Department of Medical Pharmacology and Physiology, School of Medicine, University of Missouri-Columbia, One Hospital Drive, Columbia, MO 65212, USA
* Author to whom correspondence should addressed at: Department of Medical Pharmacology and Physiology, School of Medicine, University of Missouri-Columbia, One Hospital Drive, Columbia, MO 65212, USA. Tel.: +1 573 882 2740; Fax: +1 573 884 4558; E-mail: shuklasd{at}missouri.edu
(Received 10 March 2005; first review notifed 1 April 2005; in revised form 19 April 2005; accepted 28 April 2005)
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ABSTRACT |
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INTRODUCTION |
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In eukaryotic chromatin, the nucleosomal complexes formed by the histone octamer and associated DNA are the fundamental organizational units. The precise organization of DNA in chromatin has important functional consequences in processes such as transcription, replication, repair, recombination, and segregation (Cheung et al., 2000). The N-terminal and C-terminal tails of histone, which protrude from the surface of the chromatin polymer, undergo post-translational modifications including acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation. Histone modifications may alter chromatin structure by influencing histoneDNA and histonehistone contacts. Of the modifications listed above, histone acetylation has been the most studied. In H3 from most species, the main acetylation sites include lysine 9, 14, 18 and 23 (Strahl and Allis, 2001
; Timmermann et al., 2001
). In rat hepatocytes, we reported ethanol-induced selective, post-translational Ac-H3-lys9 in a dose-dependent and time-dependent manner with maximum response at 100 mM, 24 h (Park et al., 2003
). However, whether ethanol affects HSCs is not known. Therefore, we have investigated the ethanol effect on acetylation and methylation of histone H3 in these cells.
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MATERIALS AND METHODS |
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HSC isolation and culture
Male Sprague-Dawley rats (300400 g) were obtained from Charles River Laboratories (Wilmington, MA) and were fed standard regular diet for rodent (LabDiet®). All animals received care in compliance with the guideline of Animal Care Quality Assurance, University of Missouri-Columbia (ACQA). The rats were anesthetized with ethyl ether. The liver was perfused using collagenase-perfusion protocol (Weng and Shukla, 2000), and HSCs were isolated using Nycodenz density gradient centrifugation as described previously (Vyas et al., 1995
; Lu et al., 1998
; Ramm, 1998
) with some modifications. Briefly, the rat liver was perfused with Krebs-Ringer bicarbonate buffer (KRB) containing 0.25 mM EGTA for 12 min to remove blood, and buffer was switched to KRB containing 2 mM CaCl2 and collagenase (35 mg/100 ml) for 1015 min. After digestion, the liver was carefully removed, and transferred to a beaker containing Ca++/Mg++ free KRB with 0.5% bovine serum albumin (BSA). After gentle dispersion, the hepatic nonparenchymal cells (NPC) were separated from the hepatocytes by centrifugation at 50 g for 2 min at 4°C twice. The supernatant, composed of mainly NPC but also hepatocytes, was centrifuged at 450 g for 10 min at 4°C. The pellet was resuspended in 30 ml of 17.2% Nycodenz and centrifuged at 1400 g for 20 min at 4°C. The white, diffuse top layer of Nycodenz cushion, which is enriched with HSCs, was collected and diluted with phosphate-buffered saline (PBS) with 0.3% BSA and centrifuged at 450 g for 10 min at 4°C. The cells were plated on Petri dish (100 mm) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 100 IU/ml penicillin, 100 g/ml streptomycin and 2.5 g/ml amphotericin B (antifungal). The culture medium was changed 24 h later and thereafter every 23 days. The HSCs were identified by typical stellate shaped morphology (Friedman and Roll, 1987
; Kawada et al., 1998
).
Ethanol treatment and histone isolation
After HSCs achieved a 90% confluence (usually 23 weeks), the culture medium was changed to DMEM containing 0.1% FBS with or without alcohol and sealed with Parafilm® (Pechiney plastic packaging, Chicago, IL). HSCs were divided and treated with ethanol (0, 50, 100, and 200 mM) for various times (24, 48, 72, 96, and 120 h). TSA (2 µg/ml) was used as a positive control. The ethanol-containing medium was changed every 24 h during incubation period. Histone was isolated from HSCs as follows. After the desired time, HSCs were rinsed twice with ice-cold PBS, scrapped into 800 µl ice-cold lysis buffer containing 20 mM HEPES (pH 7.9), 1 mM EDTA, 10 mM NaCl, 2 mM MgCl2, 20 mM glycerophosphate, 0.25% NP-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 1 mM dithiothreitol and transferred into Eppendorf tube at 4°C. The cells were incubated on ice for 20 min and passed through a 26-gauge syringe needle 10 times. After centrifugation at 12 000 g for 10 min at 4°C, the pellet was washed again with ice-cold lysis buffer. The pellet was mixed with 0.5 ml of 0.4 N HCl/10% glycerol, incubated for 30 min at 4°C and centrifuged at 12 000 g for 10 min. The supernatant was mixed with trichloroacetic acid (TCA) in the ratio 4:1 making 20% TCA solution, incubated for 1 h at 4°C and centrifuged at 12 000 g for 10 min. The pellet was washed by 0.5 ml of acetone/0.02 N HCl, centrifuged at 12 000 g for 5 min and the pellets were dried under the hood for 30 min. The dried pellets were resuspended in water and sonicated. After centrifugation at 12 000 g for 10 min, supernatant was collected and stored in 270°C freezer. Protein concentration was measured using the Bio-Rad DC protein assay kit.
Western blot analysis
Equal amounts (510 µg) of proteins were subjected to 15% SDSPAGE and transferred onto nitrocellulose membrane. After blocking with 5% non-fat dried milk for 1.5 h at room temperature (RT), membranes were incubated overnight at 4°C with primary antibody with the dilution of 1:1000 for anti-H3 acetyl Lys23, 1:2000 for anti-H3 acetyl Lys9 or anti-H3 acetyl Lys14, 1:10 000 for anti-H3 acetyl Lys18 or anti-H3 dimethyl Lys9. After washing with TBST solution three times, membranes were incubated with goat anti-rabbit IgG HRP conjugated secondary antibody with 1:3000 dilution for 1 h at RT. Western blots were developed with peroxidase reaction with the ECL reagents (Pierce, Supersignal West Pico Chemiluminescent Substrate). Quantitative analysis was performed by densitometry analysis. We used Quantity-1 (version 4.1.1) software for the analysis of protein bands and GraphPad Prism (version 3.03) software for graph formation and statistical analysis (one-way ANOVA).
Immunofluorescence stain
HSCs were placed and cultured on culture slides (BD FalconTM). After 24 h, HSCs were treated with ethanolcontaining medium and were incubated; ethanol-containing medium was changed every 24 h. After 72 h ethanol treatment, the slides were immersed in pre-cooled (20°C) acetone/methanol (1:1) mixture for 30 min at 20°C. The slides were next incubated in PBS containing 0.5% Triton X-100 for 10 min at RT. For blocking, the slides were incubated in 5% BSAPBS for 1 h at RT. Primary antibody for acetyl-H3 (lys9) or dimethyl-H3 (lys9), diluted 1:50 in 1% BSAPBS, was applied and incubated overnight at 4°C in a humidified chamber with light shield. On the next day, the slides were incubated with 500 µl of 1% BSAPBS containing secondary antibody conjugated with FITC (1:200 diluted) for 1 h at RT in the humidified chamber. After washing, the slides were incubated with DAPI for 5 min. The slides were washed with PBS for 10 min three times between each step. Specimens stained with DAPI and FITC were examined using Epi-fluorescence microscopy (Nikon, Japan) equipped with filters for FITC and ultra violet (x400).
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RESULTS |
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DISCUSSION |
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Exposure of HSCs to ethanol resulted in time-dependent and dose-dependent increases in selective acetylation of Lys9 in histone H3 protein. A maximum increase of 80-fold was observed at 200 mM ethanol treatment for 72 h. The in vivo peripheral blood concentration of ethanol in chronic alcoholics is normally <50 mM. Therefore, the concentrations used in the present study for HSCs may appear higher. However, the exact concentration of ethanol to which HSCs are exposed in situ in the liver remain poorly understood. It may be noted that in pigs orally administered with ethanol, portal venous blood ethanol was 2-fold higher than in peripheral blood (Elmer et al., 1982
; Luca et al., 1997
). First pass hepatic metabolism can explain this difference. Furthermore, the hepatic venous blood is diluted 45 times in the caval vein (Nuutinen et al., 1984
). The liver is also well known for its high blood supply, 70% of blood supplied from mesenteric portal venous system. In addition, acute administration of ethanol increases portal blood flow by 4060% (Orrego et al., 1988
). Taken together, liver cells are exposed to higher concentration and higher amounts of ethanol than expected by the peripheral blood level. It is also worth mentioning that the upper limit of ethanol in chronic alcoholics can be 100 or 245 mM or in one report as high as 300 mM (Deitrich and Harris, 1996
). Thus use of ethanol in the range of 50200 mM provides a reasonable range for experimental studies. We have consistently observed effects of ethanol on HSCs at 50 mM. However, to increase the sensitivity of our assay we have also used 100 and 200 mM concentrations.
The time required for the largest increment of Ac-H3-lys9 in HSCs, 72 h, was longer compared with hepatocytes where maximal acetylation occurred at 24 h ethanol exposure (Park et al., 2003). This finding suggested that HSCs are relatively slow reacting cells that might require longer ethanol exposure for H3 acetylation. The role of histone modifications by ethanol in vitro or in vivo, for example, in cell proliferation, growth, or gene expression, remains unknown at present. Lys9 in the H3 tail can be targeted for both acetylation and methylation and these modifications have opposite effects on the affinity with DNA and on transcriptional activity (Rice and Allis, 2001
). Because ethanol caused dose-dependent increase of Ac-H3-lys9, we expected methylation to decrease with increasing ethanol concentration. We monitored this by western blot and immunofluorescence stain. Although the intensity of the FITC-labelled dimethyl histone H3 at Lys9 antibody appeared to decrease slightly with increasing dose of ethanol, the levels of Me-H3-Lys9 did not appear to change when monitored by western blotting. Thus there is no significant change in the Me-H3-lys9 by ethanol in HSCs.
In conclusion, ethanol caused dose-dependent-and time-dependent increase of Ac-H3-lys9 monitored by both western blot and FITC stain in HSC cells. This is not owing to increased H3 protein expression. Levels of Ac-H3-lys14 or Ac-H3-lys18 were unaffected. Compared with hepatocytes the Ac-H3-lys9 in HSCs required longer ethanol exposure (24 vs 72 h). Levels of Me-H3-lys9 seemed to remain unaltered. Thus increase of Ac-H3-lys9 represents a nuclear-chromatin modification event in HSC exposed to ethanol.
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ACKNOWLEDGEMENTS |
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