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Selective regulation of class I and class II histone deacetylases expression by inhibitors of histone deacetylases in cultured mouse neural cells
Neuroscience Letters 365 (2004) 64–68
Selective regulation of class I and class II histone deacetylases expression by inhibitors of histone deacetylases in cultured mouse neural cells Farzam Ajamian a,b,c,1 , Antero Salminen b,c , Mati Reeben b,c,∗ a
Department of Neurobiology, A.I. Virtanen Institute for Molecular Science, University of Kuopio, P.O. Box 1627 (Neulaniementie 2), FIN 70211 Kuopio, Finland b Department of Neuroscience and Neurology, University of Kuopio, FIN 70211 Kuopio, Finland c Department of Neurology, Kuopio University Hospital, Kuopio, Finland Received 28 April 2003; received in revised form 8 April 2004; accepted 19 April 2004
Abstract The expression of 10 histone deacetylases (HDAC1–10) mRNAs in mouse neuroblastoma Neuro-2a and microglia N9 cell cultures after treatment by inhibitors of HDACs, sodium butyrate and trichostatin A was studied to elucidate whether HDAC inhibitors affect the gene expression of HDACs themselves. Northern blot analysis demonstrated two- to four-fold elevated levels of mRNAs for HDAC1, -3, -5, and -6 after drug treatment in comparison with untreated cells, while mRNA levels for HDAC2 and -7 did not change significantly. In both Neuro-2a and N9 cells the highest increase was observed for HDAC5 and -6 mRNA, whereas, HDAC4 had a prominent increase in mRNA levels after drug treatment only in N9 microglia cell line but not in Neuro-2a. Immunocytochemical examination confirmed that changes in protein levels of HDACs were similar to changes in their mRNA levels. There exists an auto-regulatory feedback loop to the expression levels of several HDACs after inhibition of their biochemical activity, adding several HDAC genes to the list of genes regulated by HDAC inhibitors. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Histone deacetylase; Epigenetic; Apoptosis; Microglial activation; Neuroblastoma; Trichostatin A; Sodium butyrate; Northern blot analysis
Reversible acetylation of internal lysine residues of core histone N-terminal domains of nucleosomal histones and the resultant changes in the chromatin structure are important processes in regulation of gene transcription. The interplay between histone acetyltransferases (HATs) and histone deacetylases (HDACs) is critical to the dynamics of chromatin structure and function, thus, regulating gene expression in all eukaryotes (for review, see [11]). Several histone acetyltransferases have been identified that act as transcriptional coactivators. In contrast, histone deacetylases (HDACs) are part of transcriptional corepressor complexes. Recently, acetylation of other substrates beside histones have been discovered for HDACs as well as other cellular functions of HDACs, like binding polyubiquitin for HDAC6 [5]. ∗
Corresponding author. Tel.: +358-17-162724; fax: +358-17-162048. E-mail address: [email protected] (M. Reeben). 1 Present address: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alta., Canada T2N 1N4.
HDAC inhibitors have been shown to inhibit cancer cell growth in vitro [20] and in vivo [18] and revert oncogene-transformed cell morphology [9,20]. They can induce cellular differentiation, as well as apoptosis in several cell lines, especially of cancer origin. The effects of TSA and butyrate on cells are presumed to be primarily mediated by inhibition of histone deacetylases. Due to the apoptosis inducing properties of HDAC inhibitors, histone deacetylases have been suggested as a new target for cancer chemotherapy [19]. HDAC inhibitors activate the transcription of certain genes by altering the acetylation status of nucleosomal histones. The list of genes whose expression is increased by HDAC inhibitors includes, e.g. catalytic subunit of telomerase [16], cyclooxygenase COX-1 [17], apoptosis-related proteins Bad and p21 [13,14]. The purpose of this study was to elucidate whether HDAC inhibitors affect the gene expression of HDACs themselves. Differential expression of three human histone deacetylase mRNAs after induction of apoptosis by trichostatin A and
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.04.087
F. Ajamian et al. / Neuroscience Letters 365 (2004) 64–68
n-butyrate in immune cells has been demonstrated earlier using peripheral blood mononuclear cells [3]. However, they studied expression of only three HDACs of class I histone deacetylases. Here using Northern blot analysis we have systematically studied the expression of 10 HDACs mRNAs belonging to class I and class II in malignant cells of nervous system origin in vitro. The differences in HDAC mRNA
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regulation upon treatment with specific inhibitors could shed light into specific roles for these enzymes and promote the design of specific HDAC targeting agents for the treatment of cancer and polyglutamine-dependent neurodegeneration (Huntington’s disease) [4,15,18,19]. Murine microglial cell line N9 was kindly provided by Dr. Paola Ricciardi-Castagnoli (University of Milano-Bicocca, Milan, Italy). Cells were cultured in Iscove’s Modified Dulbecco’s Medium with 5% heat-inactivated fetal calf serum. Murine neuroblastoma Neuro-2a cells were obtained from American Type Culture Collection (ATCC) (CCL 131) and cultured in DMEM medium supplemented with 10% FCS (Gibco Life Technologies). The cells were cultured for 24 h before adding drugs. Trichostatin A (Sigma) was added in the concentration of 30 nM for microglia N9 and 0.5 or 1 M for Neuro-2a cells and n-butyrate (Sigma) 2 mM to the cells. The effects of these inhibitors on different HDAC expression were studied after the incubation for 6, 12, and 24 h. The probes for Northern analysis were generated by PCR using cDNA from mouse total brain as described earlier [1]. Total RNA was isolated using the TRIzol reagent (Life Technologies) according to the instructions of the manufacturer. Northern blot was performed as described [1]. Signals were visualized with Storm 860 PhosphorImager (Molecular Dynamics) after 1 day of exposure. Pixel volumes of specific bands were calculated with ImageQuaNT 4.2a software (Molecular Dynamics). An 18S ribosomal probe was used to verify equivalent loading. Data were analyzed for statistical significance using two-tailed Student’s t-test for paired data. Differences with P less than 0.05 were considered as significant. We have previously shown that HDAC inhibitors TSA and n-butyrate induce a prominent neuronal apoptosis on neuroblastoma 2A cells characterized by morphological changes as well as by the activation of caspase-3 protease and subsequent cleavage of poly (ADP-ribose) polymerase, one of the caspase-3 targets [12]. Caspase-3 activities reached the highest level on the second day after treatment. TSA (1–3 M) was shown to be a more effective inducer of apoptosis than
Fig. 1. (A) Northern blot analysis shows the changes in mRNA levels for HDAC1–7 in Neuro-2A cell line treated with TSA and sodium butyrate for 12 and 24 h. Final concentration of TSA and n-butyrate were 0.5 M and 2 mM, respectively. The controls were not drug treated. HDAC8, -9, and -10 mRNA were not detectable by Northern analysis. The same filters were rehybridized with 18S ribosomal probe to normalize RNA loading (only from one filter 18S hybridization is shown). The length of transcripts is shown on the left of images in kilobases (kb). Exposition time in phosphoimager was 1 day. (B) Quantization of Northern blots performed in triplicate is presented as histogram of mRNA level changes. The bars follow as: control 12 and 24 h, TSA 12 and 24 h, n-butyrate 12 and 24 h for each HDAC, respectively. Relative mRNA values were calculated for differences in RNA abundance after normalization to intensity of 18S rRNA signal. Asterisks indicate statistically significant difference from controls, P < 0.05 (two-tailed Student’s t-test). Data is average from three separate experiments.
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n-butyrate (5–10 mM). In the case of N9 microglia cells, around 80% of cells undergo differentiation into bipolar cells, while around 20% of cells go into apoptosis. Morphologically, TSA and n-butyrate induced similar changes in N9 cells. N9 microglia cells were much more sensitive to TSA; higher than 30 nM concentrations of TSA were toxic for the cells (data not shown).
In the present study we have used lower (“sub-apoptotic”) levels of TSA (0.5 M) and n-butyrate (2 mM) for Neuro-2A cells to study whether HDAC inhibitors modulate the mRNA expression of HDACs. HDAC1 and -3 had similar profiles of expression in Neuro-2a cells. Around two- to three-fold increase in HDAC1 and 3 mRNA levels were seen 12 h after TSA treatment, with return to nearly initial levels in 24 h. In cases of HDAC2, -4, and -7 there were no significant changes in mRNA levels after both TSA and butyrate treatment and after TSA treatment even a small decrease in HDAC2 mRNA levels was detectable in comparison with untreated cells. The strongest increase was for HDAC5 and -6 mRNA, a three- to four-fold increase both after TSA and butyrate and in both time points, 12 and 24 h after treatment (Fig. 1A and B). Since in our initial studies the N9 microglia cells demonstrated greater sensitivity to TSA than Neuro-2a cells we have used lower concentration of TSA (30 nM) as “sub-apoptotic” concentration for this cell line. Following this treatment the expression pattern of HDACs in N9 cells after treatment with TSA and butyrate is rather similar to neuroblastoma cells with the exception for HDAC4. In neuroblastoma cells, there was no significant change for HDAC4 mRNA expression while in N9 microglia cell line there is two- to four-fold increase after both TSA and n-butyrate treatment (Fig. 2B). To find out whether changes in mRNA levels result also in increased protein levels we performed immunocytochemistry experiments applying specific HDAC antibodies. Cells were grown under the same conditions and TSA and n-butyrate added in the same concentrations as for the Northern blot experiments in 12-well cell culture plates (Nunc). In addition to 12 and 24 h, the cells were also treated for 36 h. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Fixed cells were permeabilized by 0.1% Triton X-100 in PBS. For staining with antibodies against HDACs, the cells were first incubated in blocking solution (0.1 M phosphate buffer, 5% bovine serum albumin, and 0.002% sodium azide) and then incubated overnight in primary antibody diluted 1:200. All antibodies were goat polyclonal IgG, 200 g/ml (Santa Cruz Biotechnology; Santa Cruz, CA), namely HDAC1 (C-19) sc-6298, HDAC5
Fig. 2. (A) Northern blot analysis of the changes in mRNA levels for HDAC1–7 in N9 cell line. The cells were treated with TSA (30 nM) and sodium butyrate (2 mM) as final concentration for 6 and 12 h. The controls were not drug treated. HDAC8, -9, and -10 mRNA were not detectable by Northern analysis. The same filters were rehybridized with 18S ribosomal probe to normalize RNA loading (only from one filter 18S hybridization is shown). The length of transcripts is shown on the left of images in kilobases (kb). Exposition time in phosphoimager was 1 day. (B) Histogram of mRNA level changes for HDAC1–7 in N9 microglial cells. The bars follow as: control 6 and 12 h, TSA 6 and 12 h, n-butyrate 6 and 12 h for each HDAC, respectively. Relative mRNA values were calculated for differences in RNA abundance after normalization to intensity of 18S rRNA signal. Asterisks indicate statistically significant difference from controls, P < 0.05 (two-tailed Student’s t-test). Data is average from three separate experiments.
F. Ajamian et al. / Neuroscience Letters 365 (2004) 64–68
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Fig. 3. HDAC1, -5, -6, and -7 protein expression (immunocytochemistry) pattern in Neuro-2a cell line 24 h after TSA and n-butyrate (BUT) treatment. CON stands for untreated cells. Similar to mRNA levels (Fig. 1), there is no significant change in HDAC7 expression, while there is a clear increase in HDAC5 and -6 immunoreactivity after both drug treatments. For HDAC1, there is slight increase after TSA treatment, the protein transient up-regulation has a certain time lapse from mRNA transient increase.
(G-18) sc-5250, HDAC6 (L-18) sc-5258, HDAC7 (G-18) sc-11492. The cells were washed in PBS then incubated with anti-goat biotinylated secondary antibody for 30 min followed by another wash and incubation with HRP-conjugated streptavidin (Vectastain Universal Elite ABC Kit, Vector Laboratories, Burlingame, CA). Finally, reactions were visualized by incubation with 3,3 -diaminobenzidine (DAB). For negative control, cells were incubated overnight with dilution buffer (no primary antibody). Photomicrographs were taken using an inverted microscope. At qualitative level immunocytochemistry experiments were in good agreement with the results of measuring mRNA levels using Northern blots. It means changes in mRNA levels resulted in increased HDAC protein amounts as well. Staining of Neuro-2a cells after 24 h treatment with TSA or n-butyrate is shown on Fig. 3. There were no significant changes in mRNA levels for HDAC7 after those treatments and there is no principal difference in HDAC7 antibody staining either. For HDAC5 and -6 for which the greatest increase in mRNA levels occurred, there was clear increase in HDAC5 and -6 immunoreactivity as well. For HDAC5 there was very prominent increase of “spotty” nuclear staining, while for HDAC6 the increase of immunoreactivity was mainly in the cytoplasm. The last could be connected with recently discovered function of HDAC6 also as a tubulin deacetylase. The only difference between Northern blot mRNA levels and immunoreactivity in this 24-h time point was for HDAC1. HDAC1 demonstrated a transient increase
of mRNA levels at 12 h after TSA treatment and mRNA levels had returned to the initial level in 24 h. The slight increase of HDAC1 immunoreactivity 24 h after TSA treatment could be explained with slower turn over rates for the HDAC1 protein than mRNA. Indeed, at 36 h after TSA treatment, the immunoreactivity does not differ from the untreated cells (data not shown). HDAC inhibitors have been demonstrated to induce severe apoptosis in cytokine dependent hematopoetic cell lines (around 80% of cell death for human IL-2-dependent adult T-cell leukemia ILT-Mat cells) and one of the possible mechanisms suggested is down-regulation of IL-2 mediated gene expression [8]. Our previous results showed an extensive apoptosis (around 20%) happening in parallel with differentiation in microglial N9 cell line [11]. This result is in certain accordance with mentioned above finding [8], because microglial cells share characteristics of mononuclear phagocytes and play an important role in proinflammatory responses in the CNS. In Neuro-2a cells, nuclear fragmentation was observed in 6% of cells at 1.0 M TSA [11]. The efficient concentrations of TSA are also significantly lower for N9 cell line (30 nM) than for neuroblastoma cell line (0.5–1 M) (Figs. 1A and 2B). TSA is proposed to block the catalytic reaction by chelating a zinc ion in the active-site pocket through its hydroxamic acid group. n-Butyrate is a less specific HDAC inhibitor than TSA. It has been suggested that the mechanism of n-butyrate action is different from that of TSA [7].
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The mechanisms of how HDAC inhibitors regulate several HDACs gene expression remain unclear. Numerous studies have reported an up or down-regulation of different genes by HDAC inhibitors [13,14,16,17]. Here we established several HDACs to be among the genes that are up-regulated by HDAC inhibitors. In the case of several genes, where the effects of HDAC inhibitors (mostly TSA has been studied) have been investigated on promoter level, the binding sequences for transcription factor Sp1 have been responsible [16,17]. One of the possible mechanisms of this action could be through complexes of HDACs with Sp1 [2]. Our data indicate that different HDACs are regulated differently by HDAC inhibitors, suggesting differential sensitivity and roles for the individual enzymes. Like HDACs of class I and class II, members of the third class of this family (SIRTs) are also differentially regulated by HDAC inhibitors [10]. HDACs have been demonstrated to participate in apoptosis pathway induced by topoisomerase II poison etoposide [6]. This links HDACs more generally to apoptosis mechanisms. Collectively, our data demonstrate that there exists an auto-regulatory feedback loop to the expression levels of HDACs after inhibition of HDAC biochemical activity, adding several HDAC genes themselves to the list of genes, regulated by histone deacetylase inhibitors. It is not clear whether this could be a mechanism set in motion by the cell in order to re-equilibrate the suppressed histone deacetylation, or more likely, an effect belonging to a more general alteration of gene transcription merely due to the inhibition of HDACs. Our findings could be important in using HDAC inhibitors as potential therapeutic agents. HDAC inhibitors have potential for certain cancer treatment, as they inhibit the growth and survival of tumor cells. Recently, HDAC inhibitors were demonstrated to be effective in prevention of progressive neurodegeneration seen in Huntington’s disease and other polyglutamine-repeat diseases first in a Drosophila [15] and subsequently in a mouse model [4]. References [1] F. Ajamian, T. Suuronen, A. Salminen, M. Reeben, Upregulation of class II histone deacetylases mRNA during neural differentiation of cultured rat hippocampal progenitor cells, Neurosci. Lett. 346 (2003) 57–60. [2] H. Choi, J. Lee, J. Park, Y. Lee, Trichostatin A, a histone deacetylase inhibitor, activates the IGFBP-3 promoter by upregulating Sp1 activity in hepatoma cells: alteration of the Sp1/Sp3/HDAC1 multiprotein complex, Biochem. Biophys. Res. Commun. 296 (2002) 1005– 1012. [3] F. Dangond, S.R. Gullans, Differential expression of human histone deacetylase mRNAs in response to immune cell apoptosis induction by trichostatin A and butyrate, Biochem. Biophys. Res. Commun. 247 (1998) 833–837.
[4] E. Hockly, V.M. Richon, B. Woodman, D.L. Smith, X. Zhou, E. Rosa, K. Sathasivam, S. Ghazi-Noori, A. Mahal, P.A. Lowden, Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington’s disease, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 2041–2046. [5] S.S. Hook, A. Orian, S.M. Cowley, R.N. Eisenman, Histone deacetylase 6 binds polyubiquitin through its zinc finger (PAZ domain) and copurifies with deubiquitinating enzymes, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 13425–13430. [6] C.A. Johnson, K. Padget, C.A. Austin, B.M. Turner, Deacetylase activity associates with topoisomerase II and is necessary for etoposide-induced apoptosis, J. Biol. Chem. 276 (2001) 4539–4542. [7] P.R. Kiela, E.R. Hines, J.F. Collins, F.K. Ghishan, Regulation of the rat NHE3 gene promoter by sodium butyrate, Am. J. Physiol. Gastrointest. Liver Physiol. 281 (2001) G947–956. [8] Y. Koyama, M. Adachi, M. Sekiya, M. Takekawa, K. Imai, Histone deacetylase inhibitors suppress IL-2-mediated gene expression prior to induction of apoptosis, Blood 96 (2000) 1490–1495. [9] H.J. Kwon, T. Owa, C.A. Hassig, J. Shimada, S.L. Schreiber, Depudecin induces morphological reversion of transformed fibroblast via the inhibition of histone deacetylase, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 3356–3361. [10] S. Kyrylenko, O. Kyrylenko, T. Suuronen, A. Salminen, Differential regulation of Sir2 histone deacetylase gene family by inhibitors of class I and II histone deacetylases, Cell. Mol. Life Sci. 60 (2003) 1990–1997. [11] C.L. Peterson, HDAC’s at work: everyone doing their part, Mol. Cell 9 (2002) 921–922. [12] A. Salminen, T. Tapiola, P. Korhonen, T. Suuronen, Neuronal apoptosis induced by histone deacetylase inhibitors, Brain Res. Mol. Brain Res. 61 (1998) 203–206. [13] H. Sawa, H. Murakami, Y. Ohshima, T. Sugino, T. Nakajyo, T. Kisanuki, Y. Tamura, A. Satone, W. Ide, I. Hashimoto, H. Kamada, Histone deacetylase inhibitors such as sodium butyrate and trichostatin A induce apoptosis through an increase of the bcl-2-related protein Bad, Brain Tumor Pathol. 18 (2001) 109–114. [14] Y. Sowa, T. Orita, S. Hiranabe-Minamikawa, K. Nakano, T. Mizuno, H. Nomura, T. Sakai, Histone deacetylase inhibitor activates the p21/WAF1/Cip1 gene promoter through the Sp1 sites, Ann. N.Y. Acad. Sci. 886 (1999) 195–199. [15] J.S. Steffan, L. Bodai, J. Pallos, M. Poelman, A. McCampbell, B.L. Apostol, A. Kazantsev, E. Schmidt, Y.Z. Zhu, M. Greenwald, R. Kurokawa, D.E. Housman, G.R. Jackson, J.L. Marsh, L.M. Thompson, Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila, Nature 413 (2001) 739–743. [16] M. Takakura, S. Kyo, Y. Sowa, Z. Wang, N. Yatabe, Y. Maida, M. Tanaka, M. Inoue, Telomerase activation by histone deacetylase inhibitor in normal cells, Nucleic Acids Res. 29 (2001) 3006–3011. [17] S. Taniura, H. Kamitani, T. Watanabe, T.E. Eling, Transcriptional regulation of cyclooxygenase-1 by histone deacetylase inhibitors in normal human astrocyte cells, J. Biol. Chem. 277 (2002) 16823– 16830. [18] D.M. Vigushin, S. Ali, P.E. Pace, N. Mirsaidi, K. Ito, I. Adcock, R.C. Coombes, Trichostatin A is a histone deacetylase inhibitor with potent antitumor activity against breast cancer in vivo, Clin. Cancer Res. 7 (2001) 971–976. [19] M. Yoshida, R. Furumai, M. Nishiyama, Y. Komatsu, N. Nishino, S. Horinouchi, Histone deacetylase as a new target for cancer chemotherapy, Cancer Chemother. Pharmacol. 48 (Suppl. 11, S) (2001) 20–26. [20] M. Yoshida, M. Kijima, M. Akita, T. Beppu, Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A, J. Biol. Chem. 265 (1990) 17174–17179.
Selective regulation of class I and class II histone deacetylases expression by inhibitors of histone deacetylases in cultured mouse neural cells Farzam Ajamian a,b,c,1 , Antero Salminen b,c , Mati Reeben b,c,∗ a
Department of Neurobiology, A.I. Virtanen Institute for Molecular Science, University of Kuopio, P.O. Box 1627 (Neulaniementie 2), FIN 70211 Kuopio, Finland b Department of Neuroscience and Neurology, University of Kuopio, FIN 70211 Kuopio, Finland c Department of Neurology, Kuopio University Hospital, Kuopio, Finland Received 28 April 2003; received in revised form 8 April 2004; accepted 19 April 2004
Abstract The expression of 10 histone deacetylases (HDAC1–10) mRNAs in mouse neuroblastoma Neuro-2a and microglia N9 cell cultures after treatment by inhibitors of HDACs, sodium butyrate and trichostatin A was studied to elucidate whether HDAC inhibitors affect the gene expression of HDACs themselves. Northern blot analysis demonstrated two- to four-fold elevated levels of mRNAs for HDAC1, -3, -5, and -6 after drug treatment in comparison with untreated cells, while mRNA levels for HDAC2 and -7 did not change significantly. In both Neuro-2a and N9 cells the highest increase was observed for HDAC5 and -6 mRNA, whereas, HDAC4 had a prominent increase in mRNA levels after drug treatment only in N9 microglia cell line but not in Neuro-2a. Immunocytochemical examination confirmed that changes in protein levels of HDACs were similar to changes in their mRNA levels. There exists an auto-regulatory feedback loop to the expression levels of several HDACs after inhibition of their biochemical activity, adding several HDAC genes to the list of genes regulated by HDAC inhibitors. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Histone deacetylase; Epigenetic; Apoptosis; Microglial activation; Neuroblastoma; Trichostatin A; Sodium butyrate; Northern blot analysis
Reversible acetylation of internal lysine residues of core histone N-terminal domains of nucleosomal histones and the resultant changes in the chromatin structure are important processes in regulation of gene transcription. The interplay between histone acetyltransferases (HATs) and histone deacetylases (HDACs) is critical to the dynamics of chromatin structure and function, thus, regulating gene expression in all eukaryotes (for review, see [11]). Several histone acetyltransferases have been identified that act as transcriptional coactivators. In contrast, histone deacetylases (HDACs) are part of transcriptional corepressor complexes. Recently, acetylation of other substrates beside histones have been discovered for HDACs as well as other cellular functions of HDACs, like binding polyubiquitin for HDAC6 [5]. ∗
Corresponding author. Tel.: +358-17-162724; fax: +358-17-162048. E-mail address: [email protected] (M. Reeben). 1 Present address: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alta., Canada T2N 1N4.
HDAC inhibitors have been shown to inhibit cancer cell growth in vitro [20] and in vivo [18] and revert oncogene-transformed cell morphology [9,20]. They can induce cellular differentiation, as well as apoptosis in several cell lines, especially of cancer origin. The effects of TSA and butyrate on cells are presumed to be primarily mediated by inhibition of histone deacetylases. Due to the apoptosis inducing properties of HDAC inhibitors, histone deacetylases have been suggested as a new target for cancer chemotherapy [19]. HDAC inhibitors activate the transcription of certain genes by altering the acetylation status of nucleosomal histones. The list of genes whose expression is increased by HDAC inhibitors includes, e.g. catalytic subunit of telomerase [16], cyclooxygenase COX-1 [17], apoptosis-related proteins Bad and p21 [13,14]. The purpose of this study was to elucidate whether HDAC inhibitors affect the gene expression of HDACs themselves. Differential expression of three human histone deacetylase mRNAs after induction of apoptosis by trichostatin A and
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.04.087
F. Ajamian et al. / Neuroscience Letters 365 (2004) 64–68
n-butyrate in immune cells has been demonstrated earlier using peripheral blood mononuclear cells [3]. However, they studied expression of only three HDACs of class I histone deacetylases. Here using Northern blot analysis we have systematically studied the expression of 10 HDACs mRNAs belonging to class I and class II in malignant cells of nervous system origin in vitro. The differences in HDAC mRNA
65
regulation upon treatment with specific inhibitors could shed light into specific roles for these enzymes and promote the design of specific HDAC targeting agents for the treatment of cancer and polyglutamine-dependent neurodegeneration (Huntington’s disease) [4,15,18,19]. Murine microglial cell line N9 was kindly provided by Dr. Paola Ricciardi-Castagnoli (University of Milano-Bicocca, Milan, Italy). Cells were cultured in Iscove’s Modified Dulbecco’s Medium with 5% heat-inactivated fetal calf serum. Murine neuroblastoma Neuro-2a cells were obtained from American Type Culture Collection (ATCC) (CCL 131) and cultured in DMEM medium supplemented with 10% FCS (Gibco Life Technologies). The cells were cultured for 24 h before adding drugs. Trichostatin A (Sigma) was added in the concentration of 30 nM for microglia N9 and 0.5 or 1 M for Neuro-2a cells and n-butyrate (Sigma) 2 mM to the cells. The effects of these inhibitors on different HDAC expression were studied after the incubation for 6, 12, and 24 h. The probes for Northern analysis were generated by PCR using cDNA from mouse total brain as described earlier [1]. Total RNA was isolated using the TRIzol reagent (Life Technologies) according to the instructions of the manufacturer. Northern blot was performed as described [1]. Signals were visualized with Storm 860 PhosphorImager (Molecular Dynamics) after 1 day of exposure. Pixel volumes of specific bands were calculated with ImageQuaNT 4.2a software (Molecular Dynamics). An 18S ribosomal probe was used to verify equivalent loading. Data were analyzed for statistical significance using two-tailed Student’s t-test for paired data. Differences with P less than 0.05 were considered as significant. We have previously shown that HDAC inhibitors TSA and n-butyrate induce a prominent neuronal apoptosis on neuroblastoma 2A cells characterized by morphological changes as well as by the activation of caspase-3 protease and subsequent cleavage of poly (ADP-ribose) polymerase, one of the caspase-3 targets [12]. Caspase-3 activities reached the highest level on the second day after treatment. TSA (1–3 M) was shown to be a more effective inducer of apoptosis than
Fig. 1. (A) Northern blot analysis shows the changes in mRNA levels for HDAC1–7 in Neuro-2A cell line treated with TSA and sodium butyrate for 12 and 24 h. Final concentration of TSA and n-butyrate were 0.5 M and 2 mM, respectively. The controls were not drug treated. HDAC8, -9, and -10 mRNA were not detectable by Northern analysis. The same filters were rehybridized with 18S ribosomal probe to normalize RNA loading (only from one filter 18S hybridization is shown). The length of transcripts is shown on the left of images in kilobases (kb). Exposition time in phosphoimager was 1 day. (B) Quantization of Northern blots performed in triplicate is presented as histogram of mRNA level changes. The bars follow as: control 12 and 24 h, TSA 12 and 24 h, n-butyrate 12 and 24 h for each HDAC, respectively. Relative mRNA values were calculated for differences in RNA abundance after normalization to intensity of 18S rRNA signal. Asterisks indicate statistically significant difference from controls, P < 0.05 (two-tailed Student’s t-test). Data is average from three separate experiments.
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n-butyrate (5–10 mM). In the case of N9 microglia cells, around 80% of cells undergo differentiation into bipolar cells, while around 20% of cells go into apoptosis. Morphologically, TSA and n-butyrate induced similar changes in N9 cells. N9 microglia cells were much more sensitive to TSA; higher than 30 nM concentrations of TSA were toxic for the cells (data not shown).
In the present study we have used lower (“sub-apoptotic”) levels of TSA (0.5 M) and n-butyrate (2 mM) for Neuro-2A cells to study whether HDAC inhibitors modulate the mRNA expression of HDACs. HDAC1 and -3 had similar profiles of expression in Neuro-2a cells. Around two- to three-fold increase in HDAC1 and 3 mRNA levels were seen 12 h after TSA treatment, with return to nearly initial levels in 24 h. In cases of HDAC2, -4, and -7 there were no significant changes in mRNA levels after both TSA and butyrate treatment and after TSA treatment even a small decrease in HDAC2 mRNA levels was detectable in comparison with untreated cells. The strongest increase was for HDAC5 and -6 mRNA, a three- to four-fold increase both after TSA and butyrate and in both time points, 12 and 24 h after treatment (Fig. 1A and B). Since in our initial studies the N9 microglia cells demonstrated greater sensitivity to TSA than Neuro-2a cells we have used lower concentration of TSA (30 nM) as “sub-apoptotic” concentration for this cell line. Following this treatment the expression pattern of HDACs in N9 cells after treatment with TSA and butyrate is rather similar to neuroblastoma cells with the exception for HDAC4. In neuroblastoma cells, there was no significant change for HDAC4 mRNA expression while in N9 microglia cell line there is two- to four-fold increase after both TSA and n-butyrate treatment (Fig. 2B). To find out whether changes in mRNA levels result also in increased protein levels we performed immunocytochemistry experiments applying specific HDAC antibodies. Cells were grown under the same conditions and TSA and n-butyrate added in the same concentrations as for the Northern blot experiments in 12-well cell culture plates (Nunc). In addition to 12 and 24 h, the cells were also treated for 36 h. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Fixed cells were permeabilized by 0.1% Triton X-100 in PBS. For staining with antibodies against HDACs, the cells were first incubated in blocking solution (0.1 M phosphate buffer, 5% bovine serum albumin, and 0.002% sodium azide) and then incubated overnight in primary antibody diluted 1:200. All antibodies were goat polyclonal IgG, 200 g/ml (Santa Cruz Biotechnology; Santa Cruz, CA), namely HDAC1 (C-19) sc-6298, HDAC5
Fig. 2. (A) Northern blot analysis of the changes in mRNA levels for HDAC1–7 in N9 cell line. The cells were treated with TSA (30 nM) and sodium butyrate (2 mM) as final concentration for 6 and 12 h. The controls were not drug treated. HDAC8, -9, and -10 mRNA were not detectable by Northern analysis. The same filters were rehybridized with 18S ribosomal probe to normalize RNA loading (only from one filter 18S hybridization is shown). The length of transcripts is shown on the left of images in kilobases (kb). Exposition time in phosphoimager was 1 day. (B) Histogram of mRNA level changes for HDAC1–7 in N9 microglial cells. The bars follow as: control 6 and 12 h, TSA 6 and 12 h, n-butyrate 6 and 12 h for each HDAC, respectively. Relative mRNA values were calculated for differences in RNA abundance after normalization to intensity of 18S rRNA signal. Asterisks indicate statistically significant difference from controls, P < 0.05 (two-tailed Student’s t-test). Data is average from three separate experiments.
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Fig. 3. HDAC1, -5, -6, and -7 protein expression (immunocytochemistry) pattern in Neuro-2a cell line 24 h after TSA and n-butyrate (BUT) treatment. CON stands for untreated cells. Similar to mRNA levels (Fig. 1), there is no significant change in HDAC7 expression, while there is a clear increase in HDAC5 and -6 immunoreactivity after both drug treatments. For HDAC1, there is slight increase after TSA treatment, the protein transient up-regulation has a certain time lapse from mRNA transient increase.
(G-18) sc-5250, HDAC6 (L-18) sc-5258, HDAC7 (G-18) sc-11492. The cells were washed in PBS then incubated with anti-goat biotinylated secondary antibody for 30 min followed by another wash and incubation with HRP-conjugated streptavidin (Vectastain Universal Elite ABC Kit, Vector Laboratories, Burlingame, CA). Finally, reactions were visualized by incubation with 3,3 -diaminobenzidine (DAB). For negative control, cells were incubated overnight with dilution buffer (no primary antibody). Photomicrographs were taken using an inverted microscope. At qualitative level immunocytochemistry experiments were in good agreement with the results of measuring mRNA levels using Northern blots. It means changes in mRNA levels resulted in increased HDAC protein amounts as well. Staining of Neuro-2a cells after 24 h treatment with TSA or n-butyrate is shown on Fig. 3. There were no significant changes in mRNA levels for HDAC7 after those treatments and there is no principal difference in HDAC7 antibody staining either. For HDAC5 and -6 for which the greatest increase in mRNA levels occurred, there was clear increase in HDAC5 and -6 immunoreactivity as well. For HDAC5 there was very prominent increase of “spotty” nuclear staining, while for HDAC6 the increase of immunoreactivity was mainly in the cytoplasm. The last could be connected with recently discovered function of HDAC6 also as a tubulin deacetylase. The only difference between Northern blot mRNA levels and immunoreactivity in this 24-h time point was for HDAC1. HDAC1 demonstrated a transient increase
of mRNA levels at 12 h after TSA treatment and mRNA levels had returned to the initial level in 24 h. The slight increase of HDAC1 immunoreactivity 24 h after TSA treatment could be explained with slower turn over rates for the HDAC1 protein than mRNA. Indeed, at 36 h after TSA treatment, the immunoreactivity does not differ from the untreated cells (data not shown). HDAC inhibitors have been demonstrated to induce severe apoptosis in cytokine dependent hematopoetic cell lines (around 80% of cell death for human IL-2-dependent adult T-cell leukemia ILT-Mat cells) and one of the possible mechanisms suggested is down-regulation of IL-2 mediated gene expression [8]. Our previous results showed an extensive apoptosis (around 20%) happening in parallel with differentiation in microglial N9 cell line [11]. This result is in certain accordance with mentioned above finding [8], because microglial cells share characteristics of mononuclear phagocytes and play an important role in proinflammatory responses in the CNS. In Neuro-2a cells, nuclear fragmentation was observed in 6% of cells at 1.0 M TSA [11]. The efficient concentrations of TSA are also significantly lower for N9 cell line (30 nM) than for neuroblastoma cell line (0.5–1 M) (Figs. 1A and 2B). TSA is proposed to block the catalytic reaction by chelating a zinc ion in the active-site pocket through its hydroxamic acid group. n-Butyrate is a less specific HDAC inhibitor than TSA. It has been suggested that the mechanism of n-butyrate action is different from that of TSA [7].
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The mechanisms of how HDAC inhibitors regulate several HDACs gene expression remain unclear. Numerous studies have reported an up or down-regulation of different genes by HDAC inhibitors [13,14,16,17]. Here we established several HDACs to be among the genes that are up-regulated by HDAC inhibitors. In the case of several genes, where the effects of HDAC inhibitors (mostly TSA has been studied) have been investigated on promoter level, the binding sequences for transcription factor Sp1 have been responsible [16,17]. One of the possible mechanisms of this action could be through complexes of HDACs with Sp1 [2]. Our data indicate that different HDACs are regulated differently by HDAC inhibitors, suggesting differential sensitivity and roles for the individual enzymes. Like HDACs of class I and class II, members of the third class of this family (SIRTs) are also differentially regulated by HDAC inhibitors [10]. HDACs have been demonstrated to participate in apoptosis pathway induced by topoisomerase II poison etoposide [6]. This links HDACs more generally to apoptosis mechanisms. Collectively, our data demonstrate that there exists an auto-regulatory feedback loop to the expression levels of HDACs after inhibition of HDAC biochemical activity, adding several HDAC genes themselves to the list of genes, regulated by histone deacetylase inhibitors. It is not clear whether this could be a mechanism set in motion by the cell in order to re-equilibrate the suppressed histone deacetylation, or more likely, an effect belonging to a more general alteration of gene transcription merely due to the inhibition of HDACs. Our findings could be important in using HDAC inhibitors as potential therapeutic agents. HDAC inhibitors have potential for certain cancer treatment, as they inhibit the growth and survival of tumor cells. Recently, HDAC inhibitors were demonstrated to be effective in prevention of progressive neurodegeneration seen in Huntington’s disease and other polyglutamine-repeat diseases first in a Drosophila [15] and subsequently in a mouse model [4]. References [1] F. Ajamian, T. Suuronen, A. Salminen, M. Reeben, Upregulation of class II histone deacetylases mRNA during neural differentiation of cultured rat hippocampal progenitor cells, Neurosci. Lett. 346 (2003) 57–60. [2] H. Choi, J. Lee, J. Park, Y. Lee, Trichostatin A, a histone deacetylase inhibitor, activates the IGFBP-3 promoter by upregulating Sp1 activity in hepatoma cells: alteration of the Sp1/Sp3/HDAC1 multiprotein complex, Biochem. Biophys. Res. Commun. 296 (2002) 1005– 1012. [3] F. Dangond, S.R. Gullans, Differential expression of human histone deacetylase mRNAs in response to immune cell apoptosis induction by trichostatin A and butyrate, Biochem. Biophys. Res. Commun. 247 (1998) 833–837.
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