Search

Cytosolic PL[A.sub.2-[alpha]] (cPL[A.sub.2]) and metabolites of arachidonic acid (AA) are key mediators of complement-dependent glomerular epithelial cell (GEC) injury. Assembly of C5b-9 increases cytosolic [Ca.sup.2+] concentration and results in transactivation of receptor tyrosine kinases and activation of PLC-[gamma]1 and the 1,2-diacylglycerol (DAG)-PKC pathway. [Ca.sup.2+] and PKC are essential for membrane association and increased catalytic activity of cPL[A.sub.2]. This study addresses the role of the actin cytoskeleton in cPL[A.sub.2] activation. Depolymerization of F-actin by cytochalasin D or latrunculin B reduced complement-dependent [[sup.3]H]AA release, as well as the complement-induced increase in cPL[A.sub.2] activity. These effects were due to inhibition of [[sup.3]H]DAG production and PKC activation, implying interference with PLC. Complement-dependent [[sup.3]H]AA release was also reduced by jasplakinolide, a compound that stabilizes F-actin and organizes actin filaments at the cell periphery, and calyculin A, which induces condensation of actin filaments at the plasma membrane. The latter drugs did not affect [[sup.3]H]DAG production, suggesting their inhibitory actions were downstream of PKC. Neither cytnchalasin D, latrunculin B, nor calyculin A affected association of cPL[A.sub.2] with microsomal membranes, and cytochalasin D and latrunculin B did not alter the localization of the endoplasmic reticulum. Stable transfection of constitutively active RhoA induced formation of stress fibers, stabilized F-actin, and attenuated the complement-induced increase in [[sup.3]H]AA. Thus in GEC, cPL[A.sub.2] activation is dependent, in part, on actin remodeling. By regulating complement-mediated activation of cPL[A.sub.2], the actin cytoskeleton may contribute to the pathophysiology of GEC injury. inflammation: lipid mediators; protein kinases; signal transduction

Glomerular epithelial cell (GEC) injury and apoptosis may contribute to sclerosis in glomerulonephritis. The present study addresses signals that regulate survival of GEC in culture and in the acute puromycin aminonucleoside nephrosis (PAN) model of GEC injury in vivo. Compared with GEC on plastic substratum, adhesion to collagen increased activation of focal adhesion kinase (FAK), c-Src, and ERK and facilitated survival (prevented apoptosis). GEC on plastic exhibited increased caspase-8 and -9 activities, increased expression of the proapoptotic protein, Bax, and decreased the antiapoptotic protein, Bcl-[X.sub.L], compared with collagen. Stable expression of constitutively active mutants of FAK (CD2-FAK) or MEK (R4F-MEK) activated the ERK pathway and supplanted the requirement of collagen for survival. In contrast, expression of a Ras mutant that activates phosphatidylinositol 3-kinase but blocks ERK activation or pharmacological inhibition of the ERK pathway decreased survival on collagen. Glomeruli isolated from rats with PAN revealed increased [[beta].sub.1]-integrin expression, along with increased activation of FAK, c-Src, and ERK, compared with controls. EGF receptor activation was undetectable in PAN. Therefore, adhesion to collagen, resulting in activation of FAK and the Ras-ERK pathway, supports GEC survival. Analogous signals for GEC survival are activated in PAN. apoptosis; glomerulonephritis; protein kinases; signal transduction

Rat fetal kidney mRNA was analyzed by RT-PCR to identify protein kinases. This screening demonstrated expression of a protein kinase consistent with SK2, a group II germinal center kinase and homolog of human Ste20-like kinase (SLK). SK2 mRNA, protein expression, and kinase activity were increased in rat fetal kidney homogenates (embryonic days 17-21) compared with adult controls. In adult kidneys subjected to cross-clamping of the renal artery, followed by reperfusion, SK2 mRNA, protein expression, and kinase activity were increased compared with untreated contralateral controls. By immunohistochemistry, SK2 expression was evident mainly in the cytoplasm of tubular epithelial cells in fetal and adult kidneys. There was also some expression in developing and mature podocytes, but staining of the interstitium was negative. In cultured renal tubular epithelial cells, SK2 kinase activity was increased after incubation with serum, or alter exposure to chemical anoxia plus reexposure to glucose. Stable overexpression of SLK reduced cell proliferation and increased apoptosis and exacerbated apoptosis and necrosis after chemical anoxia plus reexposure to glucose. Thus SK2 is a renal epithelial protein kinase whose expression and activity are increased during development and recovery from acute renal failure, where tubular epithelial regeneration may recapitulate developmental processes. The actions of SK2 appear to be antiproliferative and may facilitate cell injury. protein kinases; renal development; renal failure; signal transduction

GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD+ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD+ turnover, which makes NAD+ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD+ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD+ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers. [ABSTRACT FROM AUTHOR]

Cytosolic PLA2α (cPLA2) and metabolites of arachidonic acid (AA) are key mediators of complement-dependent glomerular epithelial cell (GEC) injury. Assembly of C5b-9 increases cytosolic Ca2+ concentration and results in transactivation of receptor tyrosine kinases and activation of PLC-γ1 and the 1,2-diacylglycerol (DAG)-PKC pathway. Ca2+ and PKC are essential for membrane association and increased catalytic activity of cPLA2. This study addresses the role of the actin cytoskeleton in cPLA2 activation. Depolymerization of F-actin by cytochalasin D or latrunculin B reduced complement-dependent [³H]AA release, as well as the complement-induced increase in cPLA2 activity. These effects were due to inhibition of [³H]DAG production and PKC activation, implying interference with PLC. Complement-dependent [³H]AA release was also reduced by jasplakinolide, a compound that stabilizes F-actin and organizes actin filaments at the cell periphery, and calyculin A, which induces condensation of actin filaments at the plasma membrane. The latter drugs did not affect [³H]DAG production, suggesting their inhibitory actions were downstream of PKC. Neither cytochalasin D, latrunculin B, nor calyculin A affected association of cPLA2 with microsomal membranes, and cytochalasin D and latrunculin B did not alter the localization of the endoplasmic reticulum. Stable transfection of constitutively active RhoA induced formation of stress fibers, stabilized F-actin, and attenuated the complement-induced increase in [³H]AA. Thus in GEC, cPLA2 activation is dependent, in part, on actin remodeling. By regulating complement-mediated activation of cPLA2, the actin cytoskeleton may contribute to the pathophysiology of GEC injury. [ABSTRACT FROM AUTHOR]

Cytosolic PLA2α (cPLA2) and metabolites of arachidonic acid (AA) are key mediators of complement-dependent glomerular epithelial cell (GEC) injury. Assembly of C5b-9 increases cytosolic Ca2+ concentration and results in transactivation of receptor tyrosine kinases and activation of PLC-γ1 and the 1,2-diacylglycerol (DAG)-PKC pathway. Ca2+ and PKC are essential for membrane association and increased catalytic activity of cPLA2. This study addresses the role of the actin cytoskeleton in cPLA2 activation. Depolymerization of F-actin by cytochalasin D or latrunculin B reduced complement-dependent [³H]AA release, as well as the complement-induced increase in cPLA2 activity. These effects were due to inhibition of [³H]DAG production and PKC activation, implying interference with PLC. Complement-dependent [³H]AA release was also reduced by jasplakinolide, a compound that stabilizes F-actin and organizes actin filaments at the cell periphery, and calyculin A, which induces condensation of actin filaments at the plasma membrane. The latter drugs did not affect [³H]DAG production, suggesting their inhibitory actions were downstream of PKC. Neither cytochalasin D, latrunculin B, nor calyculin A affected association of cPLA2 with microsomal membranes, and cytochalasin D and latrunculin B did not alter the localization of the endoplasmic reticulum. Stable transfection of constitutively active RhoA induced formation of stress fibers, stabilized F-actin, and attenuated the complement-induced increase in [³H]AA. Thus in GEC, cPLA2 activation is dependent, in part, on actin remodeling. By regulating complement-mediated activation of cPLA2, the actin cytoskeleton may contribute to the pathophysiology of GEC injury. [ABSTRACT FROM AUTHOR]

Purpose: Glucagon-like peptide-1 (GLP-1) analogues reduce ER stress and inflammation in key metabolic organs, including the liver. However, their effects on heat shock response (HSR) and mitogen-activated protein kinases (MAPKs) have not yet been elucidated. In the present study, we investigate whether the GLP-1 analogue, exendin-4, triggers the expression of HSR and increases MAPK activity under metabolic stress., Experimental Design: The effects of exendin-4 in the presence or absence of palmitic acid (PA; 400 μm) or glucose (30 mm) in the HepG2 liver cell line are assessed using Western blots, quantitative real-time PCR, and label-free proteomics., Results: Heat shock proteins (HSP60, HSP72, HSP90, and GRP78) and other chaperones are not significantly affected by exendin-4 under the conditions tested. In contrast, the presence of exendin-4 alone increases the MAPK phosphorylation levels (JNK, ERK1/2, and p38). For short incubation periods, in the presence of PA or glucose, treatment with exendin-4 exhibits limited effects but significantly attenuates MAPK phosphorylation after a 24-h incubation. Interestingly, canonical signaling pathways, such as EIF2, ILK, PKA, and Rho, are modulated by exendin-4., Conclusion and Clinical Relevance: Identifying new pathways modulated by GLP-1 analogues will provide further insights into their benefits beyond their currently recognized roles in glycemic control, such as MAPK activity, energy homeostasis, and body weight decrease., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chronic low-grade inflammation and dysregulation of the stress defense system are cardinal features of obesity, a major risk factor for the development of insulin resistance and diabetes. Dual-specificity protein phosphatase 1 (DUSP1), known also as MAP kinase phosphatase 1 (MKP1), is implicated in metabolism and energy expenditure. Mice lacking DUSP1 are resistant to high-fat diet-induced obesity. However, the expression of DUSP1 has not been investigated in human obesity. In the current study, we compared the expression pattern of DUSP1 between lean and obese nondiabetic human subjects using subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs). The levels of DUSP1 mRNA and protein were significantly increased in obese subjects with concomitant decrease in the phosphorylation of p38 MAPK (p-p38 MAPK) and PGC-1α and an increase in the levels of phospho-JNK (p-JNK) and phospho-ERK (p-ERK). Moreover, obese subjects had higher levels of circulating DUSP1 protein that correlated positively with various obesity indicators, triglycerides, glucagon, insulin, leptin, and PAI-1 (P < 0.05) but negatively with V̇O(2max) and high-density lipoprotein (P < 0.05). The observation that DUSP1 was overexpressed in obese subjects prompted us to investigate whether physical exercise could reduce its expression. In this study, we report for the first time that physical exercise significantly attenuated the expression of DUSP1 in both the SAT and PBMCs, with a parallel increase in the expression of PGC-1α and a reduction in the levels of p-JNK and p-ERK along with attenuated inflammatory response. Collectively, our data suggest that DUSP1 upregulation is strongly linked to adiposity and that physical exercise modulates its expression. This gives further evidence that exercise might be useful as a strategy for managing obesity and preventing its associated complications., (Copyright © 2015 the American Physiological Society.)