Cigarette smoke extract (CSE) time-dependently caused degradation of class I histone deacetylases (HDACs) in MonoMac6 cells. MonoMac6 cells were treated with CSE (2.5%) for indicated time points. Ten micrograms of protein from whole cell extracts was electrophoresed on SDS-7.5% polyacrylamide gel, transferred onto PVDF membranes, and immunochemically stained with HDAC1, HDAC2, and HDAC3 antibodies. (A) CSE significantly decreased class I HDAC levels as early as 1 hour after CSE exposure. (B) Relative density of HDAC levels in MonoMac6 cells at various time points of exposure to CSE. Open bars, HDAC1; solid bars, HDAC2; hatched bars, HDAC3. (C) MonoMac6 cells were treated with CSE (2.5%) for 0, 4, and 6 hours, cytospin slides were prepared, and cells were fixed in 4% paraformaldehyde and immunostained with anti-HDAC2 antibodies. DAPI stain was used to visualize the nucleus (shown by arrows). CSE showed significant loss of HDAC2 expression in the nucleus without any nucleo-cytoplasmic shuttling in MonoMac6 cells. Data are shown as mean ± SEM (n = 3). ***P < 0.001 or ^###P < 0.001, significant compared with respective control treatments.

CSE-induced HDAC2 degradation is associated with decreased specific HDAC2 activity in MonoMac6 cells. MonoMac6 cells were treated with CSE (2.5%) for 0.5, 1, and 2 hours, and immunoprecipitated HDAC2 (500 μg protein) from whole cell extracts was assayed for HDAC2 activity with a colorimetric assay kit (Biomol) at 405 nm with Color de Lys substrate (500 μM) and expressed as deacetylated standard using 1 to 500 μM standard curve. HeLa nuclear extract was used as a positive control. Data are shown as mean ± SEM (n = 4). *P < 0.05, significant compared with media alone control treatments.

CSE induces HDAC2 phosphorylation via CK2 in MonoMac6 cells. (A) MonoMac6 cells were treated with CSE (2.5%) for 0.5, 1, and 2 hours. Immunoprecipitated HDAC2 was analyzed for phosphorylation of serine and threonine residues using specific phospho-serine and phospho-threonine antibodies by SDS-polyacrylamide gel. CSE induced significant phosphorylation of HDAC2 on serine and threonine residues. NS, nonspecific bands. Relative density of (B) phospho-serine and (C) phospho-threonine levels normalized to immunoprecipitated HDAC2 and expressed as fold change relative to control. (D) MonoMac6 cells were pretreated with or without CK2 inhibitor (20 μM 4,5,6,7-tetrabromobenzotriazole [TBB]) for 2 hours. Cells were then exposed to CSE (2.5%) for 6 hours. Ten micrograms of protein from whole cell lysates was electrophoresed on SDS-polyacrylamide gel and assayed for HDAC2 levels. TBB significantly inhibited HDAC2 degradation in response to CSE. (E) Relative density of HDAC2 levels normalized to β-actin. [F(i)] MonoMac6 cells were treated with phosphatase inhibitor, okadaic acid (1 μM), for 0.5 hours. Immunoprecipitated HDAC2 was analyzed for phosphorylation of serine residues using specific phospho-serine antibody by SDS-polyacrylamide gel. Okadaic acid significantly induced phosphorylation of HDAC2 on serine residues. [F(ii)] Relative density of phospho-serine expression normalized to immunoprecipitated HDAC2. [F(iii)] MonoMac6 cells were treated with or without phosphatase inhibitor, okadaic acid (40 nM), for 24 hours. Immunoprecipitated HDAC2 was assessed for bound ubiquitin using specific ubiquitin anti-sera. Okadaic acid induced significant ubiquitination of HDAC2. [F(iv)] Relative density of ubiquitin levels normalized to immunoprecipitated HDAC2. (G) MonoMac6 cells were treated with okadaic acid (1 μM) for 0.5 hours, cytospin slides were prepared, and cells were fixed in 4% paraformaldehyde and immunostained with anti-HDAC2 antibodies. DAPI stain was used to visualize the nucleus (arrows). Okadaic acid showed an early and significant loss of HDAC2 abundance in the nucleus without any nucleo-cytoplasmic shuttling in MonoMac6 cells. (H) MonoMac6 cells were treated with okadaic acid (1 μM) for 0.5 hours. Western blots for nuclear extracts showed significant decrease in HDAC2 expression. Data expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 significant compared with media alone treatments or respective controls.

CSE-mediated degradation of HDAC2 is ubiquitin-proteasome dependent in MonoMac6 cells. (A) MonoMac6 cells were pre-treated with proteasome inhibitor N-Acetyl-Leu- Leu-Nle-CHO (ALLN, 1 μM and 5 μM) for 2 hours, followed by a 6-hour exposure to CSE (2.5%). Ten micrograms of protein from whole cell extracts was electrophoresed on SDS-7.5% polyacrylamide gel, transferred onto PVDF membranes, and immunochemically stained with HDAC2 antibodies. Dose-dependent inhibition of the proteasome significantly attenuated HDAC2 degradation. (B) Relative density of HDAC2 levels in MonoMac6 cells after proteasome inhibition of CSE-induced HDAC2 degradation normalized to β-actin. (C) MonoMac6 cells were pre-treated with or without ALLN (1 μM) and then exposed to CSE (2.5%) for 6 hours. Immunoprecipitated HDAC2 was assayed for bound ubiquitin using specific ubiquitin anti-sera. CSE induced significant increase in HDAC2 ubiquitination, which was enhanced with ALLN pretreatment. (D) Relative density of ubiquitin expression in MonoMac6 cells after proteasome inhibition of CSE-induced HDAC2 degradation normalized to immunoprecipitated HDAC2. Data shown as mean ± SEM (n = 3). ***P < 0.001, significant compared with media alone control treatments; ^##P < 0.001, significant compared with CSE+ALLN versus CSE alone.

CSE time-dependently caused degradation of HDAC2 in human bronchial epithelial and primary small airway epithelial cells. [A(i)] Bronchial epithelial cells (H292) were treated with CSE (2.5%) for 0, 1, 4, 12, and 24 hours. Ten micrograms of protein from whole cell extracts was electrophoresed on SDS-7.5% polyacrylamide gel, transferred onto PVDF membranes and immunochemically stained with HDAC2 antibodies. CSE time-dependently decreased HDAC2 levels in H292 cells. [A(ii)] Relative density of HDAC2 expression in H292 cells exposed to CSE normalized to β-actin. [B(i)] Small airway epithelial cells (SAEC) were exposed to CSE (0.5% and 1%) for 4 hours. Whole cell lysates were electrophoresed on SDS-polyacrylamide gels and membranes immunochemically stained for HDAC2 expression. CSE time-dependently induced HDAC2 degradation. [B(ii)] Relative density of HDAC2 levels in SAECs exposed to CSE normalized to β-actin. Data shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 significant compared with control treatments.

CSE induced CK2-dependent HDAC2 phosphorylation in human bronchial epithelial and small airway epithelial cells. [A(i)] H292 cells were treated with CSE (2.5%) for 0.5, 1, 2, and 4 hours. Immunoprecipitated HDAC2 was analyzed for phosphorylation of serine residues using specific phospho-serine antibodies by SDS-polyacrylamide gel. CSE induced significant cyclical phosphorylation of HDAC2 on serine residues at 0.5, 1, and 4 hours. [A(ii)] Relative density of phospho-serine level normalized to immunoprecipitated HDAC2. [B(i)] SAECs were exposed to CSE (0.1%, 0.5%, and 1%) for 4 hours. Immunoprecipitated HDAC2 from whole cell lysates was probed for phosphorylation of serine residues. CSE induced a dose-dependent increase in HDAC2 serine phosphorylation. [B(ii)] Relative density of phospho-serine level normalized to immunoprecipitated HDAC2. (C) H292 cells were treated with or without specific CK2 inhibitor, TBB, for 1 hour, and cells were washed with PBS and then treated with freshly prepared CSE (2.5%) for 0.5 hours. Immunoprecipitated HDAC2 was assayed for phosphorylation on serine residues. TBB significantly inhibited CSE-induced HDAC2 phosphorylation. (D) Relative density of phospho-serine level normalized to immunoprecipitated HDAC2. Data expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001, significant compared with control treatments, or respective controls. ^##P < 0.01 significant compared with CSE + TBB versus CSE alone.

CS exposure caused HDAC2 degradation and decreased activity in mouse lungs. Mice were exposed to CS as described in Materials and Methods. Lung tissue (100–150 mg) was homogenized and nuclear extracts prepared and frozen until analyzed. Five micrograms of protein of nuclear extracts were analyzed for HDAC2 abundance on SDS-polyacrylamide gels. (A) CS time-dependently decreased HDAC2 levels in mouse lungs. (B) Relative density of HDAC2 levels normalized to β-actin. (C) HDAC2 was immunoprecipitated and HDAC2 activity measured by colorimetric assay kit (Biomol) using Color de Lys substrate. Mice exposed to CS showed decreased specific HDAC2 activity as compared with air exposed mice. Data expressed as mean ± SEM (n = 4–5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with air-exposed mice.

CS induces HDAC2 phosphorylation in mouse lungs. Mice were exposed to CS as described in Materials and Methods. Mouse lung tissue (100–150 mg) was homogenized in RIPA buffer (100 μl) as whole tissue extracts, and HDAC2 (500 μg) was immunoprecipitated. Phosphorylation of serine and threonine residues was analyzed using specific phospho-serine and phospho-threonine antibodies on SDS-polyacrylamide gel. (A) Mice exposed to CS for 2, 4, 10, and 16 weeks showed significant phosphorylation of HDAC2 on both serine and threonine residues; however, mice exposed at 2 weeks did not show significant phosphorylation on threonine residues. Relative density of (B) phospho-serine and (C) phospho-threonine HDAC2 levels normalized to immunoprecipitated HDAC2. Data expressed as mean ± SEM (n = 4–5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with air-exposed littermates.