Involvement of autoimmunity to REG, a regeneration factor, in patients with primary Sjögren's syndrome.

The regenerating gene (Reg) was isolated originally as a gene specifically over-expressed in regenerating pancreatic islets and constitute a growth factor family. Reg gene product (Reg) is important in the pathophysiology of various human inflammatory diseases. Recently, the possible involvement of human REG in the regeneration of salivary ductal epithelial cells of patients with primary Sjögren's syndrome (SS) was reported. However, the expression of the REG family genes in minor salivary glands (MSG) and the occurrence of anti-REG Iα autoantibodies in SS patients were obscured. In this study, we examined the expression of REG family genes in the MSG of SS and screened anti-REG Iα autoantibodies in SS. The mRNA levels of REG family genes in MSG were quantified using real-time reverse transcription-polymerase chain reaction (RT-PCR) and REG Iα expression in the MSG was analysed by immunohistochemistry. The mRNA level of REG Iα in the MSG of SS patients was significantly higher than that of control. REG Iα protein was expressed highly in SS ductal epithelial cells. Anti-REG Iα autoantibodies in the sera were found in 11% of SS. All the MSG in the anti-REG Iα autoantibody-positive group showed REG Iα expression, whereas only 40% showed REG Iα expression in the anti-REG Iα autoantibody-negative group. The anti-REG Iα autoantibody-positive group showed significantly lower saliva secretion and a higher ratio of grade 4 (by Rubin-Holt) in sialography. These data suggest strongly that autoimmunity to REG Iα might play a role in the degeneration of MSG ductal epithelial cells in primary SS.

Deletion of platelet-derived growth factor receptor-ß improves diabetic nephropathy in Ca²âº/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice.

AIMS/HYPOTHESIS: The activation of platelet-derived growth factor receptor-ß (PDGFR-ß) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-ß signalling during the development of diabetic nephropathy. METHODS: We recently generated pancreatic beta cell-specific Ca(2+)/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5 mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-ß (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-ß gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16 weeks of age. RESULTS: The plasma glucose concentrations and HbA(1c) levels were elevated in the CaMKIIα mice from 4 weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16 weeks of age. The postnatal deletion of the Pdgfr-ß gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-ß gene deletion. CONCLUSIONS/INTERPRETATION: The activation of PDGFR-ß signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.

Cyclic ADP-ribose in insulin secretion from pancreatic beta cells.

Inositol 1,4,5-trisphosphate (IP3) is thought to be a second messenger for intracellular calcium mobilization. However, in a cell-free system of islet microsomes, cyclic adenosine diphosphate-ribose (cADP-ribose), a nicotinamide adenine dinucleotide (NAD+) metabolite, but not IP3, induced calcium release. In digitonin-permeabilized islets, cADP-ribose and calcium, but not IP3, induced insulin secretion. Islet microsomes released calcium when combined with the extract from intact islets that had been incubated with high concentrations of glucose. Sequential additions of cADP-ribose inhibited the calcium release response to extracts from islets treated with high concentrations of glucose. Conversely, repeated additions of the islet extract inhibited the calcium release response to a subsequent addition of cADP-ribose. These results suggest that cADP-ribose is a mediator of calcium release from islet microsomes and may be generated in islets by glucose stimulation, serving as a second messenger for calcium mobilization in the endoplasmic reticulum.

Reg I-knockout mice reveal its role in regulation of cell growth that is required in generation and maintenance of the villous structure of small intestine.

Reg I (regenerating gene product I) is a growth factor that plays a central role in the generation and regeneration of the gastric mucosal architecture. On the other hand, mouse Reg I mRNA is expressed at the highest levels in the small intestine among the gastrointestinal tissues. In the current study, with the aim to clarify the role of Reg I protein in the small intestine, the temporal and spatial pattern of Reg I expression and the phenotype of Reg I-knockout mice in the tissue were examined. In the wild-type mice, immunohistochemistry localized Reg I protein expression in absorptive cells located in the lower half of the intestinal villi. Reg I expression was undetectable until embryonic day 13 (E13), when the fetal intestine still lacks villous structure; however, it dramatically increased at E17 along with the formation and maturation of the fetal intestinal villi. In the small intestine of the adult Reg I-knockout mice, less densely packed, round-shaped aberrant morphology of the absorptive cells was observed light microscopically, and electron microscopical examination revealed a strikingly loose connection of these cells to the basement membrane. Antiproliferating cell nuclear antigen staining and anti-Ki67 staining demonstrated the marked decrease in the number of proliferating cells in the small intestinal mucosa of the knockout mice. The cell migration speed visualized by one shot labeling of 5-bromodeoxyuridine was significantly slower in the knockout mice. These phenotypes of Reg I-knockout mice emerged, in accordance with the temporal pattern of Reg I expression described above, from E17. Reg I was considered to be a regulator of cell growth that is required to generate and maintain the villous structure of the small intestine.

Cyclic ADP-ribose modulates Ca2+ release channels for activation by physiological Ca2+ entry in bullfrog sympathetic neurons.

Although Ca(2+)-induced Ca2+ release (CICR) via ryanodine receptors has been found to occur in intact neurons, little is known about the physiological processes that regulate it. We studied the effects of cyclic ADP-ribose (cADPR) on CICR in cultured bullfrog sympathetic neurons by fura-2 fluorescence recording and patch-clamp techniques. cADPR applied through a patch pipette augmented action potential- or depolarizing pulse-induced rises in intracellular Ca2+ without a change in Ca2+ entry initiating the responses, but not in the presence of ryanodine. Likewise, cADPR enhanced a single or oscillatory rise(s) in intracellular Ca2+ induced by caffeine. These results strongly suggest that cADPR can be an endogenous modulator of ryanodine receptors in neurons.

Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients.

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P

Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice.

Vascular complications arising from multiple environmental and genetic factors are responsible for many of the disabilities and short life expectancy associated with diabetes mellitus. Here we provide the first direct in vivo evidence that interactions between advanced glycation end products (AGEs; nonenzymatically glycosylated protein derivatives formed during prolonged hyperglycemic exposure) and their receptor, RAGE, lead to diabetic vascular derangement. We created transgenic mice that overexpress human RAGE in vascular cells and crossbred them with another transgenic line that develops insulin-dependent diabetes shortly after birth. The resultant double transgenic mice exhibited increased hemoglobin A(1c) and serum AGE levels, as did the diabetic controls. The double transgenic mice demonstrated enlargement of the kidney, glomerular hypertrophy, increased albuminuria, mesangial expansion, advanced glomerulosclerosis, and increased serum creatinine compared with diabetic littermates lacking the RAGE transgene. To our knowledge, the development of this double transgenic mouse provides the first animal model that exhibits the renal changes seen in humans. Furthermore, the phenotypes of advanced diabetic nephropathy were prevented by administering an AGE inhibitor, (+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (OPB-9195), thus establishing the AGE-RAGE system as a promising target for overcoming this aspect of diabetic pathogenesis.

Induction of rat pancreatic B-cell tumors by the combined administration of streptozotocin or alloxan and poly(adenosine diphosphate ribose) synthetase inhibitors.

Streptozotocin and alloxan were administered to Wistar rats in combination with poly(adenosine diphosphate ribose) synthetase inhibitors. Ten to 16 months after the injection of streptozotocin (50 mg/kg body weight i.v.) and 3-aminobenzamide (345 mg/kg i.v.), streptozotocin (50 mg/kg) and nicotinamide (350 mg/kg i.p.), streptozotocin (50 mg/kg) and picolinamide (250 mg/kg i.p.), alloxan (40 mg/kg i.v.) and nicotinamide (350 mg/kg), alloxan (40 mg/kg) and 3-aminobenzamide (345 mg/kg), and alloxan (40 mg/kg) and picolinamide (250 mg/kg), pancreatic islet cell tumors developed in 100, 98, 60, 26, 22, and 20% of surviving rats, respectively. However, after the single injection of streptozotocin and alloxan, islet cell tumors developed in 42 and 11% of surviving rats, respectively. The tumors were rich in B-granules on electron micrographs and contained as large amounts of proinsulin messenger RNA as normal pancreatic islets. The results indicate that poly(adenosine diphosphate ribose) synthetase inhibitors enhance the tumorigenic effect of streptozotocin and alloxan on islet B-cells.

The primary structure of rat rig/ribosomal protein S15 gene.

We have isolated rat rig/ribosomal protein S15 gene from a DNA library derived from a rat insulinoma and determined the complete nucleotide sequence. The rat rig/S15 gene is composed of four exons and three introns spanning 2 kbp and exhibits distinctive structural features unique for a ribosomal protein gene.

Cloning and characterization of cDNA encoding rat ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (homologue to human CD38) from islets of Langerhans.

We report the cloning and cDNA sequence of rat CD38, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase. Rat CD38 is composed of 303 amino acids and shares a high degree of homology with human and mouse CD38. Rat CD38 mRNA is expressed in various tissues including pancreatic islets but not in RINm5F cells.

Characterization of the 5'-regulatory region of rat Reg I gene.

We report here the characterization of the 5'-regulatory region of rat Reg I gene encoding a growth stimulating factor for pancreatic beta-cells. Transient expression assays of the 5'-flanking region/luciferase fusion gene in AR4-2J cells showed that the -304/-237 region contained positive cis-acting elements. Gel shift assays using AR4-2J and rat pancreas nuclear extracts showed the formation of a specific complex with the -256/-237 oligonucleotide.

Altered stoichiometry of FKBP12.6 versus ryanodine receptor as a cause of abnormal Ca(2+) leak through ryanodine receptor in heart failure.

BACKGROUND: In the pathogenesis of cardiac dysfunction in heart failure, a decrease in the activity of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase is believed to be a major determinant. Here, we report a novel mechanism of cardiac dysfunction revealed by assessing the functional interaction of FK506-binding protein (FKBP12.6) with the cardiac ryanodine receptor (RyR) in a canine model of pacing-induced heart failure. METHODS AND RESULTS: SR vesicles were isolated from left ventricular muscles (normal and heart failure). The stoichiometry of FKBP12.6 per RyR was significantly decreased in failing SR, as assessed by the ratio of the B(max) values for [(3)H]dihydro-FK506 to those for [(3)H]ryanodine binding. In normal SR, the molar ratio was 3.6 ( approximately 1 FKBP12.6 for each RyR monomer), whereas it was 1.6 in failing SR. In normal SR, FK506 caused a dose-dependent Ca(2+) leak that showed a close parallelism with the conformational change in RyR. In failing SR, a prominent Ca(2+) leak was observed even in the absence of FK506, and FK506 produced little or no further increase in Ca(2+) leak and only a slight conformational change in RyR. The level of protein expression of FKBP12.6 was indeed found to be significantly decreased in failing SR. CONCLUSIONS: An abnormal Ca(2+) leak through the RyR is present in heart failure, and this leak is presumably caused by a partial loss of RyR-bound FKBP12.6 and the resultant conformational change in RyR. This abnormal Ca(2+) leak might possibly cause Ca(2+) overload and consequent diastolic dysfunction, as well as systolic dysfunction.

Novel gene activated in rat insulinomas.

Insulinomas can be induced in experimental animals by the combined administration of diabetogenic agents with polyadenosine diphosphate (polyADP)-ribose synthetase inhibitors. A complementary DNA (cDNA) library that was constructed from streptozocin-nicotinamide-induced rat insulinomas has been found to contain a novel gene encoding a basic protein of 145 amino acids. The gene was expressed in alloxan-nicotinamide-induced insulinomas as well as in streptozocin-nicotinamide-induced insulinomas but not in normal pancreatic islets or in regenerating islets. This indicates that the activation of the gene designated rig, i.e., rat insulinoma gene, may be a general feature of pancreatic beta-cell transformation.

Autoantibodies to CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) in Caucasian patients with diabetes: effects on insulin release from human islets.

The type II transmembrane glycoprotein CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) has been proposed as a mediator of insulin secretion from pancreatic beta-cells and as a candidate for autoimmune reactions in type 2 diabetes. We evaluated the presence of anti-CD38 autoantibodies in Caucasian patients with diabetes and investigated the effect of these antibodies on insulin secretion from isolated human pancreatic islets. The presence of anti-CD38 autoantibodies was evaluated by using Western blot analysis in 236 patients with type 2 diabetes (mean age 63 years), in 160 patients with type 1 diabetes (mean age 38 years), and in 159 nondiabetic subjects. Anti-CD38 autoantibody titers at least 3 SD above the mean value of the control group were found in 9.7% of type 2 diabetic patients and in 13.1% of type 1 diabetic patients (chi2 = 15.9, P = 0.0003 vs. 1.3% of control subjects). No significant differences were observed in sex distribution, current age, age at diabetes onset, BMI, fasting serum glucose, or glycemic control between anti-CD38+ and anti-CD38-diabetic patients in either the type 2 or type 1 diabetic groups. The effect of 23 anti-CD38- and 13 anti-CD38+ sera on insulin secretion at low (3.3 mmol/l) or high (16.7 mmol/l) medium glucose concentrations was evaluated in isolated human pancreatic islets. Data are medians (interquartile range). The anti-CD38+ sera potentiated insulin release both at low [95 (64) vs. 23 (12) microU/ml of control incubations, respectively, P < 0.0001] and high [271 (336) vs. a control of 55 (37) microU/ml, respectively, P = 0.001] medium glucose concentrations, whereas the anti-CD38- sera did not. Furthermore, in the pooled data from all 36 tested sera, insulin levels in the islet incubation medium were directly related to the anti-CD38 antibody titer. We conclude that autoantibodies to CD38 are associated with both type 1 and type 2 diabetes in Caucasian subjects. These autoantibodies exert a stimulatory effect on insulin secretion by cultured human islets. The role of this autoimmune reaction in the pathogenesis of diabetes remains to be elucidated.

Transgenic CuZn-superoxide dismutase inhibits NO synthase induction in experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The expression of inducible NO synthase (iNOS) after experimental subarachnoid hemorrhage (SAH) has been postulated to play a critical role in the pathogenesis of SAH and subsequent cerebral vasospasm. The inhibitory effect of CuZn-superoxide dismutase (CuZn-SOD) on the induction of iNOS after SAH was examined by using transgenic mice overexpressing CuZn-SOD. METHODS: SOD-transgenic mice and nontransgenic littermates were subjected to SAH by endovascular perforation of the left anterior cerebral artery. The iNOS mRNA expression after SAH was determined by reverse transcription-polymerase chain reaction, and the distribution of iNOS-positive cells was immunohistochemically examined. The nuclear expression of activated nuclear factor-kappaB, a major transcription factor of iNOS gene, was also immunohistochemically examined. RESULTS: In nontransgenic mice, SAH-induced iNOS protein and mRNA expressions in the arteries of basal cistern as well as in the cerebral cortex were demonstrated by immunohistochemistry and reverse transcription-polymerase chain reaction. SAH-induced iNOS protein and mRNA expressions in those tissues were much reduced in SOD-transgenic mice compared with nontransgenic mice. Moreover, the nuclear expression of the activated form of nuclear factor-kappaB was immunohistochemically detected in the cerebral cortices of nontransgenic mice but not in those of SOD-transgenic mice. CONCLUSIONS: These results indicate that oxygen-derived free radicals, particularly superoxide, play an important role in the iNOS gene expression after SAH and provide a molecular basis for the protective role of SOD against vasospasm after SAH.

Human glutathione S-transferase P1 polymorphism and susceptibility to smoking related epithelial cancer; oral, lung, gastric, colorectal and urothelial cancer.

The A/G polymorphism at nucleotide 313 in the glutathione S-transferase P1-1 (GSTP1) gene was examined in patients with different types of smoking-related cancers (oral, lung, gastric, colorectal and urothelial cancers) and healthy control individuals. This polymorphism results in an amino acid substitution from isoleucine to valine at residue 105, which reduces catalytic activity of the enzyme. In control individuals, 23.8% of individuals had GSTP1 AG or GG genotype. This rose to 37.3% [n = 83, odds ratio = 1.93 (1.05-3.58), P = 0.035] in oral cancer patients. No increase in the frequency of the GSTP1 AG or GG genotype was obtained in lung, gastric, colorectal or urothelial cancers in this Japanese population. After grouping by smoking status, no consistent difference was observed between smoking patients and corresponding control individuals for the frequency of the GSTP1 A/G polymorphism for any cancer. However, a moderate risk (odds ratio = 2.78; 95% confidence interval 1.06-7.51) was associated with this polymorphism in the non-smoking group of oral cancer patients. The results suggest the GSTP1 polymorphism at nucleotide 313 may be associated with susceptibility to oral squamous cell carcinoma in the Japanese population.

Cloning of a cDNA encoding rat bone marrow stromal cell antigen 1 (BST-1) from the islets of Langerhans.

We have isolated the rat bone marrow stromal cell antigen 1-encoding cDNA (BST-1) from a pancreatic islet cDNA library. The cDNA encodes a 319-amino-acid (aa) protein whose aa sequence shows homology with mammalian CD38 (33%), Aplysia ADP-ribosyl cyclases (33%), as well as mouse (86%) and human (72%) BST-1.

Sequence of the chicken rig gene encoding ribosomal protein S15.

The nucleotide sequence of the chicken rig gene encoding ribosomal protein S15 was determined. The 1.6-kb gene consists of four exons and three introns. The 5'-flanking region of the gene lacks TATA- or CAAT-box sequences. Several GC-box sequences were found around the transcription start point.

The structure of the Aplysia kurodai gene encoding ADP-ribosyl cyclase, a second-messenger enzyme.

The complete nucleotide (nt) sequences of the cDNA and gene encoding the marine mollusk Aplysia kurodai (Ak) ADP-ribosyl cyclase (ADRC) which synthesizes cyclic ADP-ribose (cADP-ribose), a second messenger for Ca2+ mobilization from endoplasmic reticulum, were determined. Ak ADRC consists of 258 amino acids (aa) (29 kDa). It shares 86% aa sequence homology with that from A. californica, and 31-32% homology with the human, rat and mouse cluster of differentiation 38 (CD38) that has both ADRC and cADP-ribose hydrolase activities. The Ak ADRC-encoding gene (ADRC) spans approx. 7 kb and contains eight exons and seven introns. The transcription start point (tsp) determined by primer extension analysis and S1 mapping is 28 bp downstream from the TATA box. This gene is expressed specifically in the ovotestis, although the mammalian CD38-encoding gene is expressed in many kinds of tissues and cells. The 5'-flanking region contains several consensus sequences responsible for the germ-cell-specific expression of the mouse zona pellucida 3 (ZP3) and Drosophila melanogaster chorion genes. The existence of the consensus sequences located at nt -1649, -1161, -234 and -90 may account for the ovotestis-specific expression of the Ak ADRC gene.

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.