Claudia von Montfort

Epidermal Growth Factor Receptor Is a Common Mediator of Quinone-induced Signaling Leading to Phosphorylation of Connexin-43 ROLE OF GLUTATHIONE AND TYROSINE PHOSPHATASES

Rat liver epithelial cells were exposed to three quinones with different properties: menadione (2-methyl-1,4-naphthoquinone, vitamin K3), an alkylating as well as redox-cycling quinone, the strongly alkylating p-benzoquinone (BQ), and the non-arylating redox-cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). All three quinones induced the activation of extracellular signal-regulated kinase (ERK) 1 and ERK 2 via the activation of epidermal growth factor receptor (EGFR) and MAPK/ERK kinases (MEK) 1/2. ERK activation resulted in phosphorylation at Ser-279 and Ser-282 of the gap junctional protein, connexin-43, known to result in the loss of gap junctional intercellular communication. Another EGFR-dependent pathway was stimulated, leading to the activation of the antiapoptotic kinase Akt via phosphoinositide 3-kinase. The activation of EGFR-dependent signaling by these quinones was by different mechanisms: (i) menadione, but not BQ or DMNQ, inhibited a protein-tyrosine phosphatase regulating the EGFR, as concluded from an EGFR dephosphorylation assay; (ii) although menadione-induced activation of ERK was unimpaired by pretreatment of cells with N-acetyl cysteine, activation by BQ and DMNQ was prevented; (iii) cellular glutathione (GSH) levels were strongly depleted by BQ. The mere depletion of GSH by application of diethyl maleate EGFR-dependently activated ERK and Akt, thus mimicking BQ effects. GSH levels were only moderately decreased by menadione and not affected by DMNQ. In summary, EGFR-dependent signaling was mediated by protein-tyrosine phosphatase inactivation (menadione), GSH depletion (BQ), and redox-cycling (DMNQ), funneling into the same signaling pathway.

Singlet Oxygen-induced Attenuation of Growth Factor Signaling: Possible Role of Ceramides

Singlet oxygen, an electronically excited form of molecular oxygen, is a primary mediator of the activation of stress-activated protein kinases elicited by ultraviolet A (UVA; 320–400 nm). Here, the effects of singlet oxygen (1O2) on the extracellular signal-regulated kinase (ERK) 1/2 and Akt/protein kinase B pathways were analyzed in human dermal fibroblasts. While basal ERK 1/2 phosphorylation was lowered in cells exposed to either 1O2, UVA or photodynamic treatment, Akt was moderately activated by photochemically generated 1O2 in a phosphoinositide 3-kinase (PI3K)-dependent fashion, resulting in the phosphorylation of glycogen synthase kinase-3 (GSK3). The activation of ERK 1/2 and Akt as induced by stimulation with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) was inhibited by 1O2 generated intracellularly upon photoexcitation of rose Bengal (RB). Photodynamic therapy (PDT)-induced apoptosis is known to be associated with increased formation of ceramides. Likewise, both 1O2 and UVA induced ceramide generation in human skin fibroblasts. The attenuation of EGF- and PDGF-induced activation of ERK 1/2 and Akt by 1O2 was mimicked by stimulation of fibroblasts with the cell-permeable C2-ceramide. Interestingly, EGF-induced tyrosine phosphorylation of the EGF receptor was strongly attenuated by 1O2 but unimpaired by C2-ceramide, implying that, although ceramide formation may mediate the above attenuation of ERK and Akt phosphorylation induced by 1O2, mechanisms beyond ceramide formation exist that mediate impairment of growth factor signaling by singlet oxygen. In summary, these data point to a novel mechanism of 1O2 toxicity: the known 1O2-induced activation of proapoptotic kinases such as JNK and p38 is paralleled by the prevention of activation of growth factor receptor-dependent signaling and of anti-apoptotic kinases, thus shifting the balance towards apoptosis.

PAI-1 plays a protective role in CCl4-induced hepatic fibrosis in mice: role of hepatocyte division

Plasminogen activator inhibitor-1 (PAI-1) is an acute phase protein that has been shown to play a role in experimental fibrosis caused by bile duct ligation (BDL) in mice. However, its role in more severe models of hepatic fibrosis (e.g., carbon tetrachloride; CCl(4)) has not been determined and is important for extrapolation to human disease. Wild-type or PAI-1 knockout mice were administered CCl(4) (1 ml/kg body wt ip) 2x/wk for 4 wk. Plasma (e.g., transaminase activity) and histological (e.g., Sirius red staining) indexes of liver damage and fibrosis were evaluated. Proliferation and apoptosis were assessed by PCNA and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively, as well as by indexes of cell cycle (e.g., p53, cyclin D1). In contrast to previous studies with BDL, hepatic fibrosis was enhanced in PAI-1(-/-) mice after chronic CCl(4) administration. Indeed, all indexes of liver damage were elevated in PAI-1(-/-) mice compared with wild-type mice. This enhanced liver damage correlated with impaired hepatocyte proliferation. A similar effect on proliferation was observed after one bolus dose of CCl(4), without concomitant increases in liver damage. Under these conditions, a decrease in phospho-p38, coupled with elevated p53 protein, was observed; these results suggest impaired proliferation and a potential G(1)/S cell cycle arrest in PAI-1(-/-) mice. These data suggest that PAI-1 may play multiple roles in chronic liver diseases, both protective and damaging, the latter mediated by its influence on inflammation and fibrosis and the former via helping maintain hepatocyte division after an injury.

Singlet oxygen inactivates protein tyrosine phosphatase-1B by oxidation of the active site cysteine.

Abstract Singlet oxygen (1O2), an electronically excited form of molecular oxygen, is a mediator of biological effects of ultraviolet A radiation, stimulating signaling cascades in human cells. We demonstrate here that 1O2 generated by photosensitization or by thermodecomposition of 3,3′-(1,4-naphthylidene)dipropionate-1,4-endoperoxide inactivates isolated protein tyrosine phosphatases (PTPases). PTPase activities of PTP1B or CD45 were abolished by low concentrations of 1O2, but were largely restored by post-treatment with dithiothreitol. Electrospray ionization mass spectrometry analysis of tryptic digests of PTP1B exposed to 1O2 revealed oxidation of active-site Cys215 as the only cysteine residue oxidized. In summary, 1O2 may activate signaling cascades by interfering with phosphotyrosine dephosphorylation.

New Role of Resistin in Lipopolysaccharide-Induced Liver Damage in Mice

Studies in rodents suggest that the adipocytokine resistin causes insulin resistance via impairing normal insulin signaling. However, in humans, resistin may play a more important role in inflammation than in insulin resistance. Whether resistin contributes to inflammation in rodents is unclear. Therefore, the purpose of the present study was to determine the effect of resistin exposure on the basal and stimulated [lipopolysaccharide (LPS)] inflammatory response in mouse liver in vivo. Resistin alone had no major effects on hepatic expression of insulin-responsive genes, either in the presence or absence of LPS. Although it had no effect alone, resistin significantly enhanced hepatic inflammation and necrosis caused by LPS. Resistin increased expression of proinflammatory genes, e.g., plasminogen activator inhibitor (PAI)-1, and activity of mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase 1/2, caused by LPS, but had little effect on anti-inflammatory gene expression. Resistin also enhanced fibrin deposition (an index of hemostasis) caused by LPS. The increase in PAI-1 expression, fibrin deposition, and liver damage caused by LPS + resistin was almost completely prevented either by inhibiting the coagulation cascade, hirudin, or by blocking MAP kinase signaling, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene], indicating that these pathways play a causal role in observed enhanced liver damage caused by resistin. Taken together, the augmentation of LPS-induced liver damage caused by resistin seems to involve, at least in part, up-regulation of hepatic inflammation via mechanisms most likely involving the coagulation cascade and fibrin accumulation. These data also suggest that resistin may have proinflammatory roles in mouse liver independent of its effects on insulin signaling, analogous to previous work in humans.

Contribution of the sympathetic hormone epinephrine to the sensitizing effect of ethanol on LPS-induced liver damage in mice

It is well known that ethanol preexposure sensitizes the liver to LPS hepatotoxicity. The mechanisms by which ethanol enhances LPS-induced liver injury are not completely elucidated but are known to involve an enhanced inflammatory response. Ethanol exposure also increases the metabolic rate of the liver, and this effect of ethanol on liver is mediated, at least in part, by the sympathetic hormone, epinephrine. However, whether or not the sympathetic nervous system also contributes to the sensitizing effect of ethanol preexposure on LPS-induced liver damage has not been determined. The purpose of this study was therefore to test the hypotheses that 1) epinephrine preexposure enhances LPS-induced liver damage (comparable to that of ethanol preexposure) and that 2) the sympathetic nervous system contributes to the sensitizing effect of ethanol. Accordingly, male C57BL/6J mice were administered epinephrine for 5 days (2 mg/kg per day) via osmotic pumps or bolus ethanol for 3 days (6 g/kg per day) by gavage. Twenty-four hours later, mice were injected with LPS (10 mg/kg ip). Both epinephrine and ethanol preexposure exacerbated LPS-induced liver damage and inflammation. Concomitant administration of propranolol with ethanol significantly attenuated the sensitizing effect of ethanol on LPS-induced liver damage. These data support the hypothesis that the sympathetic nervous system contributes, at least in part, to the mechanism of the sensitizing effect of ethanol. These results also suggest that sympathetic tone may contribute to the initiation and progression of alcoholic liver disease.

Activation of ErbB2 by 2-methyl-1,4-naphthoquinone (menadione) in human keratinocytes: role of EGFR and protein tyrosine phosphatases.

Activation of ErbB receptor tyrosine kinases triggers multiple signaling pathways that regulate cellular proliferation and survival. We here demonstrate that ErbB2 is activated via the epidermal growth factor receptor (EGFR) upon exposure of cultured human keratinocytes to 2‐methyl‐1,4‐naphthoquinone (menadione). Both ErbB2 and EGFR are shown to be regulated by protein tyrosine phosphatases that are inhibited by menadione, giving rise to the hypothesis that phosphatase inhibition by menadione may result in a net activation of EGFR and an enhanced ErbB2 phosphorylation. Isolated PTP‐1B, a protein tyrosine phosphatase known to be associated with ErbB receptors, is demonstrated to be inhibited by menadione.

Signaling effects of menadione: from tyrosine phosphatase inactivation to connexin phosphorylation.

Publisher Summary The chapter explains the methods used to delineate the preceding pathway leading from protein tyrosine phosphatase (PTPase) inhibition to connexin43 (Cx43) phosphorylation to deal with (1) receptor tyrosine kinase phosphorylation and PTPase inhibition, (2) extracellular signal-regulated kinases (ERK) activation and the use of pharmacological inhibitors, and (3) Cx43 phosphorylation and gap junctional communication (GJC). These methods are helpful in analyzing menadione-induced signaling pathways in rat liver epithelial cells. The chapter shows that the exposure of cells to menadione leads to the activation of a signaling pathway that results in the activation of ERK 1/2, entailing the phosphorylation of Cx43 and a decrease in gap junctional intercellular communication in rat liver epithelial cells. These effects are brought about by the activation of the EGFR, probably because of the inactivation of a not–yet-identified PTPase regulating the receptor. Menadione is a naphthoquinone derivative (2-methyl-1, 4-naphthoquinone) that is used clinically because of its vitamin K–like properties (it is also termed vitamin K3). It is enzymatically converted to menaquinone-4(2-methyl-3-geranyl-geranyl-1, 4-naphthoquinone, a form of vitamin K2) by mammals. Menadione is capable of inducing phosphorylation of ERK 1/2 and Cx43, resulting in attenuated GJC, which is reversed in the presence of inhibitors of MAPK/ERK kinase (MEK) 1 and MEK 2 (the kinases directly upstream of ERK 1/2) and the epidermal growth factor receptor (EGFR) tyrosine kinase.

Doxorubicin induces EGF receptor-dependent downregulation of gap junctional intercellular communication in rat liver epithelial cells

Abstract Exposure of rat liver epithelial cells to doxorubicin, an anthraquinone derivative widely employed in cancer chemotherapy, led to a dose-dependent decrease in gap junctional intercellular communication (GJC). Gap junctions are clusters of inter-cellular channels consisting of connexins, the major connexin in the cells used being connexin-43 (Cx43). Doxorubicin-induced loss of GJC was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated using inhibitors of ERK activation. Furthermore, activation of the epidermal growth factor (EGF) receptor by doxorubicin was responsible for ERK activation and the subsequent attenuation of GJC. Inhibition of GJC, however, was not by direct phosphorylation of Cx43 by ERK-1/2, whereas menadione, a 1,4-naphthoquinone derivative that was previously demonstrated to activate the same EGF receptor-dependent pathway as doxorubicin, resulting in downregulation of GJC, caused strong phos-phorylation of Cx43 at serines 279 and 282. Thus, ERK-dependent downregulation of GJC upon exposure to quinones may occur both by direct phosphorylation of Cx43 and in a phosphorylation-independent manner.

Redox-active cerium oxide nanoparticles protect human dermal fibroblasts from PQ-induced damage

Recently, it has been published that cerium (Ce) oxide nanoparticles (CNP; nanoceria) are able to downregulate tumor invasion in cancer cell lines. Redox-active CNP exhibit both selective pro-oxidative and antioxidative properties, the first being responsible for impairment of tumor growth and invasion. A non-toxic and even protective effect of CNP in human dermal fibroblasts (HDF) has already been observed. However, the effect on important parameters such as cell death, proliferation and redox state of the cells needs further clarification. Here, we present that nanoceria prevent HDF from reactive oxygen species (ROS)-induced cell death and stimulate proliferation due to the antioxidative property of these particles.