| COPD is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious particles or gases. There is no a proper drug can arrest the development of COPD, nowadays. Corticosteroids are very effective in most patients with asthma, but they are much less effective in those with COPD, although in both diseases there is an active inflammatory process in the respiratory tract. It is a possible hypothesis that the corticosteroid responses are decreased in patients with COPD.In eukaryotes, the four core histones, involved in forming the nucleosome, can undergo post synthetic acetylation of one or more lysine residues in the N-terminal regions of these molecules, providing an action of gene transcription regulation. Corticosteroids produce their effects on responsive cells by activating glucocorticoid receptors to directly or indirectly regulate the transcription of target genes. Corticosteroids, by glucocorticoid receptor, act both as a direct inhibitor of CBP/p300 associated HAT activity and also by recruiting HDAC2 to the CBP/PCAF HAT complex, which is activated by pro-inflammatory transcription factors, such as NF-κB and AP-1.Many studies indicate that cigarette smoke and oxidant stress to alveolar macrophages may also increase gene expression of inflammatory mediators, with induction of histone acetylation, because of decline of HDAC activity in cells. Meanwhile, alveolar macrophages from persons with smoking are revealed increased release of IL-8 and TNF-α compared with cells from controls after stimulated by IL-1β, which is poorly suppressed by dexamethasone. Therefore, there is a relationship between the expression and activity of HDAC and steroid responses. It is unknown whether the status and its mechanism lie in pulmonary epithelial cells is similar with them in alveolar macrophages, and further study is necessary.Part 1The preparation and determination of cigarette smoke extract and its effects of proliferative inhibition on human alveolar epithelial like cells A549Objective: To establish a set of methods used to the preparation and determination of CSE and to determinate the effects and potential mechanisms of cell proliferation on human alveolar epithelial like cells A549. Methods: The apparatus for preparation cigarette smoke extract was constructed using a peristaltic pump. The concentration of nicotine, one of the relatively stable constituents in CSE, was assessed with Rp-HPLC. MTT assay and LDH assay were used for determining of A549 cells proliferation and cytotoxicity induced by CSE, respectively. AO/PI staining was determined apoptosis and necrosis in cells treated with CSE. Using Western blot detected the protein expression of the pro- and anti-apoptotic molecules. Results: The mean concentration of nicotine of six separated CSE preparations was 369.2 ± 21.6μg/ml (CV 5.9%), detected by Rp-HPLC. CSE inhibited A549 cellsproliferation in a concentration- and time- dependent manner. The IC50 and IC5 values were 37.7%(95%CI, 9.1% ～ 34.6%) and 5.1% (95%CI, 1.6% ～ 8.1%), respectively. AO/PI staining revealed that CSE induced necrosis in a concentration-dependent manner, with little evidence of apoptosis in A549 cells. The necrotic cell rate was 7.3% ± 3.3% and 34.4% ± 6.7%, and apoptotic cell rate was 3.8%±1.1%, in the groups with 10% and 25% CSE stimulation, respectively. The caspase3 and caspase9 protein levels were similar between cells incubated with CSE and blank. Interestingly, survivin and XIAP were up-regulated in a concentration-dependent manner after CSE intervention. There were different trends in expression of survivin and XIAP, such as decreased expression of survivin with 25% CSE, but XIAP not yet. The detection of IκB, phospho- IκB, and RelA/p65 expression with Western blot revealed that the levels of these molecules were not changed after CSE stimulation, and the same results of RelA/p65 in cytoplasmic extract and nuclear extract.Part 2Cigarette smoke reduced HDAC2 activity and decreased steroid responses in an alveolar type II cell-derived cell line A549 via tyrosine nitration and sumoylationObjective: To observe the effects of CSE on the expression levels of pro-inflammatory cytokines and chemokines in A549 cells. And to observe the relationship between the acetylation status of histone H4 and HDAC activity. Then, to determinate the regulation between HDAC2 activity and steroid responses. Methods: The expression levels of IL-1β, IL-8, GM-CSF, MCP-1, MIP-1α, RANTES mRNA were detected using semi-quantitative RT-PCR in A549 cells after stimulations. Indirect immunocytoflurescence staining of acetylated H4 was carriedout to determinate effects of several stimuli on acetylation of histone 4. The HDAC2 protein levels were detected using specific primary antibody by Western blot. Real time quantitative RT-PCR was used to detect the expression of HDACs and HATs mRNA. At last, HDAC2 immunoprecipitation and modified HDAC2 assay were carried out. Results: CSE could significantly increase the levels of IL-1β mRNA(.P < 0.01) but little affect gene transcription of GM-CSF, IL-8, MCP-1, MIP-1α, and RANTES (all P>0.05) in A549 cells. The changes of IL-1β mRNA expression were not statistical significant in cells treated with 10-10M and 10-8M dexamethasone (all P > 0.05). Although the treatment with 10-6M dexamethasone could inhibit the expression of IL-1β mRNA induced by CSE (P < 0.05), the results were also much higher than control (P < 0.05). CSE promoted the acetylation of histone H4 in stimulated cells(P < 0.01) by significant decline of the expression and activity of HDAC2(P < 0.01). But the influences of dexamethasone intervention on HDAC2 protein levels and activity were not found in cells treated with CSE. The results in further studies showed no alteration of HDACs mRNA levels not only in CSE incubated cells but in cells with dexamethasone intervention(P > 0.05). There were a significant increase in tyrosine nitration modification of HDAC2 and a significant decrease in SUMO-1 modification of HDAC2 after 24hr of CSE exposure, and the same results with dexamethasone intervention compared with untreated cells.Part 3The regulation of activities of HDAC2 by the modification of small ubiquitin related modifier-1Objective: To identify the SUMO-1 binding site in HDAC2 with HDAC2 cDNA cloning and site-directed point mutation, and ascertain the effect and potential mechanism of HDAC2 sumoylation on its biological functions. Methods: Theplasmids, including HDAC2 full length ORF sequences, SUMO-1 97GG sequences, and ubiquitin mature peptide sequence in their cDNA, were constructed using PCR and cloning techs. And a PCR based site-directed point mutation was carried out with HDAC2-HA DNA construction. The vectors were transfected into Hela cells using lipofectamine2000. And then SUMO-1 binding site identification, HDAC2 activity and its interactions with co-factors were determined by immunoprecipitation and Western blot. Results: The vector constructions, such as HDAC2-HA, SUMO-1-FLAG, Ub-FLAG, and HDAC2-K462R, were verified by DNA sequencing. Hela cells cotransfected with plasmids, using HDAC2-HA, HDAC2-K462R and SUMO-1-FLAG, Ub-FLAG, identified that the K462 site in HDAC2 could sumoylated by SUMO-1. The HDAC2 modification of SUMO-1 didn't affect the ubiquitination of HDAC2. Compared with wild type HDAC2, the activity of K462R mutation in HDAC2 decreased significantly (P < 0.01). The results of studies revealed that K462R mutation, compared with wild type protein, declined the interaction with CoREST and HDAC1, but did not affect the interaction with mSin3A, Mi2, RbAp46/48, and YY1.Conclusions(1) We had established a set of methods used the preparation and determination of cigarette smoke extract by an apparatus including a peristaltic pump and HPLC instrument, as the basis of further researching works, because the methods were simple and reliable to routine applications.(2) The obvious cytotoxicity in A549 cells stimulated by CSE was found in our studies, but the cytotoxic effects caused by CSE were concerned with necrosis and with little evidence of apoptosis. It was suggested that up-regulating survivin and XIAP, by a concentration dependent manner, and the inhibition of caspase3 and caspase9 cleavage played a key role in the phenomena induced byCSE. And then, the increased expression of survivin and XIAP by CSE stimulation in A549 cells could not be involved in activation of NF-κB pathway and nuclear translocation of RelA/p65.(3) CSE accelerated the acetylation of hitone H4 and regulated chromatin remodeling via inhibition of histone deacetylase activity, and consequently induced expression of pro-inflammatory cytokine, such as IL-1β, in mRNA level. The expression levels of IL-1β mRNA in cells with CSE incubation were not suppressed after dexamethasone intervention indicated that CSE could decline steroid responses. Cigarette smoke reduced HDAC2 activity and decreased steroid responses in an alveolar type Ⅱ cell-derived cell line A549 partly via tyrosine nitration and sumoylation, at least.(4) The site of K.462 in the sequence of HDAC2 was a key site during the sumoylation in HDAC2 by SUMO-1. The modification of SUMO-1 could influence on the activity of HDAC2. The results suggested that SUMO-1 didn't compete with ubiquitination of HDAC2 on a level that affected protein function. As a potential mechanism demonstrated, alteration of HDAC activity by sumoylaion was related to destroy the integrity of CoREST complex.
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