Histone deacetylases (HDACs) are important chromatin remodeling enzymes that are generally involved in transcriptional repression 1. Mammalian HDACs are classified into three main categories depending on their primary homology to Saccharomyces cerevisiae HDACs (RPD3, HDA1 and SIR2). Histone deacetylase inhibitors (HDACIs) tend to show equal effects on gene activation and repression 234. HDACIs have been shown to induce differentiation, apoptosis or growth arrest in a variety of transformed cell lines 5. This is generally attributed to the ability of these inhibitors to induce an open chromatin conformation facilitating transcription of regulatory genes like p21 which inhibit tumor cell growth 6. These qualities make HDACIs promising targets for chemotherapeutic intervention.

Recently many different types of HDAC inhibitors have been discovered (Figure 1). These include short chain fatty acids (sodium butyrate, phenylbutyrate, valproic acid) 7, hydroxamic acids (trichostatin A (TSA), suberoylanilide hydromaxic acid (SAHA), pyroxamide, cyclic hydroxamic acid-containing peptides (CHAPs), cinnamic acid bishydroxamic acid (CBHA) and scriptaid) 89, cyclic tetrapeptides (trapoxin, apicidin, depsipeptide) 1011121313, and benzamides (MS-275)1415. Most HDAC inhibitors (HDACIs) developed to date inhibit both Class I and II HDACs equally with the exceptions being valproic acid (5 fold more selective for HDAC1 vs HDACs 5 and 6) and FK-228 (Class I selective). Class I and II HDACs are inhibited by trichostatin A (TSA) and related compounds whereas Class III HDACs are not. As noted, HDACIs have been shown to promote cell cycle arrest, differentiation, and apoptosis in many transformed cultured cell types. In animal models, HDACIs have been shown to inhibit growth of breast, prostate, lung and stomach cancers, as well as neuroblastomas and leukemias, with little toxicity 1617. In a previous study looking at the combination regimen of all trans retinoic acid (RA) with the HDACI, Trichostatin A (TSA), we identified several new targets for HDACIs 18. We also identified critical differences in gene regulation subsequent to treatment with these two agents and a novel promoter module associated with the regulation of a subset of these differentially regulated genes. These analyses focused on the anticancer therapeutic potential of these compounds alone or in combination. Recent analysis of these data identified certain crucial metabolic pathways that have not previously been shown to respond to HDACI treatment and which may be critical in identifying new therapies for cardiovascular health. In this report we discuss the possible role of HDAC inhibition on cholesterol metabolism.

In a previous study we had used microarray analyses to examine the effects of RA and TSA on embryonal carcinoma cell growth and differentiation using the prototypical EC cell line F9 18. Results from these studies identified several important genes and pathways differentially regulated by these compounds. In this report we identify new target pathways for TSA treatment based on further analysis of this data. Most importantly, the regulatory pathways that are affected include pyrimidine metabolism and cholesterol biosynthesis. The pyrimidine pathway is of interest because one of the rate limiting enzymes in this pathway, dihydroorotate dehydrogenase (dhodh), has been targeted for inhibition in murine models of rheumatoid arthritis as well as in the human T-lymphoblastoma cell line (A3.01) 2122. Dhodh catalyzes the fourth committed step in the de novo biosynthesis of pyrimidines. Activated lymphocytes expand their pyrimidine pool by eightfold during proliferation 23. In rheumatoid arthritis, inflammation and degradation of synovial tissue are initiated by the influx of lymphocytes (B cells, CD4+, CD8+ and T cells) 24. Thus, inhibiting activated T-cells by decreasing their supply of pyrimidines via TSA treatment could provide an attractive alternative method for treating rheumatoid arthritis. Interestingly, HDACIs (TSA and phenylbutyrate) were used as treatments in a rat model of rheumatoid arthritis, and resulted in reduced inflammation, and inhibition both of synovial hyperplasia and bone or cartilage destruction 25. The authors also found that HDACIs inhibited the expression of tumor necrosis factor-α, which functions to stimulate matrix degradation in rheumatoid arthritis 26, therefore suggesting a mechanism by which HDACIs may alleviate some effects of rheumatoid arthritis. Further extending and supporting these results, in this study, we found that TSA itself could significantly inhibit the expression of dhodh even in non-lymphatic cells (60% in F9 and 25% in HepG2), providing an alternative (or synergistic) mechanism by which HDACI might suppress rheumatoid arthritis in both mice and men. Moreover, the mRNA levels of thymidylate synthetase (Tyms), another key enzyme in this pathway, were decreased 8 fold in HepG2 cells (2.4 fold in F9 cells) further potentiating this effect of TSA treatment. A previous study using chondrocytes showed that HDACIs such as TSA and sodium butyrate, blocked the induction of matrix metalloproteinases (MMP-1, MMP-13) as well as aggrecan-degrading enzymes (Adamts4, Adamts5 and Adamts9) 27. Both of these enzyme families mediate cartilage destruction. In our study with HepG2 cells, we also found that TSA treatment resulted in a modest decrease in the expression of MMPs (MMP-1, MMP-2, MMP-11, MMP-12) (1.2–1.5 fold) and Adamts9 (1.6 fold). This, coupled with increased expression (2.8 fold) of a collagenase inhibitor (tissue inhibitor of MMP: TIMP1, TIMP2), might further promote maintenance of a growth regulating matrix. Interestingly, TSA treatment also resulted in down regulation of LPS induced TNFα levels (2 fold) as well as suppression of the cytokines IL-12 and IL-8 (2.7 and 2 fold respectively). This result is consistent with a report by Leoni et al. 28 on the anti-inflammatory properties of SAHA (another hydroxamic acid based HDACI) in Balb/c mice and human PBMCs induced with LPS.

The pathway most significantly affected by TSA treatment in F9 EC cells is that of cholesterol biosynthesis, most specifically those steps involved in the synthesis of low density lipoprotein. There are two main types of lipoproteins that transport cholesterol in the blood: low density lipoproteins (LDL) and high density lipoproteins (HDL). HDL particles are generally considered to be "good cholesterol", while LDL is considered "bad cholesterol" 29. Several genes encoding essential enzymes in the LDL synthesis pathway are down regulated by these treatments. Pathway analysis of microarray data using genes showing statistically significant (p < 0.05) differential gene expression indicated that expression levels of 9 enzymes out of the 15 in the cholesterol biosynthesis pathway are decreased following TSA treatment. They include HMG CoA reductase (Hmgcr), mevalonate kinase (Mvk), di-p-mevalonate decarboxylase (Mvd), isopentenyl-PP isomerase (Idi1), squalene synthatase (Fdft1), squalene epoxidase (Sqle), lanosterol synthase (Lss) and lanosterol oxidase (Sc4mol) and NAD(P)-dependent steroid dehydrogenase (Nsdhl) (Figure 2). Of these only 6 of these 9, including Hmgcr, Mvd, Idi1, Sqle, Lss, Sc4mol and Nsdhl, showed a greater than two fold differential expression on TSA treatment. The z-score assigned to each category by MAPPFinder reflects the degree to which the expression of genes in that category was greater than that expected by chance. A positive z-score indicates that a large number of genes in that category are differentially expressed between the compared conditions while a negative Z score indicates that the there are fewer genes meeting the criterion than would be expected by random chance. If the MAPPFinder data truly obeyed the assumptions of the hypergeometric distribution, then a Z score or 1.96 or -1.96 would correlate with a p value of 0.05. The z-score for this pathway was highly significant with values of 4.85 associated with TSA treatment. Real-time qPCR analyses verified decreased expression of 3 of 5 genes (Hmgcr, Mvd, Lss) in this pathway but the expression of neither Mvd nor Lss was significantly down regulated by TSA. Decreasing high cholesterol levels using TSA treatments may work well since repression of few of the detected genes may be sufficient to induce the response, i.e., a reduction in cholesterol intermediates and synthesis.

Following the analysis of these pluripotent EC cells, we decided to investigate these effects in the HepG2 cell line which arose from a carcinoma of the human liver, the primary organ for cholesterol and fatty acid metabolic processes. While we realize that primary hepatocytes would be a better model to evaluate this pathway, we choose the HepG2 cell line as an means to evaluate this phenomenon but allowing for use of the known anti-cancer effects of TSA as a control. Expression data from HepG2 cells also indicated that multiple enzymes in cholesterol biosynthesis and fatty acid synthesis pathways were significantly down regulated (Figure 3). The mRNA transcript levels that were repressed at a greater than 2 fold level of significance included HMG CoA synthase (Hmgcs1), HMG CoA reductase (Hmgcr), sterol receptor binding factor-2 (Srebf2) and lanosterol 14 α-demethylase (Cyp51a1) (involved in cholesterol metabolism), and others including fatty acid synthase (Fasn), fatty acid binding protein (Fabp), farnesyl diphosphate synthase (Fdps), acetyl-coA carboxylase (Acaca), acetyl-coA dehydrogenase (Acadm), acetyl-coA acetyl transferase (Acat2), peroxisome proliferative activated receptor, gamma (Pparγ) and a variety of apolipoproteins that are involved in fatty acid and triglyceride metabolism. Quantitative PCR studies verified that TSA treatment reduced expression of Hmgcr, Hmgcs1, Srebf2, Fabp Fasn, Fdps, Acaca, Acadm, Acat2, ApoA5, C1, E and L1 as well as Cyp27a1, Ldlr, Pparγ and Tyms (Figure 4). The down regulation seems to be a complex phenomenon involving genes that regulate these pathways at different levels. Most evident is the down regulation of Srebf2 which in turn acts as a transcription factor regulating the expression of enzymes like Hmgcr (the target for the statin class of drugs) and Mvd. It is known that Srebf2 overexpression induces all 12 enzymes in the cholesterol biosynthesis pathway and inhibition of Srebf2 by TSA might inhibit the expression of these enzymes 30. In fact, our microarray data demonstrates that the levels of almost all these enzymes are down (10 genes pass all cutoff filters) following TSA treatment. The repression of Srebf2 occurs at a early time point (around 3 hrs) and continually repressed over 24 hrs (2.4 fold repression at 24 h). This effect is probably responsible for the down regulation of the cholesterol pathway since expression of Hmgcs (repressed at 6 h), Hmgcr (repressed at 12 h) and Ldlr (repressed at 24 h) are all known to be induced by Srebf2 31. Srebf-1a and -1c are more involved with regulation of fatty acid synthesis and lipogenesis 3032. While we were unable to detect the levels of Srebf1 expression in our microarray experiments, TSA treatment modestly (1.3 fold) decreased expression levels of cytosolic NADP-dependent isocitrate dehydrogenase1 (Idh1) which provides the cytosolic NADPH required for proper functioning of both the cholesterol and fatty acid biosynthetic pathways. The Idh1 promoter is activated by Srebf1a and Srebf2 in human hepatoma cells 33. Thus there appears to be a concerted down regulation of both pathways through a synergistic effect. Cyp51a1 (lanosterol 14α-demethylase) is another important intermediate in cholesterol metabolism and has in recent years gained importance as a target for the development of hypocholesterolemic agents 34. Our microarray data showed that levels of Cyp51a1 were also down regulated further adding support to the possible use of this HDACI as a way to decrease plasma cholesterol levels.

Finally, atherosclerosis is the underlying disorder associated with most cardiovascular disease 35. This disorder is characterized by deposits of fatty substances, cholesterol, cellular waste products, calcium and other substances in the inner lining of an artery (collectively known as plaques) 36. Cholesterol has been implicated as the major contributor to this condition as atherosclerosis is strongly correlated with an increase in serum cholesterol levels 3738. Generally, serum levels should be between 140 and 200 mg per deciliter (mg/dl) whereas high levels surpassing 240 mg/dl indicate one is at high risk for cardiovascular disease 39. Thus, atherosclerosis is characterized by elevated levels of LDL 40. The activity of the hepatic LDL receptor (Ldlr) is the primary determinant of plasma LDL cholesterol levels and Ldlr transcription is in turn regulated by Srebf2. When the levels of hepatocellular sterols drop, Srebf2 is activated and this process restores the normal levels by concurrent activation of de novo cholesterol synthesis and increased uptake of plasma cholesterol through Ldlr. LDL receptor is also post transcriptionally regulated by proprotein convertase subtilisn/kexin type 9a (Pcsk9) in an inverse manner 41. While our microarray and qPCR data shows decreased Ldlr expression following TSA treatment, microarray gene expression levels of Pcsk9 are also down regulated. This suggests existence of a mechanism for potential compensatory increase in Ldlr levels or activity post-transcriptionally. Our time course experiments did not show a significant repression of Ldlr levels until 24 h further highlighting the complex nature of Srebf2 regulation.

Our data with TSA treatment also showed a decrease in the levels of gene expression for a variety of apolipoproteins including apoA1, apoA5, apoB, apoC1, apoE, apoL1. This observation highlights the complex relationship of apolipoprotein levels and lipoprotein metabolism. While elevated levels of apoB and reduced levels of apoA1 are associated with increased cardiac disease, serum levels of apoB100 associated VLDL are regulated in turn by Acat2 which stimulates cholesteryl ester secretion into apoB-containing lipoproteins. Acat inhibitors are being developed as a therapeutic means to lower LDL cholesterol without affecting cholesterol uptake 4243. Also apoE deficient mice show high levels of cholesterol and develop spontaneous atherosclerosis while mice with partial or complete deficiency of high-mobility group A2 protein (Hmga2) are able to resist diet-induced obesity 44. Acat2 inhibition using antisense nucleotides was previously shown to alleviate atherosclerosis in apoB-Ldlr -/- mice 45. This study also found Acat2 inhibition to be effective in reducing plasma cholesterol, increasing plasma triglycerides, and shifting LDL cholesteryl ester fatty acids to become mainly polyunsaturated. In our study, TSA treatment showed a modest decrease in apoB, apoE and apoA1 in addition to decreased levels of Acat2, Fasn and Hmga2. This indicates that triglyceride metabolism is perturbed by TSA and further studies may be necessary to evaluate the possibility of using TSA and other HDACIs for modulating triglyceride metabolism. Cyp27 has been reported to be regulated by the nuclear receptor subfamily of which PPARγ is a member and levels of both these genes have been found to be high in atherosclerotic lesions. Levels of Cyp27a1 (maximal repression at 24 h) and Pparγ (maximal repression between 9–12 h) were found to be repressed by TSA treatment in both the microarray and qPCR data. This observation adds credence to the potential for development of TSA like HDACIs for atherosclerosis.

Our results show that cholesterol metabolism is significantly down regulated by TSA both directly and indirectly and thus HDACI therapy may be a relatively novel tool to develop for use in controlling cholesterol levels. This study only addresses the effect of TSA treatment on transcript levels of the rate limiting enzymes and transcription factors and further studies evaluating protein expression levels are necessary to derive firm conclusions on regulation of this pathway. Additional studies exploring the different classes of HDACIs with respect to their effects on regulation of the genes in the cholesterol pathway would also help dissect the details of this innovative application for these drugs.

SVC conceived the study design for the HepG2 samples and conducted all the analysis for this paper. NSG performed the microarray study on the F9 samples under the guidance of PJM.