Autoantibodies to REG, a beta-cell regeneration factor, in diabetic patients.

BACKGROUND: Regenerating gene (Reg) product, Reg, acts as an autocrine/paracrine growth factor for beta-cell regeneration. The presence of autoimmunity against REG may affect the operative of the regenerative mechanisms in beta cells of Type 1 and Type 2 diabetes patients. We screened sera from Type 1 and Type 2 diabetes subjects for anti-REG autoantibodies, searched for correlations in the general characteristics of the subjects with the presence of anti-REG autoimmunity, and tested the attenuation of REG-induced beta-cell proliferation by the autoanitibodies. MATERIAL AND METHODS: We examined the occurrence of anti-REG autoantibodies in patients' sera (265 Type 1, 368 Type 2 diabetes patients, and 75 unrelated control subjects) by Western blot analysis, and evaluated inhibitory effects of the sera on REG-stimulated beta-cell proliferation by a 5'-Bromo-2'-deoxyuridine (BrdU) incorporation assay in vitro. RESULTS: Anti-REG autoantibodies were found in 24.9% of Type 1, 14.9% of Type 2 and 2.7% of control subjects (P = 0.0004). There were significant differences between the autoantibody positive and negative groups in the duration of disease in the Type 1 subjects (P = 0.0035), and the age of onset in the Type 2 subjects (P = 0.0274). The patient sera containing anti-REG autoantibodies significantly attenuated the BrdU incorporation by REG (35.6 +/- 4.06% of the control), whereas the nondiabetic sera without anti-REG autoantibodies scarcely reduced the incorporation (88.8 +/- 5.10%). CONCLUSION: Anti-REG autoantibodies, which retard beta-cell proliferation in vitro, are found in some diabetic patients. Thus, autoimmunity to REG may be associated with the development/acceleration of diabetes in at least some patients.

Activation of Reg gene, a gene for insulin-producing beta -cell regeneration: poly(ADP-ribose) polymerase binds Reg promoter and regulates the transcription by autopoly(ADP-ribosyl)ation.

The regeneration of pancreatic islet beta cells is important for the prevention and cure of diabetes mellitus. We have demonstrated that the administration of poly(ADP-ribose) synthetase/polymerase (PARP) inhibitors such as nicotinamide to 90% depancreatized rats induces islet regeneration. From the regenerating islet-derived cDNA library, we have isolated Reg (regenerating gene) and demonstrated that Reg protein induces beta-cell replication via the Reg receptor and ameliorates experimental diabetes. However, the mechanism by which Reg gene is activated in beta cells has been elusive. In this study, we found that the combined addition of IL-6 and dexamethasone induced the expression of Reg gene in beta cells and that PARP inhibitors enhanced the expression. Reporter gene assays revealed that the -81 approximately -70 region (TGCCCCTCCCAT) of the Reg gene promoter is a cis-element for the expression of Reg gene. Gel mobility shift assays showed that the active transcriptional DNA/protein complex was formed by the stimulation with IL-6 and dexamethasone. Surprisingly, PARP bound to the cis-element and was involved in the active transcriptional DNA/protein complex. The DNA/protein complex formation was inhibited depending on the autopoly(ADP-ribosyl)ation of PARP in the complex. Thus, PARP inhibitors enhance the DNA/protein complex formation for Reg gene transcription and stabilize the complex by inhibiting the autopoly(ADP-ribosyl)ation of PARP.

Identification of a novel Reg family gene, Reg IIIdelta, and mapping of all three types of Reg family gene in a 75 kilobase mouse genomic region.

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 three subtypes (types I, II, III) based on the primary structures of the encoded proteins of the genes. We have isolated three types of mouse Reg family gene (Reg I, Reg II, Reg IIIalpha, Reg IIIbeta and Reg IIIgamma) [Unno et al. (1993) J. Biol. Chem. 268, 15974-15982; Narushima et al. (1997) Gene 185, 159-168]. In the present study, by Southern blot analysis of a mouse bacterial artificial chromosome clone containing the five Reg family genes in combination with PCR cloning of every interspace fragment between adjacent genes, the Reg family genes were mapped to a contiguous 75kb region of the mouse genome according to the following order: 5'-Reg IIIbeta-Reg IIIalpha-Reg II-Reg I-Reg IIIgamma-3'. In the process of ordering the genes, we sequenced the 6.8kb interspace fragment between Reg IIIbeta and Reg IIIalpha and encountered a novel type III Reg gene, Reg IIIdelta. This gene is divided into six exons spanning about 3kb, and encodes a 175 amino acid protein with 40-52% identity with the other five mouse Reg (regenerating gene product) proteins. Reg IIIdelta was expressed predominantly in exocrine pancreas, but not in normal islets, hyperplastic islets, intestine or colon, whereas both Reg I and Reg II were expressed in hyperplastic islets and Reg IIIalpha, Reg IIIbeta and Reg IIIgamma were expressed strongly in the intestinal tract. Possible roles of Reg IIIdelta and the widespread occurrence of the Reg IIIdelta gene in mammalian genomes are discussed.

Identification of a receptor for reg (regenerating gene) protein, a pancreatic beta-cell regeneration factor.

Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114). Rat and human Reg gene products, Reg/REG proteins, have been demonstrated to stimulate islet beta-cell growth in vitro and in vivo and to ameliorate experimental diabetes. In the present study, we isolated a cDNA for the Reg protein receptor from a rat islet cDNA library. The cDNA encoded a cell surface 919-amino acid protein, and the cells into which the cDNA had been introduced bound Reg protein with high affinity. When the cDNA was introduced into RINm5F cells, a pancreatic beta-cell line that shows Reg-dependent growth, the transformants exhibited significant increases in the incorporation of 5'-bromo-2'-deoxyuridine as well as in the cell numbers in response to Reg protein. A homology search revealed that the cDNA is a homologue to a human multiple exostoses-like gene, the function of which has hitherto been unknown. These results strongly suggest that the receptor is encoded by the exostoses-like gene and mediates a growth signal of Reg protein for beta-cell regeneration.