Correspondence to: Anjaneyulu KOWLURU, PhD, Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201, USA. Tel: 313-577-5490 Fax: 313-577-2033 E-mail: email@example.com
The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) has recently been shown to inhibit deleterious effects of cytokines on β-cells, but it is unable to protect β-cells from death due to its own cytotoxicity. Herein, we investigated novel HDAC inhibitors for their cytoprotective effects against IL-1β-induced damage to isolated β-cells. We report that three novel compounds (THS-73–44, THS-72–5 and THS-78–5) significantly inhibited HDAC activity and increased the acetylation of histone H4 in isolated β-cells. Further, these compounds exerted no toxic effects on metabolic cell viability in these cells. However, among the three compounds tested, only THS-78–5 protected against IL-1β-mediated loss in β-cell viability. THS-78–5 was also able to attenuate IL-1β-induced inducible nitric oxide synthase expression and subsequent NO release. Our data also indicate that the cytoprotective properties of THS-78–5 against IL-1β-mediated effects may, in part, be due to inhibition of IL-1β-induced transactivation of nuclear factor κB (NF-κB) in these cells. Together, we provide evidence for a novel HDAC inhibitor with a significant potential to prevent IL-1β-mediated effects on isolated β-cells. Potential implications of these findings in the development of novel therapeutics to prevent deleterious effects of cytokines and the onset of autoimmune diabetes are discussed.
The acetylation status of core histones can greatly influence the rate of transcription of ∼2% of genes. This cycle is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Generally, acetylated histones allow a more open conformation and therefore increase the transcription of the DNA. The HATs acetylate ε-lysine residues on the tails of the core histones, whereas the HDACs facilitate the removal of those acetyl groups. At least three classes of HDACs consisting of 18 known isoforms have been identified thus far . The first two classes consist of zinc-dependent HDACs 1–11, which are inhibited by known HDAC inhibitors such as TSA, MS-275, as well as polyaminohydroxamic acids and polyaminobenzamides . The third family of HDACs is the sirtuins, which are NAD+-dependent and resistant to zinc-binding HDACs. TSA, MS-275 and other zinc co-ordinating HDACs have many important functions, including the regulation of cell proliferation and inflammation [3–6]. Inhibitors of the class I and II HDACs are currently under investigation as therapeutic agents for many diseases, including cancer and rheumatoid arthritis [7–9]. As anti-cancer agents, HDAC inhibitors can induce growth arrest, differentiation and programmed cell death [3,5,6]. Also, HDAC inhibitors have been shown to exhibit anti-inflammatory effects in rheumatoid arthritis by inhibiting key transcription factors including nuclear factor κB (NF-κB) and signal transducer and activator of transcription (STAT; ).
Histone acetylation has also been shown to be important for islet β-cell function; specifically the acetylation of histone H4 has been shown to be critical for insulin gene transcription [11–13]. Emerging evidence appears to indicate that the acetylation status of specific histones plays a regulatory role in the induction of apoptosis in pancreatic β-cells. For example, using INS-1 cells and normal rat islets, Larsen et al. have recently reported significant inhibition of cytokine-induced metabolic dysfunction by HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA). These investigators demonstrated that inhibition of HDACs markedly reduced cytokine-mediated inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release and apoptosis in these cells . They also provided further evidence to suggest that cytoprotective effects of HDAC inhibitors against cytokine-mediated effects may, in part, be due to down-regulation of NF-κB activity. Along these lines, recent observations from our laboratory  have provided immunological evidence to suggest that Class 1 and 2 HDACs are localized in pancreatic islets and isolated β-cells. We also demonstrated a significant inhibition by TSA of IL-1β-mediated iNOS expression and NO release in INS 832/13 cells; such effects of TSA appear to be mediated via increasing the acetylation status of histone H4. Together, data from the above studies support the hypothesis that hypoacetylation of specific histones (e.g. histone H4) favour the induction of iNOS under the duress of cytokines in isolated β-cells. The above-mentioned studies with TSA provided useful mechanistic details with respect to the prevention of cytokine-mediated effects on β-cells; however, they could pose significant problems for future studies since these inhibitors (e.g. TSA) are by themselves cytotoxic to the β-cell. Therefore, there is an immediate need for the development of specific HDAC inhibitors with limited cytotoxicity for potential therapeutic use in prevention of cytokine-induced damage to the β-cell.
In this context, Woster and colleagues have recently reported  synthesis of a series of polyaminobenzamides, which are isoform-selective HDAC inhibitors. In the current study, we report the ability of this class of HDAC inhibitors to attenuate HDAC activity and increase the acetylation status of specific histones in INS 832/13 cells (heron referred to INS cells). We also report the efficacy of these inhibitors to attenuate IL-1β-mediated iNOS expression, NO release and loss in metabolic cell viability. Potential implications of these findings in the development of novel therapeutics to prevent the deleterious effects of cytokines and the onset of autoimmune diabetes are discussed.
Materials and methods
IL-1β was purchased from R&D Systems (Minneapolis, MN, USA), and Griess reagent, TSA and anti-actin serum were purchased from Sigma (St Louis, MO, USA). Cell viability assay (MTT) kit was purchased from Roche (Indianapolis, IN, USA). Affinity purified, monoclonal antibody directed against iNOS was purchased from Transduction Laboratories (Lexington, KY, USA). Antibodies directed against IκBα and its phosphorylated form (Ser32/36) and total and acetylated H4 (acetyl-peptide corresponding to residues surrounding Lys8 of histone H4) were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA).
Synthesis of inhibitors
Novel polyaminobenzamide inhibitors were synthesized using previously described synthetic routes [2,16].
INS 832/13 (INS) cells were kindly provided by Dr. Chris Newgard (Duke University Medical Center, Durham, NC, USA) and were cultured in RPMI 1640 medium supplemented with 1 mM sodium pyruvate, 50 μM β-mercaptoethanol and 10 mM HEPES at pH 7.4. The medium was changed twice weekly, and cells were trypsinized and subcloned weekly [17,18].
Quantitation of IL-1β-induced NO release
INS cells were cultured in 24-well plates (3 × 105 cells/well) for 2 days prior to various experimental manipulations (see text). β-cells were then cultured in 500 μL of medium/well containing either diluent alone or THS-78–5 (0–30 μM) for 24 hrs. The culture medium was then replaced with 500 μL of fresh medium containing THS-78–5 alone or in combination with IL-1β (600 pM) for 24 hrs. The medium was collected after the incubation period and centrifuged at 100g for 5 min. Equal volumes (100 μL) of medium and Griess reagent were mixed, and the absorbance was measured at 540 nm, as we previously described [19–21].
Quantitation of IL-1β-induced iNOS expression
INS cells grown in 24-well plates were treated with THS-78–5 (0–30 μM) for 24 hrs, prior to challenge with IL-1β (600 pM) for 24 hrs as described above. Extracted proteins from different conditions were separated by SDS-PAGE, and then, resolved proteins were transferred to a nitrocellulose membrane by wet transfer as we described in References [19–21]. Blots were then probed with either antibody raised against iNOS or actin and further incubated with secondary antibody conjugated to horseradish peroxidase (HRP). Immune complexes were detected using an enhanced chemiluminescence (ECL) kit.
Quantitation of IL-1β-induced phosphorylation of IκBα
INS cells grown in 24-well plates were treated with diluent or THS-78–5 (0–30 μM) for 24 hrs prior to challenge with IL-1β (600 pM) for 0–120 min. Proteins were extracted in a buffer consisting of 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 μg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride (PMSF). Extracted proteins from different conditions were separated by SDS-PAGE, and then, resolved proteins were transferred to a nitrocellulose membrane by wet transfer as we described in References [19–21]. Membranes were then probed with an antibody raised against phosphorylated IκBα and further incubated with secondary antibody conjugated to HRP. Immune complexes were detected using an ECL kit. The same membranes were further stripped and reprobed using antibodies raised against total IκBα or actin.
Identification of acetylated histones
INS cells grown in 24-well plates were treated with THS-73–44 (10 μM), THS-72–5 (10 μM) or THS-78–5 (10 μM) for 24 hrs as described above. Extracted proteins from different conditions were separated by SDS-PAGE, and then, resolved proteins were transferred to a nitrocellulose membrane by wet transfer, as we described in References [19–21]. Blots were then probed with an antibody raised against acetylated histone H4. Immune-complexes were detected using an ECL kit. The same membranes were further stripped and reprobed using rabbit polyclonal antibody raised against total histone H4 or actin.
Histone deacetylase activity assay
Nuclear extracts from INS cells were prepared using the NE-PER nuclear and cytoplasmic extraction kit from Pierce Biotechnology (Rockford, IL, USA). HDAC activity was measured using the HDAC assay kit from Upstate (Lake Placid, NY, USA). In brief, 20 μg of nuclear extracts from INS cells or HeLa cells (positive control), as the source of HDAC activity, were incubated in the presence or absence of TSA (200 nM) or THS-78–5 (30 μM) and acetylated substrate at 37°C for 60 min., allowing deacetylation of the colorimetric substrate. Subsequent addition of the activator solution after the incubation period selectively releases the colorimetric molecule from the deacetylated substrate, and the color developed was read at 405 nm. The HDAC activity was calculated as percentage of control in the absence of TSA or THS-78–5 (i.e.μM of deacetylated substrate formed/min).
Metabolic cell viability determinations
INS cells were seeded at a density of 1 × 106 cells/ml in 96-well plates and then treated with diluent or THS-73–44 (0–10 μM), THS-72–5 (0–10 μM) or THS-78–5 (0–30 μM) for 24 hrs, prior to the addition of IL-1β (600 pM; 24 hrs). Cell viability was determined by a colorimetric assay (at 550–690 nm), performed with 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyltetrazolium bromide (MTT), which measures the reduction of MTT into the blue formazan product by metabolically active cells.
Protein concentrations were determined by dye-binding method of Bradford, performed with bovine serum albumin as the standard .
Statistical analyses of data
Statistical significance of differences between the control and experimental groups was determined by ANOVA and a P value less than 0.05 was considered significant.
Novel HDAC inhibitors increase the acetylation status of histone H4 in INS cells
It is well established that chronic exposure of pancreatic β-cells to cytokines (e.g. IL-1β) culminates in β-cell death by either necrosis or apoptosis [23–25]. As stated in the Introduction, recent studies have clearly demonstrated that hypoacetylation of specific histones (e.g. histone H4) favours the induction of iNOS and subsequent NO release in isolated β-cells in the presence of cytokines (see introduction). In the present study, we investigated the potential beneficial effects of three novel HDAC inhibitors (Fig. 1) against IL-1β-mediated effects in pancreatic β-cells. At the outset, we investigated regulatory effects of these three inhibitors (Fig. 1) on histone H4 acetylation in INS cells. Data in Figure 2A and B indicate that of the three compounds tested, THS-78–5 exerted maximal effects on histone H4 acetylation (610% of control). THS-73–44 and THS-72–5 demonstrated increases in acetylation of histone H4 of 160% and 440% of control, respectively.
Novel HDAC inhibitors markedly reduce HDAC activity in nuclear extracts-derived from INS cells or HeLa cells
We next quantitated the HDAC inhibitory property of THS-78–5 in INS nuclear extracts using a commercially available kit (see Methods for additional details). As a positive control, TSA was also included in these assays. Furthermore, the HDAC inhibitory property of this compound was also determined in nuclear extracts from HeLa cells. Data described in Figure 3 demonstrate a significant inhibition of HDAC activity by TSA in nuclear extracts from both HeLa (∼83%) and INS cells (∼77%). THS-78–5 also inhibited total HDAC activity, albeit to a lesser extent (∼47%) in both INS and HeLa cell nuclear extracts. Not shown here are data to indicate significant inhibition of HDAC activity by THS-73–44 (∼42%) and THS-72–5 (∼67%) in HeLa cell nuclear extracts. Together, data in Figures 2 and 3 demonstrate that all three HDAC inhibitors significantly inhibit HDAC activity and promote the acetylation of histone H4 in pancreatic β-cells.
Of the three novel HDAC inhibitors tested, only THS-78–5 protects β-cells against IL-1β-induced loss in metabolic cell viability
To address this, we first determined the cytotoxic effects, if any, of the three novel HDAC inhibitors (0–10 μM) by assessing their effects on metabolic cell viability in INS cells (see Methods for additional experimental details). A modest, but significant increase (∼10% and 14%, respectively) in the viability was noted in cells incubated in the presence of THS-73–44 (at both 5 and 10 μM; Fig. 4). In contrast, a modest but significant reduction (∼10%) in cell viability was noted in cells incubated with 10 μM THS-72–5 (Fig. 4). No significant effects of THS-78–5 on metabolic cell viability were noted in cells treated with diluent alone (Fig. 4). As expected, treatment with IL-1β resulted in a ∼37% decrease in metabolic cell viability compared with cells treated with diluent alone (Fig. 4). Interestingly, however, only THS-78–5 (10 μM) elicited significant protective effects against IL-1β-induced loss in metabolic cell viability (bar 2 versus bar 12; Fig. 4). Data described in Figure 5 indicate that THS-78–5 alone had no cytotoxic effect in INS cells up to 30 μM. As in Figure 4, cytoprotective effects of this compound against IL-1β-induced loss in viability were demonstrable with concentrations as low as 10 μM (Fig. 5).
THS-78–5 attenuates IL-1β-induced iNOS expression and NO release in pancreatic β-cells
Since the above studies (Figs 1–5) clearly demonstrated a significant increase in acetylation of histone H4 and also a significant protection of cell viability by THS-78–5, we next investigated the regulatory (and potentially inhibitory) effects of THS-78–5 on IL-1β-mediated increase in iNOS expression (Fig. 6A and B) and subsequent NO release (Fig. 6C). Data from these studies indicated that maximal inhibition of IL-1β-induced iNOS expression was demonstrable by THS-78–5 at 30 μM (∼67% inhibition). Likewise, maximal inhibition of NO release was seen at 30 μM THS-78–5 (Fig. 6C). Taken together, these data indicate that THS-78–5 prevents IL-1β-mediated effects on iNOS expression, NO release and metabolic cell viability in INS cells in a manner akin to TSA. More importantly, however, this compound, unlike TSA, elicits no cytotoxic effects as demonstrated by no significant effects on the metabolic cell viability of the cells.
THS-78–5 attenuates IL-1β-mediated toxic effects on isolated β-cells by inhibiting transactivation of NF-κB
Since recent studies by Larsen et al. have identified NF-κB as one of the possible loci for the action of HDAC inhibitors (e.g. TSA) in isolated β-cells, we investigated, herein, potential regulatory effects of THS-78–5 on IκBα phosphorylation and degradation in INS cells following short-term incubation with IL-1β. As shown in Figure 7, THS-78–5 significantly decreased IL-1β-induced phosphorylation of IκBα, following 15 and 30 min. of challenge with IL-1β. Further, compatible with earlier observations performed with TSA , it also reduced the resynthesis of IκBα at all time-points studied (Fig. 7). In the absence of IL-1β, THS-78–5 had no effect on IκBα phosphorylation or degradation (additional data not shown). Together, these findings support the overall hypothesis that HDAC inhibitors like TSA  and THS-78–5 inhibit IL-1β-mediated effects on isolated β-cells, possibly by interfering with the transactivation of NF-κB and subsequent down-stream signaling events (see Discussion).
One of the major aims of the current investigation was to increase our current understanding of epigenetic control mechanisms (e.g. histone [de]acetylation) underlying cytokine-mediated metabolic dysfunction of the pancreatic β-cell. Salient features of our study are: (i) three novel compounds (THS-73–44, THS-72–5 and THS-78–5; synthesized in our laboratories) inhibited HDAC activity and increased the acetylation of histone H4 in isolated β-cells; (ii) they further exerted no toxic effects on metabolic cell viability in these cells; (iii) however, of the compounds tested, only THS-78–5 protected against IL-1β-mediated loss in cell viability; (iv) THS-78–5 was also able to attenuate IL-1β-induced iNOS expression and subsequent NO release; and (v) cytoprotective properties of THS-78–5 against IL-1β-mediated effects may, in part, be due to inhibition of IL-1β-induced transactivation of NF-κB in these cells. Together, we provide evidence for a novel HDAC inhibitor with a significant potential to prevent cytokine-mediated effects on isolated β-cells.
Emerging evidence from multiple laboratories appears to implicate chromatin structure remodelling in gene transcription [26,27]. One such structural modification of chromatin is through the acetylation of histones, the major protein components of chromatin . This type of modification facilitates the transcription of otherwise restricted genes. Several lines of recent evidence also suggest that a number of transcriptional factors are tightly regulated by histone modification [29–31], including the transactivation of NF-κB [32–34], which has been shown to promote the induction of iNOS in multiple cells lines, including pancreatic β-cells under the duress of various inflammatory stimuli including cytokines [14,35–39]. However, experimental evidence on the precise roles of the acetylation of histones and/or the regulation of acetylated histones in pancreatic β-cells in the context of IL-1β-induced iNOS expression and subsequent NO release has emerged only recently [14,15].
In a study published in 2007, Larsen et al. presented the first evidence to indicate that inhibition of HDACs prevents cytokine-induced toxicity in pancreatic β-cells. Using two structurally distinct HDAC inhibitors (e.g. TSA and SAHA), they reported that HDAC inhibition leads to inhibition of cytokine-induced iNOS expression and NO release in INS-1 cells and normal rat islets. They also demonstrated protection of cytokine-induced inhibition of insulin secretion by HDAC inhibitors. Further, they were able to show that HDAC inhibitors markedly down-regulate the transactivation of NF-κB induced by cytokines. In a more recent study from our laboratory, we have provided further support to Larsen et al.’s observations . First, we provided immunological evidence for the localization of Class 1 and 2 HDACs in isolated β-cells. Second, using INS cells, we were able to demonstrate a marked increase in the acetylation of histone H4 and reduction in IL-1β-induced iNOS expression and NO release by TSA in these cells. Together, these data suggest that hyperacetylation of histone H4 leads to inhibition of cytokine-induced iNOS expression and NO release.
Yu and coworkers provided evidence to suggest that HDACs augment cytokine-mediated induction of iNOS gene expression and subsequent NO release in cultured murine mesangial cells and RAW 264.7 cells . Using TSA, they observed a marked reduction in IL-1β- or lipoploysaccharide plus interferon γ-induced iNOS expression, NO release and NF-κB promoter activity. They were able to further demonstrate potentiation of IL-1β-mediated effects in cells overexpressing specific HDACs. Together, these findings provided conclusive evidence to implicate histone acetylation–deacetylation in cytokine-induced effects in renal cells. More recent studies by these investigators  have reported that TSA-mediated reduction in IL-1β-induced effects involves additional signaling mechanisms, such as phosphoinositide 3-kinase and p70s6 kinase, which increase site-specific histone H4 acetylation at the –978 to –710 region of the iNOS promoter.
The data presented in this manuscript clearly demonstrates that of the three analogues evaluated, the polyaminobenzamide THS-78–5 produces the greatest increase in histone H4 acetylation and most effectively attenuates cytokine-mediated cytotoxic effects. In vitro HDAC inhibition studies reveal that THS-72–5 is the most potent of the three with respect to global HDAC inhibition. However, in studies involving the pure HDAC isoforms 1, 3, 6 and 8, THS-78–5 appears to be most selective for HDAC 1 (data not shown). The significance of this observation in the present study is as yet unknown. It is not possible to make meaningful structure/function correlations from a library of only three compounds, but it is obvious that small changes in structure have a great effect on the ability of polyaminobenzamides to protect β-cells from IL-1β-mediated damage. Compound THS-73–44 is identical to THS-72–5, except for an amino group to methyl group structural change. The diminished effects of THS-73–44 could be attributed to a reduced ability of the benzamide moiety to co-ordinate with the zinc ion in the HDAC active site. The difference in activity between THS-72–5 and THS-78–5 is more difficult to explain since these compounds differ by a single methylene group in the linker chain portion of the molecule. Since the length of the linker chain has a direct bearing on the binding of polyaminobenzamides to the HDAC active site [2,16], it is likely that the seven carbon linker chain fits the active site channel more effectively than the six carbon linker, thus producing greater inhibition.
Additional analogues will need to be evaluated in the system described in this manuscript before any meaningful structure/activity relationships for the observed effects can be inferred. It should also be noted that the concentration of polyaminobenzamides are much higher than that of TSA to inhibit the IL-1β-induced effects, this is also true of other benzamide containing HDAC inhibitors (i.e. MS-275). This difference in the potencies between polyaminobenzamides and TSA may be due to difference in the ability to inhibit different subsets of HDACs.
In summary, based on findings in multiple cell types, including the islet β-cell [14,15,38,40], we propose that hyperacetylation of histone H4 is necessary for the attenuation of cytokine-mediated cytotoxic effects including iNOS expression, NO release and eventual loss in cell viability. It also appears that HDAC inhibitors clearly act through inhibition of transactivation of NF-κB. Our current findings of identification of THS-78–5 as one of the lead compounds to prevent cytokine-mediated metabolic dysfunction of the islet β-cell are of high translational value since this series of compounds, unlike other known HDAC inhibitors (e.g. TSA or SAHA), is by itself not cytotoxic. Studies for the development of additional structural analogues with much lower IC50 values to inhibit islet β-cell specific HDACs are in progress in our laboratories.
This research was supported by grants (to A.K.) from the Juvenile Diabetes Research Foundation, Department of Veterans Affairs and the National Institutes of Health (RO1 DK74921). Drug discovery and compound synthesis and development studies are supported by an unrestricted grant from Progen Pharmaceuticals to P.M.W. A.K. is the recipient of a Department of Veterans Affairs Senior Research Career Scientist Award. L.S. is supported by a Rumble Pre-Doctoral Fellowship from Wayne State University. The authors thank Dr. Suresh Madathilparambil for help in preparing nuclear extracts used in HDAC assays.