Differential effects of histone deacetylase inhibitors on phorbol ester- and TGF-b1 induced murine tissue inhibitor of metalloproteinases-1 gene expression David A Young*, Olivia Billingham, Clara L Sampieri, Dylan R Edwards and Ian M Clark School of Biological Sciences, University of East Anglia, Norwich, UK Keywords acetylation; c-Jun; TGFb; TIMP; trichostatin A Correspondence I M Clark, School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK Tel: +44 1603 592760 Fax: +44 1603 592250 E-mail: i.clark@uea.ac.uk *Present address Department of Rheumatology, University of Newcastle- upon-Tyne, NE2 4HH, UK (Received 16 November 2004, revised 12 January 2005, accepted 21 February 2005) doi:10.1111/j.1742-4658.2005.04622.x Expression of the tissue inhibitor of metalloproteinases-1 (Timp-1) gene can be induced by either phorbol myristate acetate (PMA) or transforming growth factor b1 (TGF-b1), although the signalling pathways involved are not clearly defined Canonically, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate (NaB) increase total cellular histone acetylation and activate expression of susceptible genes Remarkably, PMA and TGF-b1 stimulation of Timp-1 show a differential response to TSA or NaB TSA or NaB potentiate PMA-induced Timp-1 expression but repress TGF-b1-induced Timp-1 expression The repression of TGF-b1-induced Timp-1 by TSA was maximal at ngỈmL)1, while for the superinduction of PMA-induced Timp-1 expression, the maximal dose is > 500 ngỈmL)1 TSA A further HDACi, valproic acid, did not block TGF-b1-induced Timp-1 expression, demonstrating that different HDACs impact on the induction of Timp-1 For either PMA or TGF-b1 to induce Timp-1 expression, new protein synthesis is required, and the induction of AP-1 factors closely precedes that of Timp-1 The effects of the HDACi can be reiterated in transient transfection using Timp-1 promoter constructs Mutation or deletion of the AP-1 motif ()59 ⁄ )53) in the Timp-1 promoter diminishes PMA-induction of reporter constructs, however, the further addition of TSA still superinduces the reporter In c-Jun– ⁄ – cells, PMA still stimulates Timp-1 expression, but TSA superinduction is lost Transfection of a series of Timp-1 promoter constructs identified three regions through which TSA superinduces PMA-induced Timp-1 and we have demonstrated specific protein binding to two of these regions which contain either an avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets) or Sp1 binding motif Remodelling of the extracellular matrix (ECM) is an essential physiological process in, e.g development, wound healing and angiogenesis Aberrant ECM turnover is also associated with a number of pathological processes such as joint destruction in the arthritides, tumour metastasis, and fibrosis [1] Central to the turnover of ECM is the matrix metalloproteinase (MMP) family; these number 23 enzymes in man which, between them, have the capability of degrading the majority of ECM proteins [2] Four tissue inhibitors of metalloproteinases (TIMPs) safeguard ECM integrity by virtue of their ability to inhibit the MMPs [3] The TIMPs display a high degree of functional overlap, but show dramatic differences in their Abbreviations AP-1, activating protein-1; EMSA, electrophoretic mobility-shift assay; Ets, avian erythroblastosis virus E26 (v-ets) oncogene homologue; HAT, histone acetyltransferase; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; MMP, matrix metalloproteinase; NaB, sodium butyrate; PMA, phorbol myristate acetate; TIMP, tissue inhibitor of metalloproteinases; TGF, transforming growth factor; TSA, trichostatin A; VPA, valproic acid 1912 FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS D A Young et al patterns of expression both during development and in response to stimuli Timp-1 gene expression can be induced by a variety of stimuli including phorbol esters (PMA), serum, transforming growth factor b (TGFb), retinoids and interleukin-6 family members; where it has been assessed, induction is at the level of transcription [4] The expression of Timp-1 has been implicated in many disease processes including tumour progression, fibrosis, cardiovascular disease and arthritis [3] Modulation of Timp-1 may therefore have therapeutic potential in several pathologies [5], and an understanding of the mechanisms impacting upon Timp-1 gene expression is paramount Dissection of the Timp-1 gene promoter has revealed several important cis-acting sequences An AP-1 site located at )59 ⁄ )53 in the murine gene is important in both basal and inducible Timp-1 gene expression, with a neighbouring avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets)-binding site playing a more minor role [6–8] Many other regions of the gene promoter and first intron have been shown to be important, e.g a hypoxic response element has been mapped to )26 ⁄ )23 [9]; an Sp1 motif confers at least part of the repressive nature of the first intron of the Timp-1 gene by binding Sp1 and Sp3, along with an Ets-related factor [10] Recently, our laboratories have examined the induction of Timp-1 gene expression by TGF-b1, and shown that this is independent of the Smad pathway [11] This induction is dependent on the promoter proximal AP-1 site, requires at least c-fos, c-Jun and JunD, and is sensitive to extracellular signal-regulated kinase (ERK) and p38 mitogen-acivated protein kinase (p38 MAPK) inhibitors Phorbol esters (e.g phorbol myristate acetate, PMA) are also robust inducers of Timp-1 gene expression Compared to the induction of Timp-1 by TGFb, the PMA induction occurs with more rapid kinetics, is less sensitive to p38 MAPK inhibitors, and less dependent on c-fos Hence, it appears that whilst there may be some overlap in the signalling pathways used by TGFb vs PMA to impact on the Timp-1 gene, there are also some pathways exclusive to each factor ([11]; D A Young, D R Edwards and I M Clark, unpublished observation) The packaging of eukaryotic DNA into chromatin plays an important role in regulating gene expression The DNA is wound round a histone octamer consisting of two molecules each of histones H2A, H2B, H3 and H4 to form a nucleosome This unit is repeated at approximately 200 bp intervals with histone H1 associating with the intervening DNA Nucleosomes are generally repressive to transcription, hindering access of the transcriptional apparatus [12] However, two major FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS HDAC inhibitors and Timp-1 expression mechanisms exist that modulate chromatin structure to allow transcriptional activity: first, ATP-dependent nucleosome remodellers such as the Swi ⁄ Snf complex [13,14] and second, the enzymatic modification of histones, via acetylation, methylation and phosphorylation [15–18] Acetylation by histone acetyltransferases (HATs) occurs on specific lysine residues on the N-terminal tails of histone H2A, H2B, H3 and H4 This neutralization of positive charge leads to a loosening of the histone:DNA structure, allowing access of the transcriptional machinery; furthermore, the acetyl groups may associate with and recruit factors containing bromo-domains [12] Many transcriptional activators or coactivators have (or recruit) HAT activity, giving a mechanism whereby acetylation can be targeted to specific gene promoters [15,16] Conversely, histone deacetylases (HDACs) have also been characterized Hypoacetylation of histones associates with transcriptional silence, and several transcriptional repressors and corepressors have been identified which have (or recruit) HDAC activity [17,19] Non-histone substrates of HATs have also been described, e.g p53, E2F, NF-jB, Sp3 and c-Jun; these latter two transcription factors are known to be important in Timp-1 expression [20,21] Trichostatin A (TSA), sodium butyrate (NaB) and valproic acid (VPA) are HDAC inhibitors (HDACi) [22–24] Addition of these reagents to cells should therefore block histone deacetylation and result in increased acetylation of susceptible genes The prediction would be that this would lead to an increase in gene expression Here, we demonstrate for the first time that HDACi impact upon Timp-1 gene expression Furthermore, the response of the gene to HDACi is dependent upon the stimulus – either PMA or TGF-b1 – used to induce Timp-1 expression Dose–response curves and the use of the more HDAC specific HDACi, VPA, shows that at least two targets of HDACi exist which affect downstream Timp-1 expression Both TGF-b1 and PMA are known to act via the AP-1 motif to induce the Timp-1 gene We show that for HDACi to superinduce PMAinduced Timp-1, the AP-1 factor c-Jun is essential; however, the HDACi acts through both an Ets and GC-box (Sp factor binding) motif in the Timp-1 promoter itself Results The effects of HDAC inhibitors on Timp-1 gene expression As outlined above, the prediction is that HDAC inhibitors should induce expression of susceptible genes Figure 1A,B shows that both TSA and NaB 1913 HDAC inhibitors and Timp-1 expression A D A Young et al Fig Histone deacetylase inhibitors have differential effects on PMA- vs TGF-b1-induced Timp-1 expression (A and B) C3H10T1 ⁄ murine fibroblasts were serum starved for 24 h, then stimulated with 10)7 M PMA or ngỈmL)1 TGF-b1 for h in the presence or absence of (A) 250 ngỈmL)1 TSA or (B) mM NaB Total RNA was isolated and subjected to real-time qRT-PCR using a specific primer set for the Timp-1 gene [49]; data were normalized to the 18S rRNA housekeeping gene Data are plotted as mean + SEM (A) n ¼ (B) n ¼ (C) C3H10T1 ⁄ and Swiss-3T3 fibroblast cells were serum starved for 24 h, then stimulated with 10)7 M PMA or ngỈmL)1 TGF-b1 for h in the presence or absence of 500 ngỈmL)1 TSA Isolated total RNA subjected to qRT-PCR and normalized as described above Nuclear extracts (10 lg) from the C3H10T1 ⁄ cells were western blotted with an anti-(acetyl–lysine) Ig to monitor acetylation (histone band assigned by molecular mass and abundance) B C ation in the control of Timp-1 gene expression by PMA vs TGFb1 Figure 1C shows a comparison between two cell lines, C3H10T1 ⁄ and Swiss3T3, stimulated with TGF-b1 or PMA, in the presence or absence of TSA to confirm the original observations were not a cell-type dependent phenomenon The fold induction by TSA above the PMA-induced level is higher in C3H10T1 ⁄ than Swiss 3T3 as the response to PMA alone is greater in the latter cell line The effect of NaB on induced Timp-1 expression in Swiss 3T3 cells also mirrors that seen in C3H10T1 ⁄ (data not shown) Using an anti-(acetyl-lysine) Ig, TSA could be seen to cause an increase in acetylation of total histones from C3H10T1 ⁄ nuclear cell extracts (Fig 1C; lower panel) PMA- vs TGF-b1 induction of Timp-1 display differential sensitivity to HDACi superinduce PMA-induced Timp-1 expression measured by qRT-PCR in C3H10T1 ⁄ cells, but potently repress TGF-b1 induction of the gene These effects appear specific for Timp-1, as no equivalent alteration in Timp-2 or -3 expression is seen (data not shown) These unexpected data suggest a differential involvement of acetyl1914 Representative dose–response curves of the effect of HDACi’s on either PMA- or TGF-b1-induced Timp-1 gene expression are shown in Fig 2A–C For PMA induction of Timp-1 expression, TSA superinduces this expression with an optimum dose of 250 > 1000 ngỈmL)1 (0.8–3.3 lm, depending upon experiment), whilst for NaB the optimum dose is mm Another known HDACi, VPA, had no effect on PMA-induced Timp-1 expression until a concentration greater than mm was added In contrast to this, TGF-b1-induced Timp-1 expression is more sensitive to HDACi, with an optimum dose of TSA being less than 50 ngỈmL)1 (165 nm) (and in a further experiment ngỈmL)1 still potently inhibited TGF-b1 induction of Timp-1) and of NaB less than mm VPA had no effect on TGF-b1induced Timp-1 expression, but was shown to be functional as, in the same samples, even the lowest concentration of VPA (0.5 mm) repressed TGFb1induced ADAM12 expression (Fig 2C, inset panel) FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS D A Young et al HDAC inhibitors and Timp-1 expression assessed Figure shows that addition of the protein synthesis inhibitor, emetine, completely abrogates both PMA- and TGF-b1-induction of the Timp-1 gene In the presence of emetine, the HDACi have no further effect (data not shown) The action of HDACi could therefore be either on the Timp-1 gene itself, or on the expression of a protein(s) required for induction of the Timp-1 gene Time course of TSA action upon PMA- and TGF-b1-induced Timp-1 expression The time course of induction of Timp-1 gene expression by PMA and TGF-b1 measured by qRT-PCR is identical to our previous northern blot data with PMA giving a more rapid but transient induction and TGF-b1 inducing a slower more sustained stimulation of the gene [25] The effect of TSA on both PMA- and TGF-b1induced Timp-1 expression is evident as early as h after addition of reagents (Fig 4A) This represents the earliest time point that induction of the gene by PMA or TGF-b1 is measurable by qRT-PCR The magnitude of TSA superinduction of PMA-induced Timp-1 increases to 12 h, and remains at 24 h, even when the PMAinduced levels have returned to baseline TSA continues to repress TGF-b1-induced Timp-1 expression for as long as the TGF-b1 induction is measurable (> 24 h) Induction of c-fos by PMA, TGF-b1 and TSA immediately precedes that of Timp-1 Fig Histone deacetylase inhibitors display different doseresponses on PMA- vs TGF-b1-induced Timp-1 expression C3H10T1 ⁄ murine fibroblasts were serum starved for 24 h, then stimulated with 10)7 M PMA or ngỈmL)1 TGF-b1 for h in the presence or absence of (A) 0–1000 ngỈmL)1 TSA; (B) 0–10 mM NaB, or (C) 0–8 mM VPA Total RNA was isolated and subjected to real-time qRT-PCR for Timp-1; data were normalized to the 18S rRNA housekeeping gene Data is representative of at least two independent experiments in all cases As both PMA and TGF-b1 require new protein synthesis to induce Timp-1 expression, we examined the These data show that different HDACs are involved in the TGFb vs PMA induction of Timp-1 Two specific inhibitors of the Sir2 family of deacetylases (class HDACs), sirtinol and nicotinamide, had no effect on induced or basal Timp-1 expression (data not shown) showing that class HDACs are not involved Induction of the Timp-1 gene by PMA or TGF-b1 requires new protein synthesis In order to assess the possibility that the effects of HDACi are secondary, acting to modulate the expression of an intermediate, the requirement for new protein synthesis in the induction of Timp-1 was FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS Fig The induction of Timp-1 by PMA or TGF-b1 is protein synthesis dependent C3H10T1 ⁄ murine fibroblasts were serum starved for 24 h, then stimulated with 10)7 M PMA or ngỈmL)1 TGF-b1 for h in the presence or absence of the protein synthesis inhibitor emetine at 10 lgỈmL)1 Total RNA was isolated and subjected to real-time qRT-PCR for Timp-1; data were normalized to the 18S rRNA housekeeping gene Data is plotted mean + SEM, n ¼ 1915 HDAC inhibitors and Timp-1 expression D A Young et al A B C expression of the AP-1 family member c-fos, over the same time course experiment (Fig 4A) Previously, we have shown that c-fos overexpression induces Timp-1 1916 Fig Time-course of trichostatin A action on PMA- vs TGF-b1-induced Timp-1 and c-fos expression and AP-1 binding C3H10T1 ⁄ murine fibroblasts were serum starved for 24 h, then stimulated with 10)7 M PMA or ngỈmL)1 TGF-b1 in the presence or absence of 250 ngỈmL)1 TSA (A) Total RNA was isolated at timepoints 1, 3, 6, 12 and 24 h and subjected to real-time qRT-PCR for Timp-1 and c-fos; data were normalized to the 18S rRNA housekeeping gene Data plotted is representative of three independent time course experiments (B) Nuclear extracts were isolated at and h after stimulation and subjected to EMSA using the Timp-1 AP-1 motif as probe (C) Isolated nuclear extracts (2 lg) from cells stimulated with PMA or PMA and TSA for h were incubated in the presence or absence of antibodies against either acetyl–lysine and ⁄ or c-fos and subjected to EMSA expression [11]; c-fos is a known immediate early gene induced by PMA We initially confirmed this using the protein synthesis inhibitor cycloheximide, which as FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS D A Young et al expected, did not block the induction of c-fos mRNA by PMA The kinetics of c-fos induction closely precedes that of Timp-1 At h, c-fos was dramatically induced by PMA Further, this induction was superinduced by TSA; by h the PMA induction was lost, but TSA in the presence of PMA still superinduced c-fos expression Compared to PMA, TGF-b1 induction of c-fos was delayed, only becoming apparent by h; this induction remained at h but was lost by 12 h Similar to Timp-1 expression, TSA blocked TGF-b1 induced c-fos at all time points where TGF-b1 alone induced c-fos expression Interestingly, TSA alone induced c-fos but only after 12 h of stimulation The Timp-1 gene contains an AP-1 motif at )59 ⁄ )53 bp relative to the transcription start site [6] Nuclear proteins were isolated from cells stimulated for either or h with TGF-b1 or PMA, with or without TSA In an electrophoretic mobility-shift assay (EMSA), AP-1 factors could be seen to bind a 30 bp sequence encompassing the Timp-1 AP-1 motif and this binding was induced by PMA (at both and h) or TGF-b1 stimulation (at h) (Fig 4B) As with the induction of c-fos mRNA, AP-1 protein binding activity was greater with PMA than TGF-b1, and the induction by PMA occurred earlier than that by TGFb1 Stimulation with TSA alone or in the presence of PMA or TGF-b1 appeared to have little effect on the overall amount of AP-1 protein binding the Timp-1 AP-1 sequence; this was in marked contrast to that seen for c-fos mRNA levels The specificity of the AP-1 binding activity was confirmed using DNA-binding competition studies The AP-1 complex could be competed by an excess of ‘wild-type’ DNA, but not by an equivalent DNA fragment containing a mutation in the AP-1 binding sequence (DAP-1; Fig 4C) The AP-1 complex was not seen when the DAP-1 DNA was used as the radiolabelled probe (data not shown) Further, the presence of c-fos in the AP-1 complex was confirmed by supershift analysis using an anti-(c-fos) Ig (Fig 4C) As the total binding of the AP-1 complex did not significantly alter on the addition of TSA, and c-Jun, another AP-1 member, is a potential target for acetylation, we performed supershift analysis using an antibody raised against acetylated lysine When added to nuclear extracts, a low mobility ‘supershifted’ complex was evident in all extracts treated with TSA However, the binding intensity of this complex did not alter upon various stimulations and the appearance of the supershift did not coincide with the loss of another band (Fig 4C) Further analysis confirmed this complex did not appear to contain c-Jun or other AP-1 factors [i.e antibodies to these factors not alter the supershift FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS HDAC inhibitors and Timp-1 expression seen with the anti-(acetyl-lysine) Ig, data not shown] and the lack of competition for this band by the excess of cold AP-1 oligonucleotide (Fig 4C) strongly suggests that it is not related to AP-1 factor acetylation TSA superinduction of PMA-induced Timp-1 requires c-Jun To establish unequivocally the role of specific AP-1 family members upon Timp-1 expression, qRT-PCR was performed on RNA from c-fos, c-Jun or junD deficient cells (– ⁄ –) stimulated with PMA or TGF-b1 with or without TSA; Swiss-3T3 cells were used as a control Surprisingly, Swiss-3T3, c-fos– ⁄ – and junD– ⁄ – cells had the same Timp-1 expression profile as each other and as that seen previously for C3H10T1 ⁄ cells (Fig 5A) The fold induction by PMA or TGF-b1 was remarkably similar between the different cell types and for each of those three cell lines, TSA superinduced PMA-induced Timp-1 expression and repressed TGFb1-induced Timp-1 expression This shows that neither c-fos nor junD are essential for the observed affects of TSA on Timp-1 expression However, although the expression of Timp-1 in c-Jun– ⁄ – cells was induced by either PMA or TGF-b1, TSA was unable to superinduce the PMA-induced Timp-1 expression whilst retaining the ability to repress the TGF-b1 induced Timp-1 expression In fact, in c-Jun– ⁄ – cells, much like for the TGF-b1 response, TSA repressed PMAinduced Timp-1, indicating that in the absence of c-Jun (or a c-Jun-regulated factor), the default pathway for the effect of TSA on induced Timp-1 expression is repressive Many AP-1 members are differentially regulated in response to PMA or TGF-b1 with or without TSA As even in the absence of c-fos, c-Jun or junD mouse fibroblast cells are able to induce Timp-1 in response to PMA or TGF-b1, and yet the AP-1 motif in the Timp-1 promoter is important for such a response, the expression profile by qRT-PCR of all the Fos and Jun family members was determined in C3H10T1 ⁄ cells stimulated for h (Fig 5B) PMA significantly induced fosB, fra-1, fra-2, junB and c-Jun; of these only junB was further induced by TSA, while PMAinduced fra-2 was repressed by TSA TGF-b1 induced levels of fosB, fra-2 and junB, all of which were then repressed by TSA ATF2 and junD in general showed little regulation by TSA, PMA or TGF-b1 It is therefore possible that in AP-1 deficient cells, the lack of a specific factor may be compensated for by the presence 1917 HDAC inhibitors and Timp-1 expression D A Young et al Fig TSA superinduction of PMA-induced Timp-1 requires c-Jun but other AP-1 members may compensate for the loss of c-fos (A) Swiss-3T3, c-fos– ⁄ –, c-Jun– ⁄ – and junD– ⁄ – mouse fibroblast cells were serum starved for 24 h, then stimulated with 10)7 M PMA or ngỈmL)1 TGF-b1 in the presence or absence of 500 ngỈmL)1 TSA Total RNA was isolated after h and subjected to real-time qRT-PCR for Timp-1 Data were normalized to the 18S rRNA housekeeping gene Data is representative of two independent experiments with each experiment performed in triplicate and data is plotted as mean + SEM (B) C3H10T1 ⁄ murine fibroblasts were serum starved for 24 h, then stimulated with 10)7 M PMA or ngỈmL)1 TGF-b1 in the presence or absence of 250 ngỈmL)1 trichostatin A (TSA) for h before the isolation of total RNA qRT-PCR for AP-1 members fosB, fra-1, fra-2, ATF2, junB, junD and c-Jun is shown, plotted as fold-control levels for direct comparison C, control; P, PMA (10)7 M) and T, TGF-b1 (4 ngỈmL)1) Data is representative of three independent experiments of an additional, functionally overlapping, family member However, the function of c-Jun, in mediating the superinduction of PMA-induced Timp-1 expression, appears unique The effects of HDAC inhibitors on Timp-1 gene expression can be reiterated in transient transfection of Timp-1 promoter-containing plasmids Figure 6A shows that the effect of TSA on both PMA- and TGF-b1-induced Timp-1 expression can be reiterated in a )95 ⁄ +47 Timp-1 promoter-reporter construct (in the pGL2 vector), transiently transfected into C3H10T1 ⁄ cells Deletion mutation to )50 ⁄ +47, removing the promoter-proximal AP-1 site, shown 1918 previously to be important in induction of the Timp-1 gene, shows that TGF-b1-induction is lost (and hence, TSA can no longer repress this); however, some PMA induction of the deleted construct remains, and this is superinduced by TSA (Fig 6B) These data were confirmed using point mutation of the AP-1 site in a )223 ⁄ +47 Timp-1 promoter construct (in the pGL3 vector); here, some PMA- and TGF-b1-induction of the mutant construct remains TSA superinduces PMA-induced expression of the wild-type and mutant AP-1 constructs, but TSA no longer represses the residual TGF-b1-induction (Fig 6C,D) This suggests that the effect of TSA on TGF-b1-induced Timp-1 expression is mediated through the promoter proximal AP-1 site, whilst the effect of TSA on PMA-induced FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS D A Young et al Timp-1 expression is independent of the Timp-1 AP-1 motif It should be noted that TSA alone induces expression from promoter constructs made in pGL3, and similarly induces the empty pGL3-basic vector (data not shown) The more robust expression levels from this pGL3 compared to pGL2 (which is not induced by TSA) make the data more reliable despite this difference, and the effects of TSA upon PMAand TGF-b1-stimulated promoter-reporter expression remain clear TSA superinduction of PMA-induced Timp-1 is lost by mutation of a GC-box or Ets binding motif within the Timp-1 promoter In order to establish if promoter elements downstream of the AP-1 site can mediate the TSA superinduction of PMA-induced Timp-1, a series of insertion mutants were prepared in )223 ⁄ +47 In this set of constructs, the AP-1 site at )59 ⁄ )53 remains intact, but downstream of this, blocks of five bases are replaced with adenosine; these mutants overlap by two bases, giving HDAC inhibitors and Timp-1 expression a set of 20 mutant constructs Figure 7A shows that only mutants m4, m16 and m20 lose the superinduction of PMA-induced expression by TSA Mutant m4 alters a canonical Ets binding site, shown previously to be important for basal expression of the Timp-1 gene; mutant 16 alters a canonical Sp1 binding site (GCbox); mutant 20 does not alter any known consensus for transcription factor binding EMSA was used to determine the protein factors binding to the ‘wild-type’ m4, m16 and m20 or the mutated sequences Specific factors binding to both the ‘wild-type’ m4 and m16 sequences could be seen (Fig 7B,C), however, the binding of these factors did not change upon TSA stimulation (data not shown) The m4 sequence (Fig 7B) bound several complexes, although competition analysis with the ‘wild-type’ and mutant m4 revealed only one complex to be specific The identity of this, presumably Ets family member, remains to be determined The pattern of three bands (a, b and c) bound to the ‘wild-type’ m16 probe in Fig 7C is identical to that described in the literature as deriving from the binding of both Sp1 and Sp3 trans- Fig The impact of TSA on PMA- vs TGF-b1-induced Timp-1 gene expression is reiterated on Timp-1 promoter-reporter constructs and the effect of TSA on PMA-induced Timp-1 is independent of the promoter proximal AP-1 site C3H10T1 ⁄ murine fibroblasts were transiently transfected with (A) )95 ⁄ +47, (B) )50 ⁄ +47 Timp-1 promoter constructs in pGL2, (C) )223 ⁄ +47 or (D) )223 ⁄ +47 DAP-1 Timp-1 promoter constructs in pGL3 Following serum starvation for 24 h, cells were stimulated with 10)7 M PMA or ngỈmL)1 TGF-b1 for h in the presence or absence of 250 ngỈmL)1 trichostatin A (TSA) prior to harvest for luciferase assay Experiments were performed three times in triplicate Results are plotted as mean + SEM FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS 1919 HDAC inhibitors and Timp-1 expression D A Young et al A B C cription factors [26] The ‘wild-type’ dsDNA (50-fold) competed for all three Sp bands while the mutant m16 at the same concentration did not An anti-Sp1 Ig resulted in the loss of band a, and partially blocked b with the subsequent appearance of a supershifted complex; anti-Sp3 Ig blocks formation of bands b and c This suggests that band a contains Sp1, band b contains both Sp1 and Sp3, and band c contains Sp3 Discussion HDACs usually act as transcriptional repressors, therefore HDAC inhibitors should induce expression of susceptible genes, and this is the typical experimental finding However, in yeast, deletion of the HDAC 1920 Fig The impact of TSA on PMA-induced Timp-1 gene expression is mediated via three sites in the proximal promoter (A) C3H10T1 ⁄ murine fibroblasts were transiently transfected with a )223 ⁄ +47 Timp-1 promoter construct in pGL3 and 20mutant constructs as shown Following serum starvation for 24 h, cells were stimulated with 10)7 M PMA in the presence (solid bars) or absence (open bars) of 500 ngỈmL)1 trichostatin A (TSA) prior to harvest for luciferase assay Data is representative of three independent experiments, each performed in triplicate and results are plotted as mean + SEM On the Timp-1 sequence, AP-1, Ets and Sp1 binding motifs are shown in bold, position of each of the 20 (m1 to m20) d(A)5 mutation are shown underlined (B) PMA (10)7 M) + TSA (500 ngỈmL)1) stimulated nuclear were incubated with a ‘wild-type’ m4 DNA probe (Table 2) and subjected to EMSA A 50-fold excess of self and mutant m4 DNA was used to define binding specificity (C) Nuclear extracts (as in B) were incubated with a ‘wild-type’ m16 DNA probe (Table 2) and subjected to EMSA A 50-fold excess of self and mutant m16 DNA confirmed binding specificity Sp factor binding was confirmed by incubation of extracts with either lg of an anti-Sp1 Ig or anti-Sp3 Ig prior to electrophoresis Rpd3 down-regulates a subset of genes, and many of these are also repressed by treatment with TSA [27] There are also many individual instances of HDAC inhibitors acting as repressors of gene expression, e.g TSA and NaB cause a reduction in mRNA levels for the cdk1 gene [28]; TSA represses b-casein expression in mammary epithelial cells [29]; TSA represses cyclin B1 and A [30]; TSA inhibits MMTV transcription [31] One could postulate that these effects are indirect, with TSA leading to the induction of a factor (or factors) involved in the repression of a downstream target; alternatively, a direct effect on either the acetylation of a transcription factor, or recruitment of repressive factors to acetyl–histones via bromodomain interactions could be envisaged In support of the latter notion the downFEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS D A Young et al regulation of cyclin A and B1 upon TSA treatment is via diminished activity of NF-Y-associated HAT and is mediated through CCAAT motifs; further, the diminished HAT activity is mediated by phosphorylation of hGCN5 [30] Moreover, the inhibition of MMTV transcription by TSA does not depend on changes in chromatin remodelling or increased histone acetylation, but is mediated via the TATA-box region [31] Both TGF-b1- and PMA-induction of Timp-1 require new protein synthesis As both events have at least some dependency on a promoter proximal AP-1 site, this could reflect in part the synthesis of Fos and Jun family members Immediate early gene induction correlates with a nucleosomal response whereby geneassociated nucleosomes are subject to both phosphorylation and acetylation on histone H3 and acetylation on histone H4 [16] Whilst it is reported that TSA does not activate c-fos or c-Jun expression in C3H10T1 ⁄ cells [32], TSA can clearly modulate these genes in the additional presence of TGF-b1 or PMA (Figs 4A and 5B) Moreover, TSA alone did induce c-fos expression in our experiments after 12 h of stimulation As the kinetics of c-fos induction so clearly preceded that of Timp-1, we analysed the expression of Timp-1 in c-fos deficient mouse fibroblasts (Fig 5A) Surprisingly, mouse cells lacking c-fos, c-Jun or junD showed no alteration in Timp-1 induction by PMA or TGF-b1 This could be due to compensation for the lack of the factor by another AP-1 family member and our data demonstrate that expression of many AP-1 members are up-regulated in response to PMA or TGF-b1 However, Timp-1 expression in PMA-stimulated c-Jun– ⁄ – cells was not superinduced in response to TSA, and was in fact repressed, resembling the situation with TGF-b1 induction c-Jun is known to be acetylated at Lys271 by the transcriptional coactivator p300 upon its interaction with the adenoviral protein E1A [21] Overexpression of c-Jun in C3H10T1 ⁄ cells induced Timp-1 reporter expression by twofold, but mutation of Lys271 fi Arg had no effect on this induction, even in the presence of PMA and ⁄ or TSA (data not shown) Phosphorylation of c-Jun by the mitogen-activated protein kinase (MAPK) JNK on Ser63 and Ser73, as well as on Thr91 or Thr93, or both, increases its transactivating potential and DNA-binding activity by mediating its dissociation from an inhibitory complex containing HDAC3, a class HDAC [33–35] HDAC3 associates with class HDACs and c-Jun in repressor complexes such as those containing the corepressors N-CoR and SMRT [34,36] Phosphorylation by JNK causes a reduction in c-Jun ubiquitination and FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS HDAC inhibitors and Timp-1 expression subsequent protein stabilization [37,38] The proteosome inhibitor lactacystin inhibited the PMA induction of Timp-1 and no induction was seen in the additional presence of TSA (data not shown) We propose that lactacystin prevents the degradation of ubiquitinated c-Jun thus leading to its accumulation and preventing its activation by JNK and subsequent downstream activation events that would lead to Timp-1 up-regulation A possible explanation for the lack of a TSA superinduction of PMA-induced Timp-1 in c-Jun– ⁄ – cells is that the expression of one or more HDACs is c-Jundependent To test this, we monitored the expression of HDACs 1–11 in response to TGF-b1 or PMA, with or without TSA, between C3H10T1 ⁄ 2, Swiss-3T3 and c-Jun– ⁄ – cells by RT-PCR The expression of class HDACs is reported to be ubiquitous while class HDAC appear more tissue-specific [39] All three cell lines expressed the majority of HDACs, with only expression of HDAC9 and )10 being undetectable and HDAC8 was up-regulated in c-Jun– ⁄ – cells compared to C3H10T1 ⁄ or Swiss-3T3 (data not shown) Only HDAC7 and HDAC11 were regulated differentially by any of the stimuli, and this was identical between all three cell lines HDAC7 was, in all cell lines, repressed by the presence of TSA while HDAC11 expression was interestingly induced only by the combination of TGFb1 and TSA (data not shown) Hence, none of the HDACs exhibit c-Jun dependent expression Although the induction by PMA alone was partially abrogated upon mutation or deletion of the )59 ⁄ )53 AP-1 motif of Timp-1, TSA was still able to superinduce reporter expression in the presence of PMA (Fig 7) This suggests that the impact of c-Jun on the superinduction of Timp-1 is not directly on the Timp-1 gene, but via a c-Jun-dependent intermediate It should be noted that whilse transiently transfected plasmid DNA is not integrated into the host cell chromosomes, there is evidence that it can be assembled into a chromatin-like structure [40,41] If this is true in the current system, then data from transient transfection experiments could still be interpreted at the level of histone or factor acetylation Using a series of 20 overlapping mutant promoter constructs we demonstrated three mutants, m4, m16 and m20, were no longer able to superinduce reporter expression above PMA alone in the additional presence of TSA (though variation in absolute levels of induction is seen across the mutant constructs) Further, specific binding of Sp1 and Sp3 to wild-type m16 and a putative Ets factor to wild-type m4 were identified (Fig 7C,D) It is surprising that other mutants that overlap the consensus sequences for these transcription factors not impact upon the effect of TSA The 1921 HDAC inhibitors and Timp-1 expression basal and cobalt-induced expression of Timp-1 is known to be partially dependent upon Sp1, although this is via sequences upstream of the )59 ⁄ )53 AP-1 motif or within intron [10,42] Sp3 transcription factor is reported to either activate or repress gene expression in target genes [43] It has been shown that Sp3 can be acetylated potentially via p300 [20] For the TGFbRII gene at least, this acetylation acts as a switch to turn Sp3 from a transcriptional repressor into an activator [44] We were unable to detect whether Sp3 or Sp1 in our C3H10T1 ⁄ cell system were acetylated using the anti-(acetyl–lysine) Ig (data not shown) Unlike many genes, Timp-1 induction by TGF-b1 is largely independent of the Smad signalling pathway in C3H10T1 ⁄ cells and is instead AP-1-dependent [11] It has been shown previously that the AP-1 site at )59 ⁄ )53 can act in concert with the neighbouring Ets site at )45 ⁄ )41 (mutant m4) [45] TGF-b1 causes the acetylation of Ets1 and this is proposed to contribute to the ability of Ets1 overexpression to abrogate TGFb1 induction of the Timp-1 gene [46] HDACi would probably increase the level of Ets1 acetylation by TGF-b1, though the functional outcome of this is unclear The consequences of any potential PMAmediated acetylation of Ets1 are also unknown, but of future interest, as mutation of the Ets binding motif (m4) results in both a loss of protein binding and the TSA superinduction of Timp-1 Eleven NAD-independent HDACs have been described in human and mouse, although few have been characterized in detail These have been subdivided recently into three groups based upon phylogenetic analysis [47] Class HDACs are structurally related to yeast scRPD3, and contain HDAC1, )2, )3 and )8, while class HDACs, containing HDAC4, )5, )6, )7, )9 and )10 are similar to yeast scHDA1 HDAC11 alone represents class HDACs and HDAC11 related proteins have been described in all eukaryotic organisms other than fungi As described, HDACs often act in complexes with other proteins and cofactors and different HDACs are often present in the same complexes One common feature of class HDACs is that they appear to be able to homo- or heterodimerize, leading to speculation that duplication of the catalytic domain as seen in HDAC6 may have occurred as a way of ensuring self association [47] There are no inhibitors specific to a single HDAC available currently and most HDACs are believed to be equally sensitive to TSA and NaB, although HDAC6 is a possible exception [39] VPA has been shown to have some selectivity against different HDAC classes Only at concentrations > mm is VPA reported to inhibit class subclass HDACs (at 1922 D A Young et al least HDAC4, )5 and )7) [24,48] VPA was unable to inhibit the class subclass HDACs (6 and 10) even at concentrations up to 20 mm [48] From the NaB and TSA dose-curves it is clear that HDACi effects on PMA and TGF-b1-induced Timp-1 are via the inhibition of different HDACs Therefore TSA or NaB not inhibit all HDACs equally As a concentration of > mm VPA is required to stimulate PMA-induced Timp-1 expression further, it is probable that VPA is inhibiting a class subclass I enzyme in this case Even at the highest concentration used (8 mm) VPA did not affect TGF-b1 induced Timp-1 expression, although it did block TGF-b1 induced ADAM12 expression in a dose-dependent manner (Fig and inset) This would imply that a class subclass enzyme (i.e HDAC6 or 10) is involved in the induction of Timp-1 by TGF-b1 A caveat to this is the HDAC inhibition profiles of all these HDACi are incomplete and are generally based upon semipurified protein fractions and in vitro assays Finally, it is interesting to note that the HDACi not have an obvious effect on basal expression of Timp-1, only on induced gene expression In conclusion, this is to our knowledge, the only described instance of HDAC inhibitors having opposite effects on the same gene, depending upon the initial stimulus used to induce expression We have shown that different HDACs are involved in the response of the Timp-1 gene to PMA compared to TGF-b1 Moreover, c-Jun mediates the effect of HDAC inhibitors on PMA-induced Timp-1, though not via the promoter proximal AP-1 site We have also identified cis-acting promoter elements essential for the effect of HDAC inhibitors on PMA-induced Timp-1 expression Experimental procedures Cell culture Murine C3H10T1 ⁄ fibroblasts and Swiss-3T3 cells were routinely cultured in Minimal Essential Medium (MEM) with Earle’s salts and l-glutamine (2 mm) (Invitrogen, Paisley, UK) containing 10% foetal bovine serum (FBS, Invitrogen), 1% nonessential amino acids, 100 ImL)1 penicillin, 100 lgỈmL)1 streptomycin and 20 unitsỈmL)1 nystatin AP-1 knockout cells (c-Jun– ⁄ –, c-fos– ⁄ – a kind gift from E Wagner (Research Institute of Molecular Pathology, University of Vienna, Austria) and P Angel (Department of Signal Transduction and Growth Control, Deutsches Krebsforschungszentrum (DKFZ), University of Heidelberg, Germany) and junD– ⁄ –, a kind gift from M Yaniv and J Weitzman (Pasteur Institute, Paris, France) were cultured as above, but in Dulbecco’s MEM Serum-free conditions used identical medium without FBS For assays, cells were FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS D A Young et al HDAC inhibitors and Timp-1 expression grown to confluence, then serum-starved for 24 h prior to the addition of TGF-b1 (R & D Systems, Abingdon, Oxon, UK; or ngỈmL)1) or phorbol 12-myristate-13-acetate (PMA, Sigma, Poole, UK; 10)7 m) in the absence or presence of HDAC inhibitors (trichostatin A, TSA; sodium butyrate, NaB; valproic acid, VPA; Calbiochem, Nottingham, UK) at the concentrations described Experiments were repeated 2–4 times to ensure the pattern of response was reproducible and representative data are shown throughout RT-PCR RNA was isolated from monolayer cultures using Trizol reagent (Invitrogen) One microgram of total RNA was reverse transcribed using lg random hexamers (Amersham Biosciences, Chalfont St Giles, Bucks, UK) and 200 U of Superscript II reverse transcriptase (Invitrogen), according to the supplier’s instructions Quantitative RT-PCR (qRTPCR) was performed using the Applied Biosystems, Warrington, UK) ABI Prism 7700 sequence detection system (TaqManÒ) as described [49] Table contains sequences of TaqMan primers and probes for AP-1 family members with TSA (500 ngỈmL)1) and TSA (500 ngỈmL)1) was included in the all solutions for nuclear extract preparation Electrophoretic mobility-shift assays (EMSA) were performed essentially as previously described [10,11] Oligonucleotides were synthesized by MWG-Biotech (London, UK) or Sigma-Genosys (Haverhill, UK) (Table 2) Doublestranded probes were labelled with [32P]ATP[cP] using T4 polynucleotide kinase Nuclear extracts (2 lg), 0.5 lg of poly(dIdC.dIdC), and radiolabelled probe (20 000 c.p.m.) were incubated in 1· binding buffer (10 mm Tris ⁄ HCl, pH 7.5, 50 mm NaCl, 0.5 mm dithiothreitol, mm MgCl2, and 5% glycerol) with or without competitor DNA for 20 at °C in a total volume of 10 lL at °C For antibody supershift analyses, lg of the appropriate antibody [anti-(c-fos) Ig, sc-52-Gx (Santa Cruz Biotechnology, Inc., from Autogen Bioclear, Calne, UK), anti-(acetyl– lysine) Ig, clone 4G12 (Upstate Ltd., Milton Keynes, UK)] was incubated with nuclear extract for 20 at °C prior to the addition of the DNA probe Samples were separated on a 5% polyacrylamide gel in 0.5· TBE (45 mm Tris ⁄ HCl, 45 mm boric acid, mm EDTA) Gels were prerun at 10mA for h at °C and run at mA for 3–6 h at °C Gels were dried and autoradiographed Nuclear extracts and electrophoretic mobilityshift assays Plasmid construction and transient transfection Nuclear extracts were prepared essentially as described previously [11] except where indicated cells were stimulated Constructs using Timp-1 promoter driving luciferase expression were in pGL2-basic or pGL3-basic (Promega, Table Mouse AP-1 gene qRT-PCR primer and probe sets Gene c-fos c-Jun junB junD ATF2 fosB fra-1 fra-2 Sequence Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe FEBS Journal 272 (2005) 1912–1926 ª 2005 FEBS Primer Primer Primer Primer Primer Primer Primer Primer Primer Primer Primer Primer Primer Primer Primer Primer 5¢-CCTGCCCCTTCTCAACGA-3¢ 5¢-CTCCACGTTGCTGATGCTCTT-3¢ 5¢-CCCAAGCCATCCTTGGAGCCAGT-3¢ 5¢-GAAGTGACGGACCGTTCTATGAC-3¢ 5¢-GGAGGAACGAGGCGTTGAG-3¢ 5¢-AAGATGGAAACGACCTTCTACGACGATGC-3¢ 5¢-GGAGCAGGAGGGCTTTGC-3¢ 5¢-GGCGTCACGTGGTTCATCT-3¢ 5¢-ACGGTTTTGTCAAAGCCCTGGACGAC-3¢ 5¢-CGCAAGCTGGAGCGTATCTC-3¢ 5¢-GACGCCAGCTCGGTGTTCT-3¢ 5¢-CGCCTGGAGGAGAAAGTCAAGACCCTC-3¢ 5¢-CAGCCACCTCCACTACAGAAACT-3¢ 5¢-TTCTTCGACGGCCACTTGTAT-3¢ 5¢-TCTCCAGCTCACACAACTCCTCAGACCC-3¢ 5¢-GCTCCCCTATCCTCGATATTTGA-3¢ 5¢-CAGAACTCGTCTTTGGGACTGA-3¢ 5¢-TTCCCACTATCCCACTCCATCCAATTCC-3¢ 5¢-TGAACCGGAAGCACTGCATA-3¢ 5¢-GTGAAAACCAGACTCGGAGTAAAAG-3¢ 5¢-CACGCTCATGACCACACCCTCTCTGAC-3¢ 5¢-CATCACTCCCGGCACTTCA-3¢ 5¢-CGACGAAGGCGACTCCTG-3¢ 5¢-TTGTCTTCACCTACCCCAATGTCCTGGA-3¢ 1923 HDAC inhibitors and Timp-1 expression Table EMSA oligonucleotides In sequences, upper strands only depicted Mutated sequences are shown in lowercase Name Sequence Timp-1 AP-1 Timp-1 DAP-1 ‘wild-type’ m4 m4 ‘wild-type’ m16 m16 5¢-CGGTGGGTGGATGAGTAATGCGTCCAGG-3¢ 5¢-CGGTGGGTGGAgGAGTgATGCGTCCAGG-3¢ 5¢-AATGCGTCCAGGAAGCCTGGAGGCAGTGAT-3¢ 5¢-AATGCGTCCAGGAAaaaTGGAGGCAGTGAT-3¢ 5¢-GCCAACTCCGCCCTTCGCATGGACATTTAT-3¢ 5¢-GCCAACTCCGCCaaaaaCATGGACATTTAT-3¢ Southampton, UK); point mutations altered the wild-type AP-1 site (5¢-TGAGTAA-3¢) to a nonfunctional mutant AP-1 site (5¢-GgAGTgA-3¢) as described previously [11] Twenty independent overlapping mutants were all generated in pGL2-223 ⁄ +47 [11], the Timp-1 promoter regions were HindIII isolated and subcloned into pGL3-basic (Fig 7A) All mutagenesis was performed using the QuikChange method (Stratagene, Amsterdam, the Netherlands) All mutations were verified by DNA sequencing Cells were seeded in six-well or 24-well plates at a density of 8850 cellsỈcm)2 and grown overnight in medium containing 10% (v ⁄ v) FBS at 37 °C in a 5% (v ⁄ v) CO2 atmosphere Cells were transfected overnight in serumcontaining medium with lg per well (six-well plates) or 0.21 lg per well (24-well plates) reporter plasmid using FuGene6 (Roche, Lewes, UK) according to the manufacturers’ instructions The following day, cells were washed in Hank’s Balanced Salts Solution (HBSS) and 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basal expression of Timp-1, only on induced gene expression In conclusion, this is to our knowledge, the only described instance of HDAC inhibitors having opposite effects on the same gene,