Sumiyo Sakuramoto-Tsuchida

Attenuation of glucose-induced insulin secretion by intermittent hypoxia via down-regulation of CD38

AIMS Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation during sleep, the development of daytime sleepiness, and deterioration in the quality of life. Accumulating evidence suggests the association of intermittent hypoxia (IH), a hallmark of SAS, and type 2 diabetes independently on body mass index and waist circumference. In addition to insulin resistance, the progression to type 2 diabetes is dependent on the impairment of glucose-induced insulin secretion (GIS) from pancreatic β-cells. However, the direct effects of IH on GIS are elusive. MAIN METHODS HIT-T15 hamster β-cells and isolated rat islets were exposed to 64 cycles/24 h of IH (5 min hypoxia/10 min normoxia) or normoxia for 24 h. Changes of GIS and gene expression in IH-treated β-cells were analyzed by ELISA and real-time RT-PCR, respectively. KEY FINDINGS After IH treatment, GIS both from IH-treated HIT-T15 cells and isolated rat islets were significantly attenuated. The level of insulin mRNA was unchanged by IH. The mRNA levels of glucose transporter 2 (Glut2), glucokinase (GK), sulfonylurea receptor1 (SUR1), and L-type Ca2+channel1.2 (Cav1.2) in IH-treated-islets were similar to those in normoxia-treated islets. In contrast, the mRNA level of CD38 in IH-treated islets was significantly lower than that in normoxia-treated islets. The reporter gene assay revealed that the transcription of CD38 was attenuated by IH, and the transfection of CD38 expression vector recovered the attenuation of GIS by IH. SIGNIFICANCE These results indicate that IH stress directly attenuates GIS from β-cells via the down-regulation of CD38.

A novel ryanodine receptor expressed in pancreatic islets by alternative splicing from type 2 ryanodine receptor gene

Cyclic ADP-ribose (cADPR), a potent Ca(2+) mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca(2+) concentrations in pancreatic islets, resulting from Ca(2+) mobilization from RyRs as well as Ca(2+) influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [(3)H]ryanodine binding. Intracellular Ca(2+) release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling.

Pancreatic β cell proliferation by intermittent hypoxia via up-regulation of Reg family genes and HGF gene

AIMS Although accumulating evidence suggests the associations between sleep apnea syndrome (SAS) and type 2 diabetes, the direct effect of intermittent hypoxia (IH) on pancreatic β cell proliferation remains a missing piece of the puzzle. MAIN METHODS Rat RINm5F β cells, hamster HIT-T15 β cells, and human 1.1B4 β cells were exposed to normoxia (21% O2, 5% CO2, and balance N2), to sustained hypoxia (SH: 1% O2, 5% CO2, and balance N2), or to intermittent hypoxia (IH: 64 cycles of 5 min SH and 10 min normoxia) for 24 h. After the treatment, cellular proliferation and apoptosis were measured by WST-8 assay and TUNEL method, respectively. The expression of regenerating gene (Reg) family, interleukin (IL)-6, and hepatocyte growth factor (HGF) was determined by real-time RT-PCR. KEY FINDINGS The cellular proliferation of HIT-T15, RINm5F and 1.1B4 cells by IH was significantly increased, whereas apoptosis of these cells was unchanged. Real-time RT-PCR revealed that the mRNA levels of Reg family genes, IL-6, a typical Reg family gene inducer, and HGF, an inhibitor of high-concentration of Reg protein-induced apoptosis, were increased in IH-treated cells. In addition, siRNAs against rat Reg family genes except for PAP I/Reg 2 attenuated IH-induced β cell proliferation. SIGNIFICANCE IH stress stimulates pancreatic β cell to induce IL-6 gene expression. By the IL-6 stimulation, β cells over-express Reg family genes as well as HGF gene. Reg family proteins stimulate β cell proliferation and HGF inhibits apoptosis of β cells. As a result, β cell numbers are increased by IH.

REG I enhances chemo- and radiosensitivity in squamous cell esophageal cancer cells.

Identification of reliable markers of chemo‐ and radiosensitivity and the key molecules that enhance the susceptibility of squamous esophageal cancer cells to anticancer treatments would be highly desirable. To test whether regenerating gene (REG) I expression enhances chemo‐ and radiosensitivity in esophageal squamous cell carcinoma cells, we used MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) assays to compare the chemo‐ and radiosensitivities of untransfected TE‐5 and TE‐9 cells with those of cells stably transfected with REG Iα and Iβ. We then used flow cytometry to determine whether REG I expression alters cell cycle progression. No REG I mRNA or protein were detected in untransfected TE‐5 and TE‐9 cells. Transfection with REG Iα and Iβ led to strong expression of both REG I mRNA and protein in TE‐5 and TE‐9 cells, which in turn led to significant increases in both chemo‐ and radiosensitivity. Cell cycle progression was unaffected by REG I expression. REG I thus appears to enhance the chemo‐ and radiosensitivity of squamous esophageal cancer cells, which suggests that it may be a useful target for improved and more individualized treatments for patients with esophageal squamous cell carcinoma. (Cancer Sci 2008; 99: 2491–2495)

Synergistic activations of REG I α and REG I β promoters by IL-6 and Glucocorticoids through JAK/STAT pathway in human pancreatic β cells.

Reg (Regenerating gene) gene was originally isolated from rat regenerating islets and its encoding protein was revealed as an autocrine/paracrine growth factor for β cells. Rat Reg gene is activated in inflammatory conditions for β cell regeneration. In human, although five functional REG family genes (REG Iα, REG Iβ, REG III, HIP/PAP, and REG IV) were isolated, their expressions in β cells under inflammatory conditions remained unclear. In this study, we found that combined addition of IL-6 and dexamethasone (Dx) induced REG Iα and REG Iβ expression in human 1.1B4 β cells. Promoter assay revealed that a signal transducer and activator of transcription- (STAT-) binding site in each promoter of REG Iα (TGCCGGGAA) and REG Iβ (TGCCAGGAA) was essential for the IL-6+Dx-induced promoter activation. A Janus kinase 2 (JAK2) inhibitor significantly inhibited the IL-6+Dx-induced REG Iα and REG Iβ transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that IL-6+Dx stimulation increased STAT3 binding to the REG Iα promoter. Furthermore, small interfering RNA-mediated targeting of STAT3 blocked the IL-6+Dx-induced expression of REG Iα and REG Iβ. These results indicate that the expression of REG Iα and REG Iβ should be upregulated in human β cells under inflammatory conditions through the JAK/STAT pathway.

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.

Prevention of Reg I-induced β-cell apoptosis by IL-6/dexamethasone through activation of HGF gene regulation.

Reg (regenerating gene) product, Reg protein, is induced in pancreatic β-cells and acts as autocrine/paracrine growth factor for regeneration via the cell surface Reg receptor. However, high concentrations of Reg I protein induced β-cell apoptosis. In the present study, we found that hepatocyte growth factor (HGF) attenuated the β-cell apoptosis induced by the high concentrations of Reg I protein and that the combined stimulation of interleukin-6 (IL-6) and dexamethasone (Dx) induced the accumulation of HGF mRNA as well as Reg I mRNA in β-cells. The accumulation of the HGF mRNA was caused by the activation of the HGF promoter. Deletion analysis revealed that the region of -96 to -92 of the HGF gene was responsible for the promoter activation by IL-6+Dx. The promoters contain a consensus transcription factor binding sequence for signal transducer and activator of transcription (STAT). Site-directed mutations of STAT-binding motif in the region markedly attenuated the HGF promoter activity. Chromatin immunoprecipitation assay showed that STAT3 is located at the active HGF promoter in response to IL-6+Dx stimulation. These results strongly suggest that the combined stimulation of IL-6 and glucocorticoids induces the activation of both Reg and HGF genes and that the anti-apoptotic effects of HGF against the Reg I-induced apoptosis may help β-cell regeneration by Reg I protein.

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 Sjögrens 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.

Interleukin-6/STAT pathway is responsible for the induction of gene expression of REG Iα, a new auto-antigen in Sjögren׳s syndrome patients, in salivary duct epithelial cells.

The regenerating gene, Reg, was originally isolated from a rat regenerating islet cDNA library, and its human homolog was named REG Iα. Recently, we reported that REG Iα mRNA as well as its product were overexpressed in ductal epithelial cells in the minor salivary glands of Sjögren׳s syndrome (SS) patients. This study was undertaken to elucidate the role of cytokines and the subsequent intracellular mechanism for induction of REG Iα in the salivary glands of SS patients. We prepared a reporter plasmid containing REG Iα promoter (−1190/+26) upstream of a luciferase reporter gene. The promoter plasmid was introduced by lipofection into human NS-SV-DC and rat A5 salivary ductal cells. The cells were treated with interleukin (IL)-6, IL-8, and a combination of the two. Thereafter transcriptional activity of REG Iα was measured by luciferase assay. We found that IL-6 stimulation, but not IL-8, significantly enhanced the REG Iα promoter activity in salivary ductal cells. Deletion analysis revealed that the region of −141 to −117 of the REG Iα gene was responsible for the promoter activation by IL-6, which contains a consensus sequence for signal transduction and activation of transcription (STAT). The introduction of siRNA for human STAT3 abolished IL-6-induced REG Iα transcription. These results showed that IL-6 stimulation induced REG Iα transcription through STAT3 activation and binding to the consensus sequence of REG Iα promoter in salivary ductal cells. This IL-6/STAT dependent REG Iα induction might play a role in the pathogenesis of SS.

Significance of Interleukin-6/STAT Pathway for the Gene Expression of REG Iα, a New Autoantigen in Sjögren’s Syndrome Patients, in Salivary Duct Epithelial Cells

The regenerating gene, Reg, was originally isolated from a rat regenerating islet complementary DNA (cDNA) library, and its human homologue was named REG Iα. Recently, we reported that REG Iα messenger RNA (mRNA), as well as its product, was overexpressed in ductal epithelial cells in the salivary glands of Sjögren’s syndrome patients. Furthermore, autoantibodies against REG Iα were found in the sera of Sjögren’s syndrome patients, and the patients who were positive for the anti-REG Iα antibody showed significantly lower saliva secretion than antibody-negative patients. We found the mechanism of REG Iα induction in salivary ductal epithelial cells. Reporter plasmid containing REG Iα promoter (−1190/+26) upstream of a luciferase gene was introduced into human NS-SV-DC and rat A5 salivary ductal cells. The cells were treated with several cytokines (interleukin (IL)-6, IL-8, etc.), upregulated in Sjögren’s syndrome salivary ducts, and the transcriptional activity was measured. IL-6 stimulation significantly enhanced the REG Iα promoter activity in both cells. Deletion analysis revealed that the −141∼−117 region of the REG Iα gene was responsible for the promoter activation by IL-6, which contains a consensus sequence for signal transducer and activator of transcription (STAT) binding. The introduction of small interfering RNA for human STAT3 abolished IL-6-induced REG Iα transcription. These results indicated that IL-6 stimulation induced REG Iα transcription through STAT3 activation and binding to the REG Iα promoter in salivary ductal cells. This dependence of REG Iα induction upon IL-6/STAT in salivary duct epithelial cells may play an important role in the pathogenesis/progression of Sjögren’s syndrome.