Increased production of ROS has been implicated in the development of alcoholic liver injury [15
]. However, neither the source nor targets of the ROS produced during ethanol consumption are well defined. NADPH oxidase activity has been implicated as an important source of ROS during chronic ethanol exposure in rodent models of chronic ethanol exposure [15
]. Proof of principle studies using p47phox
−/− mice as well treatment of rats with DPI during chronic ethanol exposure indicate that NADPH oxidase activity is required for the development of chronic ethanol-induced liver injury [19
]. However, the mechanisms by which NADPH oxidase activity contributes to liver injury are not well understood. Here, we have demonstrated that chronic ethanol feeding enhances the ability of LPS to increase ROS production in Kupffer cells. Although baseline rates of ROS production were not affected by chronic ethanol feeding, LPS-stimulated ROS production was increased by 2.5-fold in Kupffer cells from ethanol-fed rats compared with cells from pair-fed rats. This increased ROS production was suppressed effectively by treatment with DPI, an inhibitor of NADPH oxidase. As a consequence of increased NADPH oxidase-derived ROS production, LPS-stimulated ERK1/2 phosphorylation increased in Kupffer cells after chronic ethanol feeding. Increased activation of this key MAPK signaling pathway after chronic ethanol feeding contributes to increased TNF-α production. These studies thus identify increased NADPH oxidase as an important site for ROS production and ERK1/2 signaling as an important target for ROS during LPS-mediated signal transduction by Kupffer cells after chronic ethanol feeding.
There is a growing appreciation of the specific role of ROS in the modulation/regulation of a number of signal transduction cascades [20
]. ROS contribute to cellular responses to a variety of hormones, neurotransmitters, and cytokines [20
]. Evidence also indicates that ROS contribute to LPS-stimulated signaling pathways in cells of the innate immune system (i.e., monocytes/macrophages, neutrophils) as well as nonimmune cells [35
]. In macrophages, several specific signaling pathways regulated by ROS have been identified. For example, Hsu and Wen [28
] demonstrated that LPS-stimulated ROS production acts via p38 and ERK1/2 MAPKs to enhance interleukin-1 gene expression. Here, we find that NADPH oxidase-derived ROS contribute to LPS-mediated activation of the ERK1/2, p38, and NF-κB pathways in hepatic macrophages from pair-fed control rats ( and ).
After chronic ethanol exposure, the few studies published have suggested a general role for increased ROS production in mediating the chronic effect of ethanol on LPS-mediated signaling in macrophages. For example, treatment of MonoMac6 macrophages or Kupffer cells with general antioxidant compounds, such as N-acetyl cysteine or dilinoleoylphosphatidylcholine (DLPC), abrogates the increase in LPS-stimulated TNF-α expression observed in these cells after chronic ethanol exposure during culture or in response to ethanol feeding [8
]. In particular, Cao et al. [8
] identified LPS-stimulated NF-κB, as well as ERK1/2 and p38 MAPKs, as ROS-sensitive targets for the inhibitory effects of DLPC in Kupffer cells isolated from rats fed chronic ethanol. Here, we have specifically identified NADPH oxidase-derived ROS as an important contributor to LPS-stimulated ERK1/2 phoshorylation in rat Kupffer cells. These results from Kupffer cells implicate a critical role of ROS in activation of the ERK1/2 pathway and are consistent with previous work in other cell types, demonstrating that ERK1/2 can be activated by ROS [20
]. It is interesting that chronic ethanol feeding abrogated the ability of DPI to inhibit LPS-stimulated p38 phosphorylation and IκB-α degradation in Kupffer cells (). These data suggest that additional mechanisms for p38 and NF-κB activation must come into play after chronic ethanol exposure. These additional mechanisms could include contributions from CYP2E1-derived ROS and/or additional changes in LPS-mediated signaling in response to chronic ethanol feeding.
Although ROS production by Kupffer cells after chronic ethanol feeding may be mediated by a number of pathways, such as CYP2E1, xanthine oxidase, or NADPH oxidase, only inhibitors of NADPH oxidase were able to prevent chronic, ethanol-induced increases in LPS-stimulated ERK1/2 phosphorylation in Kupffer cells (). Although NADPH oxidase-derived ROS contributed to increased LPS-stimulated ERK1/2 phosphorylation after chronic ethanol, they did not contribute to enhanced LPS-stimulated p38 MAPK phosphorylation or degradation of IκB-α, an indirect indicator of NF-κB activation, after chronic ethanol feeding (). These data suggest a compartmentalization of ROS production within hepatic macrophages, and the LPS-stimulated ERK1/2 signaling pathway was more closely linked with NADPH oxidase-derived ROS compared with other LPS-activated signaling cascades. Studies using other model systems suggest that specific signaling cascades, including ERK1/2, may be linked with NADPH oxidase-dependent ROS production. However, such specificity in ROS-mediated signaling varies between different cell types and/or activation signals (for example, see refs. [38
]), and further studies are required to define such an association in Kupffer cells after ethanol exposure.
LPS-mediated activation of NADPH oxidase involves the activation and recruitment of several cytosolic subunits to the plasma membrane [20
]. The mechanisms by which chronic ethanol feeding increased NADPH oxidase-dependent ROS production in response to LPS are not known. Although chronic ethanol feeding did not increase the total quantity of p67phox
protein in Kupffer cells, LPS-stimulated translocation of p67phox
to the plasma membrane was increased compared with pair-fed rats. Translocation of p47phox
is dependent on protein kinase C (PKC)δ-mediated phosphorylation in monocytes [41
]. As PKCδ is a target of ethanol action in a number of cell types [43
], changes in PKCδ-mediated regulation of NADPH oxidase after chronic ethanol are currently under investigation. Further, chronic ethanol exposure resulted in a constitutive activation of Rac1, the small GTP-binding protein required for activation of NADPH oxidase. Disruption of the normal GTP/guanosine 5′-diphosphate cycling of Rac1 may contribute to enhanced LPS-stimulated NADPH oxidase activity after chronic ethanol feeding. A number of studies using other cells/tissues have identified the activation of small GTP-binding proteins as a target of ethanol action, consistent with the increase in the active form of Rac1 after chronic ethanol feeding in the current study. For example, ethanol exposure increases the relative proportion of the GTP-bound forms of several guanosinetriphosphatases (GTPases) including ras in mouse liver homogenates [44
], RhoA in fetal rat astrocytes [45
], cdc42 in SVEC4-10 cells [46
], TC10 in rat adipocytes [47
], as well as RhoA and cdc42 in vitro [48
]. Potential mechanisms by which chronic ethanol targets small GTP-binding proteins include changes in the post-translational processing, such as impaired lipidation reactions [49
], impaired regulation of GTPase cycling activity [50
], and/or redox-dependent activation analagous to that observed in other ras and Rho family GTPases [51
Circulating TNF-α is increased in the blood of alcoholics and in animals chronically exposed to ethanol [4
]. Studies in transgenic mice lacking the TNF receptor I as well as treatment of mice and rats with antibodies to TNF-α during chronic ethanol exposure have demonstrated an essential role for TNF-α in the progression of chronic, ethanol-induced liver injury [1
]. We have previously demonstrated the critical role of ERK1/2 in mediating increased LPS-stimulated TNF-α production by Kupffer cells after chronic ethanol feeding [10
]. Together with our previous results, the current data suggest that chronic, ethanol-induced increases in the ERK1/2 pathway are mediated, at least in part, via increased LPS-stimulated, NADPH oxidase-derived ROS production. These data thus identify ERK1/2 signaling as a molecular target of increased ROS production in Kupffer cells, which likely contributes to increased TNF-α production and therefore liver injury during chronic ethanol feeding.