The stress- and inflammatory cytokine-induced ectodomain shedding of heparin-binding epidermal growth factor-like growth factor is mediated by p38 MAPK,distinct from the 12-O-tetradecanoylphorbol-13-acetate- and lysophosphatidic acid-induced signaling cascades

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a critical growth factor for a number of physiological and pathological processes. HB-EGF is synthesized as a membrane-anchored form (pro-HB-EGF), and pro-HB-EGF is cleaved at the cell surface to yield soluble HB-EGF by a mechanism called "ectodomain shedding." We show here that the ectodomain shedding of pro-HB-EGF in Vero cells is induced by various stress-inducing stimuli, including UV light, osmotic pressure, hyperoxidation, and translation inhibitors. The pro-inflammatory cytokine interleukin-1beta also stimulated the ectodomain shedding of pro-HB-EGF. An inhibitor of p38 MAPK (SB203580) or the expression of a dominant-negative (dn) form of p38 MAPK inhibited the stress-induced ectodomain shedding of pro-HB-EGF, whereas an inhibitor of JNK (SP600125) or the expression of dnJNK1 did not. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and lysophosphatidic acid (LPA) are also potent inducers of pro-HB-EGF shedding in Vero cells. Stress-induced pro-HB-EGF shedding was not inhibited by the inhibitors of TPA- or LPA-induced pro-HB-EGF shedding or by dn forms of molecules involved in the TPA- or LPA-induced pro-HB-EGF shedding pathway. Reciprocally, SB203580 or dnp38 MAPK did not inhibit TPA- or LPA-induced pro-HB-EGF shedding. These results indicate that stress-induced pro-HB-EGF shedding is mediated by p38 MAPK and that the signaling pathway induced by stress is distinct from the TPA- or LPA-induced pro-HB-EGF shedding pathway.     

Effect of endogenously generated nitric oxide on the energy metabolism of peritoneal macrophages

To understand the role of nitric oxide (NO) in the regulation of cellular metabolism in peritoneal macrophages under physiological low oxygen tension, its effect on the respiration and energy metabolism was examined with casein-induced peritoneal macrophages from the rat. Intraperitoneal injection of casein transiently induced peritoneal infiltration of neutrophils (peaked on day 1) followed by the migration of macrophages that peaked on day 2. Western blotting analysis using antibodies against inducible type of NO synthase (iNOS) revealed that macrophages appeared in the peritoneal cavity during an early stage (approximately day 2) but not during the late stage (day 3 approximately) of inflammation expressed iNOS and generated substantial amounts of NO by a mechanism that was inhibited by N-iminoethyl-L-ornithine (NIO), a specific inhibitor of iNOS. Although NO reversibly but strongly inhibited the respiration of macrophages from both stages particularly under physiologically low oxygen tension, NIO markedly enhanced the respiration of macrophages obtained from the early period but not from the late period of inflammation. The ATP level in the macrophages from the late period but not from the early period was markedly decreased by NO. Biochemical analysis revealed that the glycolytic activity in the macrophages obtained from the early period was significantly higher than that from the late period of inflammation. These results indicate that significant fractions of cellular ATP in iNOS-positive peritoneal macrophages are synthesized by the increased activity of glycolysis particularly under physiological low oxygen tensions where the mitochondrial respiration is strongly inhibited by endogenously generated NO by macrophages and neutrophils.     

PPAR-gamma overexpression suppresses glucose-induced proinsulin biosynthesis and insulin release synergistically with pioglitazone in MIN6 cells

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) regulates several cellular functions; however, its physiological role in pancreatic beta cell functions remains to be determined. In the present study, we investigated the synergistic effect of PPAR-gamma and its agonist, pioglitazone, on proinsulin biosynthesis and insulin release in a glucose-responsible insulinoma cell line, MIN6 cells. Expression of PPAR-gamma in MIN6 cells was not detectable by RT-PCR and immunoblot analysis. When PPAR-gamma-1 was overexpressed adenovirally in MIN6 cells, glucose-stimulated proinsulin biosynthesis and insulin release were inhibited. Pioglitazone treatment alone had no effects on these parameters of beta cell function in control MIN6 cells, although pioglitazone synergistically augmented the inhibitory effect of PPAR-gamma on proinsulin biosynthesis and insulin release under the condition of PPAR-gamma overexpression. Our results demonstrate that PPAR-gamma plays a negative role in pancreatic beta cells.     

Functional analysis of human P5, a protein disulfide isomerase homologue

Human P5 (hP5) was expressed in the Escherichia coli pET system and purified by sequential Ni(2+)-chelating resin column chromatography. Characterization of purified hP5 indicated that it has both isomerase and chaperone activities, but both activities are lower than those of human protein disulfide isomerase (PDI). Moreover, hP5 was observed to have peptide-binding ability, and its chaperone activity was confirmed with rhodanese and citrate synthase as substrates, but not with D-glyceraldehyde-3-phosphate dehydrogenase, showing that hP5 has substrate specificity with respect to chaperone activity. Mutation of two thioredoxin-related motifs in hP5 revealed that the first motif is more important than the second for isomerase activity and that the first cysteine in each motif is necessary for isomerase activity. Since thioredoxin motif mutants lacking isomerase activity retain chaperone activity with the substrate citrate synthase, the isomerase and chaperone activities of hP5 are probably independent, as was shown for PDI.     

PPAR-gamma ligands up-regulate basic fibroblast growth factor-induced VEGF release through amplifying SAPK/JNK activation in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) activates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release. In the present study, we investigated the effects of ciglitazone and pioglitazone, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, on the VEGF release by FGF-2 in MC3T3-E1 cells. The FGF-2-induced VEGF release was significantly enhanced by ciglitazone. The amplifying effect of ciglitazone was dose-dependent between 0.1 and 10 microM. Pioglitazone had a similar effect on the VEGF release. GW9662, an antagonist of PPAR-gamma, reduced the effects of ciglitazone and pioglitazone. Ciglitazone or pioglitazone markedly enhanced the phosphorylation of SAPK/JNK induced by FGF-2 without affecting both the FGF-2-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. GW9662 markedly reduced the amplification by ciglitazone of the SAPK/JNK phosphorylation. Taken together, these results strongly suggest that PPAR-gamma ligands up-regulate FGF-2-stimulated VEGF release resulting from amplifying activation of SAPK/JNK in osteoblasts.     

Regulation of Src kinase activity during Xenopus oocyte maturation

Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation.     

Amino-terminal domain of ATRIP contributes to intranuclear relocation of the ATR-ATRIP complex following DNA damage

ATM and rad3-related protein kinase (ATR), a member of the phosphoinositide kinase-like protein kinase family, plays a critical role in cellular responses to DNA structural abnormalities in conjunction with its interacting protein, ATRIP. Here, we show that the amino-terminal portion of ATRIP is relocalized to DNA damage-induced nuclear foci in an RPA-dependent manner, despite its lack of ability to associate with ATR. In addition, ATR-free ATRIP protein can be recruited to the nuclear foci. Our results suggest that the N-terminal domain of the ATRIP protein contributes to the cell cycle checkpoint by regulating the intranuclear localization of ATR.     

The novel gene encoding a putative transmembrane protein is mutated in gnathodiaphyseal dysplasia (GDD)

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of the jawbone. By linkage analysis of a large Japanese family with GDD, we previously mapped the GDD locus to chromosome 11p14.3-15.1. In the critical region determined by recombination mapping, we identified a novel gene (GDD1) that encodes a 913-amino-acid protein containing eight putative transmembrane-spanning domains. Two missense mutations (C356R and C356G) of GDD1 were identified in the two families with GDD (the original Japanese family and a new African American family), and both missense mutations occur at the cysteine residue at amino acid 356, which is evolutionarily conserved among human, mouse, zebrafish, fruit fly, and mosquito. Cellular localization to the endoplasmic reticulum suggests a role for GDD1 in the regulation of intracellular calcium homeostasis.     

PPAR-gamma overexpression selectively suppresses insulin secretory capacity in isolated pancreatic islets through induction of UCP-2 protein

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) regulates several cellular functions, but its physiological role in pancreatic islet cells remains to be investigated. In this study, we confirmed the presence of PPAR-gamma in rat isolated islets and examined its role on insulin and glucagon secretion by using PPAR-gamma-overexpressed islets. PPAR-gamma overexpression significantly suppressed insulin secretion induced by stimulatory concentration of glucose (p<0.05). In addition, insulin secretion evoked by high potassium depolarization also was significantly decreased from PPAR-gamma-overexpressed islets (p<0.05). On the other hand, no significant change in glucagon release was observed after high potassium depolarization between PPAR-gamma-overexpressed and control islets. Insulin and glucagon content in islets was not statistically different between the two groups. In addition, the expression of uncoupling protein-2 (UCP-2) was found to be induced in PPAR-gamma-overexpressed islets. This result clearly indicates that the deteriorative effect of PPAR-gamma overexpression on the secretory machinery is selective for pancreatic beta-cells. And it is possible that its site of action can be located in the energy-consuming exocytotic process of insulin secretory granules, and that the reduction of ATP production through increased UCP-2 reduces insulin exocytosis.     

Suppression of the biological activities of the epidermal growth factor (EGF)-like domain by the heparin-binding domain of heparin-binding EGF-like Growth Factor

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that has a high affinity for heparin and heparan sulfate. While interactions with heparin are thought to modulate the biological activity of HB-EGF, the precise role of the heparin-binding domain has remained unclear. We analyzed the activity of wild-type HB-EGF and a mutant form lacking the heparin-binding domain (DeltaHB) in the presence or absence of heparin. The activity of the EGF-like domain of HB-EGF was determined by measuring binding to diphtheria toxin (DT) as well as the growth factor activity in EGF receptor-expressing cells. The binding affinity of DeltaHB for DT was much higher than that of wild-type HB-EGF in the absence of heparin. The binding affinity of HB-EGF for DT was increased by addition of exogenous heparin and reached the level close to the affinity of DeltaHB, whereas that of DeltaHB was not affected. Moreover, the growth factor activity of DeltaHB was much higher than that of wild-type HB-EGF in the absence of heparin but was not affected by addition of exogenous heparin, whereas HB-EGF had increased growth factor activity with added heparin. These results indicate that the heparin-binding domain suppresses the activity of the EGF-like domain of HB-EGF and that association of heparin with HB-EGF via this domain removes the suppressive effect. Thus, we conclude that the heparin-binding domain serves as a negative regulator of this growth factor.