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. 2019 Feb 27:10:166.
doi: 10.3389/fphar.2019.00166. eCollection 2019.

Carbocisteine Improves Histone Deacetylase 2 Deacetylation Activity via Regulating Sumoylation of Histone Deacetylase 2 in Human Tracheobronchial Epithelial Cells

Yun Song  1 , Dan-Yi Chi  1 , Ping Yu  2 , Juan-Juan Lu  2 , Jian-Rong Xu  2 , Pan-Pan Tan  3 , Bin Wang  1 , Yong-Yao Cui  2 , Hong-Zhuan Chen  2
Affiliations

Carbocisteine Improves Histone Deacetylase 2 Deacetylation Activity via Regulating Sumoylation of Histone Deacetylase 2 in Human Tracheobronchial Epithelial Cells

Yun Song et al. Front Pharmacol. .

Abstract

Histone deacetylase (HDAC) 2 plays a vital role in modifying histones to mediate inflammatory responses, while HDAC2 itself is commonly regulated by post-translational modifications. Small ubiquitin-related modifier (SUMO), as an important PTM factor, is involved in the regulation of multiple protein functions. Our previous studies have shown that carbocisteine (S-CMC) reversed cigarette smoke extract (CSE)-induced down-regulation of HDAC2 expression/activity in a thiol/GSH-dependent manner and enhanced sensitivity of steroid therapy. However, the mechanism by which S-CMC regulates HDAC2 is worth further exploring. Our study aimed to investigate the relationships between HDAC2 sumoylation and its deacetylase activity under oxidative stress and the molecular mechanism of S-CMC to regulate HDAC2 activity that mediates inflammatory responses in human bronchial epithelial cells. We found that modification of HDAC2 by SUMO1 and SUMO2/3 occurred in 16HBE cells under physiological conditions, and CSE induced SUMO1 modification of HDAC2 in a dose and time-dependent manner. K462 and K51 of HDAC2 were the two major modification sites of SUMO1, and the K51 site mediated deacetylation activity and function of HDAC2 on histone H4 that regulates IL-8 secretion. S-CMC inhibited CSE-induced SUMO1 modification of HDAC2 in the presence of thiol/GSH, increased HDAC activity, and decreased IL-8 expression. Our study may provide novel mechanistic explanation of S-CMC to ameliorate steroid sensitivity treatment in chronic obstructive pulmonary disease.

Keywords: GSH/thiol; carbocisteine; cigarette smoke extract; histone deacetylase 2; small ubiquitin-related modifier.

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Figures

FIGURE 1
FIGURE 1
The interaction and colocalization of HDAC2 and SUMO1 or SUMO2/3 in 16HBE cells under physiological condition by immunoprecipitation and confocal microscopy. (A,C) Cell lysates were subjected to immunoprecipation using anti-HDAC2 antibodies. SUMOylated HDAC2 was detected by Western blot using anti-SUMO1 or SUMO2/3 antibodies. Lane 1: input (whole cell lysate without IP treatment); Lane 2: immunoprecipitation with IgG (H + L chains) but without HDAC2 antibody; Lane 3: normal IP. (B,D) Cell lysates were subjected to immunoprecipation using anti-SUMO1 or SUMO2/3 antibodies. SUMOylated HDAC2 was detected by Western blot using anti-HDAC2 antibodies. Lane 1: immunoprecipitation with IgG (H + L chains) but without SUMO1 or SUMO2/3 antibody; Lane 2: normal IP (E) Representative confocal images of fluorescently labeled nucleus (blue, channel 1: 386 nm), SUMO1 or SUMO2/3 (green, channel 2: 488 nm) and HDAC2 (red, channel 3: 555 nm) in 16HBE cells in physiological conditions. There is a colocalization of HDAC2 and SUMOs in nucleus of 16HBE cells (Merge1+2+3).
FIGURE 2
FIGURE 2
The effects of CSE in different concentrations and different incubation time on HDAC2 and SUMO1 or SUMO2/3 interaction in 16HBE by immunoprecipitation and HCS analysis. (A,B) Effects of treatment with CSE under 0, 1, 2.5, 5, and 10% concentrations for 30 min in SUMOylated HDAC2 in 16HBE cells by immunoprecipitation. (C,D) Effects of treatment with CSE under 0, 1, 2.5, 5, and 10% concentrations for 30 min in SUMOylated HDAC2 in 16HBE cells by HCS analysis. (E,F) Effects of treatment with CSE for 0, 15, 30 min, 1 and 2 h in SUMOylated HDAC2 in 16HBE cells by immunoprecipitation. (G,H) Effects of treatment with CSE for 0, 15, 30 min, 1 and 2 h in SUMOylated HDAC2 in 16HBE cells by HCS analysis. Data are expressed as the means ± SEM, n = 6. Ns means no significant difference and ∗∗p < 0.01 and ∗∗∗p < 0.001 compared to control group (0% CSE or 0 h) using one-way ANOVA with Dunnett t-test for selected pairs.
FIGURE 3
FIGURE 3
Identification of K51 and K462 as the HDAC2 SUMOylation sites. 16HBE cells were transfected with HDAC2-K462R, HDAC2-K51R, HDAC2-K145R, HDAC2-K451R using lentivirus vector. Cells lysates were subjected to immunoprecipitation using HDAC2 antibody. SUMOylated HDAC2 was detected by Western blot using anti-SUMO1 antibody.
FIGURE 4
FIGURE 4
The biological function of SUMO1 modification of HDAC2. (A) HDAC2 binding and histone acetylation were analyzed by ChIP analysis in 16HBE wild type, 16HBE-K51R and 16HBE-K462R cells after CSE incubation for 0.5 h. (B) HDAC activity in 16HBE wild type, 16HBE-K51R and 16HBE-K462R cells after CSE incubation for 0.5 h. (C) histone acetylation of the IL-8 gene promoter in 16HBE wild type, 16HBE-K51R and 16HBE-K462R cells after CSE incubation for 0.5 h. Data are presented as means ± SEM (n = 6). Ns means no significant difference and ∗∗p < 0.01 compared to wild type using one-way ANOVA with Dunnett t-test for selected pairs.
FIGURE 5
FIGURE 5
S-CMC reversed the effects of CSE on HDAC2-SUMO1 interaction, HDAC activity and IL-8 release. 16HBE cells were pre-incubated with S-CMC (10−6–10−4 M) or NAC, GSH, GSH mimetics (10−4 M) with or withnot BSO (10−4 M) for 1 h, following 5% CSE incubation for 30 min (A) Analysis for interaction between HDAC2 and SUMO1 by immunoprecipitation. (B) The activity of HDAC in 16HBE cells was detected using a HDAC activity assay kit. (C) Level of IL-8 in the indicated groups were measured by ELISA. Data are expressed as the means ± SEM, n = 6. ##p < 0.01 and ###p < 0.001 compared to control group using unpaired t-test. Ns means no significant difference, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to CSE group using one-way ANOVA with Dunnett t-test for selected pairs. †††p < 0.001 compared to the CSE+S-CMC (10−4 M) treatment group using unpaired t-test.
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