Ethanol targets nucleoredoxin/dishevelled interactions and stimulates phosphatidylinositol 4-phosphate production in vivo and in vitro

Abstract

Nucleoredoxin (NXN) is a redox-regulating protein potentially targeted by reactive oxygen species (ROS). It regulates molecular pathways that participate in several key cellular processes. However, the role of NXN in the alcohol liver disease (ALD) redox regulation has not been fully understood. Here, we investigated the effects of ethanol and ethanol plus lipopolysaccharide, a two-hit liver injury model (Ethanol/LPS), on NXN/dishevelled (DVL) interaction and on DVL-dependent phosphoinositides production both in mouse liver and in a co-culture system consisting of human hepatic stellate cells (HSC) and ethanol metabolizing-VL17A human hepatocyte cells. Ethanol and two-hit model increased Nxn protein and mRNA expression, and 4-hydroxynonenal adducts. Two-hit model promoted Nxn nuclear translocation and Dvl/Phosphatidylinositol 4-kinase type-IIα (Pi4k2a) interaction ratio but surprisingly decreased Dvl protein and mRNA levels and reverted ethanol-induced Nxn/Dvl and Dvl/frizzled (Fzd) interaction ratios. Ethanol resulted in a significant increase of Dvl protein and mRNA expression, and decreased Nxn/Dvl interaction ratio but promoted the interaction of Dvl with Fzd and Pi4k2a; formation of this complex induced phosphatidylinositol 4-phosphate [PI(4)P] production. Ethanol and LPS treatments provoked similar alterations on NXN/DVL interaction and its downstream effect in HSC/VL17A co-culture system. Interestingly, ROS and glutathione levels as well as most of ethanol-induced alterations were modified by NXN overexpression in the co-culture system. In conclusion, two-hit model of ethanol exposure disrupts NXN/DVL homeostatic status to allow DVL/FZD/PI4K2A complex formation and stimulates PI(4)P production. These results provide a new mechanism showing that NXN also participates in the regulation of phosphoinositides production that is altered by ethanol during alcoholic liver disease progression.

Keywords: Alcoholic liver disease; Nucleoredoxin; Oxidative stress; Phosphoinositide; Redox regulation.

Fig. 1.. Effect of ethanol and LPS on Nxn status in the mice liver.

Total, cytosolic and nuclear protein, and total RNA extracted from livers of female wild-type C57BL/6 mice were used for western blot and qPCR analyses, respectively. (A) Nxn mRNA expression and (B) total protein levels of Nxn. (C) Cytosolic and (D) nuclear Nxn compartmentalized levels. mRNA expression was normalized to 18s mRNA and cytosolic and nuclear protein levels were normalized to β-actin and Lamin B levels, respectively; these were used as house-keeping controls. Values are expressed as fold change compared to controls. Bars represent the mean ± SE. Statistically different from *C and from ^&E groups, p<.05. C, Control; E, Ethanol/Binge; TH, Ethanol/Binge/LPS model.

Fig. 2.. Effect of ethanol and LPS on Dvl status in the mice liver.

Total protein and RNA extracted from mice livers were used for western blot and qPCR analyses, respectively. (A) Total Dvl protein levels. This band was detected by using and anti-Dvl antibody that recognizes all the Dvl (Dvl1–3) variants (for details see Materials and methods). (B) Expression of Dvl variant genes; (C) Protein levels of Dvl1, Dvl2 and Dvl3 variants. While mRNA expression was normalized to 18s mRNA, Dvl, Dvl1, Dvl2 and Dvl3 protein levels were normalized to β-actin; these were used as house-keeping controls. Values are expressed as fold change compared to controls. Bars represent the mean ± SE. Statistically different from *C and from ^&E groups, p<.05. C, Control; E, Ethanol/Binge; TH, Ethanol/Binge/LPS model.

Fig. 3.. Effect of ethanol and LPS on Nxn/Dvl interaction and PI(4)P production in vivo.

Total protein extracts from mice livers were used for immnumoprecipitation and western blot analyses, and total lipid extracts were used for PI(4)P quantification by ELISA. (A) 4-HNE adduct levels; (B) Immunoprecipitation of Dvl coprecipitated Nxn, Fzd and Pi4k2a proteins. Dvl bands were detected by using and anti-Dvl antibody that recognizes all the Dvl (Dvl1–3) variants (for details see Materials and methods). (C) Quantification of PI(4)P levels. While β-actin expression was used to normalize 4-HNE adduct levels, that of the immunoglobulin heavy chain used for immunoprecipitation was used to normalize the immunoprecipitated. Values are expressed as fold change compared to controls. Bars represent the mean ± SE. Statistically different from *C and from ^&E groups, p<.05. C, Control; E, Ethanol/Binger; TH, Ethanol/Binge/LPS model; IgHC, Immunoglobulin heavy chain.

Fig. 4.. Ethanol and LPS effects on CYP2E1, ADH1 and NXN expression levels in the HSC/VL17A co-culture system.

For immunofluorescence, co-cultures were plated in two-well chamber slides and then cells were exposed to 100 mM ethanol for 48 h. (A) Analysis was performed in 4% formaldehyde fixed cells with mouse anti-Vinculin and rabbit anti-CYP2E1 antibodies and nuclei were staining with ToPro. Arrows show HSC and VL17A cells. (B) Efficiency of NXN transfection. Co-cultures were transfected either with 1 μg/ml of empty vector (EV) or NXN expressing vector and total protein was extracted after 48 h. (C) Co-cultures were exposed to 100 mM ethanol for 48 h and then LPS (1 μg/ml) was added and incubated for 3 h. Total protein was extracted and CYP2E1, ADH1 and NXN levels were determined by western blot. Protein levels were normalized to β-actin; this was used as house-keeping control. Three independent experiments were carried out in duplicated. Values are expressed as fold change compared to controls. Bars represent the mean ± SE. Statistically different from *C and ^#N groups, p<.05. C, Control; E, Ethanol; L, LPS; EL, Ethanol plus LPS. These groups were transfected with EV which were called untransfected cells. N, NXN-transfected cells; these cells were also exposed to E, L and EL treatments; NE, NL and NEL, respectively.

Fig. 5.. Ethanol and LPS effects on cytosolic and nuclear NXN, and DVL variants expression levels in the HSC/VL17A co-culture system.

Total, cytosolic and nuclear proteins extracted from co-cultures transfected either with EV or NXN expressing vector and exposed to ethanol and/or LPS were used for western blot analyses. While total and cytosolic protein levels were normalized to β-actin that of nuclear protein was normalized to Lamin B expression levels; these were used as house-keeping controls. Three independent experiments were carried out in duplicated. Values are expressed as fold change compared to controls. Bars represent the mean ± SE. Statistically different from *C and ^#N groups, p<.05. C, Control; E, Ethanol; L, LPS; EL, Ethanol plus LPS. These groups were transfected with EV which were called untransfected cells. N, NXNtransfected cells; these cells were also exposed to E, L and EL treatments; NE, NL and NEL, respectively.

Fig. 6.. Ethanol and LPS effects on ROS and GSH levels in the HSC/VL17A coculture system.

Co-cultures transfected either with EV or NXN expressing vector and exposed to ethanol and/or LPS were used for ROS and GSH determinations. (A) ROS levels were determined spectrofluometrically in black 96-well microplates by labeling 7×10³ cells/well approximately with DCFDA. (B) For GSH determination, after different treatments co-cultures were solubilized in 5% 5-sulfosalicylic acid as described in Methods. Three independent experiments were carried out in duplicated. Values are expressed as fold change compared to controls. Bars represent the mean ± SE. Statistically different from *C, from ^#N and from ^&the same treatment in untransfected cells groups, p<.05. C, Control; E, Ethanol; L, LPS; EL, Ethanol plus LPS. These groups were transfected with EV which were called untransfected cells. N, NXN-transfected cells; these cells were also exposed to E, L and EL treatments; NE, NL and NEL, respectively.

Fig. 7.. Ethanol and LPS effect on Nxn/Dvl interaction and PI(4)P production in vitro.

Total protein extracts from co-cultures transfected either with EV or NXN expressing vector and exposed to ethanol and/or LPS were used for DVL3 immunoprecipitation analyses. (A) Western blot analyses of NXN, FZD and PI4K2A coprecipitated from DVL3 immunoprecipitation. Densitometric quantification of (B) NXN, (C) FZD and (D) PI4K2a co-precipitated, and (E) DVL3 precipitated; (F) Quantification of PI(4)P levels by ELISA. Co- and immunoprecipitated were normalized to the heavy chain of the immunoglobulin used for immunoprecipitation. Two independent experiments were carried out in duplicated. Values are expressed as fold change compared to controls. Bars represent the mean ± SE. Statistically different from *C, from ^#N and from ^&the same treatment in untransfected cells groups, p<.05. C, Control; E, Ethanol; L, LPS; EL, Ethanol plus LPS. These groups were transfected with EV which were called untransfected cells. N, NXN-transfected cells; these cells were also exposed to E, L and EL treatments; NE, NL and NEL, respectively. Ig-HC, Immunoglobulin heavy chain.

Fig. 8.. Model of ethanol effect on NXN/DVL and FZD/DVL/PI4K interactions, PI(4)P production, and their regulation by NXN overexpression.

It is well-known that ethanol metabolism by liver cells mainly produces acetaldehyde (ACH) and ROS, among others sub-products. Our proposed model strongly suggests that elevated ROS levels disrupt NXN/DVL redox sensitive interaction allowing FZD/DVL/PI4K complex formation and binding into LPR6 transmembrane receptor. The formation of this complex in turn stimulates PI(4)P production. Chronic ethanol exposure also increases NXN and DVL protein levels and, preferentially by in vivo Ethanol/Binge model effect, NXN is translocated into the nucleus. These events are negligible in control liver cells because the endogenous antioxidant defense systems effectively neutralize oxidative stress thus maintaining the normal homeostatic functioning of NXN. Transfection of NXN expressing vector revels that the molecular alterations induced by ethanol exposure might be partially reverted by the overexpression of NXN in the co-culture system. Dotted arrows indicate the disruption of NXN/DVL interaction by high ROS levels. Molecules indicated in grey were not evaluated.