Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Abstract

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library [J. Biol. Chem. 263 (1988) 2111], encodes a secretory protein with a growth stimulating effect on pancreatic β cells that ameliorates the diabetes of 90% depancreatized rats [Proc. Natl. Acad. Sci. USA 91 (1994) 3589] and non-obese diabetic mice [Diabetes 51(Suppl. 3) (2002) S478]. 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 Iα, REG Iβ, 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 Iα 5′-5′ REG Iβ 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.

Introduction

Administration of poly(ADP-ribose) synthetase/polymerase inhibitors, such as nicotinamide, to 90% depancreatized rats induces the regeneration of pancreatic islets (Yonemura et al., 1984). We identified Regenerating gene (Reg), which was specifically expressed in regenerating islets (Terazono et al., 1988). Reg is induced in pancreatic β-cells by inflammatory stimuli such as by interleukin-6 (IL-6)/glucocoriticoids (Akiyama et al., 2001) and acts as an autocrine/paracrine growth factor for β-cell regeneration via a cell surface Reg receptor (Kobayashi et al., 2000). The rat Reg (now termed Reg I) gene product and human REG Iα gene product have been demonstrated to stimulate β-cell proliferation and ameliorate the diabetes of 90% depancreatized rats and NOD mice, respectively Watanabe et al., 1994, Unno et al., 2002. Thus, we have isolated several Reg and Reg-related genes from human Terazono et al., 1988, Watanabe et al., 1990, Moriizumi et al., 1994, Miyashita et al., 1995, rat (Suzuki et al., 1994) and mouse Unno et al., 1993, Narushima et al., 1997, Abe et al., 2000, and revealed that they constituted a multigene family, the Reg gene family Unno et al., 1993, Narushima et al., 1997, Abe et al., 2000. Based on the primary structures of the encoded proteins, the members of the family are grouped into four subclasses, types I, II, III, and IV (Okamoto and Takasawa, 2002) as follows: Type I contains human REG Iα, REG Iβ, rat RegI, and mouse RegI; type II contains mouse RegII; type III contains human HIP/PAP (gene expressed in hepatocellular carcinoma–intestine–pancreas/gene encoding pancreatitis-associated protein) Lasserre et al., 1992, Orelle et al., 1992, Dusetti et al., 1994, rat PAP/Peptide23 (Iovanna et al., 1991), rat RegIII/PAPII (Suzuki et al., 1994), rat PAP III (Frigerio et al., 1993), mouse RegIIIα, RegIIIβ, RegIIIγ (Narushima et al., 1997), mouse RegIIIδ (Abe et al., 2000), and hamster INGAP (islet neogenesis-associated protein) (Rafaeloff et al., 1997). Type IV contains human REG IV (Hartupee et al., 2001). Despite the plurality of type III Reg genes in mouse and rat genome, only one type III REG gene, HIP/PAP, has so far been found in the human genome. Recently, we examined the existence of a human counterpart of mouse RegIIIδ by moderately stringent Southern blot hybridization (zoo blot), and detected a 9 kb band in EcoRI digested human genomic DNA, whereas HIP/PAP should give a 16 kb band in the EcoRI digested DNA (Abe et al., 2000), suggesting the possible occurrence of another member of the type III REG gene in the human genome.

As human REG genes except for REG IV are clustered on chromosome 2p12, in the present study we searched a new member of type III REG gene by PCR using the YAC clone containing the human REG family gene cluster (Miyashita et al., 1995) as a template and found a novel type III REG gene, REG III, in a 140 kb region of the human REG gene cluster.