We have produced a panel of islet-specific T-cell clones from nonobese diabetic (NOD) mice. These clones proliferate and make interleukin 2 in an antigen-specific manner in response to NOD antigen-presenting cells and islet cells. Most of the clones respond to islet-cell antigen from different mouse strains but only in the presence of antigen-presenting cells bearing the class II major histocompatibility complex of the NOD mouse. In vivo, the clones mediate the destruction of islet, but not pituitary, grafts. Furthermore, pancreatic sections from a disease transfer experiment with one of the clones showed a pronounced cellular infiltration and degranulation of islets in nondiabetic (CBA x NOD)F1 recipients.
A pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP) was cloned using a subtractive cDNA expression cloning procedure from mouse insulinoma tissue. Two alternatively spliced variants that differed by the presence or absence of a 118-bp exon (exon IV) were detected in normal balb/c mice, diabetic ob/ob mice, and insulinoma tissue. The longer, 1901-bp full-length cDNA encoded a 355-amino acid protein (molecular weight 40,684) structurally related (50% overall identity) to the liver glucose-6-phosphatase and exhibited similar predicted transmembrane topology, conservation of catalytically important residues, and the presence of an endoplasmic reticulum retention signal. The shorter transcript encoded two possible open reading frames (ORFs), neither of which possessed His174, a residue thought to be the phosphoryl acceptor (Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY: Transmembrane topology of glucose-6-phosphatase. J Biol Chem 273:6144-6148, 1998). Northern blot and reverse transcription-polymerase chain reaction analysis showed that the mRNA was highly expressed in pancreatic islets and expressed more in beta-cell lines than in an alpha-cell line. It was notably absent in tissues and cell lines of non-islet neuroendocrine origin, and no other major tissue source of the mRNA was found. During development, it was expressed in parallel with insulin mRNA. The mRNA was efficiently translated and glycosylated in an in vitro translation/membrane translocation system and readily transcribed into COS 1, HIT, and CHO cells using cytomegalovirus or Rous sarcoma virus promoters. Whereas the liver glucose-6-phosphatase showed activity in these transfection systems, the IGRP failed to show glucose phosphotransferase or phosphatase activity with p-nitrophenol phosphate, inorganic pyrophosphate, or a range of sugar phosphates hydrolyzed by the liver enzyme. While the metabolic function of the enzyme is not resolved, its remarkable tissue-specific expression warrants further investigation, as does its transcriptional regulation in conditions where glucose responsiveness of the pancreatic islet is altered.
Islet-specific glucose-6-phosphatase (G6Pase) catalytic subunit-related protein (IGRP) is a homolog of the catalytic subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been defined. Since IGRP gene expression is restricted to islets, this suggests a possible role in the regulation of islet metabolism and, hence, insulin secretion induced by metabolites. We report here a comparative analysis of the human, mouse, and rat IGRP genes. These studies aimed to identify conserved sequences that may be critical for IGRP function and that specify its restricted tissue distribution. The single copy human IGRP gene has five exons of similar length and coding sequence to the mouse IGRP gene and is located on human chromosome 2q28 -32 adjacent to the myosin heavy chain 1B gene. In contrast, the rat IGRP gene does not appear to encode a protein as a result of a series of deletions and insertions in the coding sequence. Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is not expressed in islets or islet-derived cell lines, an observation that was traced by fusion gene analysis to a mutation of the TATA box motif in the mouse/human IGRP promoters to TGTA in the rat sequence. The results provide a framework for the further analysis of the molecular basis for the tissuerestricted expression of the IGRP gene and the identification of key amino acid sequences that determine its biological activity.
Cytokines are known to induce apoptosis of pancreatic -cells. Impaired expression of the anti-apoptotic gene bcl-2 is one of the mechanisms involved. In this study, we identified a defect involving transcription factor cAMP-response element-binding protein (CREB) in the expression of bcl-2. Exposure of mouse pancreatic -cell line, MIN6 cells, to cytokines (interleukin-1, tumor necrosis factor-␣, and interferon-␥) led to a significant (p < 0.01) decrease in Bcl-2 protein and mRNA levels. Cytokines decreased (56%) the activity of the bcl-2 promoter that contains a cAMP-response element (CRE) site. Similar decreases were seen with a luciferase reporter gene driven by tandem repeats of CRE and a CREB-specific Gal4-luciferase reporter, suggesting a defect at the level of CREB. The active phospho form (serine 133) of CREB diminished significantly (p < 0.01) in cells exposed to cytokines. Examination of signaling pathways upstream of CREB revealed a reduction in the active form of Akt. Cytokine-induced decrease of bcl-2 promoter activity was partially restored when cells were cotransfected with a constitutively active form of Akt. Several end points of cytokine action including decreases in phospho-CREB, phospho-Akt, and BCl-2 levels and activation of caspase-9 were observed in isolated mouse islets. Overexpression of wild-type CREB in MIN6 cells by plasmid transfection and adenoviral infection led to protection against cytokine-induced apoptosis. Adenoviral transfer of dominant-negative forms of CREB, on the other hand, resulted in activation of caspase-9 and exaggeration of cytokine-induced -cell apoptosis. Together, these results point to CREB as a novel target for strategies aimed at improving the survival of -cells.In type 1 diabetes, insulin-producing -cells are selectively destroyed by a cellular autoimmune response. Proinflammatory cytokines such as IL-1, 1 TNF-␣, and IFN-␥ are released during this autoimmune response and are believed to be important mediators of -cell destruction (1, 2). Elevated circulating levels of these cytokines have been reported in type 1 diabetic patients (3). In NOD mice and in BB rats, two genetic models for autoimmune diabetes, increased production of cytokines is observed (3). Antibodies or soluble receptors that neutralize cytokine action in these models prevent the development of diabetes (2,4). Several studies have shown that the -cell death induced by cytokines in type 1 diabetes is mainly through apoptosis (5, 6). Cytokines are known to modulate the expression of several genes in -cells (7,8). In a recent study, Cardozo et al. (7) carried out a comprehensive analysis of genes that were modulated in -cells exposed to Il-1 and IFN-␥. Genes involved in the -cell functions were down-regulated, whereas genes associated with apoptosis were up-regulated. Apoptosis can result from a variety of intracellular events or extracellular pathways such as activation of death receptors. The Bcl-2 family of proteins is important for regulation of the intrinsic mitochondrial pathw...
Islet-reactive T-cell clones from NOD mice provide an important approach to the investigation of antigens with relevance to type I diabetes. To identify a source of beta-cell antigen suitable for biochemical studies, we have used two islet-specific, diabetogenic T-cell clones to test beta-tumor cells. beta-tumor cell lines, maintained in continuous culture, were found to lose antigenicity rapidly. However, cells harvested directly from beta-tumors arising spontaneously in the transgenic NOD/Lt-Tg(RIPTag)1Lt mouse proved to be a potent source of beta-cell antigen for the T-cell clones. Subcellular fractionation of beta-tumor cells showed that the T-cell antigen was highly enriched in the beta-granule fraction and that this activity was associated with the granule membrane.
Human fibrosarcoma (HT-1080) cells, in contrast to normal fibroblasts, rapidly hydrolyze the glycoprotein, collagen, and elastin extracellular matrix (ECM) synthesized by cultured rat aortic smooth muscle cells. This degradation occurs at a rapid rate in the presence of serum, indicating that the cellular proteases responsible are relatively insensitive to serum proteinase inhibitors. Here it is shown that protease nexin I (PNI), a fibroblast-secreted inhibitor of urokinase, plasmin, and certain other serine proteinases, effectively inhibited the HT-1080 cell-mediated degradation of this ECM. PNI at 2.0 nM significantly inhibited matrix destruction for 1-2 days and at 0.2 ,uM caused a virtually complete inhibition that persisted for the entire 10-day period of observation. Inhibition of ECM destruction was accompanied by a transient arrest of HT-1080 cell proliferation that took place during the first 3 days after PNI addition. PNI did not inhibit the growth of normal fibroblasts and also did not inhibit the growth of HT-1080 cells that were seeded onto plastic dishes rather than onto ECM. Like many types of malignant cells, HT-1080 cells release large amounts of urokinase. Antibody against this plasminogen activator partially protected ECM from HT-1080 cell-mediated hydrolysis, indicating that it may have been a target of PNM. One potential physiological function ofPNI could be to help maintain the integrity of connective tissue matrices, protection that malignant cells could overcome by secreting proteinases in excessive amounts. (5, 6). Ossowski and Reich (7) recently reported that anti-urokinase antibody inhibited the metastasis of human epidermoid carcinoma cells seeded onto chicken embryo chorioallantoic membranes. In view of the ability of PNI to inhibit urokinase and plasmin, the present investigation was undertaken to determine the effect of this inhibitor on tumor-cell-mediated destruction of extracellular matrix (ECM). Jones and DeClerk (8)
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