Protein kinase CK2-mediated phosphorylation of HDAC2 regulates co-repressor formation, deacetylase activity and acetylation of HDAC2 by cigarette smoke and aldehydes

Materials

Trichostatin A (TSA) and acrolein were purchased from Sigma (St. Louis, MO). Okadaic acid was obtained from Alexis Biochemicals (Plymouth Meeting, PA). Lambda Phosphatase was obtained from Millipore (Burlington, MA). HDAC2, CK2α, CK2α′, CK2β, HDAC1, p53, p65, RbAp46/48, SAP30, CBP and Lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Mi2 Flag, monoclonal phospho-serine and β-actin antibodies were obtained from Sigma. Monoclonal anti-acetyl lysine and MBD3 antibodies were obtained from Cell Signaling (Danvers, MA).

Cell culture

Human bronchial epithelial cells (H292) were purchased from the American Type Tissue Culture Collection (Manassas, VA). 7 × 10⁵ and 4 × 10⁶ H292 cells were grown in 6-well and 100 mm dishes respectively in RPMI-1640 supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 100 μg/ml penicillin and 100 U/ml streptomycin in humidified atmosphere under 5% CO₂ at 37°C.

Plasmid constructs

pME18S-HDAC2, pGal4-HDAC2 and flag-tagged HDAC2 mutant plasmids (S³⁹⁴A, S^{394/411/422/424}A, aa1-400) were kind gifts from Dr. E. Seto (H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL). PathDetect pFR-Luc trans-reporter plasmid was purchased from Agilent Technologies (Cedar Creek, TX). CBP wild-type and CBP mutants (L¹⁶⁹⁰K/C¹⁶⁹¹L) were kind gifts from Dr S. Ghosh (Columbia University Medical Center, New York, NY). pCMX-Gal4 plasmids were a kind gift from Dr. Chawnshang Chang (University of Rochester Medical Center, Rochester, NY).

Preparation of aqueous CSE

Research-grade cigarettes (3R4F) with a filter from the Kentucky Tobacco Research and Development Centre at the University of Kentucky (Lexington, KY) were used to prepare as described [11, 18]. Extract, defined as 10% CSE, was used for all experiments within 10 minutes of preparation. Air was bubbled into 10 ml of RPMI-1640 with 0.5% serum and pH adjusted to 7.4, filtered and used as control medium.

Transfections and luciferase reporter assay

For transfection assays, H292 cells were seeded at a density of 7.0 × 10⁵ cells in 6 well culture plates or 4 × 10⁶ cells in 100 mm dishes in a total volume of 2 or 7 ml RPMI-1640 containing 10% FBS without antibiotics overnight respectively. Cells were transiently transfected with control vector (Ctrl V) or flag-tagged wild-type HDAC2 (1–488), S394A, S394/422/424A, 1–400 aa HDAC2 mutants, CBP over-expression plasmid and CBP mutant plasmids (4–8 μg) lacking intrinsic HAT activity for 24 h with Lipofectamine 2000 according to manufacturer protocol. For co-transfections, EGFP or empty pcDNA3 vector plasmids were used to control for equal amount of DNA in all transfected wells. For siRNA assays, cells were seeded at a density of 3.0–5.0 × 10⁵ cells in 6 well culture plates in a total volume of 2 ml RPMI-1640 containing 10% FBS without antibiotics overnight. 100 pmol of CK2α siRNA (Applied Biosystems, Austin, TX) or scrambled siRNA were transfected for 24 h with Lipofectamine 2000 according to manufacturer protocol. Cells were then washed once with 1X phosphate-buffered saline (PBS) and transfected with flag-tagged HDAC2 (1-488) for a further 24 h prior to treatment. Transfected cells were then washed in 1X PBS and treated with CSE (2.5 %) for 0.5 h. Cells were lysed for 30 min on ice in 50–100 μl lysis buffer (0.5 % NP-40, 25 mM Tris, 100 mM NaCl with protease and phosphatase inhibitors, pH 7.4).

For luciferase assay, cells were transiently co-transfected with either pCMX-Gal4 or pGal4-HDAC2 with pFR-luc reporter. For CBP over-expression, cells were co-transfected with pCDNA3 empty vector, CBP wild-type or CBP (HAT-) with pGal4-HDAC2 and pFR-luciferase reporter. 24 h post transfection, cells were washed once with 1 × PBS, treated with CSE (2.5 %) for 0.5 h and then lysed in 80 μl/6-well plate 1X luciferase reporter lysis buffer (Promega, Madison, WI). 20 μg protein lysates was mixed with 100 μl of luciferase assay reagent and relative light units measured using a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA).

Immunoprecipitation and immunoblotting assays

Transfected H292 cells were lysed in 100 μl lysis buffer and 250 – 500 μg cell lysate was immunoprecipitated overnight with 40 μl EZview Red anti-flag M2 affinity gel (Sigma). Beads were washed with Tris-buffered Saline and immuno-complexes separated by SDS-polyacrylamide gel. For immunoprecipitation of CK2α, 500 μg cell lysates were incubated with 2 μg polyclonal CK2α antibodies overnight at 4°C and then immunoprecipitated with protein A/G Agarose beads (Santa Cruz Biotech) for 1 h at 4°C. Beads were washed with lysis buffer, boiled for 5 minutes in 2x sample buffer and immuno-complexes separated on SDS-polyacrylamide gels. Proteins were transferred overnight unto PVDF membranes (Millipore) and immunoblotted with respective primary antibodies. Primary antibodies were detected with respective secondary antibodies and membranes visualized with enhanced chemiluminescence (PerkinElmer, Waltham, MA).

Immunofluorescence

H292 cells were treated with CSE (2.5 %) for 0.5 h, washed and fixed with 4% PFA as described previously [19]. Slides were incubated with CK2α, CK2β goat polyclonal or α-tubulin mouse monoclonal antibody (1:100) overnight at 4 °C. Slides were mounted with ProLong Gold antifade reagent (Invitrogen) and images analyzed by a Leica confocal microscope or an Olympus inverted microscope.

Phosphatase activity assay

H292 cells, plated at 4.5 × 10⁶ cells/well in 100 mm dishes overnight, were treated with CSE (2.5 %) for 0.5, 2 and 6 h or 100 nM okadaic acid for 1 h. Cells were washed once with cold 1X PBS and lysed in 300 μl phosphatase storage buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% deoxycholate without phosphatase inhibitors) and PP2A activity measured using a Promega serine/threonine phosphatase assay kit (Promega, Madison, WI) according to manufacturer’s protocol. Briefly, samples were applied to a Sephadex G-25 resin column and centrifuged at 600 × g for 5 minutes at 4°C to remove endogenous phosphate. 5 μg of protein in 35 μl phosphatase storage buffer was mixed with 5 μl of 1 mM RRA(pT)VA phosphopeptide and 10 μl PPase-2A 5X reaction buffer (250 mM imidazole pH 7.2, 1 mM EGTA, 0.1 % β-mercaptoethanol and 0.5 mg/ml BSA) in a 96-well plate. Samples were incubated for 15 minutes and reactions stopped by the addition of 50 μl molybdate dye/additive mixture, incubated for a further 15 minutes and sample optical density (OD) determined with a microplate reader using a 630 nm filter. Phosphatase activity in pmol was determined using a standard curve obtained from free phosphate standards. All experiments were performed in quadruplicates.

HDAC2 activity assay

HDAC2 activity assay was determined as described [19]. Briefly, 500 –1000 μg HDAC2 was immunoprecipitated from whole cell lysates as described above. Beads were washed thoroughly and HDAC2 activity determined using an HDAC Color de Lys activity kit (Biomol, Plymouth Meeting, PA) according to manufacturer’s protocol.

Statistical analysis

Data expressed as mean±SE of triplicate experiments. Statistical analysis of significance was calculated using one-way Analysis of Variance (ANOVA) with STATVIEW software. ImageJ software was used for densitometry analysis. *P<0.05, **P<0.01, ### ***P<0.001.

CSE induces HDAC2 phosphorylation on serine sites 394, 422 and 424 in a phosphatase-independent manner

Previously, it had been shown that phosphorylation of HDAC2 modulates deacetylase activity [16, 17, 20] and protein kinase CK2 inhibition reverses cigarette smoke extract (CSE)-induced endogenous HDAC2 phosphorylation and degradation [19], however the mechanism and physiological relevance of CSE-induced HDAC2 phosphorylation remained largely unknown. To characterize CSE-induced HDAC2 phosphorylation, full length flag-tagged HDAC2 was first expressed in bronchial epithelial H292 cells (Fig 1A) which were then exposed to CSE (2.5%) for 0.5 h. Since we had previously shown CSE-induced covalent modification of HDAC2 at 4 h [11], it was important to choose a time point at which these modifications would be negligible and any functional modulation of HDAC2 activity would be due primarily to cellular signaling mechanisms such as phosphorylation. As expected, immunoprecipitated flag-tagged HDAC2 was significantly phosphorylated on serine residues which was de-phosphorylated by pre-treating immunoprecipitated beads with λ-phosphatase for 1 h at 30 °C prior to resolving samples on an SDS-polyacrylamide gel (Fig 1B, E).

To characterize the stability of HDAC2 serine phosphorylation, we varied the time of CSE exposure from 0.5 h to time point at which HDAC2 protein degradation was first observed (6 h), HDAC2 serine phosphorylation was significant at 0.5 h, diminished by more than 50% at 2 h and was almost completely negligible at 6 h CSE treatment (Fig 1D). To determine if acrolein, a small unsaturated aldehyde and important constituent of CSE, would also induce HDAC2 phosphorylation, H292 cells were transfected with flag-tagged full length HDAC2 and then treated with acrolein (50 μM) for 1 h. Immunoprecipitated flag-tagged protein from treated cells were significantly phosphorylated on serine residues (Fig 1C).

To rule out any role for hypoxia in CSE or acrolein-induced HDAC2 phosphorylation, lysates from CSE and/or acrolein-treated cells were resolved on an SDS polyacrylamide gel and then assessed for the expression of hypoxia-inducible factor 1 alpha (HIF1α), an expression marker in response to reduced cellular oxygen concentrations. HIF1α expression was negligible in cells treated with CSE (2.5%) or acrolein (50 μM) compared to either cells exposed to media alone or 5 % oxygen for 1 h (Fig 1F).

Previous studies using radio-labeled wild-type and mutant HDAC2 constructs had identified serine sites S³⁹⁴, S⁴²² and S⁴²⁴ as potential phospho-acceptor sites on HDAC2 [17], we therefore sought to determine if these sites were also critical for CSE-induced HDAC2 phosphorylation in-vivo. H292 cells were transfected with flag-tagged full length HDAC2 (1-488), a C-terminal 88-amino acid deletion mutant (1-400) and C-terminal serine to alanine mutants. Cells were then treated with CSE (2.5%) for 0.5 h, flag proteins immunoprecipitated using anti-flag affinity gel and resolved on an SDS-polyacrylamide gel. Both wild-type and S^394A mutant HDAC2 were phosphorylated indicating the S³⁹⁴ site alone may not be crucial to CSE-induced HDAC2 phosphorylation. However, serine to alanine mutations on sites S³⁹⁴, S⁴¹¹, S⁴²² and S⁴²⁴ significantly attenuated CSE-induced HDAC2 phosphorylation while the C-terminal 88-amino acid deletion mutant (1-400) construct was only modestly phosphorylated suggesting serine sites S⁴²² and S⁴²⁴ as crucial for CSE-induced HDAC2 phosphorylation (Fig 1G).

To determine if phosphorylation of HDAC2 in response to CSE was dependent on a kinase-mediated mechanism or inhibition of phosphatase activity, PP2A serine/threonine phosphatase activity was measured in response to varying time points of exposure to CSE. Compared to non-treated controls, CSE significantly diminished PP2A activity at 6 h but had no effect at 0.5 h, time points at which CSE induced HDAC2 phosphorylation (Fig 1H).

Cigarette smoke induces CK2α phosphorylation

Since we observed that serine sites S³⁹⁴, S⁴²² and S⁴²⁴ were required for CSE-induced HDAC2 phosphorylation and are conserved protein kinase CK2 phospho-acceptor sites on HDAC2, we speculated that CK2 would be involved in HDAC2 phosphorylation in response to CSE. Despite published reports indicating that the CK2 catalytic subunit is constitutively active [21, 22], we consistently observed a doublet band for CK2α in response to CSE (2.5%) for 0.5 h (Fig 2A) with no significant change in relative protein expression. To confirm if this doublet was due to CK2α phosphorylation, immunoprecipitated CK2α from bronchial epithelial (H292) cells was determined to be phosphorylated on serine residues in response to CSE (Fig 2B). Phosphatase treatment of CK2α immunoprecipitates completely abolished the doublet band in response to CSE (Fig 2C). Okadaic acid, a specific PP2A inhibitor which had earlier been shown to induce HDAC2 serine phosphorylation [19], also induced CK2α phosphorylation as shown by the presence of doublet bands (Fig 2D).

HDAC2 serine phosphorylation is associated with binding to CK2 catalytic but not regulatory subunits in response to CSE

The presence of conserved protein kinase CK2 sites that were highly phosphorylated on HDAC2 in response to CSE and acrolein, led us to hypothesize a potential direct interaction between HDAC2 and protein kinase CK2 catalytic subunits or the holoenzyme. Flag-tagged WT HDAC2 plasmids were transfected into H292 cells and then treated with CSE (2.5%) for 0.5 h. Both catalytic subunits of protein kinase CK2; CK2α, CK2α′ and the serine-phosphorylated form of CK2α were recruited to HDAC2 in response to CSE treatment (Fig 3A and 3B). To determine if this interaction was specific only to CSE, H292 cells were also treated with acrolein (25 and 50 μM) for 1 h. Acrolein-induced HDAC2 phosphorylation was associated with increased HDAC2 CK2α′ binding (Fig 3C) which, compared with CSE treatment, suggested that the extent of HDAC2 phosphorylation correlates with the level of CK2α binding. This finding further indicated a potential role for CK2α in HDAC2 phosphorylation.

HDAC2 is a predominantly nuclear protein while CK2β is mostly localized to the cytoplasm; hence we speculated CK2β must be translocated to the nucleus in response to CSE if it is required to _target CK2 catalytic subunits to HDAC2. Using both immunoblot and immunofluorescence techniques, we did not observe any significant translocation of CK2α to the nucleus (Fig 3D and E). However, CK2β, was not detected either in the nucleus or bound to HDAC2 both by immunoblotting and confocal microscopy in response to CSE (Figs 3F and G) suggesting that independent catalytic subunits of protein kinase CK2 but not the holoenzyme was sufficient for CSE and acrolein-induced HDAC2 phosphorylation and substrate specificity.

Pharmacological inhibition and silencing of protein kinase CK2 inhibit oxidative stress-induced HDAC2 phosphorylation

To determine if protein kinase CK2 was uniquely required for CSE-induced HDAC2 phosphorylation [17, 23], bronchial epithelial H292 cells were pre-treated with a specific CK2 inhibitor, 4, 5, 6, 7-tetrabromobenzotriazole (TBB) [24] for 2 h prior to exposure to CSE (2.5%) for 0.5 h. 50 μM TBB was sufficient at completely inhibiting CSE-induced HDAC2 phosphorylation (Fig 4A–B). TBB, at doses as low as 25 μM have also been observed to partially inhibit HDAC2 phosphorylation. Using a genetic approach, H292 cells were transfected with either scrambled or a 19-nucleotide siRNA sequence _targeted to human CK2α for 24 h. Cells were then subsequently transfected with flag-tagged wild-type HDAC2 for 24 h prior to treatment with CSE (2.5%) for 0.5 h. A 56 % knock-down in CK2α but not CK2α′ expression (Fig 4C) was sufficient to partially inhibit CSE-induced serine phosphorylation of HDAC2 (Fig 4D). Partial inhibition via CK2α knock-down compared to almost complete inhibition of phosphorylation with the specific CK2 inhibitor, TBB, suggested the possibility that CK2α′, the catalytic isoform of CK2α, is also involved in CSE-mediated HDAC2 phosphorylation and compensates for loss of CK2α.

CSE-induced HDAC2 phosphorylation is required for co-repressor binding and interaction with transcription factors but not HDAC2 subcellular localization

Based on earlier studies showing a disruption in HDAC2 binding to other proteins in the co-repressor complex in response to increased intracellular phosphorylation with okadaic acid [20], we initially hypothesized that CSE-mediated oxidative stress-induced HDAC2 phosphorylation would cause a disruption of the HDAC2 co-repressor complex. However, CSE (2.5 %) treatment of H292 cells transfected with wild-type flag-tagged HDAC2 for 0.5 h showed a significant increase in HDAC2 associated with HDAC1 (Fig 5A) compared with media-alone treatments. As earlier observed with immunoprecipitation reactions showing increased p53 interaction with HDAC1/2 via mSin3A in response to doxycycline-induced DNA damage [25], we hypothesized that CSE-induced HDAC2 phosphorylation causes significantly increased HDAC2 interaction with p53. As expected, only immunoprecipitated flag-tagged wild-type HDAC2 from H292 cells exposed to CSE (2.5%) showed interaction with the p53 transcription factor (Fig 5B). To further determine if the increased interaction was dependent on phosphorylation, H292 cells were transfected with either flag-tagged HDAC2 serine to alanine mutants on sites S³⁹⁴, S⁴¹¹, S⁴²² and S⁴²⁴ or an HDAC2 C-terminal 88-amino acid deletion mutant and then exposed to CSE (2.5%) for 0.5 h. Consistent with earlier results showing abrogation of serine phosphorylation with loss of serine site mutations S³⁹⁴, S⁴¹¹, S⁴²² and S⁴²⁴ but not S⁴¹¹, S⁴²² and S⁴²⁴ alone, HDAC1 interaction was significantly lower in the S³⁹⁴, S⁴¹¹, S⁴²² and S⁴²⁴ mutant but not the C-terminal deletion mutant (Fig 5C) suggesting phosphorylation is strongly required for HDAC2 co-repressor interactions. Inhibiting HDAC2 phosphorylation by pre-treating cells with N-acetyl-L-cysteine (NAC), a cysteine source for the synthesis of the antioxidant GSH, for 2 h prior to exposure to CSE significantly attenuated HDAC2 interaction with SAP30, a Sin3-specific complex protein, MBD3, a Mi-2/nucleosome remodeling and deacetylase (NURD)-specific co-repressor complex protein, RBAp46/48, a component of both Sin3 and NURD complexes and p65, a transcription factor involved in inflammation and known to be actively deacetylated by HDAC1/HDAC2 [26] (Fig 5D). To further confirm the role of CK2-mediated HDAC2 phosphorylation in increased HDAC2 interaction with co-repressor proteins and transcription factors, H292 cells transfected with flag-tagged wild-type HDAC2 were pre-treated with either 40 or 90 μM TBB for 2 and 0.5 h respectively and then exposed to CSE (2.5%) for 0.5 h. TBB significantly inhibited CSE-induced HDAC2 interaction with HDAC1 and p65 in a dose-dependent manner (Fig 5E).

To determine whether phosphorylation was associated with HDAC2 enzymatic activity, flag-tagged wild-type HDAC2 was immunoprecipitated from H292 cells pre-treated with NAC for 2 h prior to exposure to CSE (2.5%) for 0.5 h. NAC pre-treatment significantly attenuated CSE-induced decrease in HDAC2 deacetylase activity (Fig 5F) suggesting serine phosphorylation is a possible negative regulator of HDAC2 activity by CSE-mediated oxidative stress. Since NAC is a thiol antioxidant, it was important to see whether direct modulation of the CK2-HDAC2 phosphorylation pathway would affect HDAC2 activity. H292 cells, transfected with either a scrambled sequence or 100 pmol of a siRNA sequence _targeted to CK2α, were exposed to either media alone or CSE (2.5%) for 0.5 h. Cells were then harvested and specific HDAC2 activity measured. Consistent with the pattern observed with NAC pre-treatment, partial CK2α knock-down rescued CSE-induced HDAC2 enzymatic activity almost to control levels (Fig 5G). Based on earlier findings showing reduced nuclear expression of HDAC2 following long-term exposure to CSE [19] and that cellular localization of class II HDACs such as HDAC4 is actively regulated by phosphorylation [27], we hypothesized that CSE-induced phosphorylation of HDAC2 would induce nucleo-cytoplasmic shuttling. H292 cells were exposed to CSE (2.5 %) and acrolein (50 μM) for 0.5 and 1 h respectively at time points where induction of serine phosphorylation was observed. However, HDAC2 remained exclusively localized to the nucleus (Fig 5H) suggesting localization of class I HDACs is unaffected by phosphorylation.

CBP-mediated HDAC2 acetylation is dependent on phosphorylation

Despite the presence of at least 12 conserved lysine residues on the C-terminal domain of HDAC2 and earlier findings suggesting HDAC1 is also acetylated in response to dexamethasone [28], it remained largely unknown as to whether HDAC2 was also modified by acetylation. To determine this, H292 cells were transfected with flag-tagged full length HDAC2 and then treated with or without CSE (2.5%) for 0.5 h. Consistent with our hypothesis, full length HDAC2 was strongly acetylated in response to CSE (Fig 6A). To further validate this, flag-tagged wild-type HDAC2 was transfected into H292 cells treated with acrolein (50 μM) for 1 h, using CSE as a positive control. Consistent with earlier results showing acrolein-induced HDAC2 phosphorylation, immunoprecipitated flag-tagged HDAC2 showed significant acetylation of lysine residues compared to untreated controls (Fig 6B).

Since both CREB binding protein (CBP) and HDAC2 had been recently mapped to be actively recruited to promoters and intergenic regions of active genes [29], we speculated that CBP would be involved in HDAC2 acetylation most possibly via a direct protein-protein interaction. To determine a possible interaction between HDAC2 and CBP, flag-tagged wild-type HDAC2 was transfected into H292 cells and then treated with CSE (2.5%) for 0.5 h. Immunoprecipitated flag-tagged HDAC2 showed significant interaction with CBP only in CSE-treated cells compared with media-alone controls (Fig 6C).

To further establish a role for CBP in HDAC2 interaction, we obtained both wild-type (CBP+) and HAT activity-deficient CBP mutants (CBP- L1690K and C1691L) [30] and co-transfected with flag-tagged wild-type HDAC2 in H292 cells. Cells were then exposed to CSE (2.5%) for 0.5 h and flag-tagged proteins immunoprecipitated and immunoblotted for acetylation of lysine residues. However, over-expression of wild-type CBP failed to enhance CSE-induced HDAC2 acetylation (Fig 6D) suggesting the possibility of specific CBP binding sites on HDAC2. Consistent with a prominent role for CBP in HDAC2 acetylation, over-expression of a dominant negative HAT-deficient CBP mutant completely abrogated CSE-induced HDAC2 acetylation of lysine residues (Fig 6E). The abrogation of HDAC2 acetylation, however, had no effect on HDAC2 phosphorylation in the presence of over-expressed dominant negative CBP (Fig 6F) suggesting that acetylation was more likely a consequence of CSE-induced HDAC2 phosphorylation.

To further determine whether acetylation was dependent on phosphorylation, we speculated that an HDAC2 mutant deficient in critical phosphorylation sites would not be acetylated in response to CSE. H292 cells were transfected with flag-tagged wild-type or S³⁹⁴, S⁴¹¹, S⁴²² and S⁴²⁴ mutant HDAC2 and then treated with CSE (2.5%) for 0.5 h. Consistent with our hypothesis, mutant HDAC2 was only minimally acetylated compared to the full length wild-type HDAC2 (Fig 6G).

Acetylation modulates HDAC2 transrepression

HDAC2 plays two distinct cellular roles; deacetylation of substrate proteins or histones (enzymatic activity) and repression of gene transcription (functional activity) [2]. Since CK2α knock-down and NAC-mediated inhibition of HDAC2 phosphorylation in response to CSE increased HDAC2 enzymatic activity, it was important to determine whether phosphorylation would affect HDAC2 functional activity. To determine this, H292 cells were co-transfected with plasmids encoding both Gal4-HDAC2 [17] or Gal4-DBD and a luciferase reporter plasmid containing five tandem yeast Gal4 binding site repeats (Fig 7A). Transfected cells were then treated with CSE (2.5%) or acrolein (50 μM) for 0.5 and 1 h respectively and then assayed for luciferase expression. Consistent with other reports [17], HDAC2 significantly repressed luciferase transcription in response to CSE treatment compared to untreated controls alone (Fig 7B). Acrolein, however, did not show a significant effect on luciferase expression compared to CSE-treated cells. This may be explained by data showing acrolein induces far less HDAC2 serine phosphorylation compared to CSE (data not shown) which is associated with a significant difference in CK2α′ binding to HDAC2 (Fig 3C). To determine the specific role of HDAC2 acetylation in gene transrepression, H292 cells were co-transfected with Gal4-HDAC2, a pFR-luciferase reporter plasmid and either a CBP wild-type or mutant lacking HAT activity. Cells were then treated with or without CSE (2.5%) for 0.5 h, lysed and then assayed for luciferase activity. Surprisingly, over-expression of wild-type CBP significantly increased HDAC2 transrepression activity which was attenuated by over-expression of mutant CBP with no HAT activity (Fig 7C), suggesting that acetylation is the more dominant regulator of HDAC2 transrepression activity.

Dexamethasone blocks pro-inflammatory gene expression induced by LPS but not CSE

Steroid resistance is a major hallmark of moderate to severe COPD and has been associated with loss of HDAC2 [31, 32]. As we had earlier shown that HDAC2 serine phosphorylation in response to CSE is directly associated with HDAC2 degradation [19], it was important to compare the ability of corticosteroids to inhibit IL-8 release in response to CSE or LPS which did not induce HDAC2 phosphorylation in our system (data not shown). Cells were pre-treated with low dose dexamethasone (0.1 μM) for 2 h prior to CSE or LPS exposure for 1 h. There was no significant difference in IL-8 release between samples treated with media alone or dexamethasone alone (<150±9 pg/ml). However, while dexamethasone failed to inhibit IL-8 release in response to CSE treatment (CSE 1554±54 pg/ml vs. CSE+DEX 1665±65, n=4), cells treated with dexamethasone + LPS showed a 40% decrease in IL-8 release (809±32 pg/ml, p<0.01, n=4) compared to LPS-alone treatments (1354±26 pg/ml). These results suggest that HDAC2 serine phosphorylation is a key mechanism of steroid resistance in COPD and is consistent with earlier observations showing increased HDAC2 serine phosphorylation in mouse lungs with sub-chronic CS exposure which subsequently leads to loss of nuclear HDAC2 protein [19].