RESUMEN
Regenerating gene product (Reg) is induced in pancreatic beta-cells and acts as an autocrine/paracrine growth factor for regeneration via a cell surface Reg receptor. However, the manner by which Reg induces beta-cell regeneration was unknown. In the present study, we found that Reg increased phospho-ATF-2, which binds to -57 to -52 of the cyclin D1 gene to activate the promoter. The Reg/ATF-2-induced cyclin D1 promoter activation was attenuated by PI(3)K inhibitors such as LY294002 and wortmannin. In Reg knockout mouse islets, the levels of phospho-ATF-2, cyclin D1, and phospho-Rb were greatly decreased. These results indicate that the Reg-Reg receptor system stimulates the PI(3)K/ATF-2/cyclin D1 signaling pathway to induce beta-cell regeneration.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Ciclina D1/metabolismo , Células Secretoras de Insulina/fisiología , Litostatina/metabolismo , Regeneración , Factor de Transcripción Activador 2/genética , Animales , Ciclina D1/genética , Genes Reporteros , Células Secretoras de Insulina/citología , Lectinas Tipo C/metabolismo , Litostatina/genética , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Cyclic ADP-ribose (cADPR) induces the release of Ca2+ from microsomes of pancreatic islets for insulin secretion. It has been demonstrated that cADPR binds to FK506-binding protein 12.6 (FKBP 12.6) on rat islet ryanodine receptor and that the binding of cADPR to FKBP12.6 frees the ryanodine receptor from FKBP12.6, causing it to release Ca2+ [Noguchi, N., Takasawa, S., Nata, K., Tohgo, A., Kato, I., Ikehata, F., Yonekura, H., Okamoto, H., 1997. Cyclic ADP-ribose binds to FK506-binding protein to release Ca2+ from islet microsomes. J. Biol. Chem. 272, 3133-3136.]. In this study, we cloned, characterized the structural organization of the human FKBP12.6, which is highly homologous to human FKBP12, and analyzed the promoters for FKBP12.6 and FKBP12. Human FKBP12.6 gene spanned about 16 kb in length and consisted of four exons and three introns. The positions of exon-intron junction of the FKBP12.6 gene were perfectly matched with those of FKBP12 gene except that FKBP12 has an additional exon, exon V, to code exclusively for 3'-UTR. Fluorescence in situ hybridization revealed that the FKBP12.6 gene was located on chromosome 2 p21-23, which is different from the locus (chromosome 20 p13) of the FKBP12 gene. Reporter gene analyses revealed that the regions of -58 approximately -24 of FKBP12.6 and -106 approximately -79 of FKBP12 are important for promoter activities. The promoters contain a consensus transcription factor binding sequence for Sp family in FKBP12.6 and Ets-1 in FKBP12. Electrophoretic mobility shift assays showed that nuclear proteins bind to the promoters. The DNA/protein complex on FKBP12.6 promoter was competed out by Sp1 consensus probe and the complex was supershifted by anti-Sp3 antibodies. On the other hand, the DNA/protein complex on FKBP12 promoter was competed out by Ets-1 consensus probe but not by its mutant probe, indicating that Sp3 and Ets-1 play an essential role in transcription of FKBP12.6 and FKBP12, respectively.
Asunto(s)
Cromosomas Humanos Par 2/genética , Exones/genética , Intrones/genética , Regiones Promotoras Genéticas/genética , Proteína 1A de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/genética , Regiones no Traducidas 3' , Secuencia de Bases , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Humanos , Hibridación Fluorescente in Situ , Riñón/metabolismo , Luciferasas/metabolismo , Datos de Secuencia Molecular , Proteína Proto-Oncogénica c-ets-1/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismoRESUMEN
Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.
Asunto(s)
Cromosomas Humanos Par 2/genética , Perfilación de la Expresión Génica , Proteínas/genética , Región de Flanqueo 5'/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , ADN/química , ADN/genética , ADN/aislamiento & purificación , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Familia de Multigenes/genética , Páncreas/metabolismo , Proteínas Asociadas a Pancreatitis , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Sitio de Iniciación de la TranscripciónRESUMEN
BACKGROUND: The Reg gene is known to be involved in the growth of not only pancreatic B-cells, but also epithelial cells of the gastrointestinal tract and carcinoma of its lineage. METHODS: Because, to the authors' knowledge, no studies have been reported regarding REG expression in gastric carcinoma, the authors investigated REG mRNA and REG protein expression using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemical study and correlated the results with the clinical features of gastric carcinoma. RESULTS: Using RT-PCR and Western blot analyses, reg mRNA and 16-kilodalton REG proteins were detected in two of eight human gastric carcinoma cell lines. Cytoplasmic localization of REG proteins in the cell lines was confirmed by fluorescent immunocytochemistry. The RT-PCR analysis revealed the presence of REG mRNA in as many as 77% (87 of 112 tumors) of primary gastric carcinoma tumors. Screening of a total of 195 patients with primary gastric carcinoma using immunoperoxidase staining revealed positive REG immunoreactivity in 60 of the 195 primary tumors (31%). REG expression in infiltrating tumors was found to be significantly higher compared with localized tumors (P < 0.05). Strong REG expression was noted in the cytoplasm of signet ring cell carcinoma tumors at a significantly higher incidence than in nonsignet ring cell tumors. Moreover, patients with REG-negative differentiated adenocarcinoma were found to have a significantly better prognosis compared with patients with REG-positive tumors. The incidence of venous invasion of REG-positive tumors was significantly higher than that of REG-negative tumors. CONCLUSIONS: The results of the current study suggest that the expression of the REG gene is closely related to the infiltrating property of gastric carcinoma, and may be a prognostic indicator of differentiated adenocarcinoma of the stomach.
Asunto(s)
Adenocarcinoma/genética , Proteínas de Unión al Calcio/genética , Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica/patología , Proteínas del Tejido Nervioso/genética , Neoplasias Gástricas/genética , Adenocarcinoma/patología , Secuencia de Bases , Biomarcadores de Tumor/análisis , Biopsia con Aguja , Western Blotting , Estudios de Cohortes , Técnicas de Cultivo , Femenino , Humanos , Inmunohistoquímica , Litostatina , Masculino , Datos de Secuencia Molecular , Pronóstico , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Neoplasias Gástricas/patologíaRESUMEN
Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets. We have demonstrated in vitro and in vivo that the exogenous addition of rat and human Reg gene products, Reg/REG proteins, induced beta-cell replication via the Reg receptor and thereby ameliorated experimental diabetes. In the present study, we produced Reg knockout mice by homologous recombination. The Reg gene disruption resulted in a null mutation. Knockout mice developed normally. Islets from the Reg knockout mice appeared morphologically indistinguishable from those of normal controls. However, [(3)H]thymidine incorporation in isolated islets from Reg knockout mice was decreased. When hyperplastic islets were induced by the injection of goldthioglucose, the average islet size in Reg knockout mice was significantly smaller than that of control Reg(+/+) mice. We then produced transgenic mice carrying the Reg gene under the control of the rat insulin II promoter (Ins-Reg) to express Reg in beta-cells. Isolated islets from the Ins-Reg transgenic mice showed increased [(3)H]thymidine incorporation. By intercrossing, we produced NOD mice carrying the Ins-Reg transgene and found that development of diabetes in the resultant Ins-Reg transgenic NOD mice was significantly retarded, coinciding with an increase in the pancreatic beta-cell mass. These results indicate that Reg plays an important role in beta-cell growth/regeneration.