Biomedical subjects

D J Drucker

Publications and source records attributed to D J Drucker.

At least 91 records · Page 5Linked to original sources

PubMed1994

Molecular pathophysiology of glucagon-SV40 T antigen transgenic mice.

The gene encoding proglucagon is expressed in the pancreas, intestine, and brain. The molecular determinants of proglucagon gene expression and the biological activities of the proglucagon-derived peptides (PGDPs) have been examined using transgenic mice harboring a glucagon-SV40 large T antigen (GLUTag) transgene. These experiments have delineated DNA sequences important for intestinal-specific proglucagon gene transcription. GLUTag mice develop neuroendocrine tumors of the pancreas and large bowel, leading to elevated plasma levels of the PGDPs and suppression of endogenous proglucagon gene expression. Transplantation of the large bowel tumors subcutaneously into nude mice provides additional evidence for inhibition of endogenous pancreatic proglucagon gene expression by one or more of the tumor-derived PGDPs. The large bowel GLUTag tumors exhibit abnormalities in the posttranslational processing of proglucagon. GLUTag tumors may be passaged in vivo and in vitro and have been used to generate stable cell lines that express the proglucagon gene at high levels. Taken together, these studies highlight the utility of transgenic systems for the physiological analysis of hormone action and the molecular determinants of peptide hormone gene expression.

Animals↗

PubMed1994

Inhibition of pancreatic proglucagon gene expression in mice bearing subcutaneous endocrine tumors.

The proglucagon-derived peptides (PGDPs) play key roles in the regulation of carbohydrate metabolism and insulin secretion, yet the factors important for the regulation of proglucagon gene expression remain poorly understood. Recent experiments demonstrated that nude mice carrying subcutaneous proglucagon-SV40 T antigen tumors contained markedly reduced levels of the pancreatic PGDPs. To determine if elevated circulating levels of the PGDPs are consistently associated with inhibition of endogenous pancreatic proglucagon gene expression, we have now studied islet hormone gene expression in mice bearing three different endocrine tumors (InR1-G9, RIN1056A, and STC-1) that express the proglucagon gene. All tumors synthesized large amounts of the PGDPs. Plasma levels of the PGDPs were elevated in all tumor-bearing mice (8- to 22-fold greater than controls, P < 0.001), and this was associated with marked inhibition of mouse pancreatic proglucagon gene expression. The levels of pancreatic PGDPs were also significantly lower in tumor-bearing mice compared with controls (71 +/- 4, 38 +/- 6, and 18 +/- 2% of controls for InR1-G9-, RIN1056A-, and STC-1-bearing mice, respectively, P < 0.05-0.001). This inhibition of proglucagon gene expression was highly specific, in that no consistent change in pancreatic insulin or somatostatin gene expression was detected. Examination of proglucagon posttranslational processing in the tumors demonstrated that proglucagon was processed differently in each tumor to yield a unique profile of PGDPs. These observations demonstrate that elevated circulating levels of the PGDPs are associated with reduction in islet size and inhibition of proglucagon gene expression and PGDP synthesis in the pancreatic A cell.

Animals↗

PubMed1994

The LIM domain homeobox gene isl-1: conservation of human, hamster, and rat complementary deoxyribonucleic acid sequences and expression in cell types of nonneuroendocrine lineage.

The LIM domain homeobox gene isl-1 was originally isolated by virtue of its ability to bind DNA sequences from the 5'-flanking region of the rat insulin gene. Initial experiments localized isl-1 to the islet cells of the pancreas, suggesting a possible role for this protein in the regulation of insulin gene expression and/or islet cell development. More recent studies have determined that isl-1 is also expressed in the central and peripheral nervous system. We now report that isl-1 gene expression is not restricted to cells of neuroendocrine lineage. Northern blot analysis of rat and hamster fibroblasts, rat keratinocytes, and human epidermoid carcinoma cells detected messenger RNA transcripts that hybridized, at high stringency, to different isl-1 complementary DNA probes. DNA sequence analysis of human and hamster isl-1 complementary DNAs generated by reverse transcription-polymerase chain reaction from nonneuroendocrine cell lines demonstrated 100% conservation of isl-1 amino acid sequences from rat hamster and human species. Western blot analysis with isl-1 antiserum demonstrated that immunoreactive isl-1 was detectable in nuclear extracts from islet cell lines. In contrast, immunoreactive isl-1 was only detected in cytoplasmic extracts from nonneuroendocrine cell lines. The results of these studies demonstrate striking conservation of mammalian isl-1 coding sequences and widespread expression of the isl-1 gene in heterologous cells. These experiments suggest that the biological importance of isl-1 may not be restricted to neuroendocrine cell lineages and raise the possibility that isl-1 function may be regulated by sequestration in distinct cellular compartments.

Amino Acid Sequence↗

PubMed1994

Divergent tissue-specific and developmental expression of receptors for glucagon and glucagon-like peptide-1 in the mouse.

Proglucagon mRNA transcripts are transcribed in the pancreas, bowel, and brain, after which posttranslational processing results in the liberation of a different profile of biologically active peptides in each tissue. The receptors for two of these peptides, glucagon and glucagon-like peptide-1 (GLP-1), have recently been identified, but only limited information is available concerning the tissue- and age-specific distribution of these receptors in vivo. We have investigated the expression of these receptors in the mouse using a combination of Northern blot analysis and reverse transcription-polymerase chain reaction. DNA sequence analysis of a partial mouse glucagon receptor cDNA demonstrated a high degree of sequence conservation across rodent species. Glucagon receptor mRNA transcripts were detectable by Northern blotting in poly(A)+ RNA from liver and kidney. Reverse transcription-polymerase chain reaction also detected glucagon receptor mRNA transcripts in both fetal and adult pancreas and lung, jejunum, and ileum, but not in the large intestine. In contrast, mRNA transcripts for the GLP-1 receptor were detected in both small and large intestine as well as in pancreas, liver, lung, and kidney. Both glucagon and GLP-1 receptor mRNA transcripts were identified in different regions of the fetal and adult mouse brain, but the relative levels of GLP-1 receptor mRNA transcripts were much greater in the central nervous system. Furthermore, regulation of the GLP-1 receptor (but not the glucagon receptor) gene in the brain resembled the pattern of region-specific gene expression recently defined for the mouse proglucagon gene. Taken together, these studies define novel sites for both glucagon and GLP-1 receptor gene expression in the mouse and suggest that different regulatory mechanisms have evolved for tissue-specific and developmental control of receptor gene expression.

Aging↗

PubMed1994

Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line.

The gene encoding proglucagon is expressed predominantly in the pancreas and intestine. The physiological importance of glucagon secreted from the islets of Langerhans has engendered considerable interest in the molecular control of proglucagon gene transcription in the endocrine pancreas. In contrast, little is known about the molecular control of proglucagon gene expression in the intestine. The recent demonstration that glucagon-like peptide-1 (GLP-1) secreted from the intestine is a potent regulator of insulin secretion and glucose homeostasis has stimulated renewed interest in the factors that control GLP-1 synthesis in the intestinal L-cell. To develop a model for the analysis of intestinal proglucagon gene expression, we have targeted expression of a proglucagon gene-simian virus-40 large T-antigen fusion gene to enteroendocrine cells in transgenic mice. These mice develop intestinal tumors that were used to derive a novel cell line, designated GLUTag, that expresses the proglucagon gene and secretes immunoreactive GLP-1 in vitro. GLUTag cells demonstrate morphological characteristics of enteroendocrine cells by electron microscopy and are plurihormonal, as shown by immunocytochemistry and RNA analyses. GLUTag cells express the proglucagon and cholecystokinin genes, consistent with the pattern of lineage-specific enteroendocrine differentiation described for mouse intestine. Proglucagon gene expression was induced by activators of the protein kinase-A pathway, and a combination of messenger RNA half-life and nuclear run-on experiments demonstrated that the protein kinase-A-induction is mediated by an increase in proglucagon gene transcription. In contrast, activators of protein kinase-C stimulated secretion, but not biosynthesis of the PGDPs in GLUTag cell cultures. Analysis of proglucagon processing in GLUTag cells demonstrated the liberation of glucagon, oxyntomodulin, glicentin, and multiple forms of GLP-1. These observations provide evidence for the direct induction of proglucagon gene transcription by a cAMP-dependent pathway and suggest that the GLUTag cell line represents a useful model for the analysis of the molecular determinants of enteroendocrine gene expression.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Differential expression of RNA transcripts encoding unique carboxy-terminal sequences of human parathyroid hormone-related peptide.

The human gene encoding PTH-related peptide (hPTHrP) is a complex transcriptional unit that gives rise to multiple messenger RNA (mRNA) transcripts in normal and neoplastic tissues. The utilization of three different transcription start sites and alternative splicing of exons encoding 5'-untranslated sequences accounts for some of the heterogeneity in hPTHrP gene expression; however, the pattern and potential significance of alternative exon splicing at the 3'-end of the hPTHrP gene have not been systematically examined. We have used exon-specific primers and reverse transciptase-polymerase chain reaction to analyze hPTHrP gene expression in nonneoplastic human tissues and human tumors. PTHrP mRNA transcripts encoding PTHrP-(1-139), -(1-173), or -(1-141) were identified in the majority of tissues analyzed. PTHrP mRNAs containing either exon 6 [PTHrP-(1-139)] or exon 8 [PTHrP-(1-141)] sequences were considerably more abundant than mRNAs encoding PTHrP-(1-173) (exon 7) in nonneoplastic tissues. In contrast, a switch in the profile of the different hPTHrP mRNAs was detected in some neoplastic tissues, with an increase in mRNA transcripts encoding PTHrP-(1-173) and a decrease in mRNA transcripts encoding PTHrP-(1-141) observed in several tumors. The differential properties of specific PTHrP 3'-untranslated sequences encoded by unique exons were examined by creating a series of luciferase-PTHrP fusion genes and examining the contribution of PTHrP sequences to relative luciferase activity after transfection of heterologous cell lines. LUC-PTHrP fusion genes containing exon 6 or exon 8 sequences generated consistently higher luciferase activity than fusion genes containing sequences from exon 7. Furthermore, distinct differences in the relative profiles of luciferase activities were observed after transfection of several human, but not rodent, cell lines. These studies suggest that exon splicing at the 3'-end of the hPTHrP gene may be an important determinant of the biological complexity of hPTHrP gene expression.

Alternative Splicing↗

PubMed1993

Growth factor-like properties of parathyroid hormone-related peptide in transfected rodent cell line.

Parathyroid hormone-related peptide (PTHrP) was originally isolated from tumors associated with the development of hypercalcemia in vivo. Analyses of PTHrP gene expression have demonstrated that PTHrP is also produced in a wide variety of normal fetal and adult nonneoplastic tissues. The results of recent experiments have demonstrated that PTHrP is a growth factor-regulated gene, and different molecular forms of synthetic PTHrP display variable activities in assays for growth factor-like properties in vitro. We have studied the growth factor-like activity of PTHrP in cells transfected with a human PTHrP (hPTHrP) expression vector. Transfected cell lines contained increased amounts of PTHrP mRNA transcripts as assessed by Northern blot analysis. The PTHrP mRNA transcripts were translated into immunoreactive hPTHrP as measured by radioimmunoassay, and conditioned medium from transfected cell lines stimulated cyclic AMP (cAMP) formation in ROS 17/2.8 osteosarcoma cells. The transforming growth factor-beta-like properties of hPTHrP-producing NRK 49F clones were examined using the large-colony transformation assay in soft agar. PTHrP-producing NRK 49F clones did not form large colonies in the presence of epidermal growth factor. In contrast, PTHrP-producing and wild-type NRK 49F cells formed large colonies in the presence of epidermal growth factor and transforming growth factor-beta. No effect on cell growth was observed in PTHrP-producing NRK 49F rat kidney fibroblasts or mouse NIH 3T3 fibroblasts. In contrast, RCB 2.2 osteoblast cells expressing the hPTHrP cDNA were growth inhibited. Incubation of wild-type RCB 2.2 cells with synthetic hPTHrP[1-34] (at concentrations of 1.0-10.0 nM) also produced growth inhibition. PTHrP increased cAMP formation in RCB 2.2 cells but not in NIH 3T3 or NRK 49F cells. Incubation of RCB 2.2 cells with dibutyryl cAMP was also associated with inhibition of cell growth. The results of these studies demonstrate that PTHrP may function as an autocrine growth inhibitor of specific cell types, possibly through a cAMP-dependent pathway.

3T3 Cells↗

PubMed1993

Reactive human bile ductules express parathyroid hormone-related peptide.

Various cholestatic liver diseases as well as regeneration after submassive necrosis are accompanied by a striking increase in the number of bile ductules. These reactive bile ductules are thought to arise either from proliferation of pre-existing bile ductules or bile ductule-related facultative stem cells, or from ductular metaplasia of hepatocytes. Recently, we found that reactive bile ductules display neuro-endocrine features, and speculated that the substance(s), produced in the neuro-endocrine granules, might play a role in their growth and/or differentiation through an autocrine or paracrine pathway. Parathyroid hormone-related peptide has been shown to be encoded by a growth factor-regulated gene that may play a role in cell growth and differentiation. We studied the immunohistochemical expression of this peptide in human liver, including three normal biopsies, 11 cases of cholestatic liver disease, six cases of focal nodular hyperplasia and three cases of regenerating liver. In regenerating liver, primary biliary cirrhosis, primary sclerosing cholangitis and partial or intermittent obstruction, the majority of reactive ductular cells expressing neuro-endocrine markers also expressed parathyroid hormone-related peptide. In focal nodular hyperplasia, a smaller number of bile ductular cells expressed the peptide. These findings suggest that parathyroid hormone-related peptide is localized in bile ductular cells and may indicate a role for this hormone in the growth and/or differentiation of human reactive bile ductules.

Bile Ducts↗

PubMed1993

Parathyroid hormone-related peptide expression in primary and metastatic liver tumours.

Parathyroid hormone-related peptide (PTHrP) is a major factor in the pathophysiology of hypercalcaemia of malignancy. Recent evidence suggests that PTHrP may play an important role in the growth and differentiation of neoplastic as well as non-neoplastic cells. PTHrP was originally detected in normal fetal, but not adult, liver. We have used immunocytochemistry to show that reactive human bile ductules expressing a neuroendocrine phenotype contain immunoreactive PTHrP. These observations raised the possibility that PTHrP immunoreactivity may be useful in the differential diagnosis of primary liver tumours and metastases of adenocarcinoma. A total of 24 primary liver tumours and 22 metastases of adenocarcinoma were studied. All cholangiocarcinomas showed immunopositivity for PTHrP and chromogranin A, while all hepatocellular carcinomas were negative for PTHrP and showed only focal and weak positivity for chromogranin A. Mixed types of primary liver tumour contained PTHrP immunoreactivity only in the areas of cholangiocellular differentiation. Moreover, all metastatic adenocarcinomas were negative for PTHrP and chromogranin A except for two out of five metastatic breast adenocarcinomas. These two patients had bone metastases and hypercalcaemia and thus did not yield differential diagnostic problems with cholangiocarcinoma. None of the patients with cholangiocarcinoma and hepatocellular carcinoma had hypercalcaemia. We conclude that PTHrP is a useful marker for primary cholangiocarcinoma, especially in the differential diagnosis of hepatocellular carcinoma and metastatic adenocarcinoma.

Adenocarcinoma↗

PubMed1993

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.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Region- and age-specific differences in proglucagon gene expression in the central nervous system of wild-type and glucagon-simian virus-40 T-antigen transgenic mice.

The gene encoding proglucagon is expressed in the A-cells of the endocrine pancreas and the L-cells of the gastrointestinal tract. Proglucagon-derived peptides have also been detected in different regions of the central nervous system; however, proglucagon mRNA transcripts have been localized predominantly to the brainstem and hypothalamus. We have studied proglucagon gene expression in the brain of mice using the reverse transcription-polymerase chain reaction technique. Proglucagon mRNA transcripts were detected in the cortex, cerebellum, and brainstem of embryonic day 19 mouse brain and in the cortex, cerebellum, brainstem, and hypothalamus of 2- to 12-week-old wild-type mice. Age-specific changes in proglucagon gene expression were observed, with a progressive decrease in the levels of proglucagon mRNA transcripts detected in the cortex of older mice. In contrast, a progressive increase in the levels of proglucagon mRNA transcripts was detected with increasing age in the brainstem. Proglucagon gene expression was also examined in the brain of glucagon-simian virus-40 (SV40) T-antigen transgenic mice. The levels of proglucagon gene expression in transgenic cerebellum and brainstem were relatively greater than those in wild-type mice on embryonic day 19, and increased levels of proglucagon mRNA transcripts were observed in transgenic cerebellum, brainstem, and hypothalamus at 2 weeks. In contrast, the levels of proglucagon mRNA transcripts were markedly reduced in the brain of 5-week-old transgenic mice. A reduction in the levels of SV40 T-antigen mRNA transcripts was also detected in the brain of transgenic mice at 5 weeks, but no suppression of the mRNA transcripts for the neuropeptide somatostatin was observed in 5-week-old transgenic brain. The results of these studies suggest that proglucagon mRNA transcripts are more widely distributed in the mouse central nervous system than previously demonstrated and that proglucagon gene expression in the brain appears to be regulated in a region- and age-specific manner. The coordinate region-specific expression and regulation of mRNA transcripts derived from the endogenous mouse proglucagon gene and a glucagon-SV40 T antigen transgene in the central nervous system of mice harboring a transgene containing 2.0 kilobases of rat proglucagon gene 5'-flanking sequences fused to the coding sequence of SV-4 large T-antigen suggest that neural-specific elements residing within the first 2.0 kilobases of glucagon gene 5'-flanking sequences are sufficient for the correct targeting and regulation of proglucagon gene expression in the brain.

Aging↗

PubMed1992

Alterations in proglucagon processing and inhibition of proglucagon gene expression in transgenic mice which contain a chimeric proglucagon-SV40 T antigen gene.

The proglucagon gene is expressed in A cells of the pancreas and L cells of the large and small intestine. Transgenic mice expressing SV40 large T antigen under the control of proglucagon regulatory sequences develop neuroendocrine carcinoma of the large intestine. To determine the consequences of coexpression of SV40 large T antigen and proglucagon in different cell types, the levels of proglucagon mRNA transcripts and proglucagon-derived peptides were determined in tumor-bearing transgenic mice and in age-matched paired controls. Plasma levels of proglucagon-derived peptides (glicentin, oxyntomodulin, and glucagon, as determined by high pressure liquid chromatography and radioimmunoassay) were markedly elevated in association with tumor growth (p < 0.001). Northern blot analysis demonstrated that the increased concentration of proglucagon-derived peptides was associated with significant inhibition of the endogenous proglucagon gene in pancreas, and to a lesser extent, small intestine. Concomitantly, the concentrations of proglucagon-derived peptides fell to 1-10% of control values in pancreas (p < 0.001) and to 62% of control values in small intestine (p < 0.001). Analysis of proglucagon-derived peptides in mice of different ages demonstrated that tumor growth was associated with a switch in the post-translational processing of proglucagon. Compared with normal mouse intestine, tumors contained increased proportions of glucagon and glucagon-like peptide-1(7-37) relative to glicentin, oxyntomodulin, and glucagon-like peptide-1(1-37). The results of these studies provide evidence for humorally-mediated tissue-specific inhibition of proglucagon gene expression.

Animals↗

PubMed1992

Parathyroid hormone-like peptide shares features with members of the early response gene family: rapid induction by serum, growth factors, and cycloheximide.

The parathyroid hormone-like peptide (PLP) gene is widely expressed in normal and neoplastic tissues. Previous studies have demonstrated that PLP gene expression is regulated by serum and cycloheximide, features common to the regulation of a number of different early response genes. We now report that PLP mRNA transcripts are induced within 5 min of exposure of rat keratinocytes to serum, return to control values at 20 min, and then increase and remain elevated for at least 4 h, following which they return to baseline levels. The PLP mRNA t1/2 was approximately 90 min in both serum-deprived and serum-stimulated cells. The serum induction was blocked by actinomycin D. Cycloheximide alone induced PLP gene expression; however, PLP mRNA transcripts were not superinduced in the presence of both serum and cycloheximide. Dexamethasone and 1,25-dihydroxyvitamin D3 inhibited the basal levels of PLP mRNA transcripts but did not eliminate the serum induction of PLP gene expression. Epidermal growth factor or transforming growth factor-beta alone induced PLP mRNA transcripts, but no induction was observed following exposure of cells to epidermal growth factor and transforming growth factor-beta together. Treatment with 12-O-tetradecanoylphorbol-13-acetate for 90 min did not induce PLP mRNA transcripts, but 12-O-tetradecanoylphorbol-13-acetate blocked the rapid serum induction of PLP gene expression. These features of PLP gene expression suggest that PLP is a member of the growth factor-regulated early response gene family. The rapid serum stimulation of PLP gene expression raises the possibility that PLP may contribute in an autocrine fashion to the early cellular response to growth factor stimulation.

Animals↗

PubMed1992

Glucagon gene 5'-flanking sequences direct expression of simian virus 40 large T antigen to the intestine, producing carcinoma of the large bowel in transgenic mice.

Glucagon and the glucagon-like peptides play important roles in the regulation of glucose homeostasis. Previous studies have demonstrated that approximately 1300 base pairs of rat glucagon gene 5'-flanking sequences direct transgene expression to the pancreas and brain, but not to the intestine, of transgenic mice. These observations suggested that different tissue-specific enhancer elements mediate activation of glucagon gene transcription in the pancreas and intestine. We have now generated mice that express SV40 large T antigen under the control of approximately 2000 base pairs of glucagon gene 5'-flanking sequences. Transgene expression was observed in the brain and pancreas in association with the development of pancreatic endocrine tumors. In contrast to the mice described previously, we also detected transgene expression throughout the gastrointestinal tract in endocrine cells of the stomach and small and large intestine. Focal areas of enteroendocrine cell hyperplasia in the large bowel invariably progressed to invasive and metastasizing plurihormonal endocrine carcinoma, which was clinically and pathologically evident by 4 weeks of age. In contrast, transgene expression in the small bowel and stomach was not associated with progression to either hyperplasia or carcinoma. The results of these studies provide functional evidence for the existence of an upstream cis-acting regulatory domain that directs glucagon gene transcription to the endocrine cells of the intestine in transgenic mice.

Animals↗

PubMed1992

Interaction of TPA and ultraviolet B radiation in regulation of ODC gene expression in rat keratinocytes.

Ultraviolet B radiation (UVB) and phorbol esters are known to promote tumor formation in skin; however, the interaction between UVB and phorbol esters in the regulation of gene expression remains incompletely understood. To define the interaction of UVB and phorbol esters in the control of keratinocyte gene expression, we have studied the effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and UVB on the regulation of ornithine decarboxylase (ODC) gene expression in a rat keratinocyte cell line. Both UVB and TPA alone increased ODC activity and induced the expression of the ODC gene. The combination of UVB and TPA produced a further increment in ODC gene expression at 12 h, but UVB markedly attenuated the TPA induction of ODC mRNA transcripts at 3 h. Protein synthesis inhibition with cycloheximide also induced ODC mRNA transcripts, but did not eliminate the further induction of ODC gene expression by UVB or TPA. No changes in actin gene expression following exposure to TPA/UVB were detected in the same experiments. UVB and TPA alone or in combination had no effect on the transcriptional activity of an ODC-chloramphenicol acetyltransferase fusion gene in transfected rat keratinocytes. The results of these studies suggest a complex posttranscriptional interaction of phorbol esters and UVB in the control of keratinocyte gene expression.

Animals↗

PubMed1992

Regulation of parathyroid hormone-related peptide (PTHrP) gene transcription: cell- and tissue-specific promoter utilization mediated by multiple positive and negative cis-acting DNA elements.

The gene encoding PTH-related peptide (PTHrP) is expressed in a wide variety of normal and neoplastic tissues. Increased PTHrP gene expression in and secretion of PTHrP by specific tumors directly contributes to the development of malignancy-associated hypercalcemia in vivo. To define the genetic elements important for the control of PTHrP gene transcription, we used the reverse transcription polymerase chain reaction to delineate the control of promoter utilization and the splicing patterns of the exons encoding 5'-untranslated sequences. The majority of normal and neoplastic human tissues contained PTHrP mRNA transcripts initiating from both the up-stream (P1) and down-stream (P2) human PTHrP promoters. Furthermore, the downstream promoter was preferentially used by a factor of more than 30-fold. P1-initiated transcripts contained RNA species both with and without exon 2 (E2) sequences, except in the pancreas, adrenal, and stomach, where E2-containing sequences predominated. The transcriptional activities of P1, P2, and P1 + P2 were assessed by transfection of the corresponding PTHrP-chloramphenicol acetyltransferase (CAT) fusion genes into heterologous cell lines. Fusion genes containing P2 sequences were more transcriptionally active than fusion genes containing P1 sequences. The transcriptional activities of P1 + P2 in their natural tandem orientation were additive in rat keratinocytes and human JEG choriocarcinoma cells. In contrast, the activity of P1 + P2 was less than that of P2 alone in hamster BHK fibroblasts and InR1-G9 cells, and human HeLa cells. Analysis of the transcriptional properties of 5'-deleted human PTHrP-CAT constructs revealed the presence of multiple positive and negative DNA sequences (within both P1 and P2) functionally important for human PTHrP gene transcription. Distinct positive and negative DNA elements were also identified from analysis of 5'-deleted rat PTHrP-CAT fusion genes. The results of these experiments provide evidence for cell- and tissue-specific utilization of 1) distinct human PTHrP transcription start sites and specific patterns of 5'-exon splicing and 2) multiple positive and negative DNA control elements, important for the regulation of human and rat PTHrP gene transcription.

Animals↗

PubMed1992

Inhibition of pancreatic glucagon gene expression in mice bearing a subcutaneous glucagon-producing GLUTag transplantable tumor.

Transgenic mice that express a glucagon gene-simian virus-40 large T-antigen (GLUTag) fusion gene develop neuroendocrine carcinoma of the large bowel. This glucagon-producing tumor was implanted sc and reproducibly formed tumors in nude mice. The transplanted GLUTag tumor expressed large amounts of proglucagon mRNA transcripts, and the levels of proglucagon mRNA transcripts remained constant during 2-8 weeks of tumor growth. The posttranslational processing of proglucagon in the transplantable tumor resembled that detected in the original transgenic tumor, with the liberation of glicentin, oxyntomodulin, glucagon, glucagon-like peptide (1-37) [GLP-1-(1-37)] and GLP-1-(7-37). Tumor-bearing mice demonstrated progressive elevations in the plasma levels of proglucagon-derived peptides. Elevated plasma levels of glucagon-like immunoreactive peptides and immunoreactive glucagon were associated with a marked reduction in the levels of pancreatic glucagon mRNA transcripts by 4 weeks, and after 8 weeks of tumor growth, the levels of glucagon mRNA transcripts in the pancreas were not detectable by Northern blot analysis. Synthesis of the proglucagon-derived peptides was also significantly suppressed at 4-8 weeks in the pancreas of tumor-bearing animals. Histological examination of the endocrine pancreas in mice carrying the GLUTag tumor for 6-8 weeks demonstrated a marked reduction in the number and size of the islets of Langerhans and a disproportionately greater decrease in the number of cells exhibiting glucagon immunoreactivity. By electron microscopy, the residual A-cells were small, compressed at the periphery of the islets, and had poorly developed cytoplasmic organelles. In contrast, no changes in mouse glucagon gene expression or islet morphology were detected in control animals without tumors or mice carrying a sc v-jun-induced fibrosarcoma. The suppression of pancreatic A-cell function and islet size in mice with elevated plasma levels of the proglucagon-derived peptides raises the possibility that a proglucagon-derived peptide may participate in a negative feedback loop, inhibiting expression of the glucagon gene in the A-cells of the endocrine pancreas.

Animals↗

PubMed1991

Immunocytochemical localization of parathyroid hormone-like peptide in the rat fetus.

Studies of parathyroid hormone-like peptide (PLP) have demonstrated that PLP gene expression is inducible by serum, growth factors, and cycloheximide. Rapid induction of PLP gene expression has also been observed following the induction of cell differentiation. These features of PLP gene expression are consistent with a role for PLP in the regulation of cell growth and differentiation. To understand the biology of PLP in developing cells and tissues, we have studied the distribution of PLP gene expression in the fetal and neonatal rat. PLP was localized by immunocytochemistry to skin, vascular smooth muscle, skeletal muscle, heart, liver, kidney, lung, and gastrointestinal tract in Day 14 fetal rat. By Day 18 PLP immunopositivity was also detected in both fetal pituitary and adrenal medulla, as well as in endocrine pancreas. Whereas few PLP-immunopositive cells were detected in Day 14 brain, scattered areas of PLP immunopositivity were evident in Day 18 brain, in regions such as the choroid plexus. Immunostaining for PLP was also detected in Day 18 tissues that were positive on Day 14. The pattern of staining in fetal testis, where PLP was strongly localized to seminiferous tubules, differed from adult testis, where PLP was localized predominantly in Leydig cells. PLP was localized to the hepatocytes but not to the hematopoietic elements in fetal liver. Neonatal hepatocytes were weakly PLP immunopositive, and PLP was not detected in adult rat liver. Northern blot analysis demonstrated the presence of a single 1.4-kilobase PLP mRNA transcript in fetal brain, liver, heart, lung, and intestine. The results of these studies demonstrate that the PLP gene is widely expressed in a diverse number of fetal rat tissues. The cellular and tissue localization of PLP immunopositivity remains fairly constant in the transition from fetal to neonatal and adult tissues except in the testis, where a cellular switch in PLP-producing cells occurs, and the liver, where PLP gene expression is progressively extinguished postnatally.

Adolescent↗