terminally differentiated cells, similar to the regulatory function of Id on the activity of MyoD and MyoD-related proteins important in the development of muscle cells.
3T3 Cells↗
Tumor necrosis factor-induced reversal of adipocytic phenotype of 3T3-L1 cells is preceded by a loss of nuclear CCAAT/enhancer binding protein (C/EBP).
Tumor necrosis factor (TNF)-treated 3T3-L1 adipocytes were used as a model for studying the effects of systemic inflammation on adipose tissue. Lipopolysaccharide-treated monocyte-conditioned medium or recombinant human TNF alpha induced morphological dedifferentiation of the adipocytes and led to loss of adipocyte specific gene expression. Gel shift, Southwestern and Western immunoblot analysis demonstrated that dedifferentiation was preceded by a decrease in the DNA binding activity and protein level of the transcription factor CCAAT/enhancer binding protein (C/EBP). Liver activating protein, a related protein that binds identical DNA sequences, increased during cytokine treatment. Both proteins activate specific enhancer elements located in the promoter region of many genes whose transcription is altered during systemic inflammation. Pulse-chase labeling followed by immunoprecipitation demonstrated that C/EBP is a rapidly turning over protein in adipocytes and that cytokine treatment led to a specific, time dependent decrease in its rate of synthesis. Because C/EBP binding sites have been shown to play an important role in regulating the expression of genes involved in adipocyte metabolism, we propose that the TNF-induced changes in the complement of transcription factors binding those sites may be important in the pathogenesis of inflammation-induced atrophy of adipose tissue.
3T3 Cells↗
Insulinotropic hormone glucagon-like peptide-I(7-37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma beta TC-1 cells.
Glucagon-like peptide-I(7-37) [GLP-I(7-37)] is an intestinal peptide hormone that is released in response to oral nutrients and that potently augments glucose-mediated insulin secretion. GLP-I(7-37) has potent insulin-releasing activities in vivo in response to oral nutrients, in situ in the isolated perfused pancreas, and in vitro in cultured pancreatic B-cells. As such GLP-I(7-37) is a potent hormonal mediator in the enteroinsular axis involved in the regulation of glucose homeostasis. We now show that in addition to stimulating the release of insulin, GLP-I(7-37) stimulates proinsulin gene expression at the levels of gene transcription and cellular levels of proinsulin messenger RNA as well as the translational biosynthesis of proinsulin. These findings of the positive anabolic actions of GLP-I(7-37) on the synthesis of insulin in B-cells support the notion that GLP-I(7-37) may be of therapeutic use in stimulating the production of insulin in patients with noninsulin-dependent diabetes mellitus and that overproduction of insulin with subsequent hypoglycemia will not occur in response to the administration of GLP-I(7-37). Furthermore, these positive actions of GLP-I(7-37) on insulin production obviate the possibility of B-cell exhaustion in response to such a potent secretagogue.
Animals↗
Galanin inhibits proinsulin gene expression stimulated by the insulinotropic hormone glucagon-like peptide-I(7-37) in mouse insulinoma beta TC-1 cells.
The neuropeptide hormone galanin, released by sympathetic stimulation of nerve terminals in the endocrine pancreas, inhibits insulin secretion via a receptor-linked pertussis toxin-sensitive (Gi) transmembrane signaling pathway. Glucagon-like peptide-I(7-37) [GLP-I(7-37)] is an intestinal hormone shown to have potent insulin-releasing activities in pancreatic B-cells and is believed to serve a physiological role in the augmentation of nutrient-induced insulin release. GLP-I(7-37) binds to specific Gs- and adenylate cyclase-coupled receptors on pancreatic B-cells and directly stimulates proinsulin gene transcription, thereby increasing cellular levels of proinsulin messenger RNA (mRNA) and proinsulin biosynthesis. This study examines the effects of galanin on GLP-I(7-37)-stimulated proinsulin gene expression in mouse beta TC1 cells. The degree of proinsulin gene transcription was assessed by measuring the activity of chloramphenicol acetyl transferase (CAT) expressed from a CAT reporter plasmid linked to the rat insulin-1 gene promoter transferred to beta TC1 cells and by measuring proinsulin mRNA levels by Northern blot analysis. Galanin inhibited both CAT activity and the rise in proinsulin mRNA levels stimulated by either GLP-I(7-37) or forskolin (0.1 microM). Notably, galanin was without effect on CAT activity induced by the cAMP analog, 8-bromo-cAMP, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or higher concentrations of forskolin. The inhibitory effects of galanin on GLP-I(7-37) and forskolin-induced CAT activity were reversed by the addition of pertussis toxin, a toxin that inactivates inhibitory G-proteins (Gi). We conclude that galanin inhibits GLP-I(7-37)-stimulated proinsulin gene expression by inhibiting the activation of adenylate cyclase by GLP-I(7-37) and subsequently the production of cAMP in B-cells. Further, our data suggest that these actions of galanin are mediated by a pertussis toxin sensitive pathway involving one or more Gis that inhibit adenylate cyclase. Thus, in addition to its well known inhibitory effects on insulin secretion galanin can inhibit proinsulin gene expression stimulated by GLP-I(7-37) activation of the cAMP signaling pathway. These findings may be a unique demonstration of the inhibition of proinsulin gene expression by a substance (galanin) released endogenously within the pancreas.
Adenylate Cyclase Toxin↗
Novel testis germ cell-specific transcript of the CREB gene contains an alternatively spliced exon with multiple in-frame stop codons.
The gene encoding the cAMP-responsive transcription factor CREB consists of multiple small exons some of which undergo alternative RNA splicing. We describe the finding of a novel transcript of the CREB gene expressed at high levels in the germ cells of the rat testis. The transcript contains an alternatively spliced exon inserted within the sequence encoding the transcriptional transactivation domain of CREB and this exon contains multiple in-frame stop codons. Furthermore, the exon is conserved in both rat and human genes (75% nucleotide identity). Although the function(s) of this RNA or the truncated CREB protein predicted to result from the translation of this unusual transcript is unknown, the high level of expression in the testicular germ cells and remarkable conservation of sequences in rat and human suggests that it may have a unique biological function in these cells.
Animals↗
Insulinotropic action of glucagonlike peptide-I-(7-37) in diabetic and nondiabetic subjects.
OBJECTIVE: Whether glucagonlike peptide-I-(7-37) (GLP-I-[7-37]), a naturally occurring intestinal peptide, is insulinotropic in nondiabetic and non-insulin-dependent (type II) diabetic subjects. RESEARCH DESIGN AND METHODS: GLP-I-(7-37) or saline placebo was infused (1-5 ng.kg-1.min-1 for 30 min) in 4 nondiabetic and 11 type II diabetic subjects in the fasting and prandial state. Glucose, insulin, and GLP-I-(7-37) levels were measured. RESULTS: GLP-I-(7-37) infusion resulted in a 3- to 10-fold increase in peak insulin levels and in insulin area under the curve in nondiabetic and diabetic subjects. In diabetic subjects, infusion concurrent with a standard meal eliminated the postprandial glucose excursion for 60 min after the meal. Insulin-releasing potency of GLP-I-(7-37) was attenuated at decreased glucose levels. CONCLUSIONS: GLP-I-(7-37) has potent insulinotropic effects in nondiabetic and diabetic subjects. Whether GLP-I-(7-37) is useful as a therapeutic medication in type II diabetes requires further investigation.
Adult↗
Induction of glucose competence in pancreatic beta cells by glucagon-like peptide-1(7-37).
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Animals↗
Absence of insulinotropic glucagon-like peptide-I(7-37) receptors on isolated rat liver hepatocytes.
The effects of glucagon and the glucagon-like peptide GLP-1(7-37) were compared in rat liver hepatocytes. Glucagon elevated cAMP, elevated intracellular free calcium ([Ca2+]i), activated phosphorylase and stimulated gluconeogenesis, whereas GLP-1(7-37) was without effect on any of these parameters. GLP-1(7-37) did not block any of the actions of glucagon. The glucagon analog, des His1[Glu9] glucagon amide, was a partial agonist in liver, but also was an effective antagonist of glucagon actions in liver but not those of GLP-1(7-37) in islet B cells. It was concluded that in the rat, GLP-1(7-37) is a potent insulin secretagogue [1] but is without effect on liver.
Animals↗
Functional receptors for the insulinotropic hormone glucagon-like peptide-I(7-37) on a somatostatin secreting cell line.
Glucagon-like peptide-I(7-37) [(GLP-I(7-37)] is an intestinal peptide hormone that has potent insulinotropic activities in vivo in response to oral nutrients, in the isolated perfused pancreas, and in vitro in cultured B cells. GLP-I(7-37) receptor binding and GLP-I(7-37)-induced cAMP generation and hormone secretion was studied using cell lines producing insulin/B cell (beta TC-1), glucagon/A cell (INR1G9) and somatostatin/D cell (RIN 1027-B2). [125I]GLP-I(7-37) bound specifically to both B and D cells but not to A cells. GLP-I(7-37) induced cAMP-formation in B and D cells with a maximum response at 10 nmol/l (B cells) or at 100 nmol/l (D cells). Insulin secretion from perifused B cells was stimulated by GLP-I(7-37) (maximum at 10 nmol/l) and 10 nmol/l GLP-I(7-37) released somatostatin from perifused D cells. GLP-I(7-37) did not influence cAMP or glucagon secretion from A cells. These data indicate that pancreatic B and D cells, but not the A cells are influenced directly by GLP-I(7-37) via binding to specific receptors. Our findings support a model of physiologic regulation of insulin secretion whereby GLP-I(7-37) released from the intestine in response to oral nutrients potently stimulates insulin secretion via an endocrine mechanism that in turn may be dampened by a feed-back suppression by the release of somatostatin. In addition, suppression of the secretion of glucagon, a hormone whose actions are counter-regulatory to those of insulin, may occur by paracrine mechanisms involving GLP-I(7-37)-mediated stimulation of both insulin and somatostatin secretion.
Animals↗
Angiotensinogen gene-inducible enhancer-binding protein 1, a member of a new family of large nuclear proteins that recognize nuclear factor kappa B-binding sites through a zinc finger motif.
Transcriptional activation of the rat angiotensinogen gene during the acute-phase response is dependent on a previously characterized acute-phase response element (APRE) that binds at least two types of nuclear proteins: a cytokine-inducible activity indistinguishable from nuclear factor kappa-B (NF kappa B) and a family of C/EBP-like proteins. We screened a rat liver cDNA expression library with a labeled APRE DNA probe and isolated a single clone that encodes a sequence-specific APRE-binding protein. This new protein, the angiotensinogen gene-inducible enhancer-binding protein 1 (AGIE-BP1), is encoded by a large continuous open reading frame and contains a zinc finger motif virtually identical to the DNA-binding domain of a recently described human protein, MBP-1/PRDII-BF1, and a homologous mouse protein, alpha A-CRYBP1. Outside the binding domain, the sequences diverged considerably. Southern blot analysis indicated that AGIE-BP1 and alpha A-CRYBP1 are encoded by separate genes, thus defining a new family of DNA-binding proteins. Electrophoretic mobility shift assays, methylation interference, and DNase I footprint protection assays with the bacterially expressed DNA-binding domain of AGIE-BP1 demonstrated a binding specificity indistinguishable from that of purified NF kappa B. Antiserum raised against the bacterially expressed DNA-binding domain of AGIE-BP1 detected on immunoblots of cellular proteins a large (greater than 250-kDa) nuclear protein. Northern (RNA) blot analysis of RNAs from different rat tissues and cell lines indicated different levels of expression of the large (greater than 10-kb) AGIE-BP1 transcript in different tissues. The potential role of AGIE-BP1 in the regulation of gene expression is discussed.
Amino Acid Sequence↗
Homologous desensitization of the insulinotropic glucagon-like peptide-I (7-37) receptor on insulinoma (HIT-T15) cells.
Glucagon-like peptide-I(7-37) [GLP-I(7-37)] is an intestinal peptide with potent insulinotropic activities on pancreatic beta-cells in vivo and in vitro. In earlier studies elevated concentrations GLP-I(7-37) inhibited insulin release and cAMP generation in beta-cells. We now show that the GLP-I(7-37) receptor in the glucose-responsive B-cell line HIT-T15 undergoes rapid and reversible homologous desensitization in response to supraphysiological concentrations of GLP-I(7-37). GLP-I(7-37) stimulated insulin release and cAMP generation in a glucose-dependent biphasic manner with a maximum stimulation at 10 nmol/liter. The first-phase insulin secretory response was reduced by 41% at doses of GLP-I(7-37) of 100 nmol/liter and higher. Preperifusion of B-cells with 100 nmol/liter GLP-I(7-37) for 5 or 10 min reduced a subsequent insulin secretory response to 10 nmol/liter GLP-I(7-37) after hormone washout and recovery periods of 10 min (52% and 55% reduction) or 30 min (33% reduction or full recovery). Preperifusion of HIT-T15 cells with 100 nmol/liter glucagon (10 min) or 100 nmol/liter gastric inhibitory peptide (GIP) (10 min) had no effect on the insulin secretory response to 10 nmol/liter GLP-(7-37). Prior exposure of cells to 100 nmol/liter GLP-(7-37) (10 min) did not alter the GIP-induced (10 nmol/liter) insulin release, but 100 nmol/liter GIP (10 min) reduced the insulin secretion during stimulation with 10 nmol/liter GIP by 56%. These data indicate that: 1) the GLP-I(7-37) receptor is subject to rapid and reversible homologous desensitization and, 2) the GLP-I(7-37) receptor on beta-cells is distinct from that of GIP. The recent finding of elevated GLP-I(7-36)amide levels in subjects with noninsulin-dependent diabetes suggest the possibility that a homologous desensitization of the GLP-I(7-37) receptor might contribute to the impaired insulin secretion in this disorder.
Cyclic AMP↗
Developmental stage-specific expression of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing.
Spermatogenesis is a temporally regulated developmental process by which the gonadotropin-responsive somatic Sertoli and Leydig cells act interdependently to direct the maturation of the germinal cells. The metabolism of Sertoli and Leydig cells is regulated by the pituitary gonadotropins FSH and LH, which, in turn, activate adenylate cyclase. Because the cAMP-second messenger pathway is activated by FSH and LH, we postulated that the cAMP-responsive element-binding protein (CREB) plays a physiological role in Sertoli and Leydig cells, respectively. Immunocytochemical analyses of rat testicular sections show a remarkably high expression of CREB in the haploid round spermatids and, to some extent, in pachytene spermatocytes and Sertoli cells. Although most of the CREB antigen is detected in the nuclei, some CREB antigen is also present in the cytoplasm. Remarkably, the cytoplasmic CREB results from the translation of a unique alternatively spliced transcript of the CREB gene that incorporates an exon containing multiple stop codons inserted immediately up-stream of the exons encoding the DNA-binding domain of CREB. Thus, the RNA containing the alternatively spliced exon encodes a truncated transcriptional transactivator protein lacking both the DNA-binding domain and nuclear translocation signal of CREB. Most of the CREB transcripts detected in the germinal cells contain the alternatively spliced exon, suggesting a function of the exon to modulate the synthesis of CREB. In the Sertoli cells we observed a striking cyclical (12-day periodicity) increase in the levels of CREB mRNA that coincides with the splicing out of the restrictive exon containing the stop codons. Because earlier studies established that FSH-stimulated cAMP levels in Sertoli cells are also cyclical, and the CREB gene promoter contains cAMP-responsive enhancers, we suggest that the alternative RNA splicing controls a positive autoregulation of CREB gene expression mediated by cAMP.
Animals↗
Nuclear translocation and DNA recognition signals colocalized within the bZIP domain of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB.
CREB is a cAMP-responsive nuclear DNA-binding protein that binds to cAMP response elements and stimulates gene transcription upon activation of the cAMP signalling pathway. The protein consists of an amino-terminal transcriptional transactivation domain and a carboxyl-terminal DNA-binding domain (bZIP domain) comprised of a basic region and a leucine zipper involved in DNA recognition and dimerization, respectively. Recently, we discovered a testis-specific transcript of CREB that contains an alternatively spliced exon encoding multiple stop codons. CREB encoded by this transcript is a truncated protein lacking the bZIP domain. We postulated that the antigen detected by CREB antiserum in the cytoplasm of germinal cells is the truncated CREB that must also lack its nuclear translocation signal (NTS). To test this hypothesis we prepared multiple expression plasmids encoding carboxyl-terminal deletions of CREB and transiently expressed them in COS-1 cells. By Western immunoblot analysis as well as immunocytochemistry of transfected cells, we show that CREB proteins truncated to amino acid 286 or shorter are sequestered in the cytoplasm, whereas a CREB of 295 amino acids is translocated into the nucleus. Chimeric CREBs containing a heterologous NTS fused to the first 248 or 261 amino acids of CREB are able to drive the translocation of the protein into the nucleus. Thus, the nine amino acids in the basic region involved in DNA recognition between positions 287 and 295 (RRKKKEYVK) of CREB contain the NTS. Further, mutation of the lysine at position 290 in CREB to an asparagine diminishes nuclear translocation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
The pancreatic islet-specific glucagon G3 transcription factors recognize control elements in the rat somatostatin and insulin-I genes.
The rat glucagon gene 5'-flanking region contains a pancreatic islet-specific enhancer-like element, G3. It has been shown previously that G3-binding and transactivating proteins are present in islet cell lines expressing the glucagon, somatostatin, and insulin genes, but not in several nonislet cell lines. The present study now shows that the glucagon G3 transcription factor binds to DNA sequences within cis-acting elements of the rat somatostatin and rat insulin-I genes that have been defined by others as pancreatic islet-specific transcriptional enhancers. In addition, when fused to glucagon or somatostatin minimal promoters in reporter plasmids, these enhancer elements of the three islet hormone-producing genes functionally activate transcription when transfected into islet cell lines producing glucagon, insulin, or somatostatin. The enhancer elements of the three different islet polypeptide hormone genes define a potential consensus motif that binds islet cell type-specific transcription factors.
Animals↗