Microtubules are more stable and more highly acetylated in ethanol-treated hepatic cells

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1 Journal of Hepatology 44 (2006) Microtubules are more stable and more highly acetylated in ethanol-treated hepatic cells George T. Kannarkat 1, Dean J. Tuma 2, Pamela L. Tuma 1, * 1 Department of Biology, The Catholic University of America, 620 Michigan Avenue, NE, Washington, DC 20064, USA 2 The Liver Study Unit, The Veterans Administration Medical Center, Omaha, NE 68105, USA Background/Aims: Chronic alcohol consumption can lead to serious liver disease. Although the disease progression is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not understood. Methods: We examined hepatocyte-specific, alcohol-induced alterations in microtubule dynamics in WIF-B cells. These cells provide an excellent model for studying alcohol-induced hepatotoxicity; they remain differentiated in culture and metabolize alcohol. Results: Consistent with reports in other hepatic systems, microtubule polymerization in ethanol-treated WIF-B cells was impaired. However, when viewed by epifluorescence, the microtubules in ethanol-treated cells resembled stable polymers. Antibodies to acetylated a-tubulin confirmed their identity morphologically and revealed biochemically that ethanol-treated cells had approximately three-fold more acetylated a-tubulin than control cells. Livers from ethanolfed rats also contained increased levels of acetylated a-tubulin. Consistent with increased acetylated a-tubulin levels, microtubules in ethanol-treated WIF-B cells were more stable. Because stability increased with increased time of ethanol exposure or concentration, was prevented by 4-methylpyrazole and was potentiated by cyanamide, we conclude that increased acetylation requires alcohol metabolism and is likely to be mediated by acetaldehyde. Conclusions: Ethanol metabolism impairs tubulin polymerization, but once microtubules are formed they are hyperstabilized. These ethanol-induced alterations in microtubule integrity likely have profound effects on hepatocyte function. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Alcohol; Acetaldehyde; Hepatocytes; WIF-B cells; Microtubules; Acetylation 1. Introduction The liver is the major site of ethanol metabolism, and thus, the most susceptible organ to alcohol-induced injury. In the early stages of the disease, a fatty liver develops due to the accumulation of triglycerides in hepatocytes, leading to hepatocyte injury, liver fibrosis, and ultimately to cirrhosis. Although the disease progression is well described clinically, the molecular basis for alcohol-induced hepatotoxicity is not understood. Received 21 April 2005; received in revised form 29 June 2005; accepted 2 July 2005; available online 27 July 2005 * Corresponding author. Tel.: C ; fax: C address: tuma@cua.edu (P.L. Tuma). Traditionally, animal models have been used to describe physiological responses to alcohol consumption. Although these studies have provided a wealth of information, there are disadvantages to using animal models. Not only is there considerable physiologic variation among animals, it is also difficult to quickly alter experimental parameters that are required for mechanistic studies. In addition, experimental reagents are introduced to all organs of the animal s body where they may interfere with defining hepatic-specific responses or produce severe side effects. Because these experimental barriers prevent good mechanistic studies in animals, many researchers have turned to in vitro model systems. The polarized, hepatic WIF-B cells have recently emerged as an excellent model system to investigate alcohol-induced hepatotoxicity. In culture, 70 95% of mature WIF-B cells exhibit structures that are functionally /$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi: /j.jhep

2 964 G.T. Kannarkat et al. / Journal of Hepatology 44 (2006) and compositionally analogous to bile canaliculi (BC) [1]. Domain-specific membrane proteins are localized in WIF-B cells as they are in hepatocytes in situ and many liverspecific functions are maintained [1]. Importantly, WIF-B cells efficiently metabolize ethanol into acetaldehyde using alcohol dehydrogenase (ADH) and cytochrome P4502E1 [2]. Like hepatocytes in situ, ethanol-treated WIF-B cells have a reduced redox state and increased triglycerides corresponding to the clinically observed fatty liver [2]. As ethanol is metabolized, acetaldehyde is produced. This highly reactive metabolite can readily covalently modify proteins, DNA and lipids [3 13]. Many proteins have been shown to be modified by acetaldehyde including tubulin, actin, calmodulin, hemoglobin, hepatic enzymes and plasma proteins [8 10,12 14]. In general, acetaldehyde is thought to form stable adducts with the 3-amino group of lysine residues [11,15,16]. The hypothesis is that these covalent modifications disrupt the normal functioning of hepatic proteins leading to cell injury. One of the best-studied target proteins for acetaldehyde is a-tubulin [11]. In vitro, this protein was found to be preferentially modified on a highly reactive lysine which led to drastically impaired microtubule polymerization [11,17]. Similarly, in hepatocytes isolated from ethanol-fed rats, microtubule regrowth after nocodazole washout was inhibited [17]. We chose to extend these studies on alcohol-induced changes in microtubule dynamics in polarized, hepatic WIF-B cells. We initiated our studies by examining microtubule polymerization in ethanoltreated WIF-B cells and found that it was impaired as shown for other hepatic systems. We also made the exciting finding that polymerizing and steady state populations of microtubules were more highly acetylated and more stable. Importantly, these changes were also observed in livers from alcohol-fed rats indicating the physiologic importance of our WIF-B observations. By using inhibitors of ethanol metabolism, we further determined that this response required alcohol metabolism and was likely mediated by acetaldehyde. Because microtubules are central to many cellular processes including mitosis, vesicular transport, organelle placement and transport, we propose that altered microtubule dynamics in ethanol-exposed cells have farreaching effects on numerous hepatocyte functions Ethanol and drug treatments In general, WIF-B cells were treated with 50 mm ethanol in medium buffered with 10 mm Hepes (HyClone, Logan, Utah), ph 7.0 at 37 8C for 72 h as described [2]. Cells were treated with nocodazole, 4-methypyrazole (4-MP), cyanamide or taxol (all from Sigma-Aldrich) as described in the figure legends Immunofluorescence microscopy In general, WIF-B cells were fixed on ice with PBS containing 4% paraformaldehyde for 1 min and permeabilized with methanol for 10 min [18]. To detect acetylated tubulin, cells were fixed and permeabilized with methanol at K20 8C for 5 min. Cells were processed for indirect immunofluorescence as described [18] using anti-a-tubulin (1:500) or anti-acetylated-a-tubulin (1:100) monoclonal antibodies (Sigma-Aldrich). Alexa-568-conjugated secondary antibodies (Molecular Probes, Eugene, OR) were used at 5 mg/ml Imaging Labeled cells were visualized by epifluorescence using an Olympus BX60 Fluorescence Microscope (OPELCO, Dulles, VA). Images were taken using a SPOT digital camera and software (Diagnostic Instruments, Sterling Heights, MI). Images were processed and figures compiled using Adobe Photoshop (Adobe Systems, Inc., Mountain View, CA) Immunoblot analysis Cells were washed in prewarmed PBS and lysed directly in SDS-PAGE sample buffer. To determine relative amounts of soluble and insoluble tubulin, cells were washed with prewarmed PEM (80 mm Pipes, 1 mm MgCl 2, 2 mm EGTA, ph 6.8) and extracted in PEM containing 0.15% Triton X-100 at 37 8C for 1 min. The supernatant was collected and SDS- PAGE sample buffer added. The permeabilized cells were washed in prewarmed PEM and immediately lysed in SDS-PAGE sample buffer. Liver homogenates were prepared from rats that were pair-fed control and ethanol (36% of calories) Lieber-DeCarli liquid diets for 5 weeks as described [19]. Excised livers were Dounce homogenized in 0.25 M sucrose, 10 mm Tris, ph 7.4 (20%, w/v). Samples were immunoblotted with anti-a-tubulin (1:7500) or anti-acetylated-a-tubulin (1:4000). Immunoreactivity was detected using enhanced chemiluminescence (Pierce, Rockford, IL). Relative levels of the tubulin species were determined by densitometric analysis of immunoreactive bands. The levels of acetylated a-tubulin were normalized against the total level of a-tubulin present in each sample Statistical analysis Results were expressed as the meangsem Comparisons between control and ethanol-treated cells were made using the Student s t test for paired data. P values of %0.05 were considered significant. 3. Results 2. Materials and methods 2.1. Cell culture WIF-B cells were grown in a humidified 7% CO 2 incubator at 37 8C in Coon s-modified F-12 medium (Sigma-Aldrich, St Louis, MO), ph 7.0, supplemented with 5% fetal bovine serum (Gemini Bio-Products, Woodland, CA) [1]. Cells were seeded onto glass coverslips at 1.3!10 4 cells/cm 2 and cultured for 8 12 days until they reached maximum density and polarity Ethanol impairs microtubule polymerization Acetaldehyde has been shown to covalently modify tubulin which impairs microtubule polymerization in vitro [11]. Similarly, microtubule regrowth after nocodazole washout was impaired in hepatocytes isolated from ethanol-fed rats, [17]. To further confirm that WIF-B cells are a good model system for studying alcohol-induced hepatic injury, we tested microtubule regrowth using both

3 G.T. Kannarkat et al. / Journal of Hepatology 44 (2006) morphological (data not shown) and biochemical methods. Microtubules were first depolymerized by addition of the microtubule-disrupting agent, nocodazole (Fig. 1A, 0 min). After 60 min of re-incubation in nocodazole-free medium, w70% of total tubulin was polymeric in control cells, equivalent to the amount of steady-state polymeric tubulin (data not shown). In contrast, only 35% of tubulin was polymeric after 60 min in ethanol-treated cells (Fig. 1A). Thus, O40% of the total tubulin repolymerized after nocodazole treatment in control cells as compared to!10% in ethanol-treated cells (Fig. 1B). Thus, microtubule polymerization was significantly impaired in ethanoltreated WIF-B cells Microtubules are hyperacetylated and more stable in ethanol-treated cells We next examined the morphology of microtubules at steady state in control and ethanol-treated WIF-B cells by indirect immunofluorescence. Numerous phase-lucent BCs were evident in both control and ethanol-treated cells indicating that the polarized phenotype was maintained after treatment (Fig. 2a and b). In polarized hepatic cells, microtubules emanate from centrosomal structures located near the apical surfaces. Such an arrangement was observed in polarized WIF-B cells with microtubules radiating from BCs (several are marked with asterisks; Fig. 2c). In ethanoltreated cells, microtubules also emanated from BCs, but appeared shorter and more gnarled than in control cells (Fig. 2d). Similar alterations in microtubule morphology have been reported for ethanol-exposed hepatocytes in situ [20] and cultured Caco-2 cells [21]. Because this gnarled phenotype is a hallmark of stable microtubules, we stained ethanol-treated cells for acetylated a-tubulin, a post-translational modification present on stable microtubules [22 24]. As shown in Fig. 3A, the specific anti-acetylated-a-tubulin antibodies recognized microtubules at or near BCs in both control and ethanol-treated cells, but staining was more intense in ethanol-treated cells. This apparent increase in acetylation was confirmed biochemically (Fig. 3B). Cell lysates were immunoblotted Fig. 1. Ethanol impairs microtubule polymerization. WIF-B cells were treated with 50 mm ethanol (EtOH) for 72 h. In the continued absence or presence of ethanol, 5 mm nocodazole was added for 1 h at 37 8C to depolymerize microtubules. To allow repolymerization, the nocodazole was removed and the cells reincubated at 37 8C for the indicated times. Cells were extracted in a microtubule-stabilizing buffer containing 0.15% Triton X-100 for 1 min at 37 8C. Cells were then lysed in a microtubule stabilizing buffer containing 0.15% Triton X-100. Soluble tubulin was released into the supernatant while polymeric tubulin remained cell-associated. Samples were immunoblotted for a-tubulin (A and B) or acetylated a-tubulin (C). A, The percent of polymeric tubulin at each time point is plotted. B, From the values in A, the percent of total tubulin that repolymerized was calculated. C, The percent of polymeric, acetylated a-tubulin at each time point is plotted. In A and C, values are the average GSEM from four independent experiments. *P!0.004, **P!0.04. Fig. 2. Microtubules are shorter and gnarled in ethanol-treated cells. WIF-B cells were treated in the absence or presence of 50 mm ethanol (EtOH) for 72 h. Phase images (a and b) and a-tubulin labeling are shown (c and d). Asterisks are marking selected BCs.

4 966 G.T. Kannarkat et al. / Journal of Hepatology 44 (2006) Fig. 3. Microtubules are more highly acetylated in ethanol-treated cells. A, WIF-B cells were treated in the absence or presence of 50 mm ethanol (EtOH) for 72 h. Cells were labeled for acetylated a-tubulin. Asterisks are marking selected BCs. B, Cell extracts from control or ethanol-treated WIF-B cells and liver homogenates from control and ethanol pair-fed rats were immunoblotted for a-tubulin and acetylated a-tubulin. with specific antibodies to detect the total a-tubulin pool or the acetylated population. Importantly, no change in the total amount of a-tubulin was detected consistent with reports from ethanol-exposed hepatocytes [17]. We normalized the levels of acetylated a-tubulin to the amount of total a-tubulin in each sample and found that ethanoltreated cells had 2.62G0.48-fold more acetylated a-tubulin than control cells. We confirmed this result in livers of ethanol-fed rats. As for the WIF-B cells, no changes in the total a-tubulin pool were observed in ethanol-treated livers. However, both ethanol-fed rats shown had increased levels of acetylated tubulin, and 2.65-fold more than their pair-fed controls indicating our WIF-B findings have physiologic importance. We next tested whether increased acetylated tubulin correlated with increased microtubule stability in ethanoltreated cells. Because stable populations are reported to be more resistant to microtubule poisons, we monitored soluble and polymeric tubulin amounts after nocodazole treatment. Cells were treated with 33 mm nocodazole for the indicated times and processed for indirect immunofluorescence (Fig. 4). In control cells, the majority of microtubules were depolymerized 5 min after nocodazole addition (compare Fig. 4a and c). Only diffuse, cytosolic staining was detected after 15 min (Fig. 4e). As described above, the microtubules in ethanol-treated cells in the absence of nocodazole were gnarled and shorter than in control (Fig. 4b). These microtubules were more resistant to depolymerization; even after 30 (Fig. 4h) or 60 min (data not shown) of nocodazole treatment, polymeric tubulin was detected emanating from the apical centrosomes. We confirmed these results using a biochemical assay. Cells were treated with 33 mm nocodazole for the indicated Fig. 4. Microtubules are more stable in ethanol-treated cells. Control (a, c, e and g) or ethanol-treated cells (b, d, f and h) were incubated with 33 mm nocodazole for the indicated times and labeled for a-tubulin. Asterisks mark selected BCs. times (Fig. 5) and then lysed in a microtubule stabilizing buffer containing 0.15% Triton X-100. Soluble (S) tubulin was released into the supernatant while polymeric (P) tubulin remained cell-associated. In control cells, acetylated Fig. 5. Microtubules are more stable in ethanol-treated cells. WIF-B cells were treated with nocodazole for the indicated times then extracted in a microtubule-stabilizing buffer containing 0.15% Triton X-100 for 1 min at 37 8C. Supernatants (S) containing the released soluble tubulin were collected and cells containing intact polymers (P) were lysed. Samples were immunoblotted for acetylated a-tubulin. The immunoblots shown are representative of three independent experiments.

5 G.T. Kannarkat et al. / Journal of Hepatology 44 (2006) microtubule polymers were virtually depleted after 15 min of nocodazole treatment, consistent with our morphological observations. In striking contrast, the polymers in ethanoltreated cells were highly resistant to nocodazole. Even after 60 min of nocodazole treatment, significant levels of acetylated, polymeric tubulin remained. Thus, increased microtubule acetylation correlated with increased microtubule stability in ethanol-treated cells. To further confirm that microtubules from ethanoltreated cells were more stable, we treated cells with the microtubule-stabilizing drug, taxol (Fig. 6). In control cells, taxol induced the formation of bundles located adjacent to the apical and basolateral domains that were most apparent after 120 min (Fig. 6e). Taxol-induced microtubule bundles were similarly localized in ethanol-treated cells, but bundle formation was more extensive and rapid. Within 30 min, extensive bundling was seen near the apical surface (Fig. 6d) and by 120 min, no diffuse tubulin staining was observed (Fig. 6f) indicating complete taxol-induced bundling. In contrast, diffuse tubulin staining was observed in control cells after 120 min (Fig. 6e) Repolymerizing microtubules are more highly acetylated in ethanol-treated cells Our results demonstrate that microtubules at steady state were more stable and more highly acetylated in Fig. 6. Taxol induces more extensive microtubule bundling in ethanoltreated cells. Control (a, c and e) or ethanol-treated cells (b, d, and f) were incubated with 10 mm taxol for the indicated times and labeled for a-tubulin. ethanol-treated cells, yet they were unable to efficiently polymerize. To reconcile these disparate results, we examined the acetylation levels of polymerizing microtubules. After nocodazole depolymerization (Fig. 1C, 0 min), microtubules in ethanol-treated cells were wthreefold more acetylated than control, consistent with results shown in Figs. 4 and 5. As expected, acetylation levels in both control and ethanol-treated cells increased as microtubules repolymerized (Fig. 1C) and acetylated tubulin was detected only in the polymeric fraction (data not shown). From Fig. 1A, we determined that approximately half as much of tubulin repolymerized in ethanol-treated cells than in control. Thus, the polymeric tubulin in ethanol-treated cells is two-fold more acetylated than in control (i.e. same level of acetylation on half as much tubulin). These results further confirm that microtubules in ethanol-treated cells are more highly acetylated and suggest that acetylation may also impair repolymerization kinetics Increased microtubule stability requires ethanol metabolism and is mediated by acetaldehyde To determine whether increased microtubule acetylation and stability were dependent on ethanol metabolism, we examined the time- and dose-dependence of ethanol treatment on microtubule acetylation by quantitative immunoblotting. Microtubule acetylation increased nearly linearly with increased days in culture in the presence of 50 mm alcohol (Fig. 7A). After 3 d, a wtwo-fold increase in tubulin acetylation was observed which began to plateau on day 4. Similarly, a saturable ethanol dose-dependence was observed for tubulin acetylation (Fig. 7B). Maximal tubulin acetylation (Otwo-fold increase) was observed at 10 mm ethanol. Increasing ethanol concentrations up to 250 mm led to no further acetylation (data not shown). These results suggest saturation of alcohol metabolizing enzymes, such that maximal metabolite concentrations were achieved, resulting in maximal microtubule acetylation. More direct evidence for the metabolism-dependence of microtubule acetylation and increased stability came from experiments using 4-MP (an ADH inhibitor that leads to decreased acetaldehyde levels) and cyanamide (an aldehyde dehydrogenase inhibitor that leads to increased acetaldehyde levels). As predicted, the agents had opposite effects on microtubule acetylation. 4-MP co-incubation prevented tubulin acetylation to near control levels in ethanol-treated cells (Fig. 8A). Less than 10% increase in microtubule acetylation was observed in cells treated with both ethanol and 4-MP compared to O150% increase observed in cells treated with ethanol alone. In contrast, cyanamide enhanced microtubule acetylation beyond that observed in cells treated with alcohol alone (Fig. 8B). For these experiments, cells were treated with 5 mm ethanol such that sub-optimal acetylation was achieved, thus allowing cyanamide potentiation to be observed. The cyanamide-induced acetylation was dose-dependent,

6 968 G.T. Kannarkat et al. / Journal of Hepatology 44 (2006) Fig. 7. Increased microtubule acetylation and stability are time- and dose-dependent. A, WIF-B cells were treated for 0 4 days with 50 mm ethanol. B, Cells were treated with the indicated ethanol concentrations for 72 h. Samples in A and B were immunoblotted for a-tubulin and acetylated a-tubulin. Acetylated a-tubulin values were normalized to the total a-tubulin present in each sample and their relative levels calculated. The graph in A is representative of four independent experiments. Values in B are the averagegsem from five independent experiments. saturating at 50 mm of the drug with peak acetylation levels of w2.5-fold over control. Together these results indicate that alcohol metabolism is required for increased microtubule acetylation and stability and that these effects are likely to be mediated by acetaldehyde. 4. Discussion In this study, we determined that ethanol metabolism impairs microtubule dynamics in two major ways. First, microtubule polymerization was significantly impaired in ethanol-treated WIF-B cells. Secondly, repolymerizing and steady-state microtubules were more stable and acetylated 2 3-fold more in ethanol-treated cells than in control. We confirmed these results in hepatocytes from ethanol-fed rats indicating that the findings have physiologic importance. Fig. 8. Increased microtubule acetylation and stability are dependent on ethanol metabolism. Microtubule acetylation was monitored by immunoblotting WIF-B cell extracts after the following treatments: A, incubation in the absence or presence of 4-MP (1 3 mm) or 50 mm ethanol for 72 h or B, incubation with 5 mm ethanol and increasing cyanamide concentrations (as indicated) for 72 h. In A, values are the averagegsem from three independent experiments. In B, a representative immunoblot is shown from three independent experiments. *P!0.03 (4-MP vs. EtOH); **P!0.025 (EtOH vs. EtOH C4- MP). We further determined that increased microtubule acetylation in WIF-B cells is dependent on ethanol metabolism and is likely to be mediated by acetaldehyde. These microtubule alterations likely have profound effects on hepatic structure and function Possible mechanisms for increased microtubule stability and acetylation Two populations of microtubules have been described: stable and dynamic [25]. Unlike dynamic microtubules, the stable population is characterized by a longer half-life (several hours vs. 10 min), by its resistance to microtubule poisons and by specific post-translational modifications on a-tubulin including the removal of a carboxy-terminal tyrosine, polyglutamylation, polyglycation and acetylation of lysine 40 [22]; the latter modification is emphasized in this study. Microtubule acetylation is regulated by the coordinated activities of acetyltransferases and deacetylases [26,27]. At present, no microtubule-specific acetyltransferase has been identified. However, there are two known microtubulespecific deacetylases, sirtuin T2 (SirT2) and histone

7 G.T. Kannarkat et al. / Journal of Hepatology 44 (2006) deacetylase 6 (HDAC6). Both enzymes colocalize with microtubules, and when overexpressed, microtubules are specifically deacetylated and destabilized [28 31]. In contrast, when the enzymes are inactivated by pharmacological agents or their expression is knocked down, microtubules are more stable and hyperacetylated up to five-fold [28 31]. One possibility is that ethanol treatment inhibits these deacetylases leading to increased microtubule acetylation. Both enzymes are good candidates for the ethanol-induced changes in acetylation. HDAC6 expression is enriched in liver [32] while SirT2 deacetylase activity is NAD C -dependent [30]. Thus, in ethanol-treated cells where NAD C amounts are limiting, we propose SirT2 activity is inhibited. Because alcohol metabolism is required for increased microtubule acetylation and stability, another attractive hypothesis is that acetaldehyde inactivates the deacetylases via adduction. Studies are currently underway in our laboratory to discriminate among these and other possibilities Why is increased microtubule stability harmful to hepatocytes? Because microtubules are central to multiple cellular processes, any changes in their dynamics will alter proper hepatic function. Of particular interest is the emerging evidence that indicates that different microtubule populations support specific protein transport steps [33]. Especially intriguing are studies performed in WIF-B cells that used a novel microtubule depolymerizing drug, 201-F. Pous and colleagues [34] showed that 201-F specifically depolymerized deacetylated microtubules indicating it is a specific poison for dynamic microtubules. They further examined specific protein transport steps and found that secretion and transcytosis were dependent on dynamic (deacetylated) microtubules while sinusoidal delivery of glycoproteins was dependent on stable (acetylated) microtubules. Currently, numerous proteins are known to have alcoholinduced alterations in their dynamics [35 37]. In general, two transport pathways appear to be affected: transport of newly synthesized secretory or membrane proteins from the Golgi to the plasma membrane and receptor-mediated endocytosis from the sinusoidal surface. Our recent work in WIF-B cells indicates similar defects in protein trafficking (our unpublished data). We propose the observed alcohol-induced defects may likely be explained by alterations in microtubule dynamics, i.e. increased microtubule stability interferes with vesicle delivery on microtubule tracks thereby impairing protein trafficking. Consistent with this hypothesis is the finding that the microtubule-associated motor, kinesin, preferentially binds the stable, modified population of microtubules [38]. Furthermore, acetaldehyde robustly binds purified hepatic microtubule associated proteins (MAPs) and motors, 1.5- fold more than tubulin [39]. 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