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Hepatic steatosis in transgenic mice overexpressing human histone deacetylase 1
BBRC Biochemical and Biophysical Research Communications 330 (2005) 461–466 www.elsevier.com/locate/ybbrc
Hepatic steatosis in transgenic mice overexpressing human histone deacetylase 1 Ai-Guo Wang a, Sang-Beom Seo b, Hyung-Bae Moon c, Hye-Jun Shin a, Dong Hoon Kim f, Jin-Man Kim d, Tae-Hoon Lee e, Ho Jeong Kwon f, Dae-Yeul Yu a,*, Dong-Seok Lee a,* a
c
Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea b Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756, Republic of Korea Department of Pathology, School of Medicine, Institute of Medical Science, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea d Department of Pathology, College of Medicine, Chungnam National University, Daejeon 301-131, Republic of Korea e Department of Oral Biochemistry, College of Dentistry, Chonnam National University, Gwangju 500-757, Republic of Korea f Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747, Republic of Korea Received 24 February 2005 Available online 16 March 2005
Abstract It is generally thought that histone deacetylases (HDACs) play important roles in the transcriptional regulation of genes. However, little information is available concerning the specific functions of individual HDACs in disease states. In this study, two transgenic mice lines were established which harbored the human HDAC1 gene. Overexpressed HDAC1 was detected in the nuclei of transgenic liver cells, and HDAC1 enzymatic activity was significantly higher in the transgenic mice than in control littermates. The HDAC1 transgenic mice exhibited a high incidence of hepatic steatosis and nuclear pleomorphism. Molecular studies showed that HDAC1 may contribute to nuclear pleomorphism through the p53/p21 signaling pathway. 2005 Elsevier Inc. All rights reserved. Keywords: Histone deacetylase 1; Steatosis; Nuclear pleomorphism; p53; p21
Chromatin structure has a central role in the regulation of gene expression [1]. One of the mechanisms that control chromatin remodeling events is the modification of the amino termini of histone proteins that extend from the core of the chromatin structure. Among these modifications, the acetylation of histones has gained importance since the first demonstration that the activities of certain genes were correlated with histone acetylation [2]. The acetylation status of histones is controlled by competition between two classes of enzymes, histone deacetyltransferases (HDACs) and histone acetyltransferases (HATs) [3]. As these enzymes participate in a *
Corresponding authors. Fax: +82 42 860 4608 (D.-S. Lee). E-mail addresses: [email protected] (D.-Y. Yu), lee10@kribb. re.kr (D.-S. Lee). 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.179
variety of cellular processes including transcription, DNA replication, and cell cycle progression, it has been suggested that they may be associated with proliferative diseases such as cancer [4]. Consequently, HDACs have recently attracted interest as novel targets for anti-tumor therapies. HDAC inhibitors (HDACIs) have been shown to induce growth arrest, differentiation, or apoptosis of cancer cells in vitro and in vivo [5]. In addition, few or no side effects of HDACIs have been observed in animal experiments and clinical trials, within the therapeutic range studied, enhancing the potential use of these agents for therapeutic applications [6]. However, it is thought that almost all HDACs are approximately equally sensitive to different HDACIs [6]. Little information is currently available concerning the specific functions of individual HDACs.
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The characterization of individual HDACs and the development of new HDACIs targeting specific HDACs may make the tailored therapeutic use of HDACIs possible. The class I HDAC enzyme HDAC1 was the first mammalian deacetylase identified [7]. Numerous transcription factors, including regulators of the cell cycle, differentiation, and development, have been shown to be associated with HDAC1, thereby mediating the repression of specific target genes [8–10]. The disruption of both HDAC1 alleles results in embryonic lethality attributable to severe proliferation defects and retardation in development [11]. Moreover, the loss of HDAC1 leads to significantly reduced overall deacetylase activity, and there is no alternate pathway that can substitute for its functions [11]. On the other hand, HDAC1 activity is known to be elevated in several types of human cancer, as compared with the activity in normal cells [12]. A new HDACI that preferentially inhibits HDAC1, MS-27-275, was recently identified [13]. MS-27-275 increased histone H4 acetylation and induced apoptosis in the human colon cancer cell line SW620, thus exhibiting effects similar to those of trichostatin A (TSA). Collectively, these findings indicate that HDAC1 plays important roles in development and in proliferative diseases such as cancer and that its functions cannot be replaced by alternate enzymes. However, the question of whether overexpressed or overactive HDAC1 contributes to cytotoxicity in normal cells in vivo has not been resolved. To address this issue, we produced a transgenic mouse model expressing the human HDAC1 gene under the control of the HBV enhancer and examined the pattern and effects of HDAC1 overexpression in these transgenic mice.
Materials and methods Generation and identification of transgenic mice. To generate the expression vector pHE/HDAC1, a 1.5-kb fragment containing the human HDAC1 cDNA was sub-cloned into the NcoI/BglII cloning site of the pHEX vector to replace the DNA encoding the hepatitis B virusX protein (HBX; Fig. 1). The pHEX plasmid was previously reported to express the HBX gene under the control of its authentic promoter [14]. Injectable DNA containing the hepatitis B virus enhancer (HBV), the human HDAC1 cDNA, and the SV40 poly(A) sequences was obtained by removal of the phagemid sequences from the pBluescript II SK vector. The DNA was microinjected into fertilized, inbred C57BL/ 6J mouse eggs, and the injected eggs were transferred into pseudopregnant recipients as previously described [14]. Potential founders were analyzed using polymerase chain reaction (PCR) and Southern blots of DNA extracted from tail samples. To confirm the presence and integrity of the transgene, the PCR products were purified and sequenced. RT-PCR analysis. We performed RT-PCR to detect the expression of the transgene in transgenic mouse tissues and to determine whether the expression of HDAC1 influenced the expression of other HDACs or of cell cycle-regulated genes in the liver. Total RNA was isolated from mouse tissues using the TRIzol reagent (Invitrogen Life Technologies,
Carlsbad, CA). RT-PCR was performed using a reverse transcription system (Promega, Madison, WI) according to manufacturerÕs instructions. The primers used to detect the expression of the transgene were the sense primer, 5 0 -GTACCACAGCGATGACTACAT-3 0 , and the anti-sense primer, 5 0 -CTGGGAAGTACTCTCCATACT-3 0 . A separate RT-PCR using primers for the detection of GAPDH was used as a loading control. The primers for other members of the mouse HDAC family (HDAC1, 2, 3, 4, 5, 6, and 8) and the primers for the detection of cell cycle-regulated genes (p53, p21Waf1/Cip1) were designed based on the gene sequences obtained from the PubMed databases. The production of a single band of the expected size on RT-PCR was recognized as amplification of the target genes. Western blotting. For the detection of HDAC1 expression, the nuclear proteins were extracted from transgenic mouse tissues using a nuclear extraction kit (Panomics, Redwood City, CA) according to the manufacturerÕs instructions. The protein concentrations of the cell extracts were determined using the Bradford protein assay (Bio-Rad, Hercules, CA). Aliquots of the nuclear and cytosolic extracts were separated by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore, Bradford, MA). The membranes were probed with anti-HDAC1 antibody (ab12168m; Abcam, Cambridge, UK) as the primary antibody and horseradish peroxidase (HRP)-conjugated rabbit polyclonal antibody to mouse IgG (ab6728; Abcam) as the secondary antibody. Positive bands were detected with an enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL). For the detection of p53, whole tissue lysates were prepared by homogenizing freshly collected or flash-frozen tissues in extraction lysis buffer. Aliquots containing equal amounts of protein (30 lg) were separated by SDS–PAGE and transferred to membranes for Western blotting using anti-p53 antibody (2524; Cell Signaling, Frankfurt, Germany) as the primary antibody. HDAC activity assay. The HDAC activity in the nuclear extracts (described above) was determined using a fluorometric HDAC assay and detection kit (Upstate USA, New York) according to the manufacturerÕs instructions. Briefly, the control HeLa cell nuclear extracts or the sample extracts were incubated with the HDAC assay substrate at 37 C for 30 min before addition of the activator solution. The mixtures were incubated for an additional 10 min at room temperature, and fluorescence was measured using a Spectra Max Gemini XS fluorescence plate reader (Molecular Devices, San Diego, CA), with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Histopathological studies and immunohistochemical staining. Freshly collected tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard methods. Immunohistochemical studies were performed on formalin-fixed and paraffin-embedded 3-lm-thick sections, using rabbit anti-HDAC1 antibody (Upstate Biotechnology) as the primary antibody and the avidin–biotin peroxidase complex detection method, as previously described [14]. As the HDAC1 antibody does not distinguish between the mouse and human HDAC1 proteins, the specimens from transgenic animals were examined in tandem with specimens from normal littermates as controls. Statistical analysis. The differences between the experimental groups were tested for statistical significance using the v2 test. P values <0.05 were considered to be significant.
Results Establishment of transgenic mice To generate transgenic mice overexpressing HDAC1, cDNA encoding the human HDAC1 gene under the
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Fig. 1. (A) Construction of the HDAC1 expression vector. The transgenic vector contains the HBV enhancer followed by the human HDAC1 cDNA (1.5 kb) and the SV40 poly(A) signal. (B) RT-PCR analysis of the expression of human HDAC1 in tissues from transgenic mice of lineage 18 at 8 weeks of age. GAPDH was used as an internal control for RT-PCR. M, marker; P, positive control; and Wt, normal wild-type littermate.
control of the HBV enhancer (Fig. 1A) was microinjected into fertilized oocytes obtained from C57BL/6J inbred mice. Two transgenic lineages were established which stably transmitted the transgene through the germ
line to subsequent generations. No developmental defects were detected in the transgenic mice throughout their normal life span. Total RNAs isolated from the livers and other tissues of control and transgenic mice were analyzed by RT-PCR to determine the expression of the human HDAC1 gene, and the identities of the products were further confirmed by DNA sequencing. The results showed that the expression of the human HDAC1 gene sequence was restricted exclusively to the transgenic mice and was not found in the control littermates. The HDAC1 transgene was widely expressed in the transgenic mouse tissues and was detected in both lines (Fig. 1B). Steatosis and nuclear pleomorphism in the livers of HDAC1 transgenic mice To determine whether the overexpression of HDAC1 was cytotoxic, gross and microscopic examinations were
Fig. 3. (A) Western blot analysis of nuclear and cytosolic extracts of livers from transgenic mice of lineage 18 and normal littermates at 6 months of age. The relative molecular weight of the human HDAC1 band (60 kDa) was determined by reference to standard molecular weight markers (Invitrogen). (B) Comparison of the HDAC1 activity in the livers of transgenic and normal littermates at 6 months of age. Treatment with trichostatin A was used as an inhibition control. TSA, trichostatin A; Wt, normal wild-type littermates; and Tg, HDAC1 transgenic mice. N P 4 for each treatment.
Fig. 4. The expression of p53 and p21Waf1/Cip1 in the livers of transgenic mice. (A) The expression of p53 protein in the livers of transgenic mice as shown by Western blotting. (B) The expression of p21Waf1/Cip1 mRNA in the livers of transgenic mice as shown by RTPCR. Wt, normal wild-type littermate; Tg, HDAC1 mouse.
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performed on the transgenic mice and on the control littermates. Hepatomegaly was the only gross abnormality observed in the HDAC1 transgenic mice up to 18 months of age. Microscopic examination of the livers revealed that steatosis (Figs. 2A and B) and nuclear pleomorphism (Figs. 2C and D) occurred more frequently in the HDAC1 transgenic mice than in the control littermates (Table 1). In addition, one hepatic tumor and two livers with dysplastic nodules were detected in transgenic mice of lineage 18, but there was no significant difference in the incidence of these abnormalities between the transgenic mice and the control littermates. As pathological changes were detected only in the livers of transgenic mice, the subsequent studies were restricted to the liver. Expression, location, and enzymatic activity of human HDAC1 protein in the livers of transgenic mice Previous studies have shown that the HDAC1 protein is located exclusively in the nucleus [6]. In order
to confirm the localization of the transgenically expressed HDAC1 protein in our animal model, nuclear and cytosolic extracts were prepared from the mouse livers and examined by Western blotting using an anti-HDAC1 antibody. The nuclear extracts contained much larger amounts of the HDAC1 protein than did the cytosolic extracts, and the levels of HDAC1 protein were much higher in the nuclear extracts of the transgenic mice than in those of the controls (Fig. 3A). To determine whether the overexpressed human HDAC1 protein contributed to an enhanced level of HDAC activity, fluorometric enzymatic activity assays were performed. The results showed that the livers of transgenic mice had significantly higher HDAC activity compared with those of the normal littermates (Fig. 3B). No differences in the HDAC activity levels attributable to age or transgenic lineage were detected (data not shown). To detect the possible induction of other HDACs by the overexpression of the human HDAC1 transgene, the
Fig. 2. Immunostaining and pathological analysis of the livers of HDAC1 transgenic mice. Severe steatosis was found in the livers of 14-month-old transgenic mice (A) as compared with those of non-transgenic littermates of the same age (B). Nuclear pleomorphism, with nuclear enlargement and prominent nucleoli, was found in the livers of 14-month-old transgenic mice (C) as compared with those of non-transgenic littermates of the same age (D). The expression of HDAC1 was increased in nuclei of liver from 6-month-old transgenic mice (E) as compared with expression in non-transgenic littermates of the same age (F). The tissues were stained with HE (A–D) or with immunohistochemical staining for HDAC1 (E,F). 20· magnification (A, B, E, and F), 40· magnification (C,D). Table 1 Summary of pathological changes in HDAC1 transgenic lineages Transgenic lines 6 18 Total Control littermates a *
No. of mice examined 35 18 53 27
Age group (months) 6–18 6–18 6–18 6–18
Hepatic steatosis (%) a
8 (22.9) 5 (27.8)a 13 (24.5)a 1 (3.7)
Indicates a significant difference within the same column compared to control mice. P < 0.05. P = 0.08.
Nuclear pleomorphism (%) 15 (42.9) 5 (27.8) 20 (37.7)* 5 (18.5)
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expression levels of the endogenous mouse HDAC genes were examined by RT-PCR. The results showed that the overexpression of the HDAC1 transgene did not induce the expression of the mouse HDAC genes (HDAC1, 2, 3, 4, 5, 6, and 8; data not shown). This confirmed that the significantly higher levels of HDAC activity detected in the transgenic mice were mainly attributable to the overexpressed HDAC1 transgene. In addition, immunohistochemical staining confirmed the nuclear location of the transgenic HDAC1 protein; the nuclei of hepatocytes of the transgenic mice were stained more intensely with anti-HDAC1 antibody than were those of the normal littermates (Figs. 2E and F). The expression of p53 and p21Waf1/Cip1 in the liver of transgenic mice The deacetylation of p53 protein by HDAC1 has been shown to play an important role in the regulation of p53 functions [15]. In order to determine whether this was the case in vivo in our transgenic mice, the protein level of p53 was examined. The p53 protein level was significantly decreased compared with the levels in the control mice (Fig. 4A). This finding indicates that the activated HDAC1 promoted the degradation of p53 protein in vivo. The downregulation of the p53 protein level is consistent with results obtained in vitro [15]. Furthermore, the level of p21Waf1/Cip1 mRNA was significantly decreased in the livers of the transgenic mice compared with the level in the control mice, as shown by RT-PCR (Fig. 4B). The decreased level of p21Waf1/Cip1 mRNA may be related to the decreased p53 protein level. Another possibility is that HDAC1 may regulate the transcription of p21Waf1/Cip1 directly [5]. These findings indicate that HDAC1 may contribute to the observed nuclear pleomorphism in the livers of the transgenic mice through the regulation of cell proliferation by means of the p53/p21 signaling pathway.
Discussion Nonalcoholic fatty liver disease (NAFLD) is currently a major cause of liver-related morbidity and mortality in developed countries. NAFLD encompasses several histological patterns, from benign steatosis to nonalcoholic steatohepatitis, the latter having a significant risk of progression to fibrosis and the development of cirrhosis [16]. Various compounds that influence cellular lipid metabolism can also inhibit the growth of malignant cells; consequently, the modulation of lipid metabolism has been proposed as a possible approach to cancer prevention and treatment [17]. It has been reported that lipid metabolism may be regulated by genes that are affected by regulatory complexes requiring HDAC activity [18,19]. The hepatic steatosis observed
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in our transgenic mice was not the result of stimulation with alcohol or the uptake of lipids from the blood, because the mice were not exposed to alcohol and did not exhibit any elevation in the levels of serum triglycerides or cholesterol (data not shown). The significantly higher frequency of steatosis in the transgenic mice indicates that HDAC1 may be an important factor in the regulation of lipid metabolism. In addition, the overexpression of PPAR-c was detected in mice with fatty livers, and the ablation of liver PPAR-c reduced the occurrence of hepatic steatosis in lipoatrophic mice [20]. However, we detected a significant decrease in the level of PPAR-c protein in the HDAC1 transgenic mice (data not shown). Although the mechanism remains to be elucidated, this finding indicates that the development of fatty liver in HDAC1 transgenic mice may be the result of disturbances in pathways other than the PPAR-c pathway. HDAC1 levels have been found to be elevated in highly proliferative tissues, embryonic stem cells, and several transformed cell lines [11,21], suggesting a link between HDAC1 functions and proliferation. Consistent with this possibility, the disruption of the HDAC1 gene resulted in a reduction in proliferation of mouse embryos and of embryonic stem cells [11], whereas the overexpression of HDAC1 led to impaired proliferation of murine fibroblasts [21]. Collectively, these results suggest that the tightly controlled cell-type-specific expression of HDAC1 may be crucial for the regulation of unrestricted proliferation. Our in vivo studies indicate that the overexpression of HDAC1 as a transgene induced significantly higher levels of HDAC1 activity (data not shown). A large body of literature suggests that the functions of multiple nuclear factors are dependent on the recruitment of HDAC1 activity [8]. The significantly higher HDAC activity detected in the tissues of our transgenic mice indicates that the overexpressed human HDAC1 effectively formed a functional complex with other cofactors to produce its activity. However, this high level of HDAC1 enzymatic activity was not cytotoxic to most cell types in vivo, as indicated by the absence of developmental defects in our transgenic mice. In addition, most tissues examined in our experiments were largely normal histologically through 18 months of age, except for the liver. Consequently, we propose that HDAC proteins, or HDAC1 in particular, might act primarily as an important modulator of other mechanisms involved in development and in proliferative diseases such as tumorigenesis. As such, the overexpression of HDAC alone would not directly induce disease, but the activity of other molecular regulators would be required. This hypothesis is also supported by the observation that HDAC is overexpressed in most cancer cells, as compared with the expression levels in normal cells [6]. The HDACIs with anticancer activity may disrupt the functions of HDACs in tumor cells that lead to
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cancer progression. In addition, the observation that HDACIs produce few or no side effects at doses within the therapeutic range in animal experiments and clinical trials indicates that the HDACs are more critical to cancer cell progression than to normal cellular homeostasis. However, some clinical trials using HDACIs have shown that limited amounts of the compounds reach the target cells owing to the rapid metabolism of HDACIs; this suggests that new drugs need to be designed in order to increase the efficacy of HDACIs. Our work provides an in vivo model that can be used to screenspecific HDAC1 inhibitors for efficacy and to determine whether the compounds reach the target tissues at inhibitory concentrations. In conclusion, this is the first description of the phenotypes produced by overexpression of transgenic HDAC1 in vivo. Our transgenic mice offer a valuable model for the examination of potential etiological mechanisms that function through the recruitment of HDAC1 activity. Specifically, our work offers new insight into the function of HDAC1 as related to hepatic steatosis and the cell cycle.
Acknowledgment This work was supported by the 21st Century Frontier Functional Human Genome Project of Korea, Grant No. HGC0300324 and the Ministry of Health and Welfare of Korea, Grant No. 02-PJ2-PG1-CH120002.
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[6] A.J. de Ruijter, A.H. van Gennip, H.N. Caron, S. Kemp, A.B. van Kuilenburg, Histone deacetylases (HDACs): characterization of the classical HDAC family, Biochem. J. 370 (2003) 737– 749. [7] J. Taunton, C.A. Hassig, S.L. Schreiber, A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p, Science 272 (1996) 408–411. [8] W.D. Cress, E. Seto, Histone deacetylases, transcriptional control, and cancer, J. Cell Physiol. 184 (2000) 1–16. [9] J. Ahringer, NuRD and SIN3 histone deacetylase complexes in development, Trends Genet. 16 (2000) 351–356. [10] H.H. Ng, A. Bird, Histone deacetylases: silencers for hire, Trends Biochem. Sci. 25 (2000) 121–126. [11] G. Lagger, D. OÕCarroll, M. Rembold, H. Khier, J. Tischler, G. Weitzer, B. Schuettengruber, C. Hauser, R. Brunmeir, T. Jenuwein, Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression, EMBO J. 21 (2002) 2672– 2681. [12] J.H. Choi, H.J. Kwon, B.I. Yoon, J.H. Kim, S.U. Han, H.J. Joo, D.Y. Kim, Expression profile of histone deacetylase 1 in gastric cancer tissues, Jpn. J. Cancer Res. 92 (2001) 1300– 1304. [13] E. Hu, E. Dul, C.M. Sung, Z. Chen, R. Kirkpatrick, G.F. Zhang, K. Johanson, R. Liu, A. Lago, G. Hofmann, Identification of novel isoform-selective inhibitors within class I histone deacetylases, J. Pharmacol. Exp. Ther. 307 (2003) 720–728. [14] D.Y. Yu, H.B. Moon, J.K. Son, S. Jeong, S.L. Yu, H. Yoon, Y.M. Han, C.S. Lee, J.S. Park, C.H. Lee, Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein, J. Hepatol. 31 (1999) 123–132. [15] A. Ito, Y. Kawaguchi, C.H. Lai, J.J. Kovacs, Y. Higashimoto, E. Appella, T.P. Yao, MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation, EMBO J. 21 (2002) 6236– 6245. [16] A.D. Clouston, E.E. Powell, Nonalcoholic fatty liver disease: is all the fat bad?, Intern. Med. J. 34 (2004) 187–191. [17] K.J. Tronstad, K. Berge, R.K. Berge, O. Bruserud, Modified fatty acids and their possible therapeutic targets in malignant diseases, Expert Opin. Ther. Targets 7 (2003) 663–677. [18] Y. Shi, M. Hon, R.M. Evans, The peroxisome proliferatoractivated receptor delta, an integrator of transcriptional repression and nuclear receptor signaling, Proc. Natl. Acad. Sci. USA 99 (2002) 2613–2618. [19] A. Aranda, A. Pascual, Nuclear hormone receptors and gene expression, Physiol. Rev. 81 (2001) 1269–1304. [20] O. Gavrilova, M. Haluzik, K. Matsusue, J.J. Cutson, L. Johnson, K.R. Dietz, C.J. Nicol, C. Vinson, F.J. Gonzalez, M.L. Reitman, Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis, triglyceride clearance, and regulation of body fat mass, J. Biol. Chem. 278 (2003) 34268–34276. [21] S. Bartl, J. Taplick, G. Lagger, H. Khier, K. Kuchler, C. Seiser, Identification of mouse histone deacetylase 1 as a growth factor-inducible gene, Mol. Cell. Biol. 17 (1997) 5033– 5043.
Hepatic steatosis in transgenic mice overexpressing human histone deacetylase 1 Ai-Guo Wang a, Sang-Beom Seo b, Hyung-Bae Moon c, Hye-Jun Shin a, Dong Hoon Kim f, Jin-Man Kim d, Tae-Hoon Lee e, Ho Jeong Kwon f, Dae-Yeul Yu a,*, Dong-Seok Lee a,* a
c
Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea b Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756, Republic of Korea Department of Pathology, School of Medicine, Institute of Medical Science, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea d Department of Pathology, College of Medicine, Chungnam National University, Daejeon 301-131, Republic of Korea e Department of Oral Biochemistry, College of Dentistry, Chonnam National University, Gwangju 500-757, Republic of Korea f Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747, Republic of Korea Received 24 February 2005 Available online 16 March 2005
Abstract It is generally thought that histone deacetylases (HDACs) play important roles in the transcriptional regulation of genes. However, little information is available concerning the specific functions of individual HDACs in disease states. In this study, two transgenic mice lines were established which harbored the human HDAC1 gene. Overexpressed HDAC1 was detected in the nuclei of transgenic liver cells, and HDAC1 enzymatic activity was significantly higher in the transgenic mice than in control littermates. The HDAC1 transgenic mice exhibited a high incidence of hepatic steatosis and nuclear pleomorphism. Molecular studies showed that HDAC1 may contribute to nuclear pleomorphism through the p53/p21 signaling pathway. 2005 Elsevier Inc. All rights reserved. Keywords: Histone deacetylase 1; Steatosis; Nuclear pleomorphism; p53; p21
Chromatin structure has a central role in the regulation of gene expression [1]. One of the mechanisms that control chromatin remodeling events is the modification of the amino termini of histone proteins that extend from the core of the chromatin structure. Among these modifications, the acetylation of histones has gained importance since the first demonstration that the activities of certain genes were correlated with histone acetylation [2]. The acetylation status of histones is controlled by competition between two classes of enzymes, histone deacetyltransferases (HDACs) and histone acetyltransferases (HATs) [3]. As these enzymes participate in a *
Corresponding authors. Fax: +82 42 860 4608 (D.-S. Lee). E-mail addresses: [email protected] (D.-Y. Yu), lee10@kribb. re.kr (D.-S. Lee). 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.179
variety of cellular processes including transcription, DNA replication, and cell cycle progression, it has been suggested that they may be associated with proliferative diseases such as cancer [4]. Consequently, HDACs have recently attracted interest as novel targets for anti-tumor therapies. HDAC inhibitors (HDACIs) have been shown to induce growth arrest, differentiation, or apoptosis of cancer cells in vitro and in vivo [5]. In addition, few or no side effects of HDACIs have been observed in animal experiments and clinical trials, within the therapeutic range studied, enhancing the potential use of these agents for therapeutic applications [6]. However, it is thought that almost all HDACs are approximately equally sensitive to different HDACIs [6]. Little information is currently available concerning the specific functions of individual HDACs.
462
A.-G. Wang et al. / Biochemical and Biophysical Research Communications 330 (2005) 461–466
The characterization of individual HDACs and the development of new HDACIs targeting specific HDACs may make the tailored therapeutic use of HDACIs possible. The class I HDAC enzyme HDAC1 was the first mammalian deacetylase identified [7]. Numerous transcription factors, including regulators of the cell cycle, differentiation, and development, have been shown to be associated with HDAC1, thereby mediating the repression of specific target genes [8–10]. The disruption of both HDAC1 alleles results in embryonic lethality attributable to severe proliferation defects and retardation in development [11]. Moreover, the loss of HDAC1 leads to significantly reduced overall deacetylase activity, and there is no alternate pathway that can substitute for its functions [11]. On the other hand, HDAC1 activity is known to be elevated in several types of human cancer, as compared with the activity in normal cells [12]. A new HDACI that preferentially inhibits HDAC1, MS-27-275, was recently identified [13]. MS-27-275 increased histone H4 acetylation and induced apoptosis in the human colon cancer cell line SW620, thus exhibiting effects similar to those of trichostatin A (TSA). Collectively, these findings indicate that HDAC1 plays important roles in development and in proliferative diseases such as cancer and that its functions cannot be replaced by alternate enzymes. However, the question of whether overexpressed or overactive HDAC1 contributes to cytotoxicity in normal cells in vivo has not been resolved. To address this issue, we produced a transgenic mouse model expressing the human HDAC1 gene under the control of the HBV enhancer and examined the pattern and effects of HDAC1 overexpression in these transgenic mice.
Materials and methods Generation and identification of transgenic mice. To generate the expression vector pHE/HDAC1, a 1.5-kb fragment containing the human HDAC1 cDNA was sub-cloned into the NcoI/BglII cloning site of the pHEX vector to replace the DNA encoding the hepatitis B virusX protein (HBX; Fig. 1). The pHEX plasmid was previously reported to express the HBX gene under the control of its authentic promoter [14]. Injectable DNA containing the hepatitis B virus enhancer (HBV), the human HDAC1 cDNA, and the SV40 poly(A) sequences was obtained by removal of the phagemid sequences from the pBluescript II SK vector. The DNA was microinjected into fertilized, inbred C57BL/ 6J mouse eggs, and the injected eggs were transferred into pseudopregnant recipients as previously described [14]. Potential founders were analyzed using polymerase chain reaction (PCR) and Southern blots of DNA extracted from tail samples. To confirm the presence and integrity of the transgene, the PCR products were purified and sequenced. RT-PCR analysis. We performed RT-PCR to detect the expression of the transgene in transgenic mouse tissues and to determine whether the expression of HDAC1 influenced the expression of other HDACs or of cell cycle-regulated genes in the liver. Total RNA was isolated from mouse tissues using the TRIzol reagent (Invitrogen Life Technologies,
Carlsbad, CA). RT-PCR was performed using a reverse transcription system (Promega, Madison, WI) according to manufacturerÕs instructions. The primers used to detect the expression of the transgene were the sense primer, 5 0 -GTACCACAGCGATGACTACAT-3 0 , and the anti-sense primer, 5 0 -CTGGGAAGTACTCTCCATACT-3 0 . A separate RT-PCR using primers for the detection of GAPDH was used as a loading control. The primers for other members of the mouse HDAC family (HDAC1, 2, 3, 4, 5, 6, and 8) and the primers for the detection of cell cycle-regulated genes (p53, p21Waf1/Cip1) were designed based on the gene sequences obtained from the PubMed databases. The production of a single band of the expected size on RT-PCR was recognized as amplification of the target genes. Western blotting. For the detection of HDAC1 expression, the nuclear proteins were extracted from transgenic mouse tissues using a nuclear extraction kit (Panomics, Redwood City, CA) according to the manufacturerÕs instructions. The protein concentrations of the cell extracts were determined using the Bradford protein assay (Bio-Rad, Hercules, CA). Aliquots of the nuclear and cytosolic extracts were separated by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore, Bradford, MA). The membranes were probed with anti-HDAC1 antibody (ab12168m; Abcam, Cambridge, UK) as the primary antibody and horseradish peroxidase (HRP)-conjugated rabbit polyclonal antibody to mouse IgG (ab6728; Abcam) as the secondary antibody. Positive bands were detected with an enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL). For the detection of p53, whole tissue lysates were prepared by homogenizing freshly collected or flash-frozen tissues in extraction lysis buffer. Aliquots containing equal amounts of protein (30 lg) were separated by SDS–PAGE and transferred to membranes for Western blotting using anti-p53 antibody (2524; Cell Signaling, Frankfurt, Germany) as the primary antibody. HDAC activity assay. The HDAC activity in the nuclear extracts (described above) was determined using a fluorometric HDAC assay and detection kit (Upstate USA, New York) according to the manufacturerÕs instructions. Briefly, the control HeLa cell nuclear extracts or the sample extracts were incubated with the HDAC assay substrate at 37 C for 30 min before addition of the activator solution. The mixtures were incubated for an additional 10 min at room temperature, and fluorescence was measured using a Spectra Max Gemini XS fluorescence plate reader (Molecular Devices, San Diego, CA), with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Histopathological studies and immunohistochemical staining. Freshly collected tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard methods. Immunohistochemical studies were performed on formalin-fixed and paraffin-embedded 3-lm-thick sections, using rabbit anti-HDAC1 antibody (Upstate Biotechnology) as the primary antibody and the avidin–biotin peroxidase complex detection method, as previously described [14]. As the HDAC1 antibody does not distinguish between the mouse and human HDAC1 proteins, the specimens from transgenic animals were examined in tandem with specimens from normal littermates as controls. Statistical analysis. The differences between the experimental groups were tested for statistical significance using the v2 test. P values <0.05 were considered to be significant.
Results Establishment of transgenic mice To generate transgenic mice overexpressing HDAC1, cDNA encoding the human HDAC1 gene under the
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Fig. 1. (A) Construction of the HDAC1 expression vector. The transgenic vector contains the HBV enhancer followed by the human HDAC1 cDNA (1.5 kb) and the SV40 poly(A) signal. (B) RT-PCR analysis of the expression of human HDAC1 in tissues from transgenic mice of lineage 18 at 8 weeks of age. GAPDH was used as an internal control for RT-PCR. M, marker; P, positive control; and Wt, normal wild-type littermate.
control of the HBV enhancer (Fig. 1A) was microinjected into fertilized oocytes obtained from C57BL/6J inbred mice. Two transgenic lineages were established which stably transmitted the transgene through the germ
line to subsequent generations. No developmental defects were detected in the transgenic mice throughout their normal life span. Total RNAs isolated from the livers and other tissues of control and transgenic mice were analyzed by RT-PCR to determine the expression of the human HDAC1 gene, and the identities of the products were further confirmed by DNA sequencing. The results showed that the expression of the human HDAC1 gene sequence was restricted exclusively to the transgenic mice and was not found in the control littermates. The HDAC1 transgene was widely expressed in the transgenic mouse tissues and was detected in both lines (Fig. 1B). Steatosis and nuclear pleomorphism in the livers of HDAC1 transgenic mice To determine whether the overexpression of HDAC1 was cytotoxic, gross and microscopic examinations were
Fig. 3. (A) Western blot analysis of nuclear and cytosolic extracts of livers from transgenic mice of lineage 18 and normal littermates at 6 months of age. The relative molecular weight of the human HDAC1 band (60 kDa) was determined by reference to standard molecular weight markers (Invitrogen). (B) Comparison of the HDAC1 activity in the livers of transgenic and normal littermates at 6 months of age. Treatment with trichostatin A was used as an inhibition control. TSA, trichostatin A; Wt, normal wild-type littermates; and Tg, HDAC1 transgenic mice. N P 4 for each treatment.
Fig. 4. The expression of p53 and p21Waf1/Cip1 in the livers of transgenic mice. (A) The expression of p53 protein in the livers of transgenic mice as shown by Western blotting. (B) The expression of p21Waf1/Cip1 mRNA in the livers of transgenic mice as shown by RTPCR. Wt, normal wild-type littermate; Tg, HDAC1 mouse.
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performed on the transgenic mice and on the control littermates. Hepatomegaly was the only gross abnormality observed in the HDAC1 transgenic mice up to 18 months of age. Microscopic examination of the livers revealed that steatosis (Figs. 2A and B) and nuclear pleomorphism (Figs. 2C and D) occurred more frequently in the HDAC1 transgenic mice than in the control littermates (Table 1). In addition, one hepatic tumor and two livers with dysplastic nodules were detected in transgenic mice of lineage 18, but there was no significant difference in the incidence of these abnormalities between the transgenic mice and the control littermates. As pathological changes were detected only in the livers of transgenic mice, the subsequent studies were restricted to the liver. Expression, location, and enzymatic activity of human HDAC1 protein in the livers of transgenic mice Previous studies have shown that the HDAC1 protein is located exclusively in the nucleus [6]. In order
to confirm the localization of the transgenically expressed HDAC1 protein in our animal model, nuclear and cytosolic extracts were prepared from the mouse livers and examined by Western blotting using an anti-HDAC1 antibody. The nuclear extracts contained much larger amounts of the HDAC1 protein than did the cytosolic extracts, and the levels of HDAC1 protein were much higher in the nuclear extracts of the transgenic mice than in those of the controls (Fig. 3A). To determine whether the overexpressed human HDAC1 protein contributed to an enhanced level of HDAC activity, fluorometric enzymatic activity assays were performed. The results showed that the livers of transgenic mice had significantly higher HDAC activity compared with those of the normal littermates (Fig. 3B). No differences in the HDAC activity levels attributable to age or transgenic lineage were detected (data not shown). To detect the possible induction of other HDACs by the overexpression of the human HDAC1 transgene, the
Fig. 2. Immunostaining and pathological analysis of the livers of HDAC1 transgenic mice. Severe steatosis was found in the livers of 14-month-old transgenic mice (A) as compared with those of non-transgenic littermates of the same age (B). Nuclear pleomorphism, with nuclear enlargement and prominent nucleoli, was found in the livers of 14-month-old transgenic mice (C) as compared with those of non-transgenic littermates of the same age (D). The expression of HDAC1 was increased in nuclei of liver from 6-month-old transgenic mice (E) as compared with expression in non-transgenic littermates of the same age (F). The tissues were stained with HE (A–D) or with immunohistochemical staining for HDAC1 (E,F). 20· magnification (A, B, E, and F), 40· magnification (C,D). Table 1 Summary of pathological changes in HDAC1 transgenic lineages Transgenic lines 6 18 Total Control littermates a *
No. of mice examined 35 18 53 27
Age group (months) 6–18 6–18 6–18 6–18
Hepatic steatosis (%) a
8 (22.9) 5 (27.8)a 13 (24.5)a 1 (3.7)
Indicates a significant difference within the same column compared to control mice. P < 0.05. P = 0.08.
Nuclear pleomorphism (%) 15 (42.9) 5 (27.8) 20 (37.7)* 5 (18.5)
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expression levels of the endogenous mouse HDAC genes were examined by RT-PCR. The results showed that the overexpression of the HDAC1 transgene did not induce the expression of the mouse HDAC genes (HDAC1, 2, 3, 4, 5, 6, and 8; data not shown). This confirmed that the significantly higher levels of HDAC activity detected in the transgenic mice were mainly attributable to the overexpressed HDAC1 transgene. In addition, immunohistochemical staining confirmed the nuclear location of the transgenic HDAC1 protein; the nuclei of hepatocytes of the transgenic mice were stained more intensely with anti-HDAC1 antibody than were those of the normal littermates (Figs. 2E and F). The expression of p53 and p21Waf1/Cip1 in the liver of transgenic mice The deacetylation of p53 protein by HDAC1 has been shown to play an important role in the regulation of p53 functions [15]. In order to determine whether this was the case in vivo in our transgenic mice, the protein level of p53 was examined. The p53 protein level was significantly decreased compared with the levels in the control mice (Fig. 4A). This finding indicates that the activated HDAC1 promoted the degradation of p53 protein in vivo. The downregulation of the p53 protein level is consistent with results obtained in vitro [15]. Furthermore, the level of p21Waf1/Cip1 mRNA was significantly decreased in the livers of the transgenic mice compared with the level in the control mice, as shown by RT-PCR (Fig. 4B). The decreased level of p21Waf1/Cip1 mRNA may be related to the decreased p53 protein level. Another possibility is that HDAC1 may regulate the transcription of p21Waf1/Cip1 directly [5]. These findings indicate that HDAC1 may contribute to the observed nuclear pleomorphism in the livers of the transgenic mice through the regulation of cell proliferation by means of the p53/p21 signaling pathway.
Discussion Nonalcoholic fatty liver disease (NAFLD) is currently a major cause of liver-related morbidity and mortality in developed countries. NAFLD encompasses several histological patterns, from benign steatosis to nonalcoholic steatohepatitis, the latter having a significant risk of progression to fibrosis and the development of cirrhosis [16]. Various compounds that influence cellular lipid metabolism can also inhibit the growth of malignant cells; consequently, the modulation of lipid metabolism has been proposed as a possible approach to cancer prevention and treatment [17]. It has been reported that lipid metabolism may be regulated by genes that are affected by regulatory complexes requiring HDAC activity [18,19]. The hepatic steatosis observed
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in our transgenic mice was not the result of stimulation with alcohol or the uptake of lipids from the blood, because the mice were not exposed to alcohol and did not exhibit any elevation in the levels of serum triglycerides or cholesterol (data not shown). The significantly higher frequency of steatosis in the transgenic mice indicates that HDAC1 may be an important factor in the regulation of lipid metabolism. In addition, the overexpression of PPAR-c was detected in mice with fatty livers, and the ablation of liver PPAR-c reduced the occurrence of hepatic steatosis in lipoatrophic mice [20]. However, we detected a significant decrease in the level of PPAR-c protein in the HDAC1 transgenic mice (data not shown). Although the mechanism remains to be elucidated, this finding indicates that the development of fatty liver in HDAC1 transgenic mice may be the result of disturbances in pathways other than the PPAR-c pathway. HDAC1 levels have been found to be elevated in highly proliferative tissues, embryonic stem cells, and several transformed cell lines [11,21], suggesting a link between HDAC1 functions and proliferation. Consistent with this possibility, the disruption of the HDAC1 gene resulted in a reduction in proliferation of mouse embryos and of embryonic stem cells [11], whereas the overexpression of HDAC1 led to impaired proliferation of murine fibroblasts [21]. Collectively, these results suggest that the tightly controlled cell-type-specific expression of HDAC1 may be crucial for the regulation of unrestricted proliferation. Our in vivo studies indicate that the overexpression of HDAC1 as a transgene induced significantly higher levels of HDAC1 activity (data not shown). A large body of literature suggests that the functions of multiple nuclear factors are dependent on the recruitment of HDAC1 activity [8]. The significantly higher HDAC activity detected in the tissues of our transgenic mice indicates that the overexpressed human HDAC1 effectively formed a functional complex with other cofactors to produce its activity. However, this high level of HDAC1 enzymatic activity was not cytotoxic to most cell types in vivo, as indicated by the absence of developmental defects in our transgenic mice. In addition, most tissues examined in our experiments were largely normal histologically through 18 months of age, except for the liver. Consequently, we propose that HDAC proteins, or HDAC1 in particular, might act primarily as an important modulator of other mechanisms involved in development and in proliferative diseases such as tumorigenesis. As such, the overexpression of HDAC alone would not directly induce disease, but the activity of other molecular regulators would be required. This hypothesis is also supported by the observation that HDAC is overexpressed in most cancer cells, as compared with the expression levels in normal cells [6]. The HDACIs with anticancer activity may disrupt the functions of HDACs in tumor cells that lead to
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cancer progression. In addition, the observation that HDACIs produce few or no side effects at doses within the therapeutic range in animal experiments and clinical trials indicates that the HDACs are more critical to cancer cell progression than to normal cellular homeostasis. However, some clinical trials using HDACIs have shown that limited amounts of the compounds reach the target cells owing to the rapid metabolism of HDACIs; this suggests that new drugs need to be designed in order to increase the efficacy of HDACIs. Our work provides an in vivo model that can be used to screenspecific HDAC1 inhibitors for efficacy and to determine whether the compounds reach the target tissues at inhibitory concentrations. In conclusion, this is the first description of the phenotypes produced by overexpression of transgenic HDAC1 in vivo. Our transgenic mice offer a valuable model for the examination of potential etiological mechanisms that function through the recruitment of HDAC1 activity. Specifically, our work offers new insight into the function of HDAC1 as related to hepatic steatosis and the cell cycle.
Acknowledgment This work was supported by the 21st Century Frontier Functional Human Genome Project of Korea, Grant No. HGC0300324 and the Ministry of Health and Welfare of Korea, Grant No. 02-PJ2-PG1-CH120002.
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