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One of the obstacles in irradiation therapy is cytoresistance, acquired by activation of self-defense systems, such as antioxidant or molecular chaperone systems, to cope with stress. We investigated whether irradiation preconditioning (IP) rendered resistance of the kidney against subsequent ischemia-reperfusion (I/R) and attempted tel elucidate any such protective mechanisms. Mice were irradiated with a total of 4, 6, or 8 Gy using a cesium-137 source irradiator and then, 6 days later, were subjected to 28 mm of bilateral renal ischemia followed by reperfusion. Eight Gy of IP significantly attenuated the increases in plasma creatinine (PCr) and blood urea nitrogen (RUN) concentration, structural damage, lipid peroxidation, superoxide formation, expression and activity of NADPH oxidase (NOX)-2, nitrotyrosine level, and hydrogen peroxide production after I/R in kidney tissues, indicating that IP protects the kidneys from I/R injury. IP markedly increased the activity of NOX, resulting in increased superoxide formation, manganese superoxide dismutase (MnSOD) activity and expression, and heat shock protein (HSP)-27 expression in kidneys. However, it did not change expressions of catalase, copper-zinc superoxide dismutase (CuZnSOD), and HSP-72. To investigate whether the protection afforded by IP was associated with increases in MnSOD and HSP-27 expression triggered by increased superoxide formation after IP, we administered manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin, a superoxide scavenger, to IP mice. This administration blocked superoxide formation and subsequent increases in MnSOD and HSP-27 expression and accelerated the post-I/R increases in PCr and BUN. In conclusion, IP renders kidney resistance to I/R injury, and this resistance is mediated by increased superoxide formation, which activates MnSOD activity and expression as well as HSP-27 expression. [ABSTRACT FROM AUTHOR]

A soluble protein from Saccharomyces cerevisiae acts as a peroxidase but requires a NADPH-dependent thioredoxin system and was named thioredoxin peroxidase (TPx). The role of TPx in aging of stationary cultures of S. cerevisiae was investigated in a wild-type strain and a mutant yeast strain in which the tsa gene that encodes TPx was disrupted by homologous recombination. The occurrence of oxidative stress during aging of stationary cultures of the yeast has been proposed. Comparison of 5-day-old (young) stationary cultures of S. cerevisiae and of cultures aged for 3 months (old) revealed decreased viability, increased generation of reactive oxygen species, modulation of cellular redox status, and increased cellular oxidative damage reflected by increased protein carbonyl content and lipid peroxidation. The magnitude of this stress was augmented in yeast mutant lacking TPx. These results suggest that TPx may play a direct role in cellular defense against aging of stationary cultures presumably, functioning as an antioxidant enzyme. [ABSTRACT FROM AUTHOR]

Singlet oxygen ([sup1]O[sub2]) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP[sup+]-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP[sup+] dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury. [ABSTRACT FROM AUTHOR]

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O[sub2] from oxygen using NADPH as the electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47[supphox] and p67[supphox] migrate to the plasma membrane, where they associate with cytochrome b[sub558], a membrane-integrated flavorhemoprotein, to assemble the active oxidase. In whole cells and under certain circumstances in the cell-free system, the phosphorylation of p47[supphox] mediates the activation process. It has been proposed that conformational changes in the protein structure of cytosolic factor p47[supphox] may be an important part of the activation mechanism. The total protein steady-state intrinsic fluorescence (an emission maximum of 338 nm) exhibited by the tryptophan residues of p47[subphox] was substantially decreased, reflecting on the conformational change that occurs when p47[supphox] was phosphorylated with protein kinase C. We show here that the phosphorylation of p47[supphox] by protein kinase A or mitogen-activated protein kinase, however, had little effect on the intrinsic fluorescence of p47[supphox]. In addition, the present experiments indicate that in the mutant p47[supphox]S379A, only the single S → A mutation appears to be a major importance for the function of p47[supphox], which is able to undergo the change in conformation that takes place when p47[supphox] is phosphorylated by protein kinase C. [ABSTRACT FROM AUTHOR]

A response by Jinu Kim et al. to a letter to the editor about their article "Increased Superoxide Formation Induced by Irradiation Preconditioning Triggers Kidney Resistance to Ischemia-Reperfusion Injury in Mice" in a 2009 issue is presented.

Background. Progression of acute kidney injury to chronic kidney disease (CKD) is associated with inadequate recovery of damaged kidney. Hydrogen sulfide (H2S) regulates a variety of cellular signals involved in cell death, differentiation and proliferation. This study aimed to identify the role of H2S and its producing enzymes in the recovery of kidney following ischemia/reperfusion (I/R) injury. Methods. Mice were subjected to 30 min of bilateral renal ischemia. Some mice were administered daily NaHS, an H2S donor, and propargylglycine (PAG), an inhibitor of the H2Sproducing enzyme cystathionine gamma-lyase (CSE), during the recovery phase. Cell proliferation was assessed via 50-bromo- 20-deoxyuridine (BrdU) incorporation assay. Results. Ischemia resulted in decreases in CSE and cystathionine beta-synthase (CBS) expression and activity, and H2S level in the kidney. These decreases did not return to sham level until 8 days after ischemia when kidney had fibrotic lesions. NaHS administration to I/R-injured mice accelerated the recovery of renal function and tubule morphology, whereas PAG delayed that. Furthermore, PAG increased mortality after ischemia. NaHS administration to I/R-injured mice accelerated tubular cell proliferation, whereas it inhibited interstitial cell proliferation. In addition, NaHS treatment reduced post-I/R superoxide formation, lipid peroxidation, level of GSSG/GSH and Nox4 expression, whereas it increased catalase and MnSOD expression. Conclusions. Our findings demonstrate that H2S accelerates the recovery of I/R-induced kidney damage, suggesting that the H2S-producing transsulfuration pathway plays an important role in kidney repair after acute injury. [ABSTRACT FROM AUTHOR]

Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP + -dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition. [ABSTRACT FROM AUTHOR]

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP[sup +]-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. We investigated whether the ICDH would be a vulnerable target of peroxynitrite anion (ONOO[sup -]) as a purified enzyme, in intact cells, and in liver mitochondria from ethanol-fed rats. Synthetic peroxynitrite and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a peroxynitrite-generating compound, inactivated ICDH in a dose- and time-dependent manner. The inactivation of ICDH by peroxynitrite or SIN-1 was reversed by dithiothreitol. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. Immunoblotting analysis of peroxynitrite-modified ICDH indicates that S-nitrosylation of cysteine and nitration of tyrosine residues are the predominant modifications. Using electrospray ionization mass spectrometry (ESI-MS) with tryptic digestion of protein, we found that peroxynitrite forms S-nitrosothiol adducts on Cys[sup 305] and Cys[sup 387] of ICDH. Nitration of Tyr[sup 280] was also identified, however, this modification did not significantly affect the activity of ICDH. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by peroxynitrite. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and binding of the hydrophobic probe 8-anilino1-naphthalene sulfonic acid. When U937 cells were incubated with 100 µM SIN-1 bolus, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Using immunoprecipitation and ESI-MS, we were also able to isolate and positively identify S-nitrosylated and nitrated mitochondrial ICDH from SIN-1-treated U937 cells as well as liver from ethanol-fed rats. Inactivation of ICDH resulted in the prooxidant state of cells reflected by an increased level of intracellular reactive oxygen species, a decrease in the ratio of [NADPH]/[NADPH + NADP[sup +]], and a decrease in the efficiency of reduced glutathione turnover. The peroxynitrite-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition. [ABSTRACT FROM AUTHOR]

SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined. Here, we show that SAG possesses a potent peroxidase property to decompose hydrogen peroxide in the presence of dithiothreitol (DTT). However, without DTT as a reducing equivalent, SAG was not able to destroy hydrogen peroxide. The peroxidase activity was completely abolished by the reaction of SAG with N -ethylmaleimide (NEM), a chemical modification agent for the sulfhydryl of proteins. These observations suggested that the sulfhydryl of cysteines in SAG could function as strong nucleophiles to destroy hydrogen peroxide. In addition to the peroxidase activity used to remove hydrogen peroxide, SAG also showed t -butylhydroperoxide ( t -BOOH) and fatty acid hydroperoxide-selective peroxidase activity. [ABSTRACT FROM AUTHOR]

Ischemic preconditioning (IPC) by ischemia/reperfusion (I/R) renders resistance to the kidney. Strong IPC triggers kidney fibrosis, which is involved in angiotensin II (AngII) and its type 1 receptor (AT1R) signaling. Here, we investigated the role of AngII/AT1R signal pathway in the resistance of IPC kidneys to subsequent I/R injury. IPC of kidneys was generated by 30 minutes of bilateral renal ischemia and 8 days of reperfusion. Sham-operation was performed to generate control (non-IPC) mice. To examine the roles of AngII and AT1R in IPC kidneys to subsequent I/R, IPC kidneys were subjected to either 30 minutes of bilateral kidney ischemia or sham-operation following treatment with AngII, losartan (AT1R blocker), or AngII plus losartan. IPC kidneys showed fibrotic changes, decreased AngII, and increased AT1R expression. I/R dramatically increased plasma creatinine concentrations in non-IPC mice, but not in IPC mice. AngII treatment in IPC mice resulted in enhanced morphological damage, oxidative stress, and inflammatory responses, with functional impairment, whereas losartan treatment reversed these effects. However, AngII treatment in non-IPC mice did not change I/R-induced injury. AngII abolished the resistance of IPC kidneys to subsequent I/R via the enhancement of oxidative stress and inflammatory responses, suggesting that the AngII/AT1R signaling pathway is associated with outcome in injury-experienced kidney. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress generally occurs in secretory cell types. It has been reported that Leydig cells, which produce testosterone in response to human chorionic gonadotropin (hCG), express key steroidogenic enzymes for the regulation of testosterone synthesis. In this study, we analyzed whether hCG induces ER stress via three unfolded protein response (UPR) pathways in mouse Leydig tumor (mLTC-1) cells and the testis. Treatment with hCG induced ER stress in mLTC-1 cells via the ATF6, IRE1a/XBP1, and eIF2α/GADD34/ATF4 UPR pathways, and transient expression of 50 kDa protein activating transcription factor 6 (p50ATF6) reduced the expression level of steroidogenic 3β-hydroxysteroid dehydrogenase ▵5-▵4-isomerase (3β-HSD) enzyme. In an in vivo model, high-level hCG treatment induced expression of p50ATF6 while that of steroidogenic enzymes, especially 3b-HSD, 17α-hydroxylase/C17-20 lyase (CYP17), and 17β-hydrozysteroid dehydrogenase (17β-HSD), was reduced. Expression levels of steroidogenic enzymes were restored by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Furthermore, lentivirus-mediated transient expression of p50ATF6 reduced the expression level of 3β-HSD in the testis. Protein expression levels of phospho-JNK, CHOP, and cleaved caspases-12 and -3 as markers of ER stress-mediated apoptosis markedly increased in response to high-level hCG treatment in mLTC-1 cells and the testis. Based on transmission electron microscopy and H&E staining of the testis, it was shown that abnormal ER morphology and destruction of testicular histology induced by high-level hCG treatment were reversed by the addition of TUDCA. These findings suggest that hCG-induced ER stress plays important roles in steroidogenic enzyme expression via modulation of the ATF6 pathway as well as ER stress-mediated apoptosis in Leydig cells. [ABSTRACT FROM AUTHOR]

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (1/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to hR. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. hR significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/ (GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-PK1 cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that hDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with hR. [ABSTRACT FROM AUTHOR]

Ultraviolet (UV) radiation has been shown to generate reactive oxygen species (ROS), such as singlet oxygen, superoxide radicals, hydroxyl radicals and hydrogen peroxide in a variety of cells. These ROS have the potential to damage critical cellular components such as DNA, proteins, and lipids and eventually result in physical and chemical damage to tissues that may lead to cell death. The steamed root of Rehmannia glutinosa (Saeng Jihuang, SJH) is reported to have an antioxidant activity. We investigated the effect of SJH on UV-induced apoptosis in U937 cells. Upon exposure to UV, there was a distinct difference between untreated cells and cells pre-treated with 0.5–2 mg/ml SJH for 12 hours in regard to cellular redox status and morphological change to cells. SJH pre-treated cells showed significant suppression of apoptotic features such as DNA fragmentation, damage to mitochondrial function, and modulation of apoptotic marker proteins upon exposure to UV. This study indicates that SJH may play an important role in regulating the apoptosis induced by UV presumably through scavenging of reactive oxygen species. [ABSTRACT FROM AUTHOR]

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. By supplying NADPH for antioxidant systems, we recently demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are some of the primary functions of mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm). In this study, we demonstrate that modulation of IDPm activity in U937 cells regulates ionizing radiation-induced apoptosis. When we examined the regulatory role of IDPm against ionizing radiation-induced apoptosis in U937 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. Upon exposure to 2 gray γ-irradiation, there was a distinct difference between the IDPm transfectant cells in regard to the morphological evidence of apoptosis, DNA fragmentation, cellular redox status, oxidative damage to cells, mitochondrial function, and the modulation of apoptotic marker proteins. In addition, transfection of HeLa cells with an IDPm small interfering RNA decreased the activity of IDPm, enhancing the susceptibility of radiation-induced apoptosis. Taken together, these results indicate that IDPm may play an important role in regulating the apoptosis induced by ionizing radiation, and the effect of IDPm small interfering RNA on HeLa cells offers the possibility of developing a modifier of radiation therapy. [ABSTRACT FROM AUTHOR]

Males are much more susceptible to ischemia/reperfusion (I/R)-induced kidney injury when compared with females. Recently we reported that the presence of testosterone, rather than the absence of estrogen, plays a critical role in gender differences in kidney susceptibility to I/R injury in mice. Although reactive oxygen species and antioxidant defenses have been implicated in I/R injury, their roles remain to be defined. Here we report that the orchiectomized animal had significantly less lipid peroxidation and lower hydrogen peroxide levels in the kidney 4 and 24 h after 30 min of bilateral renal ischemia when compared with intact or dihydrotestosterone-treated orchiectomized males. The post-ischemic kidney expression and activity of manganese superoxide dismutase (MnSOD) in orchiectomized mice was much greater than in intact or dihydrotestosterone-administered orchiectomized mice. Four hours after 30 min of bilateral ischemia, superoxide formation was significantly lower in orchiectomized mice than in intact mice. In Madin-Darby canine kidney cells, a kidney epithelial cell line, 1 mM H2O2 decreased MnSOD activity, an effect that was potentiated by pretreatment with dihydrotestosterone. Orchiectomy prevented the post-ischemic decrease of catalase activity. Treatment of male mice with manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a SOD mimetic, reduced the post-ischemic increase of plasma creatinine, lipid peroxidation, and tissue hydrogen peroxide. These results suggest that orchiectomy accelerates the post-ischemic activation of MnSOD and reduces reactive oxygen species and lipid peroxidation, resulting in reduced kidney susceptibility to I/R injury. [ABSTRACT FROM AUTHOR]

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP+-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46–48 population doubling level (PDL) and then gradually decreased at later PDL. 2′,7′-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon. [ABSTRACT FROM AUTHOR]

NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP+-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor γ were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis. [ABSTRACT FROM AUTHOR]

Deals with a study which investigated the role of nicotinamide adenine dinucleotide phosphate-dependent isocitrate dehydrogenase (ICDH) in cellular defense against heat shock using an Escherichia coli wild-type strain and the ICDH-deficient mutant strain. Materials and methods; Results; Discussion.

The Notch signaling pathway appears to perform an important function in inflammation. Here, we present evidence to suggest that lipopolysaccharide (LPS) suppresses Notch signaling via the direct modification of Notch by the nitration of tyrosine residues in macrophages. In the RAW264.7 macrophage cell line and in rat primary alveolar macrophages, LPS was found to inhibit Notch1 intracellular domain (Notch1-IC) transcription activity, which could then be rescued by treatment with N(G)-nitro-l-arginine, a nitric oxide synthase (NOS) inhibitor. Nitric oxide (NO), which was produced in cells that stably express endothelial NOS (eNOS) and brain NOS (bNOS), also induced the inhibition of Notch1 signaling. The NO-induced inhibition of Notch1 signaling remained unchanged after treatment with 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a guanylyl-cyclase inhibitor, and was not found to be mimicked by 8-bromo-cyclic GMP in the primary alveolar macrophages. With regards to the control of Notch signaling, NO appears to have a significant negative influence, via the nitration of Notch1-IC, on the binding that occurs between Notch1-IC and RBP-Jk, both in vitro and in vivo. By intrinsic fluorescence, we also determined that nitration could mediate conformational changes of Notch1-IC. The substitution of phenylalanine for tyrosine at residue 1905 in Notch1-IC abolished the nitration of Notch1-IC by LPS. Overall, our data suggest that an important relationship exists between LPS-mediated inflammation and the Notch1 signaling pathway, and that this relationship intimately involves the nitration of Notch1-IC tyrosine residues. [ABSTRACT FROM AUTHOR]

Integrin-linked kinase (ILK) is a scaffold and protein kinase that acts as a pivotal effector in integrin signaling for various cellular functions. In this study, we found that ILK remarkably reduced the protein stability of Notch1 through Fbw7. The kinase activity of ILK was essential for the inhibition of Notch1 signaling. Notably, the protein level and transcriptional activity of the endogenous Notch1 intracellular domain (Notch1-IC) were higher in ILK-null cells than in ILK wild-type cells, and the level of endogenous Notch1-IC was increased by the blocking of the proteasome, suggesting that ILK enhances the proteasomal degradation of Notch1-IC. ILK directly bound and phosphorylated Notch1-IC, thereby facilitating proteasomal protein degradation through Fbw7. Furthermore, we found down-regulation of Notch1-IC and up-regulation of ILK in basal cell carcinoma and melanoma patients but not in squamous cell carcinoma patients. These results suggest that ILK down-regulated the protein stability of Notch1-IC through the ubiquitin-proteasome pathway by means of Fbw7. [ABSTRACT FROM AUTHOR]