Objective and designReduced expression of histone deacetylase 2 (HDAC2) in alveolar macrophages and epithelial cells may account for reduced response of chronic obstructive pulmonary disease (COPD) patients to glucocorticoids. HDAC2 expression and its role in mediating glucocorticoid effects on fibroblast functions, however, has not been fully studied. This study was designed to investigate whether HDAC2 mediates glucocorticoid effects on release of inflammatory cytokines and matrix metalloproteinases (MMPs) from human lung fibroblasts.MethodsHuman lung fibroblasts (HFL-1 cells) were stimulated with interleukin (IL)-1 β plus tumor necrosis factor (TNF)-α in the presence or absence of the glucocorticoid budesonide. Cytokines (IL-6 and IL-8) were quantified by enzyme linked immunosorbent assay (ELISA) and MMPs (MMP-1 and MMP-3) by immunoblotting in culture medium. The role of HDAC2 was investigated using a pharmacologic inhibitor as well as a small interfering ribonucleic acid (siRNA) targeting HDAC2.ResultsWe have demonstrated that budesonide concentration-dependently (10−10–10−7 M) inhibited IL-6, IL-8, MMP-1, and MMP-3 release by HFL-1 cells in response to IL-1β plus TNF-α. While an HDAC inhibitor significantly blocked the inhibitory effect of budesonide on human bronchial epithelial cells (HBECs) and monocytes (THP-1 cells), it did not block the inhibitory effect of budesonide on release of cytokines and MMPs from HFL-1 cells. Similarly, an HDAC2-siRNA blocked budesonide inhibition of cytokine release in HBECs, but it did not block the inhibitory effect of budesonide on HFL-1 cytokine and MMP release. Furthermore, budesonide significantly blocked release of cytokines and MMPs to a similar degree in normal and COPD lung fibroblasts as well as in HFL-1 cells exposed or not exposed to cigarette smoke extract.ConclusionThese findings suggest that, in contrast to airway epithelial cells and monocytes/macrophages, HDAC2 is not required for budesonide to inhibit MMP and cytokine release by lung fibroblasts and this inhibitory pathway appears to be intact in cultured fibroblasts from COPD patients. These results also suggest that budesonide has the potential to modulate fibroblast-mediated tissue remodeling following airway inflammation in COPD, which is mediated via an HDAC2 independent pathway.

MicroRNAs and other small noncoding RNAs regulate nearly 50% of genes at either the transcriptional or Rationale and Objectives: post-transcriptional levels. It is therefore not surprising that the expression of the miRNA genes themselves is tightly regulated. We previously implicated differential expression of miRNA 146a in COPD and control fibroblasts and showed that its target site in the 3'-UTR of COX-2 regulates levels of this protein, and consequent PGE production. We hypothesize that differential expression of miRNA 146a in COPD and control fibroblasts may be due to single nucleotide polymorphisms in this gene.Genomic DNA was isolated from 10 fibroblast strains obtained from the human lungs of control and COPD subjects. Material and Methods: The complete coding region of the pre-miRNA146a (99bp) as well as 5 kb of 5' genomic and 1 kb of 3'genomic was resequenced.A total of 3 single nucleotide polymorphisms were identified, all of which are novel. The SNPs were located at -853A/C, -1135G/A Results: and -3014G/A relative to the pre-miRNA 146a start site. Within 25 bp of the -1135 and -3014 polymorphic sites, the OCT1 transcription factor was predicted to have multiple binding sites (MatInspector prediction software 8.0). The polymorphism at -853 had no predicted transcription factor binding sites.These data suggest that single nucleotide polymorphisms are common in the miRNA146a regulatory region. In the future we Conclusion: intend to screen a total of 20 control and 20 COPD fibroblast strains for single nucleotide polymorphisms and relate these polymorphisms to the clinical phenotypes of COPD. This abstract is funded by: State of Nebraska LB506 Am J Respir Crit Care Med 183;2011:A3526 Internet address: www.atsjournals.org Online Abstracts Issue