Dahn L. Clemens

Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice

BACKGROUND & AIMS Alcohol abuse is a major cause of liver injury. The pathologic features of alcoholic liver disease develop over prolonged periods, yet the cellular defense mechanisms against the detrimental effects of alcohol are not well understood. We investigated whether macroautophagy, an evolutionarily conserved cellular mechanism that is commonly activated in response to stress, could protect liver cells from ethanol toxicity. METHODS Mice were acutely given ethanol by gavage. The effects of ethanol on primary hepatocytes and hepatic cell lines were also studied in vitro. RESULTS Ethanol-induced macroautophagy in the livers of mice and cultured cells required ethanol metabolism, generation of reactive oxygen species, and inhibition of mammalian target of rapamycin signaling. Suppression of macroautophagy with pharmacologic agents or small interfering RNAs significantly increased hepatocyte apoptosis and liver injury; macroautophagy therefore protected cells from the toxic effects of ethanol. Macroautophagy induced by ethanol seemed to be selective for damaged mitochondria and accumulated lipid droplets, but not long-lived proteins, which could account for its protective effects. Increasing macroautophagy pharmacologically reduced hepatotoxicity and steatosis associated with acute ethanol exposure. CONCLUSIONS Macroautophagy protects against ethanol-induced toxicity in livers of mice. Reagents that modify macroautophagy might be developed as therapeutics for patients with alcoholic liver disease.

Interferon Gamma Enhances Proteasome Activity in Recombinant Hep G2 Cells that Express Cytochrome P4502E1: Modulation by Ethanol

We tested the influence of IFNgamma on proteasome activity in parental Hep G2 cells that do not metabolize ethanol, as well as in recombinant Hep G2-derived cells that express either or both alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1). IFNgamma treatment increased proteasome activity in VL-17A (ADH(+), CYP2E1(+)) and E-47 (CYP2E1(+)) cells, but not in Hep G2, VI-R2 (parental cells with empty vectors) or in VA-13 (ADH(+)) cells. Proteasome activation by IFNgamma correlated positively with the level of CYP2E1 activity. Treatment of VL-17A cells with agents that inhibit CYP2E1 or the inducible nitric oxide synthase (iNOS) or that prevent the formation of peroxynitrite also blocked proteasome activation by IFNgamma, indicating that the proteasome may be directly activated by products of CYP2E1 and iNOS catalysis. While IFNgamma treatment increased proteasome activity, it also decreased CYP2E1 activity. Both effects were mediated via the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) pathway, as both were blocked by the JAK2 inhibitor, tyrphostin AG 490. Ethanol treatment of VL-17A cells also caused a similar blockage of these same IFNgamma-mediated effects, by inhibiting STAT1 phosphorylation. This inhibition was largely due to ethanol metabolism, as 4-methylpyrazole, an ethanol metabolism inhibitor, restored IFNgamma-mediated STAT1 phosphorylation in ethanol-treated cells. Our results lead us to propose that IFNgamma initiates signal transduction, which alters the activities of CYP2E1 and iNOS, thereby producing reactive oxygen species. One of these oxidants, possibly peroxynitrite, may be directly involved in proteasome activation. Ethanol metabolism by VL-17A cells suppresses IFNgamma-mediated induction of proteasome activity, in part, by preventing STAT1 phosphorylation.

Role of MAPK Phosphatase-1 in Sustained Activation of JNK during Ethanol-induced Apoptosis in Hepatocyte-like VL-17A Cells

Ethanol metabolism plays a central role in activating the mitogen-activated protein kinase (MAPK) cascade leading to inflammation and apoptosis. Sustained activation of c-Jun N-terminal kinase (JNK), one of the MAPKs, has been shown to induce apoptosis in hepatocytes. MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate MAPKs in several cells. The aim of the study is to evaluate the role of MKP-1 in sustained JNK activation as a mechanism to explain ethanol-induced hepatocyte apoptosis. VL-17A cells (HepG2 cells overexpressing alcohol dehydrogenase and cytochrome P450-2E1) were exposed to ethanol for different time periods. Western blots were performed for MKP-1, phospho-JNK, phosphotyrosine, and protein kinase Cδ (PKCδ). Electrophoretic mobility shift assays for AP-1 were performed. Apoptosis was measured by caspase-3 activity assay, TUNEL, and 4′,6-diamidino-2-phenylindole staining. Reactive oxygen species were neutralized by overexpressing both superoxide dismutase-3 and catalase genes using lentiviral vectors in VL-17A cells. Ethanol incubation markedly decreased the MKP-1 protein levels to 15% of control levels and was associated with sustained phosphorylation of p46 JNK and p54 JNK, as well as increased apoptosis. VL-17A cells overexpressing superoxide dismutase-3 and catalase, treatment with a tyrosine kinase inhibitor, or incubation of the cells with PKCδ small interference RNAs significantly inhibited the ethanol-induced MKP-1 degradation and apoptosis. Ethanol-induced oxidative stress enhanced the tyrosine phosphorylation of PKCδ, which in turn caused the proteasomal degradation of MKP-1, leading to sustained JNK activation and increased apoptosis in VL-17A cells.

Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells

We previously showed that IFNγ signal transduction was suppressed by ethanol in recombinant HepG2 cells (VL‐17A cells), which express alcohol dehydrogenase (ADH) and CYP2E1. We examined the mechanisms by which STAT1 phosphorylation is blocked by ethanol treatment in VL‐17A cells. Cells were exposed to 0 or 100 mmol/L ethanol for 72 hours. STAT1 phosphorylation was determined by Western blot after 1 hour IFNγ exposure. Reduction of STAT1 phosphorylation by ethanol was prevented in the presence of 4MP, DAS, or uric acid, indicating that the oxidative products from ethanol metabolism were partly responsible for suppression of STAT1 phosphorylation. Ethanol exposure decreased STAT1 tyrosine phosphorylation, whereas serine phosphorylation on the protein was unchanged. These effects of ethanol were mimicked by the peroxynitrite (PN) donor, SIN‐1, which also blocked tyrosine, but not serine phosphorylation, on STAT1. When cells expressing either ADH (VA‐13 cells) or CYP2E1 (E‐47 cells) were exposed to ethanol, both ADH‐ and CYP2E1‐generated products reduced STAT1 phosphorylation. In addition, SOCS1, a negative regulator of IFNγ signaling and which is degraded by the proteasome, was stabilized by ethanol treatment, presumably because of inhibited proteasome activity. Furthermore, SIN‐1 treatment elevated SOCS1 levels in VL‐17A cells, indicating that PN has a role in SOCS1 elevation. In conclusion, under conditions of ethanol‐elicited oxidative stress, PN prevents STAT1 phosphorylation by stabilization of SOCS1, and possibly by nitration of tyrosine residues in STAT1 protein. (HEPATOLOGY 2005;42:1109–1117.)

Sulfation of minoxidil by multiple human cytosolic sulfotransferases

Minoxidil is an antihypertensive agent and hair growth promoter that is metabolized by sulfation to the active compound, minoxidil sulfate. Thermostable phenol sulfotransferase (TS PST or P-PST) was initially thought to catalyze the reaction, and the enzyme was designated minoxidil sulfotransferase (MNX-ST). Information about human ST activities toward minoxidil would be useful in developing the capacity to predict individual responses to minoxidil based on tissue levels of STs. Therefore, human STs were studied from platelet homogenates, partially purified platelets, scalp skin high speed supernatants and COS-1 cell cDNA expressed preparations using a radiochemical enzymatic assay with minoxidil as the substrate. Studies showed the presence of TS PST, TL (thermolabile) PST and MNX-ST activities in human scalp skin. Biochemical properties and correlation studies suggested that in addition to TS PST, the TL PST activity, another ST activity or both were involved in the reaction. Partially purified human platelet TL PST tested with minoxidil and dopamine showed identical thermal stabilities and similar responses to the inhibitors 2,6-dichloro-4-nitrophenol (DCNP) and NaCl. To characterize the activity of TL PST toward minoxidil, several biochemical properties of the enzyme expressed from a human liver cDNA clone were investigated. When assayed with minoxidil and dopamine, thermal stabilities of the expressed enzyme were identical and IC50 values for the inhibitors DCNP and NaCl were similar. It was also demonstrated that cDNA encoded human liver dehydroepiandrosterone sulfotransferase and estrogen sulfotransferase contributed to the sulfation of minoxidil. The results confirm that at least four human STs contribute to minoxidil sulfation. MNX-ST activity represents a combination of ST activities. The data indicate that multiple ST activities should be taken into account in attempts to predict the regulation of minoxidil sulfation and individual responses to minoxidil.

Hepatocyte growth factor protects hepatocytes against oxidative injury induced by ethanol metabolism.

Hepatocyte growth factor (HGF) is involved in many cellular responses, such as mitogenesis and apoptosis protection; however, its effect against oxidative injury induced by ethanol metabolism is not well understood. The aim of this work was to address the mechanism of HGF-induced protection against ethanol-generated oxidative stress damage in the human cell line VL-17A (cytochrome P450 2E1/alcohol dehydrogenase-transfected HepG2 cells). Cells were pretreated with 50 ng/ml HGF for 12 h and then treated with 100 mM ethanol for 0-48 h. Some parameters of oxidative damage were evaluated. We found that ethanol induced peroxide formation (3.3-fold) and oxidative damage as judged by lipid peroxidation (5.4-fold). Damage was prevented by HGF. To address the mechanisms of HGF-induced protection we investigated the cellular antioxidant system. We found that HGF increased the GSH/GSSG ratio, as well as SOD1, catalase, and gamma-glutamylcysteine synthetase expression. To explore the signaling pathways involved in this process, VL-17A cells were pretreated with inhibitors against PI3K, Akt, and NF-kappaB. We found that all treatments decreased the expression of the antioxidant enzymes, thus abrogating the HGF-induced protection against oxidative stress. Our results demonstrate that HGF protects cells from the oxidative damage induced by ethanol metabolism by a mechanism driven by NF-kappaB and PI3K/Akt signaling.

Ethanol-induced oxidative stress suppresses generation of peptides for antigen presentation by hepatoma cells

Processing of peptides for antigen presentation is catalyzed by antigen‐trimming enzymes, including the proteasome and leucine aminopeptidase. Oxidative stress suppresses proteasome function. We hypothesized that in liver cells, processing of antigenic peptides is altered by ethanol metabolism. To address this issue, soluble extracts of ethanol‐metabolizing VL‐17A cells treated with 100 mM ethanol or left untreated were incubated with C‐extended or N‐extended 18‐27 HBV core peptides. Peptide cleavage was measured by recovery after HPLC. Ethanol exposure to VL‐17A cells increased CYP2E1 and decreased proteasome peptidase activities. The latter effect was prevented by treatment of cells with inhibitors, 4‐methylpyrazole and diallyl sulfide. Ethanol treatment of VL‐17A cells also reduced the activity of leucine aminopeptidase (LAP). Consequently, cleavage of both C‐extended and N‐extended peptides by cytosolic extracts was suppressed by pretreatment of cells with ethanol. Treatment of cells with interferon gamma, which enhances proteasome activity, did not reverse the effects of ethanol. Ethanol exerted similar effects on WIFB cells, indicating that its effects are not unique to one cell type. Conclusion: Ethanol metabolism suppresses activities of antigen‐trimming enzymes, thereby decreasing the cleavage of C‐extended and N‐extended peptides. This defect may potentially result in decreased MHC class I–restricted antigen presentation on virally infected liver cells. (HEPATOLOGY 2007;45:53–61.)

Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells

Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH+/CYP2E1+) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.

Animal model of alcoholic pancreatitis: role of viral infections.

Pancreatitis is clearly associated with alcohol abuse, but only a relatively small percentage of people who abuse alcohol develops obvious pancreatitis. These observations have led to the concept that the development of alcoholic pancreatitis requires cofactors. Although diet and smoking have been studied, a clear cofactor has not been identified. The study results presented in this paper were obtained to determine whether viral infection of the pancreas would be a cofactor for alcoholic pancreatitis similar to the role of hepatitis virus infections in the development of alcoholic liver disease. To test this hypothesis, mice were fed ethanol with a liquid diet protocol and infected with coxsackievirus B3 (CVB3). It was found that consumption of alcohol alone did not result in pancreatitis as determined by serum levels of amylase or histologic changes in the pancreas. Two strains of CVB3 that are tropic for the pancreas were used; a virulent and an avirulent strain. Infection of alcohol-fed animals with the virulent CVB3 strain 28 resulted in a more severe pancreatitis than the pancreatitis noted in control animals. Alcohol-fed mice infected with the avirulent strain (GA) showed severe pancreatitis, whereas the infection of control mice did not result in obvious pathologic effects in the pancreas. This model allows mechanistic studies to define the role of viral infection as a cofactor for alcoholic pancreatitis.

Ethanol inhibits the JAK-STAT signaling pathway in freshly isolated rat hepatocytes but not in cultured hepatocytes or HepG2 cells: evidence for a lack of involvement of ethanol metabolism.

OBJECTIVES To understand the molecular mechanism underlying alcoholic liver injury, effects of acute ethanol on the Janus kinase-signal transducer and activator transcription factor (JAK-STAT) signaling in hepatic cells were studied. DESIGNS AND METHODS Effects of acute ethanol on the JAK-STAT signaling in freshly isolated, cultured rat hepatocytes, and HepG2 cells were explored. RESULTS Acute ethanol exposure inhibited IL-6- or IFN-activated STAT in freshly isolated hepatocytes but not in cultured hepatocytes, HepG2 cells, or HepG2 cells transfected with alcohol dehydrogenase (ADH) or cytochrome P450(2E1). The inhibitory action of ethanol in freshly isolated hepatocytes was not antagonized by the ADH inhibitor 4-methylpyrazole (4-MP). Acute exposure of hepatocytes to acetaldehyde or hydrogen peroxide did not suppress STAT activation. Further studies indicated that the loss of response to the inhibitory effect of ethanol was not due to hepatocyte proliferation and collagen contact. CONCLUSIONS Freshly isolated hepatocytes are more susceptible to the inhibitory action of ethanol on the JAK-STAT signaling than cultured hepatocytes or HepG2 cells, which may be implicated in pathogenesis and progression of alcoholic liver disease.