The primary sequences of ERK-2 from H. sapiens (top row), M. musculus (2nd row), R. norvegicus (3rd row), and X. laevis (4th row) were obtained from NCBI (P28482, P63085, P63086, and P26696, respectively) and aligned to the human primary sequence. Highlighted regions of these sequences indicate homologous sequences across the species that are greater than 98% identical to human for mouse and rat and greater than 96% identical to human for Xenopus, indicating that experimental alternatives to human are appropriate for studies focused on the extracellular signal-regulated kinase pathway.

Immunoblot analysis using hepatocytes isolated from animals consuming control and ethanol-containing diets for 45 days is shown. A, immunoblot analysis of hepatocyte total ERK-1/2 concentrations, showing no effect of ethanol when compared with control (stripped blots reprobed for B-actin shows equal loading). B, immunoblot analysis of phosphorylated ERK-1/2 indicates that chronic ethanol ingestion ablates constitutive ERK-1/2 phosphorylation (stripped blots reprobed for B-actin shows equal loading). C, immunoblot and corresponding densitometry demonstrating chronic ethanol administration results in the significant increase in 4-HNE-ERK-2 adduct concentration as indicated by immunoprecipitated ERK-1/2, probed for 4-HNE-protein adducts. Corresponding densitometry analysis normalized to the total ERK-2 immunoprecipitated (D) indicates a significantly elevated level of adduct concentrations in ethanol-treated animals (p < 0.0042). D, total ERK-2 protein concentrations demonstrated by Coomassie Blue staining of immunoprecipitated ERK-2 and corresponding densitometry using hepatocyte lysates from control and ethanol-treated animals indicating increased concentrations of ERK-2 purified from control samples when compared with ethanol-treated samples (p < 0.05). (*, p < 0.05, n = 4.)

A, Western blot analysis of nuclear phosphorylated ERK-1/2 shows a loss of positive staining with increasing 4-HNE concentrations used to treat primary hepatocytes. B, unlike the analysis of total cell lysates, analysis of total nuclear ERK-1/2 indicates that the loss of nuclear phospho-ERK-1/2 results from the 4-HNE-mediated decrease in total ERK-1/2 protein concentrations, a change indicative of cytosolic modification and inhibition. C, immunoblot analysis of total hepatocyte lysates probed against the ERK-1/2 substrate, ELK-1, shows no effect of 4-HNE on total nuclear ELK-1 concentrations. D, immunoblot analysis and densitometry of nuclear phosphorylated ELK-1 demonstrating a concentration-dependent inhibition of ERK-1/2 activity in situ as shown by a loss of phosphorylated substrate with increasing 4-HNE concentrations. (*, p < 0.05, n = 5.)

In normal non-ethanol-exposed and non-aldehyde-exposed hepatocytes, upstream kinase events (solid arrows) cascade down through ERK-1/2 to phosphorylate and activate this signaling intermediate, resulting in the active transport of ERK-1/2 into the nucleus where phosphorylation of downstream ELK-1 and subsequently AP-1 (c-Fos, and to a lesser extent c-Jun) result in transcriptional activity. During ethanol-induced lipid peroxidation, 4-HNE forms covalent adducts with inactive ERK-1/2 in the cytosol, preventing its phosphorylation, activation, and nuclear translocation, which results in the loss of downstream ELK-1 activity (dotted arrows). The 4-HNE-mediated inactivation of ERK-1/2 and the subsequent loss of ELK-1 activity could potentially result in the loss of normal transcriptional activity through the c-Fos and c-Jun AP-1 transcription factors associated with proliferation, cell survival, and homeostasis (dashed arrows).

Representative serial sections of rat liver from animals fed a control liquid diet or a diet containing 35% ethanol-derived calories for 60 days. A, hematoxylin and eosin staining of a control liver section showing normal hepatolobular architecture with chords of hepatocytes radiating out from the central vein (CV) to the portal triad (PV), with clear linear sinuses between the chords (×10 magnification). B, hematoxylin and eosin staining of a liver section from an ethanol-treated rat demonstrating the loss of normal hepatic architecture and the accumulation of lipid (steatosis) within hepatocytes surrounding the central vein (×10 magnification). B2, ×80 magnification of the ethanol-treated liver section from B, showing the cytosolic accumulation of lipid within hepatocytes (microvesicular = small arrowheads, macrovesicular = large arrowheads). C, control liver section stained against 4-HNE-modified proteins that demonstrate a faint, pan-lobular positive staining (×10 magnification). D, 4-HNE-adduct staining indicating the ethanol-induced accumulation of protein adducts within the central lobular and mid-zonal regions and clearly absent in areas proximal to the central veins (×10 magnification). D2, ×80 magnification of D indicating the cytosolic accumulation of 4-HNE-protein adducts and the relative lack of positive nuclear staining (hematoxylin counterstain). E, control liver section stained against total ERK-1/2, indicating a diffuse pan-lobular staining (×10 magnification). F, total ERK-1/2 staining indicating the ethanol-induced accumulation of ERK protein within the central lobular and mid-zonal regions and clearly absent in areas proximal to the central veins, similar to observations made for 4-HNE-adduct accumulation in D (×10 magnification). F2, ×80 magnification of F demonstrating the cytosolic accumulation of ERK-1/2 and the distinct lack of positive nuclear staining in most cells (27/×40 field compared with 63/×40 field in control liver sections), also similar to observations made for the cellular accumulation of 4-HNE adducts in D2. G, control liver section stained against phospho-ERK-1/2, showing distinct positive staining nuclei (64/×40 field) throughout the tissue and moderate cytosolic staining (×40 magnification). H, phospho-Erk-1/2 staining indicating the ethanol-induced loss of positive nuclear staining (25/×40 field) with moderate cytosolic staining absent in the midzonal region (center of image, ×40 magnification). I, control liver section stained against total ELK-1 showing distinct positive nuclear staining (51/×40 field) throughout the tissue and diffuse cytosolic staining (×40 magnification). J, total ELK-1 staining of liver from an ethanol-treated animal. Although there is obvious loss of structural organization within the tissue, the number of positive staining nuclei per ×40 field is maintained (52/×40 field) when compared with control I. K, control liver section stained against phosphorylated ELK-1 demonstrating distinct positive nuclei (47/×40 field) throughout the tissue and diffuse cytosolic staining (×40 magnification). L, phospho-ELK-1 staining of ethanol-treated animal liver showing a significant loss of positive staining nuclei per ×40 field (18/×40 field) when compared with control (×40 magnification).

The cytotoxicity of 4-HNE after 4 h of treatment on primary rat hepatocytes was determined over a range of physiologic concentrations. A, cytotoxicity shown as the ratio of release LDH activity over total cellular LDH activity, indicating a lack of cytotoxicity from 0 to 100 μm 4-HNE. These sub-cytotoxic concentrations were used for subsequent primary culture experiments. B, immunoblot indicating a concentration-dependent decrease in ERK-1/2 phosphorylation when compared with constitutive control levels (lane 1, 0 μm 4-HNE) (B-actin reprobe of stripped blots confirms equal loading). C, immunoblot analysis of total ERK-1/2 protein indicating no change with increasing concentrations of 4-HNE treatment (B-actin reprobe of stripped blots indicates equal loading). D, ERK-1/2 activity assay demonstrating the concentration-dependent suppression of immunoprecipitated ERK-1/2 collected from primary cultures exposed to increasing levels of 4-HNE to phosphorylate the ELK-1 substrate. D, inset, active, phosphorylated IP-ERK-1/2 from control hepatocytes is not affected by increasing concentrations of 4-HNE, indicating 4-HNE-mediated effects on ERK-1/2 result from interactions with the inactive, unphosphorylated ERK monomers. E, Western blot analysis of activity assay components probed for 4-HNE-protein adducts indicating an inverse correlation between ERK-1/2 phosphorylation and activity and the concentration-dependent increase in 4-HNE-adducted ERK-1/2 levels in primary hepatocytes. (*, p < 0.05, n = 9.)

A, immunoblot analysis of inactive ERK-2 protein incubated with increasing physiologic concentrations of 4-HNE and probed for 4-HNE-protein adducts shows the formation of the 4-HNE-ERK-2 monomer adducts as the predominant adducted species (lower, 42-kDa bands). At the highest concentration (100 μm), 4-HNE adduction of unphosphorylated ERK-2 results in chemical cross-linking, as demonstrated by the partial shift from 42 to 82 and 126 kDa (arrowheads) for 4-HNE-adduct-positive staining bands. B, immunoblot analysis of the blot in A, when stripped and reprobed for total ERK-1/2 protein, that shows a partial loss of positive signal that correlates with the cross-linking of ERK-2 monomers at 100 μm 4-HNE. These data suggest that two distinct adduct species exist, where only the 100 μm 4-HNE treatment results in cross-linked adducts that preclude the recognition of the epitope by the total ERK-1/2 antibody.

A, representative immunoblot analysis and corresponding densitometry analysis of PCNA using nuclear extracts of hepatocytes isolated from control animals (1st to 4th lanes, left to right) and animals consuming ethanol for 45 days (5th to 8th lanes, left to right) showing a 36.7% ethanol-dependent decrease in constitutive cell proliferation when compared with control levels (n = 12, p < 0.005). B, preliminary data showing an immunoblot for PCNA using nuclear extracts from primary hepatocytes treated with increasing concentrations of 4-HNE, suggesting a concentration-dependent decrease in cell proliferation with increasing 4-HNE.