Elisabet Wieslander

Novel conserved hydrolase domain in the CLCA family of alleged calcium-activated chloride channels.

Advanced protein structure prediction methods combined with structure modeling show that the mammalian proteins, described until now as calcium‐activated chloride channels (CLCAs), appear in fact to be membrane anchored metal‐dependent hydrolases, possibly proteases. A metallohydrolase structural domain was predicted, unexpectedly, in the CLCA sequences. The well‐conserved active site in the modeled structure of this hydrolase domain allows the prediction of catalytic action similar to that of metalloproteases. A number of protein structure prediction methods suggest the overall fold of the N‐terminal hydrolase domain to be most similar to that of zinc metalloproteases (zincins), notably matrixins. This is confirmed by analysis of the three‐dimensional structure model of the predicted CLCA1 hydrolase domain built using the known structure of the MMP‐11 catalytic domain. Fragments of CLCA1 corresponding to the modeled hydrolase domain were expressed in Escherichia coli, and the resulting proteins were readily refolded into monomeric soluble protein, indicating formation of stable independent domains. The homology model was used to predict putative substrate sequences. Homologs of mammalian CLCA genes were detected in the genomes of a vast array of multicellular animals: lower vertebrates, tunicates, insects, crustaceans, echinoderms, and flatworms. The hydrolase prediction is discussed in the context of published experimentally determined effects of CLCA proteins on chloride conductance. Altered proteolytic processing of full‐length CLCA1 containing a mutation abolishing the predicted hydrolase activity is shown as initial experimental evidence for a role of the hydrolase domain in processing of mature full‐length CLCA1. The hydrolase prediction together with the presented experimental data add to doubts about the function of CLCAs as chloride channels and strengthen the hypothesis of channel‐activating and/or channel‐accessory roles. Proteins 2006.

Agonist-specific patterns of beta(2)-adrenoceptor responses in human airway cells during prolonged exposure.

Background and purpose:u2002 β2‐Adrenoceptor agonists (β2‐agonists) are important bronchodilators used in the treatment of asthma and chronic obstructive pulmonary disease. At the molecular level, β2‐adrenergic agonist stimulation induces desensitization of the β2‐adrenoceptor. In this study, we have examined the relationships between initial effect and subsequent reduction of responsiveness to restimulation for a panel of β2‐agonists in cellular and in vitro tissue models.

Enhanced ROCK1 dependent contractility in fibroblast from chronic obstructive pulmonary disease patients

BackgroundDuring wound healing processes fibroblasts account for wound closure by adopting a contractile phenotype. One disease manifestation of COPD is emphysema which is characterized by destruction of alveolar walls and our hypothesis is that fibroblasts in the COPD lungs differentiate into a more contractile phenotype as a response to the deteriorating environment.MethodsBronchial (central) and parenchymal (distal) fibroblasts were isolated from lung explants from COPD patients (n = 9) (GOLD stage IV) and from biopsies from control subjects and from donor lungs (n = 12). Tissue-derived fibroblasts were assessed for expression of proteins involved in fibroblast contraction by western blotting whereas contraction capacity was measured in three-dimensional collagen gels.ResultsThe basal expression of rho-associated coiled-coil protein kinase 1 (ROCK1) was increased in both centrally and distally derived fibroblasts from COPD patients compared to fibroblasts from control subjects (pu2009<u20090.001) and (pu2009<u20090.01), respectively. Distally derived fibroblasts from COPD patients had increased contractile capacity compared to control fibroblasts (pu2009<u20090.01). The contraction was dependent on ROCK1 activity as the ROCK inhibitor Y27632 dose-dependently blocked contraction in fibroblasts from COPD patients. ROCK1-positive fibroblasts were also identified by immunohistochemistry in the alveolar parenchyma in lung tissue sections from COPD patients.ConclusionsDistally derived fibroblasts from COPD patients have an enhanced contractile phenotype that is dependent on ROCK1 activity. This feature may be of importance for the elastic dynamics of small airways and the parenchyma in late stages of COPD.

Anti-inflammatory effects of budesonide in human lung fibroblasts are independent of histone deacetylase 2

Objective and design Reduced 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. Methods Human 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. Results We 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. Conclusion These 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.

Effect of budesonide on fibroblast-mediated collagen gel contraction and degradation.

Background The balance between production and degradation of extracellular matrix is crucial in maintaining normal tissue structure. This study was designed to investigate the effect of budesonide on fibroblast-mediated tissue repair and remodeling. Methods Using human fetal lung fibroblasts in a three-dimensional collagen gel culture system, we investigated the effect of budesonide (1-1000 nM) on collagen gel contraction and degradation in the presence or absence of Inflammatory cytokines (interleukin-1β and tumor necrosis factor α; 5 ng/mL each) and, in order to activate latent proteases, serine protease trypsin 0.25 μg/mL. The effects of budesonide on metalloproteinase production and activation were also investigated. Results Inflammatory cytokines significantly inhibited collagen gel contraction mediated by lung fibroblasts. Budesonide counteracted the effect of cytokines in a concentration-dependent manner (to 50%, P< 0.01). Budesonide 100 nM almost completely inhibited the release and mRNA expression of metalloproteinase-1, metalloproteinase-3, and metalloproteinase-9 induced by the cytokines (P< 0.05). Exposure to the cytokines plus trypsin increased collagen degradation and conversion of the metalloproteinases to lower molecular weight forms corresponding to their active forms. Budesonide blocked both enhanced collagen degradation (P< 0.01) and suppressed trypsin-mediated conversion of cytokine-induced metalloproteinase-9 and metalloproteinase-3 to lower molecular weight forms. Similar effects were observed with dexamethasone 1 μM, suggesting a class effect. Conclusion These findings demonstrate that budesonide directly modulates contraction of collagen gels and can decrease collagen degradation under Inflammatory conditions. The mechanism of this effect is through suppressing gene expression, release, and activation of metalloproteinases. By modulating the release and activity of metalloproteinases, inhaled budesonide may be able to modify airway tissue repair and remodeling.