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Stabilization of tumor necrosis factor-alpha mRNA in macrophages in response to chronic ethanol exposure
Alcohol 33 (2004) 229–233
Stabilization of tumor necrosis factor-alpha mRNA in macrophages in response to chronic ethanol exposure Laura E. Nagy* Department of Nutrition, Case Western Reserve University, 2123 Abington Road, Room 201, Cleveland, OH 44106-4906, USA Received 9 March 2004; received in revised form 23 June 2004; accepted 29 June 2004
Abstract Tumor necrosis factor-alpha (TNF-α) is one of a number of cytokines implicated in the progression of alcohol-induced liver disease. Activation of hepatic macrophages by lipopolysaccharide (LPS) during exposure to ethanol is thought to be an important mechanism for stimulation of TNF-α expression. Chronic exposure of macrophages to ethanol, both in vivo after ad libitum feeding of ethanol for 4 weeks and in culture for 48 h, has an impact on specific elements within the LPS-stimulated signaling cascade, disrupting both transcriptional and posttranscriptional regulation of TNF-α biosynthesis. Stabilization of TNF-α mRNA after chronic exposure to ethanol is one important mechanism for increased TNF-α production by hepatic macrophages. Increased LPS stimulation of p38 mitogen-activated protein kinase contributes to this stabilization of TNF-α mRNA in macrophages. Stabilization of TNF-α mRNA after chronic exposure to ethanol requires both cis-acting elements in the TNF-α mRNA and trans-acting mRNA-binding proteins. The adenosine plus uridine–rich element in the 3′ untranslated region of the TNF-α mRNA is an important regulator of TNF-α mRNA stability. Its activity is required for stabilization of TNF-α mRNA induced by chronic exposure to ethanol. Moreover, results from studies have demonstrated that at least one mRNAbinding protein, HuR, is also involved in stabilization of TNF-α mRNA stability after chronic exposure to ethanol. Taken together, the results from these studies identify the regulation of TNF-α mRNA stability as a novel mechanism by which chronic exposure to ethanol increases the expression of TNF-α. 쑖 2004 Elsevier Inc. All rights reserved. Keywords: Kupffer cells; Lipopolysaccharide; HuR; 3′ untranslated regions; mRNA stability
1. Role of tumor necrosis factor-alpha in the progression of alcohol-induced liver disease Alcohol-induced liver disease develops in approximately 20% of all alcohol-dependent individuals, with a higher prevalence in females (Lieber, 1994). Tumor necrosis factor-alpha (TNF-α) is thought to play a particularly critical role in the pathogenesis of alcohol-induced liver disease. Tumor necrosis factor-alpha is one of the principal mediators of the inflammatory response in mammals, transducing differential signals that regulate cellular activation and proliferation, cytotoxicity, and apoptosis (Beutler, 1995; Jacob, 1992). In addition to its role in acute septic shock, TNF-α has been implicated in the pathogenesis of a wide variety of inflammatory diseases (Jacob, 1992; Keffer et al., 1991; Reimund et al., 1996; Shalaby et al., 1989), as well as in
* Corresponding author. Tel.: ⫹1-216-368-6230; fax: ⫹1-216-3686644. E-mail address: [email protected] (L.E. Nagy). Editor: T.R. Jerrells 0741-8329/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2004.09.002
the progression of alcohol-induced liver disease (Thurman, 1998; Tilg & Diehl, 2000). Production of TNF-α by hepatic macrophages (Kupffer cells) is one of the earliest responses of the liver to injury (Tilg & Diehl, 2000). Circulating TNF-α is increased in the blood of alcohol-dependent human beings and in animals chronically exposed to ethanol (Khoruts et al., 1991; McClain & Cohen, 1989). Antibiotic treatment decreases TNF-α expression and ethanol-induced liver injury in rats exposed to ethanol by means of intragastric infusion for 4 weeks (Thurman, 1998), supporting the suggestion that increased TNF-α after exposure to ethanol is due, at least in part, to increased exposure to lipopolysaccharide (LPS) derived from intestinal bacteria. In addition to increasing LPS exposure, chronic exposure to ethanol increases the sensitivity of both the whole animal and isolated macrophages to LPS. For example, long-term ethanol consumption increases the susceptibility of rats to endotoxin-induced liver injury (Honchel et al., 1992; Mathurin et al., 2000). Moreover, LPS-stimulated TNF-α secretion is increased in Kupffer cells isolated from rats fed ethanol in their diet for 4 weeks compared with findings for pair-fed control rats (Aldred & Nagy, 1999; Kishore et al., 2001, 2002).
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2. Regulation of lipopolysaccharide-stimulated tumor necrosis factor-alpha production Production of inflammatory cytokines is a highly regulated process. Regulation of TNF-α production has been reported at the levels of transcription, translation, and secretion (Papadakis & Targan, 2000). The precise quantity of TNF-α secreted in response to activation is modulated by the complex signal transduction cascades evoked in response to LPS exposure. Lipopolysaccharide binds to a cell surface receptor, CD14, which, through interactions with the Toll-like receptor 4 (Poltorak et al., 1998), stimulates a complex array of signal-transduction cascades (Sweet & Hume, 1996; Ulevitch & Tobias, 1995). Stimulation of macrophages with LPS activates tyrosine kinases, protein kinase C, nuclear factor-kappa B, as well as members of the mitogenactivated protein (MAP) kinase family, including extracellular signal-regulated kinase (ERK)1/2, p38, and NH2–c-jun terminal kinase (Sweet & Hume, 1996). Ethanol disrupts a number of hormone- and neurotransmitter-dependent signaling pathways (Diamond & Gordon, 1997), including many of the same signaling pathways activated by LPS in macrophages. Findings from studies in Kupffer cells isolated from rats after chronic exposure to ethanol (4–6 weeks of ethanol feeding) (Cao et al., 2002; Kishore et al., 2001, 2002), in peripheral mononuclear cells obtained from alcohol-dependent human beings (Szabo & Mandrekar, 2002), as well as in macrophage-like cell lines exposed to ethanol in culture (25 mM ethanol for 48 h) (Kishore et al., 2001; Shi et al., 2002) have demonstrated that chronic exposure to ethanol disrupts specific LPS-stimulated signal transduction pathways, which regulate both TNF-α transcription and mRNA stability. The impact of these disruptions of LPS-mediated signal transduction, induced by chronic exposure to ethanol, on the transcription of TNF-α has been reviewed (Nagy, 2004). In addition to the impact of chronic exposure to ethanol on the regulation of TNF-α transcription, studies have been conducted to determine whether chronic exposure to ethanol impairs the regulation of TNF-α mRNA stability. Chronic exposure to ethanol, either by in vivo ad libitum feeding of ethanol to rats for 4 weeks or in vitro exposure to 25 mM ethanol for 48 h during culture of a macrophage-like cell line, RAW 264.7, results in a stabilization of the LPS-stimulated TNF-α mRNA transcripts (Kishore et al., 2001). In Kupffer cells isolated from rats fed ethanol-containing diets for 4 weeks, the halflife of TNF-α mRNA after LPS stimulation is increased to ⬎100 min, compared with ⬍40 min in Kupffer cells isolated from pair-fed control rats (Kishore et al., 2001). Interestingly, Motomura et al. (2001) reported a stabilization of TNF-α mRNA transcripts in Kupffer cells isolated from ethanolfed rats even when they were not stimulated with exogenous LPS. This stabilization was associated with a depletion of retinoic acid during in vivo exposure to ethanol (Motomura et al., 2001). Taken together, these findings indicate that stabilization of TNF-α mRNA, induced by chronic exposure
to ethanol, contributes significantly to increased basal and LPS-stimulated TNF-α secretion by macrophages. 3. Tumor necrosis factor-alpha mRNA stability 3.1. Stimulus-induced stabilization: role of lipopolysaccharide-stimulated signal transduction Modulation of mRNA stability is an important mechanism in the regulation of TNF-α biosynthesis (Hel et al., 1996; Jacob et al., 1996). Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory process. The signaling pathways involved in the control of mRNA stabilization are not well defined, but there is a growing body of evidence indicating the participation of MAP kinases in stimulus-induced stabilization of various short-lived transcripts. Findings of studies on the signaling pathways involved in mRNA stability have demonstrated that p38, ERK1/2, stress-activated protein kinase/NH2–c-jun terminal kinase MAP kinases, as well as cyclic AMP–dependent protein kinase A are important contributors to this posttranscriptional regulatory mechanism (Chen et al., 1998; Xu & Murphy, 2000; Xu et al., 2000). The emerging picture from the results of these studies indicates that stabilization of mRNAs can be achieved by different signaling events. Effectors of multiple signaling cascades can have an impact on the pathways regulating mRNA decay. These effectors can, therefore, connect changes in the extracellular environment to the posttranscriptional control of gene expression. Stabilization of TNF-α mRNA, induced by chronic exposure to ethanol, requires p38 MAP kinase activity (Kishore et al., 2001). Inhibition of p38 MAP kinase activity in Kupffer cells by pretreatment with SB203580, a specific inhibitor of p38 activation, or by transient transfection of RAW 264.7 macrophages with a dominant negative p38 MAP kinase, specifically abrogates the stabilization of the TNF-α transcript mediated by chronic exposure to ethanol (Kishore et al., 2001). In contrast, there was no effect of inhibition of ERK1/2 on stabilization of TNF-α mRNA induced by chronic exposure to ethanol (Kishore et al., 2001). The p38 MAP kinase has been implicated in the regulation of mRNA stability in other systems as well. For example, whereas maximal interleukin-8 gene expression requires activation of all three MAP kinases, the p38 MAP kinase pathway selectively stabilizes interleukin-8 mRNA (Holtmann et al., 1999). Similarly, cyclooxygenase-2 mRNA stabilization in HeLa cells involves activation of p38 MAP kinase (Lasa et al., 2001). Stimulus-induced stabilization of mRNAs encoding for cyclooxygenase-2 (Jang et al., 2000; Ridley et al., 1998), interleukin-6 (Winzen et al., 1999), platelet-derived growth factor receptor alpha (Wang et al., 2000b), monocyte chemoattractant protein-1 (Rovin et al., 1999), erythropoietin (Tamura et al., 2000), and TNF-α (Brook et al., 2000) is mediated by means of activation of the p38 MAP kinase. The specific protein effectors acting downstream of p38
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MAP kinase to regulate mRNA stability are not known. However, findings from a study by Carballo et al. (2001) seem to indicate that tristetraprolin, a protein that modulates the stability of TNF-α mRNA, acts in the p38 MAP kinase– signaling cascade. 3.2. Cis- and trans-acting elements involved in stabilization of tumor necrosis factor-alpha mRNA induced by chronic exposure to ethanol The TNF-α mRNA, like other short-lived mRNAs such as granulocyte–macrophage colony-stimulating factor (Bickel et al., 1992), contains adenosine plus uridine–rich elements (AREs) in its 3′ untranslated region (3′UTR) that function as destabilizing elements, as demonstrated in transgenic mice in which the TNF-α–ARE is deleted (Kontoyiannis et al., 1999), as well as in various in vitro systems (Hel et al., 1996; Lagnado et al., 1994). In addition to the destabilizing activity of the TNF-α 3′UTR, the AREs in the 3′UTR allow for stabilization of the TNF-α mRNA in response to activation (Brook et al., 2000; Dean et al., 2001). To identify the cisacting domains of the TNF-α mRNA required for stabilization induced by chronic exposure to ethanol, my colleagues and I have made use of luciferase reporter constructs. RAW 264.7 macrophages were transfected with a luciferase reporter construct containing the TNF-α promoter, 5′ untranslated region (5′UTR), and 3′UTR (pTNF-α-5′UTR-LUC-3′UTR) and subsequently cultured with or without 25 mM ethanol for 48 h and stimulated or not with LPS at 0 to 10 ng/ml (McMullen et al., 2003). Lipopolysaccharide-stimulated luciferase expression driven by the TNF-α promoter alone is not affected by chronic exposure to ethanol in this model system, consistent with findings that chronic exposure to ethanol has no net effect on LPS-stimulated transcription of TNF-α (Kishore et al., 2001; Shi et al., 2002). Of importance is the finding that when the TNF-α 5′UTR and 3′UTR were included in the luciferase reporter construct, chronic exposure to ethanol increased LPS-stimulated luciferase expression (McMullen et al., 2003). Furthermore, with the use of a reporter construct with the cytomegalovirus promoter to drive luciferase expression, the 3′UTR of murine TNF-α mRNA was sufficient to mediate increased LPSstimulated expression of a luciferase reporter in RAW 264.7 macrophages after chronic exposure to ethanol. The results from these studies thus identify the 3′UTR of the TNF-α mRNA as the essential cis-acting element mediating TNFα mRNA accumulation after chronic exposure to ethanol in murine macrophages. Stability of the TNF-α mRNA is controlled by trans-acting factors, which bind to the TNF-α mRNA. A large number of mRNA-binding proteins regulate both stabilization and destabilization (Brennan & Steitz, 2001). Multiple proteins bind to the TNF-α mRNA 3′UTR, including tristetraprolin (Carballo et al., 1998) and HuR (Dean et al., 2001), which act to regulate mRNA stability, as well as T cell–restricted intracellular antigen-1 and T cell–restricted intracellular
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antigen-related protein, which regulate translational efficiency (Gueydan et al., 1999; Piecyk et al., 2000). Tristetraprolin is a zinc finger protein induced by LPS in macrophages that destabilizes TNF-α mRNA (Carballo et al., 1998; Lai et al., 2000). In contrast, HuR, a member of the embryonic lethal abnormal vision family of RNA-binding proteins, is a nuclear-cytoplasmic shuttling protein, which is postulated to bind to specific mRNAs in the nucleus, shuttling with them to the cytoplasm, providing protection from mRNAdegradation machinery (Brennan & Steitz, 2001). HuR functions to stabilize the TNF-α mRNA (Dean et al., 2001; Sakai et al., 1999). Cytoplasmic localization of HuR is associated with conditions of cellular stress, including heat shock (Gallouzi et al., 2000), ultraviolet irradiation (Wang et al., 2000a), and amino acid starvation (Yaman et al., 2002), as well as stimulation of macrophages with LPS (Dean et al., 2001). In a series of experiments, my colleagues and I have found that HuR is required for increased expression of TNF-α after chronic feeding of ethanol for 4 weeks. In Kupffer cells, HuR was localized primarily to the nucleus and translocated subsequently to the cytosol in response to LPS in both pair-fed and ethanol-fed rats (McMullen et al., 2003). After chronic feeding of ethanol, HuR quantity in the cytosol was greater, both at baseline and in response to LPS, compared with findings in pair-fed control rats. By using RNA gel shift assays, it was observed that LPS treatment increased HuR binding to the 65 nucleotide ARE of the TNF-α 3′UTR by twofold over baseline in Kupffer cells obtained from pair-fed rats. After chronic feeding of ethanol, HuR binding to the TNF-α ARE was increased by more than fivefold at baseline and in response to LPS, compared with findings in pair-fed control rats (McMullen et al., 2003). Down-regulation of HuR expression in RAW 264.7 macrophages by RNA interference prevented the increase in expression of luciferase reporters containing the TNF-α 3′UTR after culture with 25 mM ethanol for 48 h (McMullen et al., 2003). Taken together, these results demonstrate that increased binding of HuR to the TNF-α 3′UTR contributes to increased LPS-stimulated TNF-α expression in macrophages after chronic exposure to ethanol. 4. Does chronic exposure to ethanol regulate the stability of other mRNA? Very little information is available in the literature regarding the effects of chronic exposure to ethanol on stabilization of mRNA. In addition to the results cited above demonstrating that chronic exposure to ethanol stabilizes TNF-α mRNA, chronic exposure of cultured fetal cortical neurons to ethanol stabilizes the NR1 receptor subunit of the N-methyl-D-aspartate receptor (Kumari et al., 2003). Future studies are required to assess whether chronic exposure to ethanol acts specifically to stabilize TNF-α mRNA in macrophages, or whether chronic exposure to ethanol also regulates stability of other mRNAs by targeting common
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elements in the molecular machinery regulating mRNA stability. HuR is involved in the stabilization of a number of short-lived mRNAs and is very sensitive to cellular stress, including chronic exposure to ethanol (McMullen et al., 2003), heat shock (Gallouzi et al., 2000), ultraviolet irradiation (Wang et al., 2000a), and amino acid starvation (Yaman et al., 2002). Therefore, HuR function is a potential target of chronic exposure to ethanol that may have pleiotropic effects on mRNA stability and thus contribute to the increased production of other cytokines and inflammatory mediators during chronic exposure to ethanol. For example, HuR has been identified in the stabilization of cyclooxygenase2 mRNA (Sengupta et al., 2003), an enzyme considered to be an important contributor to liver injury induced by chronic exposure to ethanol (Nanji et al., 1997). Further studies are required to determine whether disruption of mRNA stability induced by chronic exposure to ethanol is specific to regulation of TNF-α expression, or is a more common mechanism of ethanol action.
5. Summary Results of studies in Kupffer cells isolated from rats chronically exposed to ethanol in their diet, as well as in the macrophage-like cell line, RAW 264.7, exposed to ethanol during culture, have identified TNF-α mRNA stabilization as a novel mechanism contributing to up-regulation of TNF-α biosynthesis mediated by chronic exposure to ethanol. These findings support the suggestion that regulation of TNF-α mRNA stability contributes to increased TNF-α production during ethanol consumption and thus contributes to the progression of inflammation during alcohol-induced liver disease.
Acknowledgments This work was supported by grant AA 11975 from the National Institutes of Health.
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Stabilization of tumor necrosis factor-alpha mRNA in macrophages in response to chronic ethanol exposure Laura E. Nagy* Department of Nutrition, Case Western Reserve University, 2123 Abington Road, Room 201, Cleveland, OH 44106-4906, USA Received 9 March 2004; received in revised form 23 June 2004; accepted 29 June 2004
Abstract Tumor necrosis factor-alpha (TNF-α) is one of a number of cytokines implicated in the progression of alcohol-induced liver disease. Activation of hepatic macrophages by lipopolysaccharide (LPS) during exposure to ethanol is thought to be an important mechanism for stimulation of TNF-α expression. Chronic exposure of macrophages to ethanol, both in vivo after ad libitum feeding of ethanol for 4 weeks and in culture for 48 h, has an impact on specific elements within the LPS-stimulated signaling cascade, disrupting both transcriptional and posttranscriptional regulation of TNF-α biosynthesis. Stabilization of TNF-α mRNA after chronic exposure to ethanol is one important mechanism for increased TNF-α production by hepatic macrophages. Increased LPS stimulation of p38 mitogen-activated protein kinase contributes to this stabilization of TNF-α mRNA in macrophages. Stabilization of TNF-α mRNA after chronic exposure to ethanol requires both cis-acting elements in the TNF-α mRNA and trans-acting mRNA-binding proteins. The adenosine plus uridine–rich element in the 3′ untranslated region of the TNF-α mRNA is an important regulator of TNF-α mRNA stability. Its activity is required for stabilization of TNF-α mRNA induced by chronic exposure to ethanol. Moreover, results from studies have demonstrated that at least one mRNAbinding protein, HuR, is also involved in stabilization of TNF-α mRNA stability after chronic exposure to ethanol. Taken together, the results from these studies identify the regulation of TNF-α mRNA stability as a novel mechanism by which chronic exposure to ethanol increases the expression of TNF-α. 쑖 2004 Elsevier Inc. All rights reserved. Keywords: Kupffer cells; Lipopolysaccharide; HuR; 3′ untranslated regions; mRNA stability
1. Role of tumor necrosis factor-alpha in the progression of alcohol-induced liver disease Alcohol-induced liver disease develops in approximately 20% of all alcohol-dependent individuals, with a higher prevalence in females (Lieber, 1994). Tumor necrosis factor-alpha (TNF-α) is thought to play a particularly critical role in the pathogenesis of alcohol-induced liver disease. Tumor necrosis factor-alpha is one of the principal mediators of the inflammatory response in mammals, transducing differential signals that regulate cellular activation and proliferation, cytotoxicity, and apoptosis (Beutler, 1995; Jacob, 1992). In addition to its role in acute septic shock, TNF-α has been implicated in the pathogenesis of a wide variety of inflammatory diseases (Jacob, 1992; Keffer et al., 1991; Reimund et al., 1996; Shalaby et al., 1989), as well as in
* Corresponding author. Tel.: ⫹1-216-368-6230; fax: ⫹1-216-3686644. E-mail address: [email protected] (L.E. Nagy). Editor: T.R. Jerrells 0741-8329/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2004.09.002
the progression of alcohol-induced liver disease (Thurman, 1998; Tilg & Diehl, 2000). Production of TNF-α by hepatic macrophages (Kupffer cells) is one of the earliest responses of the liver to injury (Tilg & Diehl, 2000). Circulating TNF-α is increased in the blood of alcohol-dependent human beings and in animals chronically exposed to ethanol (Khoruts et al., 1991; McClain & Cohen, 1989). Antibiotic treatment decreases TNF-α expression and ethanol-induced liver injury in rats exposed to ethanol by means of intragastric infusion for 4 weeks (Thurman, 1998), supporting the suggestion that increased TNF-α after exposure to ethanol is due, at least in part, to increased exposure to lipopolysaccharide (LPS) derived from intestinal bacteria. In addition to increasing LPS exposure, chronic exposure to ethanol increases the sensitivity of both the whole animal and isolated macrophages to LPS. For example, long-term ethanol consumption increases the susceptibility of rats to endotoxin-induced liver injury (Honchel et al., 1992; Mathurin et al., 2000). Moreover, LPS-stimulated TNF-α secretion is increased in Kupffer cells isolated from rats fed ethanol in their diet for 4 weeks compared with findings for pair-fed control rats (Aldred & Nagy, 1999; Kishore et al., 2001, 2002).
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2. Regulation of lipopolysaccharide-stimulated tumor necrosis factor-alpha production Production of inflammatory cytokines is a highly regulated process. Regulation of TNF-α production has been reported at the levels of transcription, translation, and secretion (Papadakis & Targan, 2000). The precise quantity of TNF-α secreted in response to activation is modulated by the complex signal transduction cascades evoked in response to LPS exposure. Lipopolysaccharide binds to a cell surface receptor, CD14, which, through interactions with the Toll-like receptor 4 (Poltorak et al., 1998), stimulates a complex array of signal-transduction cascades (Sweet & Hume, 1996; Ulevitch & Tobias, 1995). Stimulation of macrophages with LPS activates tyrosine kinases, protein kinase C, nuclear factor-kappa B, as well as members of the mitogenactivated protein (MAP) kinase family, including extracellular signal-regulated kinase (ERK)1/2, p38, and NH2–c-jun terminal kinase (Sweet & Hume, 1996). Ethanol disrupts a number of hormone- and neurotransmitter-dependent signaling pathways (Diamond & Gordon, 1997), including many of the same signaling pathways activated by LPS in macrophages. Findings from studies in Kupffer cells isolated from rats after chronic exposure to ethanol (4–6 weeks of ethanol feeding) (Cao et al., 2002; Kishore et al., 2001, 2002), in peripheral mononuclear cells obtained from alcohol-dependent human beings (Szabo & Mandrekar, 2002), as well as in macrophage-like cell lines exposed to ethanol in culture (25 mM ethanol for 48 h) (Kishore et al., 2001; Shi et al., 2002) have demonstrated that chronic exposure to ethanol disrupts specific LPS-stimulated signal transduction pathways, which regulate both TNF-α transcription and mRNA stability. The impact of these disruptions of LPS-mediated signal transduction, induced by chronic exposure to ethanol, on the transcription of TNF-α has been reviewed (Nagy, 2004). In addition to the impact of chronic exposure to ethanol on the regulation of TNF-α transcription, studies have been conducted to determine whether chronic exposure to ethanol impairs the regulation of TNF-α mRNA stability. Chronic exposure to ethanol, either by in vivo ad libitum feeding of ethanol to rats for 4 weeks or in vitro exposure to 25 mM ethanol for 48 h during culture of a macrophage-like cell line, RAW 264.7, results in a stabilization of the LPS-stimulated TNF-α mRNA transcripts (Kishore et al., 2001). In Kupffer cells isolated from rats fed ethanol-containing diets for 4 weeks, the halflife of TNF-α mRNA after LPS stimulation is increased to ⬎100 min, compared with ⬍40 min in Kupffer cells isolated from pair-fed control rats (Kishore et al., 2001). Interestingly, Motomura et al. (2001) reported a stabilization of TNF-α mRNA transcripts in Kupffer cells isolated from ethanolfed rats even when they were not stimulated with exogenous LPS. This stabilization was associated with a depletion of retinoic acid during in vivo exposure to ethanol (Motomura et al., 2001). Taken together, these findings indicate that stabilization of TNF-α mRNA, induced by chronic exposure
to ethanol, contributes significantly to increased basal and LPS-stimulated TNF-α secretion by macrophages. 3. Tumor necrosis factor-alpha mRNA stability 3.1. Stimulus-induced stabilization: role of lipopolysaccharide-stimulated signal transduction Modulation of mRNA stability is an important mechanism in the regulation of TNF-α biosynthesis (Hel et al., 1996; Jacob et al., 1996). Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory process. The signaling pathways involved in the control of mRNA stabilization are not well defined, but there is a growing body of evidence indicating the participation of MAP kinases in stimulus-induced stabilization of various short-lived transcripts. Findings of studies on the signaling pathways involved in mRNA stability have demonstrated that p38, ERK1/2, stress-activated protein kinase/NH2–c-jun terminal kinase MAP kinases, as well as cyclic AMP–dependent protein kinase A are important contributors to this posttranscriptional regulatory mechanism (Chen et al., 1998; Xu & Murphy, 2000; Xu et al., 2000). The emerging picture from the results of these studies indicates that stabilization of mRNAs can be achieved by different signaling events. Effectors of multiple signaling cascades can have an impact on the pathways regulating mRNA decay. These effectors can, therefore, connect changes in the extracellular environment to the posttranscriptional control of gene expression. Stabilization of TNF-α mRNA, induced by chronic exposure to ethanol, requires p38 MAP kinase activity (Kishore et al., 2001). Inhibition of p38 MAP kinase activity in Kupffer cells by pretreatment with SB203580, a specific inhibitor of p38 activation, or by transient transfection of RAW 264.7 macrophages with a dominant negative p38 MAP kinase, specifically abrogates the stabilization of the TNF-α transcript mediated by chronic exposure to ethanol (Kishore et al., 2001). In contrast, there was no effect of inhibition of ERK1/2 on stabilization of TNF-α mRNA induced by chronic exposure to ethanol (Kishore et al., 2001). The p38 MAP kinase has been implicated in the regulation of mRNA stability in other systems as well. For example, whereas maximal interleukin-8 gene expression requires activation of all three MAP kinases, the p38 MAP kinase pathway selectively stabilizes interleukin-8 mRNA (Holtmann et al., 1999). Similarly, cyclooxygenase-2 mRNA stabilization in HeLa cells involves activation of p38 MAP kinase (Lasa et al., 2001). Stimulus-induced stabilization of mRNAs encoding for cyclooxygenase-2 (Jang et al., 2000; Ridley et al., 1998), interleukin-6 (Winzen et al., 1999), platelet-derived growth factor receptor alpha (Wang et al., 2000b), monocyte chemoattractant protein-1 (Rovin et al., 1999), erythropoietin (Tamura et al., 2000), and TNF-α (Brook et al., 2000) is mediated by means of activation of the p38 MAP kinase. The specific protein effectors acting downstream of p38
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MAP kinase to regulate mRNA stability are not known. However, findings from a study by Carballo et al. (2001) seem to indicate that tristetraprolin, a protein that modulates the stability of TNF-α mRNA, acts in the p38 MAP kinase– signaling cascade. 3.2. Cis- and trans-acting elements involved in stabilization of tumor necrosis factor-alpha mRNA induced by chronic exposure to ethanol The TNF-α mRNA, like other short-lived mRNAs such as granulocyte–macrophage colony-stimulating factor (Bickel et al., 1992), contains adenosine plus uridine–rich elements (AREs) in its 3′ untranslated region (3′UTR) that function as destabilizing elements, as demonstrated in transgenic mice in which the TNF-α–ARE is deleted (Kontoyiannis et al., 1999), as well as in various in vitro systems (Hel et al., 1996; Lagnado et al., 1994). In addition to the destabilizing activity of the TNF-α 3′UTR, the AREs in the 3′UTR allow for stabilization of the TNF-α mRNA in response to activation (Brook et al., 2000; Dean et al., 2001). To identify the cisacting domains of the TNF-α mRNA required for stabilization induced by chronic exposure to ethanol, my colleagues and I have made use of luciferase reporter constructs. RAW 264.7 macrophages were transfected with a luciferase reporter construct containing the TNF-α promoter, 5′ untranslated region (5′UTR), and 3′UTR (pTNF-α-5′UTR-LUC-3′UTR) and subsequently cultured with or without 25 mM ethanol for 48 h and stimulated or not with LPS at 0 to 10 ng/ml (McMullen et al., 2003). Lipopolysaccharide-stimulated luciferase expression driven by the TNF-α promoter alone is not affected by chronic exposure to ethanol in this model system, consistent with findings that chronic exposure to ethanol has no net effect on LPS-stimulated transcription of TNF-α (Kishore et al., 2001; Shi et al., 2002). Of importance is the finding that when the TNF-α 5′UTR and 3′UTR were included in the luciferase reporter construct, chronic exposure to ethanol increased LPS-stimulated luciferase expression (McMullen et al., 2003). Furthermore, with the use of a reporter construct with the cytomegalovirus promoter to drive luciferase expression, the 3′UTR of murine TNF-α mRNA was sufficient to mediate increased LPSstimulated expression of a luciferase reporter in RAW 264.7 macrophages after chronic exposure to ethanol. The results from these studies thus identify the 3′UTR of the TNF-α mRNA as the essential cis-acting element mediating TNFα mRNA accumulation after chronic exposure to ethanol in murine macrophages. Stability of the TNF-α mRNA is controlled by trans-acting factors, which bind to the TNF-α mRNA. A large number of mRNA-binding proteins regulate both stabilization and destabilization (Brennan & Steitz, 2001). Multiple proteins bind to the TNF-α mRNA 3′UTR, including tristetraprolin (Carballo et al., 1998) and HuR (Dean et al., 2001), which act to regulate mRNA stability, as well as T cell–restricted intracellular antigen-1 and T cell–restricted intracellular
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antigen-related protein, which regulate translational efficiency (Gueydan et al., 1999; Piecyk et al., 2000). Tristetraprolin is a zinc finger protein induced by LPS in macrophages that destabilizes TNF-α mRNA (Carballo et al., 1998; Lai et al., 2000). In contrast, HuR, a member of the embryonic lethal abnormal vision family of RNA-binding proteins, is a nuclear-cytoplasmic shuttling protein, which is postulated to bind to specific mRNAs in the nucleus, shuttling with them to the cytoplasm, providing protection from mRNAdegradation machinery (Brennan & Steitz, 2001). HuR functions to stabilize the TNF-α mRNA (Dean et al., 2001; Sakai et al., 1999). Cytoplasmic localization of HuR is associated with conditions of cellular stress, including heat shock (Gallouzi et al., 2000), ultraviolet irradiation (Wang et al., 2000a), and amino acid starvation (Yaman et al., 2002), as well as stimulation of macrophages with LPS (Dean et al., 2001). In a series of experiments, my colleagues and I have found that HuR is required for increased expression of TNF-α after chronic feeding of ethanol for 4 weeks. In Kupffer cells, HuR was localized primarily to the nucleus and translocated subsequently to the cytosol in response to LPS in both pair-fed and ethanol-fed rats (McMullen et al., 2003). After chronic feeding of ethanol, HuR quantity in the cytosol was greater, both at baseline and in response to LPS, compared with findings in pair-fed control rats. By using RNA gel shift assays, it was observed that LPS treatment increased HuR binding to the 65 nucleotide ARE of the TNF-α 3′UTR by twofold over baseline in Kupffer cells obtained from pair-fed rats. After chronic feeding of ethanol, HuR binding to the TNF-α ARE was increased by more than fivefold at baseline and in response to LPS, compared with findings in pair-fed control rats (McMullen et al., 2003). Down-regulation of HuR expression in RAW 264.7 macrophages by RNA interference prevented the increase in expression of luciferase reporters containing the TNF-α 3′UTR after culture with 25 mM ethanol for 48 h (McMullen et al., 2003). Taken together, these results demonstrate that increased binding of HuR to the TNF-α 3′UTR contributes to increased LPS-stimulated TNF-α expression in macrophages after chronic exposure to ethanol. 4. Does chronic exposure to ethanol regulate the stability of other mRNA? Very little information is available in the literature regarding the effects of chronic exposure to ethanol on stabilization of mRNA. In addition to the results cited above demonstrating that chronic exposure to ethanol stabilizes TNF-α mRNA, chronic exposure of cultured fetal cortical neurons to ethanol stabilizes the NR1 receptor subunit of the N-methyl-D-aspartate receptor (Kumari et al., 2003). Future studies are required to assess whether chronic exposure to ethanol acts specifically to stabilize TNF-α mRNA in macrophages, or whether chronic exposure to ethanol also regulates stability of other mRNAs by targeting common
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elements in the molecular machinery regulating mRNA stability. HuR is involved in the stabilization of a number of short-lived mRNAs and is very sensitive to cellular stress, including chronic exposure to ethanol (McMullen et al., 2003), heat shock (Gallouzi et al., 2000), ultraviolet irradiation (Wang et al., 2000a), and amino acid starvation (Yaman et al., 2002). Therefore, HuR function is a potential target of chronic exposure to ethanol that may have pleiotropic effects on mRNA stability and thus contribute to the increased production of other cytokines and inflammatory mediators during chronic exposure to ethanol. For example, HuR has been identified in the stabilization of cyclooxygenase2 mRNA (Sengupta et al., 2003), an enzyme considered to be an important contributor to liver injury induced by chronic exposure to ethanol (Nanji et al., 1997). Further studies are required to determine whether disruption of mRNA stability induced by chronic exposure to ethanol is specific to regulation of TNF-α expression, or is a more common mechanism of ethanol action.
5. Summary Results of studies in Kupffer cells isolated from rats chronically exposed to ethanol in their diet, as well as in the macrophage-like cell line, RAW 264.7, exposed to ethanol during culture, have identified TNF-α mRNA stabilization as a novel mechanism contributing to up-regulation of TNF-α biosynthesis mediated by chronic exposure to ethanol. These findings support the suggestion that regulation of TNF-α mRNA stability contributes to increased TNF-α production during ethanol consumption and thus contributes to the progression of inflammation during alcohol-induced liver disease.
Acknowledgments This work was supported by grant AA 11975 from the National Institutes of Health.
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