MAP kinase signaling in diverse effects of ethanol

Life Sciences 74 (2004) 2339 – 2364 www.elsevier.com/locate/lifescie

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MAP kinase signaling in diverse effects of ethanol Annayya R. Aroor a,b, Shivendra D. Shukla a,* a

Department of Medical Pharmacology and Physiology, School of Medicine, University of Missouri, Columbia, MO 65212, USA b Kempegowda Institute of Medical Sciences, Bangalore, India Received 19 August 2003; accepted 12 November 2003

Abstract Chronic ethanol abuse is associated with liver injury, neurotoxicity, hypertension, cardiomyopathy, modulation of immune responses and increased risk for cancer, whereas moderate alcohol consumption exerts protective effect on coronary heart disease. However, the signal transduction mechanisms underlying these processes are not well understood. Emerging evidences highlight a central role for mitogen activated protein kinase (MAPK) family in several of these effects of ethanol. MAPK signaling cascade plays an essential role in the initiation of cellular processes such as proliferation, differentiation, development, apoptosis, stress and inflammatory responses. Modulation of MAPK signaling pathway by ethanol is distinctive, depending on the cell type; acute or chronic; normal or transformed cell phenotype and on the type of agonist stimulating the MAPK. Acute exposure to ethanol results in modest activation of p42/44 MAPK in hepatocytes, astrocytes, and vascular smooth muscle cells. Acute ethanol exposure also results in potentiation or prolonged activation of p42/44MAPK in an agonist selective manner. Acute ethanol treatment also inhibits serum stimulated p42/44 MAPK activation and DNA synthesis in vascular smooth muscle cells. Chronic ethanol treatment causes decreased activation of p42/44 MAPK and inhibition of growth factor stimulated p42/44 MAPK activation and these effects of ethanol are correlated to suppression of DNA synthesis, impaired synaptic plasticity and neurotoxicity. In contrast, chronic ethanol treatment causes potentiation of endotoxin stimulated p42/44 MAPK and p38 MAPK signaling in Kupffer cells leading to increased synthesis of tumor necrosis factor. Acute exposure to ethanol activates pro-apoptotic JNK pathway and anti-apoptotic p42/44 MAPK pathway. Apoptosis caused by chronic ethanol treatment may be due to ethanol potentiation of TNF induced activation of p38 MAPK. Ethanol induced activation of MAPK signaling is also involved in collagen expression in stellate cells. Ethanol did not potentiate serum stimulated or Gi-protein dependent activation of p42/44 MAPK in normal hepatocytes but did so in embryonic liver cells and transformed hepatocytes leading to enhanced DNA synthesis. Ethanol has a ‘triangular effect’ on MAPK that involve direct effects of ethanol, its metabolically derived mediators and oxidative stress. Acetaldehyde, phosphatidylethanol,

* Corresponding author. Tel.: +1-573-882-2740; fax: +1-573-884-4558. E-mail address: [email protected] (S.D. Shukla). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0024-3205(03)01196-2

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fatty acid ethyl ester and oxidative stress, mediate some of the effects seen after ethanol alone whereas ethanol modulation of agonist stimulated MAPK signaling appears to be mediated by phosphatidylethanol. Nuclear MAPKs are also affected by ethanol. Ethanol modulation of nuclear p42/44 MAPK occurs by both nuclear translocation of p42/44 MAPK and its activation in the nucleus. Of interest is the observation that ethanol caused selective acetylation of Lys 9 of histone 3 in the hepatocyte nucleus. It is plausible that ethanol modulation of cross talk between phosphorylation and acetylations of histone may regulate chromatin remodeling. Taken together, these recent developments place MAPK in a pivotal position in relation to cellular actions of ethanol. Furthermore, they offer promising insights into the specificity of ethanol effects and pharmacological modulation of MAPK signaling. Such molecular signaling approaches have the potential to provide mechanism-based therapy for the management of deleterious effects of ethanol or for exploiting its beneficial effects. D 2004 Elsevier Inc. All rights reserved. Keywords: Ethanol; MAP kinase; Alcohol; Cell signaling; Signal transduction

Introduction The association of ethanol abuse and organ damage is known since the beginning of recorded medical history. Ethanol abuse leads to dementia, neuro-behavioral problems, liver injury, modulation of immune responses, hypertension and enhances the risk for cancer (Alcohol and Health, 2000). In contrast, moderate alcohol consumption produces protective effect on coronary heart disease (Schenker and Bay, 1998; Zakhari and Gordis, 1999; Meister et al., 2000). Although spectrum of ethanol cytotoxicty is well known, the signal transduction pathways underlying cytotoxicity of ethanol remain poorly defined (Lands, 1995; Baker and Kramer, 1999). The mitogen activated protein kinase (MAPK) pathway is one of the primordial signaling system that exists in all eukaryotic organisms (Pearson et al., 2001). MAP kinase modules are involved in the signal transduction

Fig. 1. A model depicting the pivotal role of MAP Kinase in diverse effects of ethanol

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of a variety of cellular responses including proliferation, differentiation, survival, apoptosis and execution of inflammatory response (Cross et al., 2000; Pearson et al., 2001). At least three distinct and parallel MAP kinase cascades have been identified, including p42/44 MAPK (also called extracellular signal regulated kinases 1 and 2: ERK1/2), the p38 MAP kinases, and c-jun N-terminal kinase or stress activated protein kinases (JNK/SAPK). Although the biochemistry of these pathways is being increasingly appreciated, their contribution to ethanol related tissue injury remains far less clear. In this context recent reports from several laboratories demonstrate that ethanol influences MAPK in diverse cell/organ systems (e.g. liver, pancreas, neuronal etc.) exhibiting different patho-physiological consequences (eg. liver injury, pancreatitis, neuronal toxicity etc., see Fig. 1). This review aims to summarize the progress in this area and also

Fig. 2. A simplified diagram demonstrating the upstream and downstream elements invlolved in MAPK modulations by ethanol. The model also shows the key effector components i.e. ethanol and its metabolites, that influence MAPK. Ethanol effects on nuclear MAPK is also presented to highlight the translocation and consequential effects. Abbreviations: PEth, phosphatidylethanol; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; Please see text for other common abbreviations.

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Table 1 Ethanol modulation of MAPK signaling in diverse cell systems Cells/tissues

Kinase affected Ethanol Characteristics of altered responses treatment

Reference

Hepatocytes/ liver

p42/44 MAPK Acute

Tombes et al., 1998; Lee et al., 2002 Weng and Shukla, 2000a Tombes et al., 1998

Chronic

JNK

Acute Chronic

p38 MAPK

Embryonic BNLCL2 liver cells Transformed hepatocytes Kupffer cells

Acute Chronic

p42/44 MAPK

p42/44 MAPK p42/44 MAPK Chronic p38 MAPK

Stellate cells

Pancreas

Astrocytes

JNK

Acute

p42/44 MAPK P42/44 MAPK p38 MAPK JNK p42/44 MAPK Acute

Ethanol induced slow activation Potentiation of Ang II stimulated MAPK Prolongation of EGF, NGF stimulated MAPK: inhibition of DNA synthesis Potentation of cytokine stimulated MAPK Inhibition of EGF or insulin stimulated MAPK: Inhibition of DNA synthesis Inhibition of MAPK activated by hepatectomy Suppression of potentiation effect on Ang II stimulated MAPK Potentiation of endotoxin stimulated MAPK Increased MAPK activation and protein expression in alcoholic liver disease Ethanol induced activation of JNK Prolongation of TNF stimulated JNK Blunting of response to ethanol Blunting of response after endotoxin stimulation in vivo Increased JNK and increased DNA synthesis and reversal by retinoic acid Prolongation of TNF stimulated MAPK Inhibition of TNF stimulated MAPK Blunting of p38 activation after hepatectomy at later time points Inhibition after hepatectomy Delayed increase after TNF stimulation and induction of apoptosis Potentiation of serum and PMA stimulated MAPK Potentiation of Gi stimulated MAPK and stimulation of DNA synthesis Potentaition of endotoxin stimulated MAPK And increased TNF synthesis Potentaition of endotoxin stimulated MAPK and increased TNF synthesis Stimulation of JNK and increased collagen synthesis Stimulation and increased collagen synthesis Stimulation but no effect on collagen synthesis Stimulation and increased collagen synthesis Stimulation Prolongation of TGF h stimulated MAPK and inhibition of DNA synthesis Increased basal MAPK and potentiation of MAPK activation by PDGF

Nguyen et al., 2000 Chen et al., 1998 Chen et al., 1998 Weng and Shukla, 2003 Koteish et al., 2002a Nguyen and Gao, 2002 Lee et al., 2002 Chen et al., 1998 Lee et al., 2002 Koteish et al., 2002a Chung et al., 2002 Chen et al., 1998 Chen et al., 1998 Koteish et al., 2002a Chen et al., 1998 Pastorino et al., 2003 Reddy and Shukla, 1996

McKillop et al., 1999 Kishore et al., 2002; Cao et al., 2002a Cao et al., 2002a Chen and Davis, 2000 Svegliati-Baroni et al., 2001 Masamune et al., 2002a Masamune et al., 2002a Masamune et al., 2002a Luo and Miller, 1999a Luo and Miller, 1999b

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Table 1 (continued) Cells/tissues

Kinase affected Ethanol Characteristics of altered responses treatment Chronic

Brain

Chronic

Neurons

p42/44 MAPK Acute

Cerebral arteries

p42/44 MAPK Acute p38 MAPK

Aortic rings

p42/44 MAPK

Increased basal activation of MAPK and potentaition of EGF, PDGF and FGF stimulated MAPK Suppression of BDNF induced MAPK During brain development Suppression of MAPK activation during PTP and correlation to synaptic plasticity Suppression of MAPK stimulation induced by picrotoxin and role in synaptic plasticity Increased p42/44 MAPK during ethanol withdrawal Prolongation of NGF stimulated MAPK and enhanced neurite outgrwoth Inhibition of cerebral artery constriction by p38 MAPK and p42/44 MAPK inhibition: Risk for stroke Suppression of contraction of isolated aortic rings by p42/44 MAPK inhibition: Risk for hypertension

Reference Smith and Navratilova, 2003

Climent et al., 2002 Roberto et al., 2003

Kalluri and Ticku, 2002a

Sanna et al., 2002 Roivainen et al., 1995 Yang et al., 2001a,b

Yang et al., 2002

Vascular smooth muscle cells p42/44 MAPK

Heart

JNK

MCF – 7 breast cancer cells

p42/44 MAPK

Hendrickson et al., 1998 Suppression of serum stimulated MAPK and correlation to decreased DNA synthesis: Cardiovascular protection Sato et al., 2002 Suppression of JNK activation during post-ischemic period by wine and ethanol: decreased infarct size: cardioprotection Activation of MAPK and subsequent cell growth: Izevbigie et al., 2002 Increased risk for breast cancer

highlights our current molecular and mechanistic understanding of the MAPK modulations by ethanol (Fig. 2) and its relevance to cellular responses (Table 1).

Diversity of MAPK modulations by ethanol Ethanol, MAPK and alcoholic liver disease The spectrum of alcoholic liver disease includes fatty liver (steatosis), alcoholic hepatitis, cirrhosis and hepatocellular carcinoma (Lieber, 2000). Fatty liver can occur even after moderate occasional

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drinking but usually resolves quickly with abstinence. The important consequences of heavy ethanol consumption on hepatocytes during early alcoholic injury are induction of apoptosis and initiation of fibrogenesis. The alcoholic liver injury is caused by direct effects of ethanol on hepatocytes as well as effects of ethanol on Kupffer cells and stellate cells (Purohit and Russo, 2002; Nanji, 2002). This pattern of early alcoholic liver injury appears to be an important step in the evolution of progressive liver injury and is a direct precursor of cirrhosis. However, signal modulation by chronic ethanol consumption concomitant with initiation or transformation of hepatocytes may result in progression to hepatocelluar carcinoma. MAPK activation and agonist selectivity In rat primary hepatocyte culture, ethanol caused moderate and sustained activation of p42/44 MAPK. Activation of MAPK was observed at 1hr (Lee et al., 2002) and 24 hr (Tombes et al., 1998). In contrast to p42/44 MAPK, activation of JNK by ethanol is robust and prolonged (Lee et al., 2002). Although acute treatment of hepatocytes for 1 hr with ethanol results in activation of p42/44 MAPK, ethanol does not modulate agonist-induced activation of p42/44 MAPK unless hepatocytes are treated with ethanol for at least 16 hr. Furthermore, a 16 hr exposure of hepatocytes to ethanol caused either prolonged activation of p42/44 MAPK (Chen et al., 1998) or potentiation of p42/44 MAPK activation in an agonist selective manner (Weng and Shukla, 2000a). Ethanol potentiated angiotensin II and epinephrine stimulated MAPK but not that activated by EGF or serum. Vasopressin stimulated p42/44 MAPK activation was not affected by ethanol treatment (24 hr) demonstrating further agonist selectivity in ethanol modulation of G-protein mediated p42/44 MAPK stimulation (Weng and Shukla, 2000a). Moreover, stimulation of hepatocytes after 16 hr of ethanol treatment with insulin or EGF or HGF (hepatocyte growth factor) caused prolonged activation of p42/44 MAPK and p38 MAPK but not potentiation of p42/44 MAPK activation (Chen et al., 1998). Similar results were also observed in another study on NGF stimulated p42/44 MAPK in hepatocytes in the presence of ethanol (Tombes et al., 1998). Acute ethanol treatment of freshly isolated hepatocytes but not cultured hepatocytes, resulted in potentiation of interferon h or interferon g induced activation of p42/44 MAPK (Nguyen et al., 2000). Thus ethanol modulation of p42/44 MAPK exhibits differences in agonist selectivity. In contrast to acute effects, chronic ethanol consumption inhibited the activation of p42/44 MAPK in liver induced either by partial hepatectomy or in vitro by agonist stimulation of p42/44 MAPK by EGF and insulin; although basal p42/44 MAPK activity and protein levels were not significantly altered in ethanol treated rats from that of controls (Chen et al., 1998). However, angiotensin II stimulated MAPK activation was not altered after chronic ethanol treatment (Weng and Shukla, 2003). Since angiotensin II infusion caused MAPK activation in rat liver, and angiotensin II levels are increased in alcoholic liver disease, angiotensin modulation of MAPK signaling in hepatocytes may play a role in alcoholic liver injury. In humans, phosphorylation and protein levels of p42/44 MAPK were increased about 3.9 and 3.2 fold respectively in alcoholic liver disease patients compared to healthy control liver tissues (Nguyen and Gao, 2002). Acute exposure to ethanol resulted in prolonged activation of JNK (Chen et al., 1998; Lee et al., 2002) but this response was attenuated in hepatocytes obtained from rats chronically treated with ethanol for 6 weeks (Lee et al., 2002). Interestingly, JNK activation in hepatocytes was also decreased after endotoxin administration in mice that were chronically treated with ethanol for four weeks compared to endotoxin alone treated mice (Koteish et al., 2002a,b). The significance of loss of responses to JNK activation in the progression of liver injury needs further investigation.

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Ethanol, MAPK signaling and liver regeneration In contrast to other cell types (e.g. intestinal epithelial cells) hepatocytes do not terminally differentiate and can enter and exit cell cycle during liver regeneration. Recently, signaling pathways leading to increased DNA synthesis in primary hepatocytes were shown to involve all three types of MAPKs depending on agonist used (Tombes et al., 1998; Chen et al., 1998). Precise involvement of p42/ 44 MAPK on hepatocyte DNA synthesis depends on whether the activation is transient or sustained. Transient activation of MAPK by EGF or NGF correlated with enhanced DNA synthesis in hepatocytes (Tombes et al., 1998). Role of p42/44 MAPK signaling in stimulation of hepatocyte DNA synthesis after partial hepatectomy has also been demonstrated (Talarmin et al., 1999). Chronic treatment with ethanol for 2 months caused inhibition of partial hepatectomy induced p42/44 MAPK activation. Similarly, in vitro experiments also demonstrated inhibition of EGF stimulated MAPK activation after chronic ethanol treatment (Chen et al., 1998). Prolonged activation of p42/44 MAPK pathway was shown to play a prominent role in cell cycle arrest in hepatocytes (Tombes et al., 1998; Chen et al., 1998). The ability of p42/44 MAPK to cause cell cycle arrest was correlated to the expression of the cyclin dependent kinase inhibitor protein p21 Cip-1/WAFI/mda6 (p21). In hepatocytes, NGF alone causes transient activation of p42/44 MAPK and increased DNA synthesis. However, treatment of hepatocytes with ethanol for 24 hr resulted in prolonged activation of p42/44 MAPK and increased expression of p21 with concomitant suppression of DNA synthesis (Tombes et al., 1998). Similarly, prolonged activation of p42/44 MAPK by EGF in the presence of ethanol has been correlated to suppression of DNA synthesis (Chen et al., 1998). p38 MAPK plays a permissive role in hepatocyte proliferation/regeneration (Spector et al., 1997). Chronic ethanol consumption inhibited the activation of p38 MAPK induced either by partial hepatectomy or by various agonists. A selective inhibitor of p38 MAPK decreased hepatocyte proliferation (Chen et al., 1998). In this study, p38 MAPK activation was examined during early periods after partial hepatectomy. In another study, ethanol prevented decrease in the levels of phosphorylated p38 MAPK that occurred at 24 and 48 hr after partial hepatectomy. This is accompanied by reduced expression of cyclin D1 messenger RNA and protein, and increases in other cell-cycle regulators (such as signal transducer and activator of transcription-3 and p27) that are normally inhibited by cyclin D1 (Koteish et al., 2002b). Since p38 MAPK down regulates cyclin D1 expression, ethanol effects on activation of p38 MAPK has been suggested to be responsible for ethanol induced cell cycle arrest and cell-cycle inhibition. This may be an adaptive response that helps ethanol-exposed liver survive eg. in partial hepatectomy. Pro- and anti-apoptotic roles of MAPK signaling in hepatocytes exposed to ethanol Until recently, cell death in most forms of toxin induced liver injury was attributed to necrosis. However, it is now apparent that apoptosis plays an important role in many forms of toxin induced liver injury. The limited data available from both experimental models and human liver pathology suggest increased apoptosis in alcoholic liver disease (Kurose et al., 1997) but the mechanisms by which ethanol induces apoptosis is far from clear. Recent studies suggest the role for activation of p42/44 MAPK, p38 MAPK and JNK depending on the nature of cell insult and the presence and absence of growth factors (Webster and Anwer, 1998; Roberts et al., 2000; Crenesse et al., 2000). TNF induced activation of p38 MAPK was demonstrated to protect hepatocytes against TGF h induced apoptosis (Roberts et al., 2000). In contrast, exposure of hepatocytes to ethanol is associated

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with increased sensitivity of hepatocytes to TNF induced cell killing. This was attributed to enhanced oxidative stress dependent and independent increases in mitochondrial permeability transition after ethanol exposure of hepatocytes (Pastorino and Hoek, 2000). Recently, ethanol-enhanced cell killing after TNF treatment of hepatocytes was noted to be dependent on p38 MAPK signaling which results in caspase-3 activation, mitochondrial depolarization, accumulation of cytochrome c release and translocation of Bax to mitochondria. Interestingly, chronic treatment with ethanol caused a delayed and sustained activation of p38 MAPK that is distinct from early activation of p38 by TNF alone and the early activation of p38 MAPK was not altered by ethanol treatment (Pastorino et al., 2003). JNK activation has been linked to both hepatocyte proliferation and apoptosis (Czaja, 2003; Schwabe et al., 2003). The mechanism (s) by which JNK/AP-1 induces hepatocyte death may depend on time course of activation or modulation of survival through other signal transduction pathways. Sustained JNK activation is required for apoptotic signaling (Chen et al., 1996) and is sufficient for apoptosis (Lei et al., 2002). Transient JNK activation normally occurs in response to growth stimuli and is involved in cell proliferation (Xu et al., 1998). Prolonged activation of JNK/ AP-1 activation is associated with hepatocyte apoptosis and necrosis, particularly resulting from bile acid induced injury (Graf et al., 2002), oxidative stress (Czaja, 2003) and ischemia/reperfusion injury (Bradham et al., 1997). Ethanol causes apoptosis of hepatocytes in vitro and in vivo (Kurose et al., 1997). Ethanol causes persistent activation of JNK and ethanol induced activation of caspase 3 was blocked by JNK inhibitor SP600125 (Lee and Shukla, 2003b). A second mechanism that may account for the different roles of JNK in apoptosis signaling is that biological consequence of JNK may depend on the activation of other signal transduction pathways. Recently, JNK mediated survival signaling was shown to be mediated by Jun D that collaborated with NF-kB to increase antiapoptotic gene expression (Lamb et al., 2002). Target genes that are induced by the antiapoptotic pathways may contain JNK responsive elements in their promoter site. The cIAP-2 gene represents as an example for this class of gene. JNK increases the expression of these genes in cells with activated NF-kB and thus increases cell survival. In contrast, in the absence of a survival pathway that can cooperate with JNK, sustained activation of JNK may lead to apoptosis. Indeed, inhibition of NF-kB in hepatocytes sensitizes hepatocytes for apoptosis induced by TNF (Liu et al., 2002). However, activation of NF-kB in hepatocytes after endotoxin stimulation was blunted by chronic treatment of rats with ethanol (Koteish et al., 2002a) but apoptosis was not enhanced suggesting mechanisms independent of NF-kB may contribute to ethanol induced apoptosis. Since ethanol potentiated LPS induced necrosis, it would be of interest to know whether suppression of NF-kB sensitizes hepatocytes for TNF-induced necrosis. Similarly, increased activation of NFkB caused by stimulation of PI-3 kinase pathway, can suppress the apoptotic effects of activated JNK (Liu et al., 2002). Ethanol also suppressed TNF induced AKT activation in hepatocytes after chronic ethanol treatment (Pastorino et al., 2003). These results suggest that chronic ethanol treatment causes apoptosis by compromising the survival pathway. In contrast to JNK, p42/44 MAPK serves as a protective signaling pathway in bile acid induced apoptosis of hepatocytes. Ethanol also caused activation of p42/44 MAPK; and inhibition of MAPK resulted in increased activation of caspase 3 suggesting the role of p42/44 MAPK as a survival pathway in ethanol induced apoptosis (Lee and Shukla, 2003b). The modulatory effects of MAPK may result from either negative regulation of JNK or JNK independent mechanisms (Czaja, 2003).

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Hepatocytes express two JNK genes (JNK1 and JNK2) and bile acids cause activation of both JNK1 and JNK2 but JNK1 activation caused apoptosis and JNK2 activation protected against apoptosis (Qiao et al., 2003). Interestingly, ethanol caused more pronounced activation of JNK 1 compared to JNK2 (11 fold versus 4.5 fold) suggesting a possible role for this preferential activation of JNK1 in ethanol induced apoptosis of hepatocytes (Lee et al., 2002). Role of MAPK signaling in Kupffer and stellate cells in alcoholic liver injury Increased TNF production from activated Kupffer cells is one of the important cytokine imbalance in alcoholic liver injury (Bird et al., 1990; Hill et al., 1992; Tilg and Diehl, 2000). Recently, treatment of Kupffer cells from ethanol fed rats with endotoxin resulted in increased production of TNF that was blocked by inhibition of p42/44 MAPK signaling pathways (Kishore et al., 2002; Cao et al., 2002a ). Moreover, treatment of Kupffer cells with acetaldehyde resulted in increased production of TNF that was blocked by inhibition of p42/44 MAPK and p38 MAPK signaling pathways (Cao et al., 2002b). These results implicate p42/44 MAPK and p38 MAPK pathways in ethanol and/or acetaldehyde induced activation of Kupffer cells and cytokine mediated hepatocyte dysfunction in alcoholic liver injury. Although activation of p38 MAPK and JNK have been implicated in acetaldehyde induced collagen production by stellate cells (Chen and Davis, 2000), recent studies also demonstrate p42/44 MAPK dependent production of collagen induced by acetaldehyde (Svegliati-Baroni et al., 2001). These data provide support for the role of MAPK signaling in early alcoholic liver injury progressing to cirrhosis. Role of p42/44 MAPK in angiotensin II stimulated stellate cell proliferation and acetaldehyde induced collagen expression in stellate cells is also noteworthy. Studies have shown that angiotensin II stimulated stellate cell proliferation was associated with activation of p42/ 44 MAPK, and inhibition of MAPK signaling resulted in its suppression (Bataller et al., 2000, 2003). Stellate cell proliferation and collagen production by stellate cells are one of the hallmarks of alcoholic liver injury leading to initiation of hepatic fibrogenesis (Reeves et al., 1996). The relevance of angiotensin II stimulated stellate cell proliferation in alcoholic liver disease is more intriguing because of elevated plasma angiotensin II levels in chronic alcoholic patients (Wright et al., 1986). Ethanol, MAPK and pancreatitis Alcohol is one of the causative factors for both acute and chronic pancreatitis (Schenker and Bay, 1998). Activated stellate cells have been implicated in the pathogenesis of pancreatic inflammation and fibrosis (Apte et al., 2000). Exposure of cultured rat pancreatic stellate cells to ethanol led to cell activation and intracellular lipid peroxidation (Masamune et al., 2002a). Stellate cells also exhibited ethanol inducible alcohol dehydrogenase (ADH) activity. Inhibition of ADH by 4-methylpyrazole prevented ethanol-induced stellate cell activation. Ethanol and acetaldehyde also activated AP-1 but not NF-kB. In addition, they activated all three classes of MAP kinases. Ethanol and acetaldehyde induced activation of AP-1 and MAPKs was blocked by the antioxidant N-acetylcysteine, suggesting a role of oxidative stress in MAPK signal transduction. However, ethanol and acetaldehyde induced alpha(1) procollagen gene expression was inhibited by p38 MAPK inhibitor but not by p42/44 MAPK inhibitor. These results suggest involvement of p38 MAPK in the pathogenesis of ethanol induced pancreatic injury (Masamune et al., 2002a,b).

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Ethanol, MAPK and increased risk for cancer Cirrhosis caused by chronic ethanol abuse remains a common clinical precursor for the development of hepatocellular carcinoma (Lieber, 2000). Depending on the proliferation status of hepatocytes, ethanol differentially regulates p42/44 MAPK activation and mitogenesis. While serum alone was not a potent stimulus for normal hepatocyte DNA synthesis, it did stimulate proliferation of embryonic nontransformed hepatocyte BNLCL2 cells (Reddy and Shukla, 1996) or transformed hepatocytes (McKillop et al., 1999). These findings suggest distinct mechanisms in normal versus transformed hepatocytes. More intriguing is the involvement of G-proteins. While mastoparan G, an activator of Gi, is ineffective in causing activation of p42/44 MAPK or DNA synthesis in normal rat hepatocytes, it caused both activation of p42/44 MAPK and stimulation of DNA synthesis in transformed rat hepatocytes (McKillop et al., 1999). Ethanol also potentiated mastoparan G induced p42/44 MAPK activation and DNA synthesis in transformed hepatocytes. Moreover, ethanol significantly increased the expression of Gi in transformed hepatocytes compared to normal hepatocytes. In contrast to normal hepatocytes (Weng and Shukla, 2000a), ethanol potentiated p42/44 MAPK activation induced by 10% FBS or PMA in embryonic liver cells and the potentiation effect was pertussis toxin sensitive. However, ethanol did not potentiate EGF stimulated p42/44 MAPK activation in embryonic liver cells (Reddy and Shukla, 1996). In this context, it is interesting to note that hepatitis C virus core protein, synergistically activated p42/44 MAPK cascade with tumor promoter PMA but not with TGFa or EGF in HepG2 cells (Hayashi et al., 2000). This is similar to studies in embryonic liver cells with ethanol. Thus ethanol potentiation of serum or PMA (tumor promoter) activated p42/44 MAPK in transformed hepatocytes argues for the role of p42/44 MAPK signaling as one of the targets for the proliferation of already intiated hepatocellular carcinoma after ethanol exposure. Chronic ethanol consumption in humans is also accompanied by increased p42/44 MAPK activation. Furthermore, human hepatocellular carcinoma is also characterized by increased p42/44MAK activation and Gi protein expression (McKillop et al., 1998; Schmidt et al., 1997). In hepatocytes from rats chronically treated with ethanol for 6 months (as opposed to 6 weeks, see Lee et al., 2002), the basal activity of JNK was increased and ethanol induced increase in JNK activation was reversed by retinoic acid administration (Chung et al., 2002). Ethanol induced increase in JNK was correlated to increased activation of SEK as well as decreased expression of MAPK phosphatase (MKP-1) (Chung et al., 2002). Retinoic acid induced the expression of MAPK phosphatase. The findings of rats chronically treated with ethanol for several months also included enhanced liver proliferation and these results suggest the role for JNK in ethanol induced proliferation of hepatocytes which may increase the risk for hepatcocellular carcinoma. Numerous epidemiological studies support the existence of a positive association between ethanol intake and risk of breast cancer (Singletary, 1997). However, the role of ethanol or its metabolites on breast neoplasm has not been characterized. Increased activation of p42/44 MAPK was found in breast cancer in several studies. (Sivaraman et al., 1997; Mueller et al., 2000; Santen et al., 2002). In a large recent study, an elevated p42/44 MAPK activity was found in 131 samples from primary breast tumors when compared to 18 normal tissues adjacent to tumors (Mueller et al., 2000). In this group, higher activity of p42/44 MAPK correlated to more severe form of disease. Recent studies on the effect of ethanol on growth of MCF-7 human breast cancer line indicate activation of p42/44 MAPK by pathophysiologically relevant concentration (65 mM) of ethanol, accompanied by 200% increase in the cell growth in a MEK inhibitor sensitive fashion. These results suggest that MAPK signaling pathways are

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crucial for ethanol induced MCF-7 cell growth (Izevbigie et al., 2002). However, MCF-7 is an advanced tumor cell line and the relationship of its response to ethanol and that of normal breast may be of interest but remains to be established. Ethanol, MAPK and neurotoxicity Ethanol effects on neurons and glia include reactive astrogliosis, neuronal apotosis and significant reduction in glia after long term ethanol consumption (Goodlett et al., 1997; Davis and Vernadakis, 1984; Korbo, 1999). Ethanol both inhibits as well as potentiates proliferation of cultured atsrocytes depending on the presence of serum or specific growth factors in human as well as rat astrocytes (Kane et al., 1996; Luo and Miller, 1998; Aroor and Baker, 1997). Recent studies have examined the effects of ethanol on MAPK signaling in astrocytes. TGF h1 caused sustained activation p42/44 MAPK in B104 neuroblastoma cells that correlated to inhibition of DNA synthesis. Ethanol caused potentiation of TGF h1 induced inhibition of DNA synthesis which was correlated to ethanol induced prolongation of TGF h induced activation of p42/44 MAPK (Luo and Miller, 1999a). Basal MAPK as well as activation of MAPK by platelet derived growth factor (PDGF) are increased by acute exposure of astrocytes to ethanol in vitro (Luo and Miller, 1999b). Exposure to ethanol for 4 days resulted in increase in the basal activity of p42/44 MAPK and potentiation of its activation by EGF, PDGF and FGF in astrocytes (Smith and Navratilova, 2003). These results suggest a role of MAPK in reactive astrogliosis induced by ethanol. During brain development BDNF (brain-derived neutrotrophic factor) activates p42/44 MAPK and PI3 K and this signaling is significantly reduced by ethanol exposure (Climent et al., 2002). However, activation of p42/44 MAPK by carbachol may be necessary but not sufficient for its mitogenic effect in 1321N1 human astrocytoma cells and it does not represent a target for ethanol induced inhibition of DNA synthesis elicited by muscarinic receptors(Yagle et al., 2001). These results suggest agonist selective effects of ethanol on modulation of astrocyte proliferation and brain development. Process of learning and memory in mammalian brains involve the establishment of new synaptic connections regulated by several intracellular signaling pathways and there is an emerging role for p42/44 MAPK signaling in synaptic plasticity and memory (Sweatt, 2001). Chronic intermittent ethanol treatment caused suppression of p42/44 MAPK activation during post tetanic potentiation (PTP) as well as impairment of PTP. However, MAPK activation was enhanced significantly during long term potentiation (LTP) in rats after 5 days of ethanol withdrawal. Therefore, a role of MAPK signaling in ethanol modulation of depression of hippocampal CA1 LTP can be expected. Alterations in MAPK signaling could play an important role in ethanol induced changes in synaptic plasticity associated with the effects of ethanol abuse on learning and memory process (Roberto et al., 2003). Intraperitoneal administration of ethanol into mice inhibited phosphorylation of MAPK in both the cytosolic and nuclear fractions of the cerebral cortex (Kalluri and Ticku, 2002a) and may involve the modulation of GABA A receptor function (Kalluri and Tichku, 2002b). It was further demonstrated that acute ethanol inhibited while chronic treatment increased basal phosphorylation of MAPK (Kalluri and Ticku, 2003). The nature of alcohol consumption appears to affect magnitude of MAPK responses in brain. Activation of p42/44 MAPK was decreased in brain after both intermittent and continuous exposure to ethanol. However, increase in p42/44 MAPK activation during ethanol withdrawal was higher after intermittent ethanol exposure compared to continuous exposure of rats to ethanol (Sanna et al., 2002).

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Aberrant neurite outgrowth caused by neurotoxicants can affect neuronal function, neuron-glia interactions and impaired neuronal function. Ethanol has been shown to potentiate neurite outgrowth induced by nerve growth factor (NGF) and this process was dependent on PKC mediated ethanol potentiation of p42/44 MAPK activated by NGF (Roivainen et al., 1995). These results underscore the importance of MAPK signaling in aberrant neurite outgrowth causing dysregulated neuronal function after ethanol abuse. MAPK, ethanol and cardiovascular diseases Chronic alcohol abuse is associated with a higher incidence of cardiovascular disorders such as stroke, high blood pressure and alcoholic cardiomyopathy (Hanna et al., 1997; Alcohol and Health, 2000). Ethanol, MAPK, hypertension and stroke The link between ethanol and hypertension is well established, yet the mechanism by which ethanol raises blood pressure remains elusive. Acute elevation of blood pressure in vivo caused by hypertensive agents such as phenylephrine or angiotensin II was shown to cause transient activation of p42/44 MAPK and JNK in rat aorta, carotid and femoral arteries (Takahashi and Berk, 1998) and contraction of rat aortic strips was associated with p42/44 MAPK activation (Pyles et al., 1997). Ethanol induces concentration dependent contractions of isolated aortic rings in vitro and this effect is suppressed by treatment with MEK inhibitor suggesting a possible role for MAPK signaling in hypertension in humans associated with chronic ethanol consumption (Yang et al., 2002). The risk for developing cerebral vascular diseases such as stroke, clearly increases with increase in alcohol consumption (Truelsen et al., 1998; Altura and Altura, 1999). Acute treatment with ethanol produces prolonged constriction of cerebral blood vessels, suggesting that such vasoconstrictive actions are involved in hypoxic, ischemic and hemorrhagic actions of ethanol in the brain (Zhang et al., 1993). Recently, role of MAPK signaling in ethanol induced vasoconstriction in intact canine basilar arteries was reported. In this case, ethanol induced contractions and elevated increase in intracellular calcium were attenuated by specific antagonist of p38 MAPK and p42/44 MAPK (Yang et al., 2001a,b; see also, Sachinidis et al., 1999). The other side of the coin: Cardioprotection by ethanol and MAPK signaling Both epidemiological and experimental studies indicate that moderate alcohol consumption is associated with a reduced incidence of mortality and morbidity from coronary heart disease (Criqui and Ringel, 1994; Kannel and Ellison, 1996; Zakhari and Gordis, 1999; Klatsky, 2003). Ethanol induced reduction in neointimal formation has been reported in both rabbit and pig models (Merritt et al., 1997; Liu et al., 1996) and ethanol was shown to suppress serum stimulated p42/44 MAPK activation and DNA synthesis in vascular smooth muscle cells (Hendrickson et al., 1998). The consumption of red wine imparts a greater benefit in the prevention of coronary heart disease than the consumption of other alcoholic beverages (Klatsky, 2003; Sato et al., 2002). The cardioprotective effects of red wine have been attributed to several polyphenolic antioxidants including resveratrol and proanthocyanidines. Recently, feeding rats with wine extract or its polyphenolic antioxidants as well as alcohol resulted in the improvement of post-ischemic ventricular function. Additionally, both wine and ethanol triggered a signal transduction cascade by reducing proapoptotic factors such as JNK-1 and c-jun thereby

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potentiating an antideath signal. This resulted in reduction of myocardial infarct size and cardiomyocyte apoptosis (Sato et al., 2002).

Upstream pathways mediating ethanol modulation of p42/44 MAPK signaling Role of tyrosine kinases Ethanol effects on p42/44 MAPK signaling may occur through modulation of several upstream components including receptor and non-receptor tyrosine kinases (Fig. 2). Ethanol effect on receptor tyrosine kinases was first demonstrated with EGF. In A431 cells, ethanol treatment in vitro caused a dose dependent biphasic effect on EGF-R tyrosine kinase activity; stimulatory at low (0.1 mM) and inhibitory at high concentrations (100 mM) of ethanol (Thurston and Shukla, 1992; Shukla et al., 1993). Chronic ethanol treatment results in suppression of p42/44 MAPK activation induced by EGF. In primary rat hepatocytes, EGF-receptor binding activity and EGF-induced receptor autophosphorylation were suppressed at all EGF concentrations in hepatocytes obtained from ethanol fed rats compared to pair fed rats (O’Rourke et al., 1997). However, there were no changes in total EGF-R protein and EGF-receptor tyrosine kinase activity in hepatocytes form ethanol fed rats (Zhang et al., 1996). Chronic ethanol consumption also disrupted complex formation between EGF-R and PLCg (Zhang and Farrel, 1999). Chronic ethanol treatments also inhibited TGF-a induced EGF-R autophosphorylation and binding activity at different TGF-a concentrations (Tuma et al., 1998). Ethanol alone enhances tyrosine kinase activity in cerebral vessels and inhibition of tyrosine kinase activation abolished ethanol induced p42/44 MAPK activation (Yang et al., 2001a,b). In primary cultures of hepatocytes, ethanol induced p42/44 MAPK activation was inhibited by the tyrosine kinase inhibitor, genistein (Weng and Shukla, 2002). Ethanol also inhibited tyrosine autophosphorylation of IGF-I receptor in SH-SY5Y neuroblastoma cells (Seiler et al., 2001), c6 rat glioblastoma cells (Resnicoff et al., 1994) and Swiss 3T3 cells (Resnicoff et al., 1993). Ethanol did not affect EGF-stimulated p42/44 MAPK, but potentiated serum stimulated p42/44 MAPK in embryonic BNLCL2 liver cells (Reddy and Shukla, 1996). This implies involvement of non-receptor tyrosine kinases such as src. Indeed, treatment of hepatocytes with ethanol resulted in angiotensin II stimulated p42/44 MAPK with concomitant activation of src and focal adhesion kinase (Weng and Shukla, 2000b). In canine cerebral arteries, ethanol induced contraction was inhibited by SH2 domain inhibitor peptide (Yang et al., 2001a,b). Thus, ethanol modulation of p42/44 MAPK signaling occurs through ethanol effects on both receptor and non-receptor tyrosine kinases. Role of protein kinase C and G-protein coupling distal to protein kinase C activation Other pathways activating p42/44 MAPK involve seven transmembrane receptors coupled to Gproteins Gi and Gq. The PKC activates ras, raf or MEK1/2 leading to the activation of p42/44 MAPK. Ethanol caused activation of p21 ras and its stimulation by angiotensin II was potentiated by ethanol in hepatocytes (Park et al., 2002). In hepatocytes, ethanol stimulated PKC and ethanol induced stimulation of p42/44 MAPK was inhibited by PKC inhibitors (Nguyen et al., 2000). Ethanol induced contraction of cerebral arterial smooth muscle is inhibited by PKC and PI-3 kinase inhibitors (Yang et al., 2001a,b) but the role of these kinases as upstream regulators for MAPKs have not been well examined. Ethanol

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activated PKC and potentiated NGF induced p42/44 MAPK activation by a PKC dependent mechanism in PC 12 cells (Roivainen et al., 1995). Ethanol also caused potentiation of PDGF mediated PKC dependent activation of p42/44 MAPK in cultured cortical astrocytes (Luo and Miller, 1999b). However, ethanol induced prolonged activation of EGF stimulated p42/44 MAPK activation in hepatocytes has been shown to be insensitive to PKC inhibition (Chen et al., 1998), thus also suggesting PKC independent mechanisms for ethanol modulation of p42/44 MAPK in hepatocytes. G-proteins coupled to different second messenger systems are affected by ethanol. In neuroblastoma cells, acute doses of ethanol activated adenylate cyclase, whereas in cells chronically treated with ethanol, adenylate cyclase and G-protein coupled PLC activation were inhibited (Kelly et al., 1995). Ethanol potentiation of p42/44 MAPK activation by serum or PMA in embryonic BNLCL2 liver cells and ethanol potentiation of angiotensin II stimulated p42/44 MAPK activation in primary cultured hepatocytes, both were suppressed by pertussis toxin treatments (Reddy and Shukla, 1996; Weng and Shukla, 2000a). This suggests a potential role for Gi proteins in ethanol modulation of p42/44 MAPK signaling in hepatocytes. It is therefore likely that, in relation to MAPK activation, ethanol modulates a novel component of protein kinase C pathway that is Gi dependent but distal to protein kinase C activation. The mechanism of action of ethanol on G-proteins is not understood clearly. Ethanol has been proposed to act both at the level of function and expression of G-proteins. The longer time required for the effect of ethanol in embryonic liver cells and hepatocytes favors the view that changes in the expression of G-proteins may be involved. It was demonstrated that ethanol causes an increase in the expression of Gia subunits, whereas it inhibits the expression of Gsa subunits in regenerating rat liver (Diehl et al., 1992). Ethanol treatment also causes increased expression of Gi in a rat model of hepatocellular carcinoma (McKillop et al., 1999).

Relevance of ethanol metabolism on MAPK signaling Oxidative stress and MAPK Signaling Although molecular mechanisms underlying ethanol- induced cellular effects are not clearly understood, there is evidence that ethanol and/or its metabolites are directly injurious to the liver and other tissues. The mediators that are important for ethanol-induced effects include both oxidative and non-oxidative metabolites (Shukla et al., 2001; Hoek and Pastorino, 2002). Increased oxidative stress occurs in liver after both acute and chronic ethanol exposure (Bailey and Cunningham, 1998). Ethanol induced oxidative stress in hepatocytes can occur acutely through ethanol metabolism or chronically through overexpression of Cytochrome P450 isoform 2E1 (CYP2E1). The oxidative metabolism of ethanol within hepatocytes elicits a range of mediators including the generation of reactive oxygen species (ROS) and the formation of acetaldehyde. The microsomal ethanol oxidizing system (MEOS) appears to produce active hydroxyl radicals. CYP2E1 has been shown to be able to produce superoxide anion, hydrogen peroxide and ethanol-derived hydroxyethyl free radicals. Reactive oxygen intermediates are also produced when acetaldehyde is oxidized to acetate via xanthine oxidase or mitochondrial aldehyde dehydrogenase (Lieber, 2000). Other sources of free radical generation include NADH oxidation by aldehyde oxidase during ethanol oxidation (Kono et al., 2000). In addition to the generation of oxidative stress in hepatocytes by various pathways, significant production of reactive

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oxygen species occurred due to ethanol effects on endothelial cells and Kupffer cells (Hasegawa et al., 2002). Oxidative stress has been shown to activate all three MAPKs but the magnitude of stimulation can depend on dose and potency of stimulus (Owuor and Kong, 2002). The upstream pathways regulating oxidative stress induced activation of MAPKs are redox sensitive. Growing evidence indicates that EGFR and PDGF-R trans-activation is redox sensitive (Griendling et al., 2000). In hepatocytes, ethanol causes accumulation of ROS in vitro (Kurose et al., 1997) and hydrogen peroxide induced activation of p42/44 MAPK is inhibited by tyrosine kinase inhibitors and PKC inhibitors whereas hydrogen peroxide induced activation of JNK was independent of tyrosine kinase and PKC activation (Lee and Shukla, 2003a). Since ras signaling pathway is directly activated by oxidative stress (Lander et al., 1995), ethanol may cause ras dependent activation of MAPK. Indeed, ethanol causes activation of ras in primary cultured hepatocytes (Park et al., 2002). ASK-1 is another upstream kinase that is redox senstive and causes activation of p38MAPK and JNK (Matsuzawa et al., 2002). Hepatocyte CYP2E1 overexpression occurs in alcoholic and non-alcoholic steatohepatitis and has been implicated as a causal factor in liver injury. Stable overexpression of CYP2E1 sensitized hepatocytes to necrosis by TNF. CYP2E1 overexpression itself caused a slight increase in JNK activity and led to a prolonged JNK activation in response to TNF. TNF induced necrosis in hepatocytes was markedly decreased when AP-1 function was blocked by the expression of TAM67 (Czaja, 2003). Ethanol induced oxidative stress also affects other tissues such as pancreas (Masamune et al., 2002a,b), brain (Sun and Sun, 2001) and heart (Sato et al., 2002). Exposure of cultured rat pancreatic stellate cells to ethanol or acetaldehyde led to cell activation and intracellular lipid peroxidation. Ethanol and acetaldehyde induced activation of AP-1 and MAPK was blocked by the antioxidant Nacetylcysteine, suggesting the role of oxidative stress in MAPK signal transduction induced by ethanol and acetaldehyde in pancreatic cells. Acetaldehyde Acetaldehyde is a highly reactive product of the oxidative metabolism of ethanol. In the hepatocytes, there are three known enzymes capable of ethanol oxidation to acetaldehyde: cytosolic alcohol dehydrogenase (ADH), microsomal ethanol oxidizing system (MEOS, e.g., CYP 2E1), and peroxisomal catalase. While ADH appears to be the major pathway of ethanol oxidation under normal conditions and at low ethanol concentrations (Crabb et al., 1987); MEOS activity increases after chronic ethanol administration and catalase may play a significant role in ethanol oxidation at high blood portal ethanol levels (Perrot et al., 1989). Many of the functional and structural alterations in the liver produced by ethanol consumption have been attributed to ethanol oxidation since liver is the major organ of ethanol metabolism. In addition to endogenously produced acetaldehyde, hepatocytes are exposed to further burden of acetaldehyde present in portal vein that is derived from intracolonic production and accumulation of acetaldehyde. Ethanol ingested orally is oxidized to acetaldehyde in the colon by a bacteriocolonic pathway. Due to the low aldehyde dehydrogenase activity of colonic mucosa, acetaldehyde accumulates in the colon. Accordingly, during ethanol oxidation, the highest acetaldehyde levels (up to 3.0 mM) are found in colon (up to 3.0 mM) (Salaspuro, 1996). Intracolonic acetaldehyde can be absorbed by the portal vein. Acetaldehyde administration in drinking water has been shown to cause ethanol-like liver injury including steatosis (Salaspuro, 1996). Acetaldehyde is considered as an important mediator of ethanol induced deleterious effects on hepatocytes including inhibition of

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hormone-stimulated hepatocyte DNA synthesis (Carter and Wands, 1988) and induction of apoptosis (Kurose et al., 1997). Acute treatment of rat hepatocytes with acetaldehyde causes activation of p42/44 MAPK (Lee et al., 2002). The effects of acetaldehyde on p42/44 MAPK were also seen with ethanol treatment but ethanol caused peak activation of p42/44 MAPK at 1 hr compared to 10 min by acetaldehyde. The delayed time in activation of p42/44 MAPK by ethanol compared to that by acetaldehyde suggests that acetaldehyde might be a critical mediator of the effects of ethanol on MAP kinases. However, it is possible that ethanol can cause activation of p42/44 MAPK by both acetaldehyde dependent and independent manner. The role of acetaldehyde in the modulation of ethanol effects is further supported by increased expression of Gi proteins in hepatocellular carcinoma cell lines by acetaldehyde and this effect is blocked by alcohol dehydrogenase inhibition (Kovach et al., 2001). Acetaldehdye is a potential mediator of ethanol induced activation of pancreatic stellate cells and fibrogenesis in the pancreas (Apte et al., 2000; Gukovskaya et al., 2002; Masamune et al., 2002a,b). Stellate cells also exhibited ethanol inducible ADH activity. Inhibition of ADH by 4-methylpyrazole prevented ethanol-induced stellate cell activation. Ethanol and acetaldehyde also activated AP-1 but not NF-kB. In addition, they activated all three classes of MAP kinases. Phosphatidylethanol Although direct acute effects of ethanol, ethanol induced oxidative stress and acetaldehyde can account for some of the effects of ethanol on hepatocytes, pathways that are independent of direct acute effects of ethanol or acetaldehyde accumulation have been implicated in hepatocytes exposed to longer periods of time, especially for agonist stimulated responses. For example, ethanol potentiation of TNF-a induced apoptosis of primary cultured hepatocytes occurred after two days of ethanol treatment and the effects were not significantly suppressed by inhibition of alcohol dehydrogenase. Similarly, exposure for 24 hrs with ethanol (100 mM) potentiated p42/44 MAPK activation by angiotensin II in rat hepatocytes but potentiation effects were not observed during shorter (1–8 hrs) exposures to ethanol or acetaldehyde (Weng and Shukla, 2000a). In this regard, phosphatidylethanol (PEth) merits attention here. It is a novel ethanol-derived bioactive lipid formed by phospholipase D catalyzed transphosphatidylation reaction (Shukla and Halenda, 1991). This abnormal phospholipid is formed in vivo in various animal tissues, including brain, during ethanol exposure (Alling et al., 1984). Chronic ethanol use has also been reported to cause activation of phospholipase D. Formation of PEth, in response to PMA stimulation, is increased in lymphocytes from chronic alcoholics as compared to lymphocytes obtained from individuals who use alcohol moderately (Mueller et al., 1988). Phosphatidylethanol accumulates in hepatocytes after ethanol administration in vivo and in vitro (Alling et al., 1984; Gustavsson, 1995). The accumulation of PEth may account for some of the patho-physiology associated with chronic ethanol use as PEth is degraded slowly and may accumulate in the tissues after long-term ethanol use. PEth induces tolerance to fluidization by ethanol in artificial bilayers and inhibits ethanol activation of Na-K ATPase in crude brain membranes (OmodeoSale et al., 1991), all of which have been attributed to chronic ethanol treatment. PEth also stimulated phosphoinositide hydrolysis in neuronal cells, as did ethanol (Lundqvist et al., 1993). PEth activates protein kinase C (Asaoka et al., 1988; Asaoka, 1989; Aroor and Baker, 1996) and phospholipase A2 (Chang et al., 2000) suggesting a mediatory role for it in ethanol modulation of signal transduction. Recently, the effects of PEth on angiotensin II stimulated p42/44 MAPK activation were reported (Aroor et al., 2002). Treatment of hepatocytes with ethanol for 24 hr caused accumulation of PEth and

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exogenously added PEth potentiated angiotensin II stimulated p42/44 MAPK activation in a dose dependent manner. However, PEth was not effective in potentiation of p42/44 MAPK activated by either EGF or vasopressin thus mimicking the known agonist selectivity for this ethanol effect (Weng and Shukla, 2000a). The effects of PEth were seen at pathophysiological concentrations reported in blood cells of humans. PEth levels remained stable after two hours of ethanol removal and potentiating effects of ethanol also persisted for 2 hr after the removal of ethanol. These results support a role for endogenously accumulated PEth in mediating the ethanol potentiation of agonist stimulated p42/44 MAPK signaling (Aroor et al., 2002). Based on these studies a potential role for PEth in withdrawal or tolerance can be proposed. Fatty acid ethyl ester Fatty acid ethyl esters (FAEE) are metabolites of ethanol formed in significant amounts through nonoxidative pathway in liver, pancreas and heart (Beckemeier and Bora, 1998; Best and Laposata, 2003). FAEEs can accumulate in mitochondria and impair cell function in heart (Beckemeier and Bora, 1998) and pancreas (Kaphalia and Ansari, 2001). Since significant amounts of FAEEs are formed in heart after ethanol consumption, it may contribute in the development of alcohol-induced heart muscle disease (Beckemeier and Bora, 1998). Recent studies have provided compelling evidence for the role of FAEEs in the development of hepatic fibrosis. Cultured hepatic stellate cells form linolenic acid ethyl ester (LAEE), which has promitogenic and activating effects on hepatic stellate cells (Li et al., 1998). LAEE also induced cyclin E expression and cyclin E/CDK2 activity which may underlie the pro-mitogenic effects of this compound. In addition, LAEE increased p42/44 MAPK and JNK activity with concomitant increase in AP-1 dependent gene expression. AP-1 dependent gene expression was significantly decreased by inhibition of p42/44 MAPK activation as well as JNK activation. These results implicate MAPK activation in ethanol induced hepatic fibrosis through FAEE (LAEE) formation (Li et al., 2003).

Ethanol and Nuclear MAPK signaling cascade Ethanol induced translocation of cyclic AMP dependent protein kinase (PKA) into the nucleus and its consequences have been elegantly demonstrated (Constantinescu et al., 1999). A key step in the signaling mechanism in the p42/44 MAPK cascade is its translocation into the nucleus where it phosphorylates transcription factors including c-myc, c-jun, c-fos and Elk-1 involved in cell growth, differentiation and apoptosis (Chen et al., 1992; Pearson et al., 2001). Although nuclear activation of p42/44 MAPK was considered to be the consequence of nuclear translocation of p42/44 MAPK (Chen et al., 1992), other studies indicate translocation of MEK1/2 followed by MEK1/2 dependent nuclear activation of p42/44 MAPK (Fukuda et al., 1997; Tolwinski et al., 1999). MEK1/2 may translocate to the nucleus independent of activation, but seems to be rapidly exported from the nucleus by its nuclear export signal (NES), although the timing and role of its translocation are still controversial (Fukuda et al., 1996). Studies have shown that nuclear import of MEK1/2 is independent of both nuclear export signal and of regulation of nuclear import of MEK1/2 in response to signaling by phosphorylation of MEK at the activation lip and signaling downstream of MEK1/2 (Tolwinski et al., 1999). Rapid translocation of MEK1/2 to the nucleus may play an important role after stimulation with insulin (Kim

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and Kahn, 1997). Recently, phosphorylated MEK 1/2 was observed mainly in the nucleus 15 min after post-ischemic reoxygenation in Hgc2 cells derived from rat cardiomyoctyes, whereas phospho MEK 1/2 was present in the cytoplasm in serum stimulated cardiomyocytes (Mizukami et al., 2000). The potential importance of nuclear accumulation of MEK1/2 for cell regulation has been shown by studies on NIH 3T3 cell transformation where high levels of active nuclear MEK1/2 led to high levels of active p42/44 MAPK in the nucleus (Brunet et al., 1999). Ethanol modulates nuclear p42/44 MAPK signaling in embryonic liver cells and primary cultured rat hepatocytes. In embryonic liver cells, levels of p42/44 MAPK proteins increased in nuclear fractions from cells treated with ethanol for 24 hr. Concurrent with modest increase in p42/44 MAPK activity, serum stimulated nuclear translocation of p42/44 MAPK was potentiated in ethanol treated cells (Reddy and Shukla, 2000). In BNLCL2 liver cells, ethanol has dual effects: it triggers nuclear p42/44 MAPK translocation without significant nuclear activation of p42/44 MAPK. Second, it potentiated serum stimulated nuclear translocation and activation of p42/44 MAPK. However, ethanol has been shown to inhibit BDNF induced nuclear translocation of p42/44 MAPK in hippocampal neurons (Davis et al., 1999). In ethanol treated (100 mM, 24 hr) rat hepatocyte cultures, nuclear MAPK activity increased but the translocation of MAPK to the nucleus was not significant. However, ethanol treatment of hepatocytes caused significant increases in the levels of phospho-MEK1/2 and MEK1/2 protein in the nucleus (Aroor et al., 2001). Treatment of hepatocytes with angiotensin II (Ang II, 100 nM, 5 min) caused both activation of MAPK and translocation of MAPK into the nucleus. Ang II also caused increased accumulation of phospho-MEK1/2 and MEK1/2 protein in the nucleus. Ratio of phospho-MEK to MEK protein in the nucleus after Ang II treatment was 4 to 5 times greater than that with control or ethanol alone suggesting phosphorylation of MEK inside the nucleus. Ethanol potentiated Ang II stimulated accumulation of phospho-MEK in the nucleus. The results indicate that ethanol modulation of nuclear MAPK in hepatocytes may involve both (a) translocation of activated MAPK from cytosol into the nucleus and (b) MEK dependent activation within the nucleus (Aroor et al., 2001). Whether increased phospho-MEK1/2 in the nucleus is due to nuclear translocation of MEK1/2 alone or nuclear activation of MEK by PKC ( eg, PKC zeta ) is unknown. Angiotensin II does induce nuclear translocation of PKC zeta in rat liver epithelial cell line (Goetze et al., 1999). Nuclear JNK signaling is modulated in several ways. In islet cells of pancreas, IL-1 stimulation of JNK in the cytosol occurs without nuclear translocation. The nuclear accumulation of phosphorylated JNK is determined by nuclear translocation of SEK1 into the nucleus causing JNK activation (Mizukami et al., 1997) or changes in the expression of nuclear JNK phosphatase such as MKP-1(Chung et al., 2002). Ethanol treatment for 6 months in rats was accompanied by activation of JNK in the nucleus with concomitant increase in SEK1 and decreased expression of MKP-1 (Chung et al., 2002). In cultured hepatocytes, acetaldehyde and ethanol treatments caused translocation of JNK into the nucleus and phosphorylation of c-Jun in the nucleus (Lee and Shukla, 2003b). Nuclear MAPK signaling, phosphoacteylation of histone 3 and chromatin remodeling Mitogen and stress activated protein kinases 1 and 2 (MSK 1 and 2) are newly identified family of serine/threonine protein kinases (Deak et al., 1998; Soloaga et al., 2003). MSK1/2 have a bipartite nuclear localization signal and are localized mainly in the nucleus. MSK 1/2 are activated in vitro by p42 MAPK (ERK2) and in vivo by mitogens in a manner inhibitable by the p42/44 MAPK inhibitor

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PD 98059. MSK1/MSK2 are also substrates for the p38 MAPK. MSK1 is activated in vivo by stress stimuli such as arsenite, ultraviolet light and oxidative stress and is sensitive to p38 MAPK inhibitor SB203580. MSK1/2 may be involved in the phosphorylation of components involved in chromatin remodeling (Clayton and Mahadevan, 2003). The loosening of chromatin structure in target genes enables the transcriptional machinery to gain access to the genes for expression. This alteration in chromatin structure involves both acetylation and phosphorylation of histones. Recent evidence indicates that histone 3 phosphorylation at ser-10 and ser-28 is mediated by MSK1/2 (Deak et al., 1998; Zhong et al., 2001). Enhanced nucleosomal response leading to the expression of immediate early genes fos, jun and myc has been shown to be mediated by MSK1 (Thompson et al., 1999). Phosphorylation of histone 3 has been correlated to apoptosis in thymocytes (Enomoto et al., 2002). These findings suggest that ethanol modulates MAPK and nucleosomal responses. MSK1/2 also phosphorylate CREB which in turn may recruit acetyltransferase to enhance histone acetylation (Yuan and Gambee, 2001). Thus phosphoacetylation of histone 3 may represent a pathway modulated by nuclear MAPK signaling through MSK1/2. However, phosphorylation and acetylation may also occur as independent dynamic modifications (Thompson et al., 2001). Recently, ethanol has been shown to cause selective Lys 9 acetylation of histone 3 (Park et al., 2003). Moreover, this effect of ethanol was decreased by 4-methyl pyrazole and cyanamide thus providing evidence for the possible role of ethanol metabolism in chromatin remodeling in hepatocytes. Thus, ethanol induced, MAPK mediated histone phosphorylation and acetylation may underlie the mechanism involved in ethanol induced alterations in gene expression. In summary, it is abundantly clear that ethanol alters MAP kinase signaling ‘grid’ (Fig. 2) in diverse cells and organs (Table 1). Such modulations in the activation of MAPK pathways, after acute or chronic ethanol exposure, can profoundly alter cellular adaptations to its environment. These developments now necessitate to define the importance of MAPK cascade in the molecular actions of ethanol and its patho-physiological consequences on cellular systems. Future also awaits targeting of the components of MAPK grid to develop mechanism-based therapeutic tools useful for alcohol related health problems.

Acknowledgements Authors acknowledge the excellent contributions of researchers in this field whose work have been cited or were inadvertently not cited. We also acknowledge the scientific input of Ms.Youn Ju Lee, Mr. Pil hoon Park and technical assistance of Ms. Elizabeth Klemme in the preparation of this review. Work in authors laboratory is supported by NIAAA (AA11962).

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