1 Department of Medicine and 2 Department of Oncology and Immunology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2195, USA
* Author to whom correspondence should be addressed at: Department of Medicine, 921 Ross Research Building, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205-2195, USA. Tel.: +1 410 955 7856; Fax: +1 410 955 9677; E-mail: emezey{at}jhmi.edu
(Received 27 July 2004; first review notified 16 September 2004; in revised form 18 October 2004; accepted 19 October 2004)
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ABSTRACT |
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INTRODUCTION |
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Hepatic stellate cells are the source of collagen and other extracellular matrix proteins that accumulate during hepatic fibrosis. Acetaldehyde, the product of alcohol oxidation, enhances 1(I) and
2(I) gene transcription and type I collagen production by hepatic stellate cells (Casini et al., 1991
; Anania et al., 1996
). TNF
increases hepatic inflammation, but inhibits
1(I) and
2(I) gene transcription by stellate cells (Hernandez et al., 2000
; Novitskiy et al., 2004
). NF-
B consists principally of homo- and heterodimers of p50 and p65 protein units, which in their inactive state are present in the cytoplasm, bound to the inhibitors of NF-
B activity (I
B). Phosphorylation of I
B by kinases results in the degradation of I
B with release of NF-
B, which translocates to the nucleus where it is active in regulation of transcription gene.
Acetaldehyde (Roman et al., 2000; Novitskiy et al., 2004
) and TNF
(Nehls et al., 1991
; Novitskiy et al., 2004
) independently increase NF-
B (p65) and its binding to the
2(I) collagen promoter (Novitskiy et al., 2004
).
The activities of both the 2(I) (Novitskiy et al., 2004
) and
1(I) (Rippe et al., 1999
) collagen promoters are inhibited by transfection of the NF-
B (p65) expression vector. Hence, the inhibitory effect of TNF
on the
1(I) collagen promoter may be mediated by NF-
B (p65), whereas the activating effect of acetaldehyde on the collagen promoters is unrelated to its effect on NF-
B (p65).
The aim of this study was to determine the time course and mechanism of the effects of acetaldehyde in comparison to the effects of TNF on NF-
B activation in hepatic stellate cells.
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MATERIALS AND METHODS |
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Animals and stellate cell culture
Adult male SpragueDawley rats were obtained from Charles River Laboratories (Wilmington, MA). All animals received humane care in compliance with the guidelines of the Animal Care and Use Committee of the Johns Hopkins University. Stellate cells were isolated in situ, under xylazine and ketamine anaesthesia, by perfusion of the portal vein under sterile conditions with 0.2% pronase E and 0.015% collagenase in DMEM in a sequential manner. The liver cell suspension obtained was centrifuged at 1400 g in a two-step discontinuous Nycodenz gradient (Anania et al., 1996). The isolated cells were suspended in DMEM and seeded in 25 cm2 tissue culture flasks maintained in DMEM containing 10% FBS, fungizone (2.5 mg/ml), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37°C with a humidified atmosphere of 5% CO2 and 95% air. The medium was changed every 48 h, while the cells transform into activated cells after 1014 days in culture. The experiments with TNF
and acetaldehyde were in the activated stellate cells at 7080% confluence. The media was removed, the cells were washed with serum-free DMEM, and after 1 h the medium was changed to serum-free DMEM containing the following six supplemental growth factors (SGF6): epidermal growth factor (10 µg/l), transferrin (0.5 mg/l), selenous acid (5 µg/l), linoleic acid (0.5 mg/l), bovine serum albumin (0.5 mg/l) and fetuin (0.5 mg/l). TNF
was added at a concentration of 0.6 nM, while the concentration of added acetaldehyde was 200 µM. The flasks were tightly capped.
Western Blot Analysis
Cells were lysed in NP-40 lysis buffer containing 50 mM TrisHCl, pH 8.0, 400 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM Na3VO4, 1 mM PMSF, 1 µg/ml of leupeptin, and 1 µg/ml aprotinin for 1 h at 4°C, and then centrifuged at 12 000 g for 15 min at 4°C. The cytosolic protein in the supernatant was initially stored at 80°C. Protein concentration was determined by the method of Lowry et al. (1951). The protein for western blot was separated on mini-SDS gels at 100 V for 1 h and electrotransferred to polyvinylidine difluoride membranes (Hybond-P, Amersham Biosciences, Piscataway, NJ) at 100 V for 1 h. The membranes were washed in PBS, pH 7.6, containing 0.1% Tween 20 (PBS-T), blocked with 5% (w/v) dried nonfat milk in PBS-T for 1 h, then incubated with rabbit polyclonal antibodies to NF-
B (p50), NF-
B (p65), I
B-
and phosphorylated I
B-
(Santa Cruz Biotechnology) at 4°C overnight. After repeated washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10 000 dilution, Amersham Life Science) at room temperature for 1 h. The membranes were then subsequently washed and visualized by enhanced chemiluminescence reaction (ECL-plus, Amersham Biosciences).
IB Kinase Assay
IB-
kinase activity was determined by the method of Mercurio et al. (1997)
. The cytosolic protein was incubated with antibody to IKK-
(Santa Cruz Biotechnology) overnight. Twenty microlitres of protein A Sepharose (Rockland, Gilbertsville, PA) was then added. After 2 h of incubation, the antigenantibodyprotein A Sepharose complex was precipitated, washed and then assayed for kinase activity. The reaction mixture for the kinase activity consisted of 20 mM HEPES buffer, pH 7.4, 20 mM MgCl2, 2 mM MnCl2, 10 mM ß-glycerophosphate, 10 mM NaF, 10 mM p-nitrophenyl phosphate, 300 µM Na3VO4, 1 mM benzamidine, 1 mM DTT, 2 µM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 10 µg/ml of aprotinin, 20 µCi [
-32P] ATP, 10 µM ATP and 2 µg GST I
B-
(Santa Cruz Biotechnology). The total volume of reaction mixture was 25 µl. After incubation at 30°C for 30 min, the reaction was terminated by boiling with 5 µl of 6x SDS sample buffer for 5 min. The protein was resolved on a 10% polyacrylamide gel under reducing conditions. The gel was dried and the radioactive bands were visualized by PhosphorImaging.
Data analysis
Data were analysed with the Student's t-test when appropriate, or by two-way analysis of variance when comparing means of more than two groups.
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RESULTS |
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Inhibitory B kinase activity, using GST-I
B-
as a substrate (I
-B
kinase), was maximally increased by acetaldehyde at 10 min of exposure, with a subsequent fall at 20 min (Fig. 3A). This is in contrast to the effect of TNF
, which caused a higher I
B-
kinase activity after 20 min than after 10 min of exposure (Fig. 3B).
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DISCUSSION |
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This study also shows that acetaldehyde results in an earlier and more transient increase in IB-
kinase activity and decrease in I
B-
than found with TNF
indicating that the effect of acetaldehyde in decreasing I
B-
, which results in increased nuclear NF-
B, is not mediated by TNF
. Furthermore, this study explains the previous contradictory data on the effect of acetaldehyde, which either did not change (Roman et al., 2000
) or increased I
B-
(Novitskiy et al., 2004
) after 30 min of exposure, since those experiments missed the early decrease in I
B-
occurring at 10 min after exposure to acetaldehyde. Despite the findings that degradation of I
B-
after exposure to acetaldehyde is followed by its recovery to control or higher I
B-
levels, nuclear NF-
B(p65) remained elevated for up to 24 h (Novitskiy et al., 2004
). Similarly, after stimulation of various cells by lipopolysaccaride (LPS) and cytokines such as interleukin-1b or TNF
, NF-
B (p65) remains elevated for up to 24 h although I
B-
is newly synthesized (Lee et al., 2003
).
In this study, TNF in the stellate cells results in a greater increase in I
B-
kinase after 20 min than after 10 min of exposure. This result differs to some extent from the effect of TNF
in other cell types (DiDonato et al., 1997
; Kirillova et al., 1999
), where maximum increase in I
B-
kinase occurred after 5 min of exposure, with subsequent decreases after 1520 min. The decrease in I
B-
kinase is followed by a further slight decrease at 30 min, which then remains steady for at least 90 min (DiDonato et al., 1997
).
The effect of acetaldehyde on NF-B activation is possibly mediated by increased oxidative stress. Acetaldehyde increases the formation of oxygen radicals in hepatocytes (Putarulo and Cederbaum, 1989
; Shaw and Jayatilleke, 1990
) and in stellate cells (Novitskiy et al., 2004
, and our unpublished data.). Reactive oxygen species in turn have been shown to mediate the effects of cytokines or to lead directly to NF-
B activation (Kutuk and Basaga, 2003
; Ryan et al., 2004
).
The effects of NF-B differ with respect to liver injury and fibrogenesis. The activation of NF-
B increases liver injury in hepatocytes, whereas it inhibits fibrogenesis in stellate cells (Novitskiy et al., 2004
). As expected, the effects of TNF
in activating NF-
B are associated with increased liver injury and decreased fibrogenesis. In contrast, the effect of acetaldehyde in activating NF-
B is associated with increases in both liver injury and fibrogenesis, indicating that the effects of acetaldehyde on fibrogenesis are mediated by trans-acting factors other than NF-
B or by cytokines that stimulate fibrogenesis such as TGFß. The activation of murine type I collagen promoters has been shown to be mediated by NF-I (Anania et al., 1995
) and C/EBPß (Attard et al., 2001
). TGFß is a strong stimulator of fibrogenesis (Penttinen et al., 1988
). A number of studies indicate that the effects of acetaldehyde on enhancing Type I collagen transcription and collagen formation are, at least in part, mediated by TGFß (Anania et al., 1996
; Chen, 2002
). Acetaldehyde increases the secretion and activation of TFGß by stellate cells and increases the expression of the type II TGF-ß1 receptor (Chen, 2002
). The effect of acetaldehyde in activating the rat
1(I) collagen promoter is partially blocked by neutralizing anti-TGFß antibodies and by antisense Tß-RII oligonucleotides (Chen, 2002
), and neutralizing anti-TGFß antibody is known to suppress the effect of acetaldehyde in increasing mouse
1(I) collagen mRNA (Anania et al., 1996
).
The additional finding in this study that acetaldehyde increased both nuclear NF-B (P65) and (p50), whereas TNF
merely increased NF-
B (p65), probably contributes to the lack of suppressive effect of acetaldehyde on fibrogenesis as well. NF-
B (p50) has been described to be inactive or less active in transcriptional regulation of genes, and in some cases suppresses the effects of NF-
B (p65) (Udalova et al., 2000
). Furthermore, the NF-
B (p65), but not the NF-
B (p50) expression vector resulted in inhibition of the
2(I) collagen promoter in transfected stellate cells (Novitskiy et al., 2004
). In contrast, other studies with different cell types also found that TNF
increased only NF-
B (p65) (Aoudjit et al., 1997
). The activating effect of acetaldehyde on NF-
B (p50) with a lack of effect and/or suppression of an enhancing effect of NF-
B (p65) may allow other trans-activating factors to mediate the activating effect of acetaldehyde on the collagen promoter.
In conclusion, TNF and acetaldehyde independently activate NF-
B by rapid enhancement of I
B-
kinase activity with degradation of the I
B-
protein. Increased TNF
is the principal mechanism for the elevation of NF-
B in severe alcoholic hepatitis. The elevation of NF-
B due to TNF
enhances liver injury but inhibit fibrogenesis. In contrast, the effect of acetaldehyde in activating NF-
B is associated with increases in both liver injury and fibrogenesis, indicating that the effects of acetaldehyde on fibrogenesis are mediated by cytokines and by trans-acting factors other than NF-
B.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Anania, F. A., Potter, J. J., Rennie-Tankersley, L. and Mezey, E. (1996) Activation by acetaldehyde of the promoter of the mouse 2 (I) collagen gene when transfected into rat activated stellate cells. Archives of Biochemistry and Biophysics 331, 187193.[CrossRef][ISI][Medline]
Aoudjit, F., Brochu, N., Belanger, B., Stratowa, C., Hiscott, J. and Audette, M. (1997) Regulation of intracellular adhesion molecule-1 gene by tumor necrosis factor- is mediated by the nuclear factor
B heterodimers p65/p65 and p65/c-Rel in the absence of p50. Cell Growth and Differentiation 8, 335342.[Abstract]
Attard, F. A., Wang, L., Potter J. J., Rennie-Tankersley, L. and Mezey, E. (2001) Identification of new sites of binding and activation of the murine 1(I) collagen promoter by CCAAT/enhancer binding protein ß. DNA and Cell Biology 20, 445463.
Bird, G. L., Sheron, N., Goka, A. K., Alexander, G. J. and Williams, R. S. (1990) Increased plasma tumor necrosis factor in severe alcoholic hepatitis. Annals of Internal Medicine 112, 917920.[ISI][Medline]
Casini, A., Cunningham, M., Rojkind, M. and Lieber, C. S. (1991) Acetaldehyde increases procollagen type I and fibronectin gene transcription in cultured rat fat-storing cells through a protein synthesis-dependent mechanism. Hepatology 13, 758765.[ISI][Medline]
Chen, A. (2002) Acetaldehyde stimulates the activation of latent transforming growth factor-ß1 and induces the expression of the type II receptor of the cytokine in rat cultured hepatic stellate cells. Biochemical Journal 368, 683693.[CrossRef][ISI][Medline]
DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E. and Karin, M. (1997) A cytokine-responsive IB kinase that activates the transcription factor NF-
B. Nature 388, 548554.[CrossRef][ISI][Medline]
Felver, M. E., Mezey, E., McGuire, M., Mitchell, M. C., Herlong, H. F., Veech, G. A. and Veech, R. L. (1990) Plasma tumor necrosis factor alpha predicts long-term survival in severe alcoholic hepatitis. Alcoholism: Clinical and Experimental Research 14, 355259.
Ghosh, S., May, M. J. and Kopp, E. B. (1998) NF-B and REL proteins evolutionary conserved mediators of immune responses. Annual Review of Immunology 16, 225260.[CrossRef][ISI][Medline]
Hernandez, I., de la Torre, P., Rey-Campos, J., Garcia, I., Sanchez, J. A., Munoz., R., Rippe, R. A., Munoz-Yague, T. and Solis-Herruzo, J. A. (2000) Collagen 1(I) gene contains an element responsive to tumor necrosis factor-alpha located in the 5' untranslated region of its first exon. DNA and Cell Biology 19, 341352.[CrossRef][ISI][Medline]
Hill, D. B., Barve, S., Joshi-Barve, S. and McClain, C. (2000) Increased monocyte nuclear factor-B activation and tumor necrosis factor in alcoholic hepatitis. Journal of Laboratory and Clinical Medicine 135, 387395.[CrossRef][ISI][Medline]
Hirano, F., Komura, K., Fukawa, E. and Makino, I. (2003) Tumor necrosis factor (TNF-
) induces RANTES chemokine expression via activation of NF-
B and p38 MAP kinase: roles of TNF-
in alcoholic liver disease. Journal of Hepatology 38, 483489.[CrossRef][ISI][Medline]
Karin, M. and Ben-Neriah, Y. (2000) Phosphorylation meets ubiquitination: the control of NF-B activity. Annual Review of Immunology 18, 621663.[CrossRef][ISI][Medline]
Kirillova, I., Chaisson, M. and Fausto, N. (1999) Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor B activation. Cell Growth and Differentiation 10, 819828.
Kono, H., Wheeler, M. D., Rusyn, I., Lin, M., Seabra, V., Rivera, C. A., Bradford, B. U., Forman, D. T. and Thurman, R. G. (2000) Gender differences in early alcohol-induced liver injury: role of CD14, NF-B. and TNF
. American Journal of Physiology Gastrointestinal and Liver Physiology 278, G652661.
Kutuk, O. and Basaga, H. (2003) Aspirin prevents apoptosis and NF-kappaB activation induced by H2O2 in hela cells. Free Radical Research 37, 12671276.[CrossRef][ISI][Medline]
Lee, Y., Allport, V., Sykes, A., Lindstrom, T., Slater, D. and Bennett, P. (2003) The effect of labour and of interleukin 1 beta upon the expression of nuclear factor kappa B related proteins in human amnion. Molecular Human Reproduction 9, 213218.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265275.
Mercurio, F., Zhu, H., Murray, B. W. et al. (1997) IKK-1 and IKK-2: cytokine-activated IB kinases essential for NF-
B activation. Science 278, 860866.
Mezey, E., Potter, J. J., Rennie-Tankerley, L., Caballeria, J. and Pares A. (2004) A randomized placebo controlled trial of vitamin E for alcoholic hepatitis. Journal of Hepatology 40, 4046.[Medline]
Nehls, M. C., Rippe, R. A., Veloz, L. and Brenner, D. A. (1991) Transcription factors nuclear factor I and Sp1 interact with the murine collagen 1(I) promoter. Molecular and Cellular Biology 11, 40654073.[ISI][Medline]
Novitskiy, G., Potter, J. J., Rennie-Tankersley, L. and Mezey, E. (2004) Identification of a novel NF-B binding site with regulation of the murine
2(I) collagen promoter. Journal of Biological Chemistry 279, 1563915644.
Penttinen, R. P., Kobayashi, S. and Bornstein, P. (1988) Transforming growth factor ß increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability. Proceedings of the National Academy of Sciences of the United States of America 85, 11051108.[Abstract]
Potter, J. J., Rennie-Tankersley, L. and Mezey, E. (2003) Endotoxin enhances liver alcohol dehydrogenase by action through upstream stimulatory factor but not by nuclear factor-B. Journal of Biological Chemistry 278, 43534357.
Putarulo, S. and Cederbaum, A. I. (1989) Chemiluminescence from acetaldehyde oxidation by xanthine oxidase involves generation of and interactions with hydroxy radicals. Alcoholism: Clinical and Experimental Research 13, 8490.[ISI][Medline]
Rippe, R. A., Schrum, L. W., Stefanovic, B., Solis-Herruzo, J. A. and Brenner, D. A. (1999) NF-kappaB inhibits expression of the 1(I) collagen gene. DNA and Cell Biology 18, 751761.[CrossRef][ISI][Medline]
Roman, J., Gimenez, A., Lluis, J. M., Gasso, M., Rubio, M., Caballeria, J., Pares, A., Rodes, J. and Fernandez-Checa, J. C. (2000) Enhanced DNA binding and activation of transcription factors NF-B and AP-1 by acetaldehyde in HEPG2 cells. Journal of Biological Chemistry 275, 1468414690.
Ryan, K. A., Smith, M. F. Jr, Sanders, M. K. and Ernst, P. B. (2004) Reactive oxygen and nitrogen species differentially regulate Toll-like receptor 4-mediated activation of NF-B and interleukin-8 expression. Infection and Immunity 72, 21232130.
Shaw, S. and Jayatilleke, E. (1990) The role of aldehyde oxidase in ethanol-induced hepatic lipid peroxidation in the rat. Biochemical Journal 268, 579583.[ISI][Medline]
Udalova, I. A., Richardson, A., Denys, A., Smith, C., Ackerman, H., Foxwell, B. and Kwiatkowski, D. (2000) Functional consequences of a polymorphism affecting NF-B p50-p50 binding to the TNF promoter region. Molecular and Cellular Biology 20, 91139119.
Uesugi, T., Froh, M., Arteel, G. E., Bradford, B. U., Gabele, E., Wheeler, M. D. and Thurman, R. G. (2001) Delivery of IB superrepressor gene with adenovirus reduces early alcohol-induced injury in rats. Hepatology 34, 11491157.[CrossRef][ISI][Medline]
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