Abstract
BackgroundExposure to cigarette smoke is considered a major risk factor for the development of lung diseases, since its causative role has been assessed in the induction and maintenance of an inflamed state in the airways. Lung fibroblasts can contribute to these processes, due to their ability to produce proinflammatory chemotactic molecules and extracellular matrix remodelling proteinases. Among proteolytic enzymes, gelatinases A and B have been studied for their role in tissue breakdown and mobilisation of matrix-derived signalling molecules. Multiple reports linked gelatinase deregulation and overexpression to the development of inflammatory chronic lung diseases such as COPD.MethodsIn this study we aimed to determine variations in the gelatinolytic pattern of human lung fibroblasts (HFL-1 cell line) exposed to cigarette smoke extract (CSE). Gelatinolytic activity levels were determined by using gelatin zymography for the in-gel detection of the enzymes (proenzyme and activated forms), and the subsequent semi-quantitative densitometric evaluation of lytic bands. Expression of gelatinases was evaluated also by RT-PCR, zymography of the cell lysates and by western blotting.ResultsCSE exposure at the doses used (1–10%) did not exert any significant cytotoxic effects on fibroblasts. Zymographic analysis showed that CSE exposure resulted in a linear decrease of the activity of gelatinase A. Control experiments allowed excluding a direct inhibitory effect of CSE on gelatinases. Zymography of cell lysates confirmed the expression of MMP-2 in all conditions. Semi-quantitative evaluation of mRNA expression allowed assessing a reduced transcription of the enzyme, as well as an increase in the expression of TIMP-2. Statistical analyses showed that the decrease of MMP-2 activity in conditioned media reached the statistical significance (p = 0.0031 for 24 h and p = 0.0012 for 48 h), while correlation analysis showed that this result was independent from CSE cytotoxicity (p = 0.7833 for both exposures).ConclusionPresent work describes for the first time that, apart well characterized proinflammatory responses, human lung fibroblasts may react to CSE with a significant reduction of extracellular MMP-2 lytic activity. Therefore, fibroblasts may actively participate to the alteration of the proteolysis/antiproteolysis balance, which reflects the defective repair of the extracellular matrix. Such event should provide a further contribution to the maintenance of the inflamed state in the lungs.
Highlights
Exposure to cigarette smoke is considered a major risk factor for the development of lung diseases, since its causative role has been assessed in the induction and maintenance of an inflamed state in the airways
cigarette smoke extract (CSE) exposure of HFL-1 cells and cell viability Exposure of HFL-1 fibroblasts to aqueous extract of cigarette smoke was accomplished after a starvation period of 24 h, exposing cells to increasing (1–10%) concentrations of CSE for 24 h and 48 h
The negative regulation of matrix metalloproteinases (MMPs)-2 extracellular activity in CSE exposed cells is a result which may integrate the current models of COPD progression, which are focused on an increase of some extracellular lytic activities [14,34]
Summary
Exposure to cigarette smoke is considered a major risk factor for the development of lung diseases, since its causative role has been assessed in the induction and maintenance of an inflamed state in the airways. The generation of proteolytic fragments (matrikins) of ECM molecules by the proteolytic enzymes secreted by different cell types, may contribute to prolong the effects of inflammation even after the cessation of the causative stimulus. Different subgroups of MMPs have been characterised, on the basis of their substrate specificity (e.g. collagenases, elastases and gelatinases), even if different enzymes may share similar substrates This overlap of target molecules, both ECM structural proteins and regulatory ones, reflects the complex organisation of matrix microenvironmental regulation. As recently stated, gelatinases produced by structural cells (as demonstrated for mouse lung fibroblasts) may play a role in the pathogenesis of COPD, and this process seems to be regulated by CSE exposure [18]. The observed modulator effect of CSE on fibroblast-secreted gelatinases may in part explain the effects due to cigarette smoke exposure in vivo, and confirm the recent hypotheses on the central role of fibroblasts in the development of chronic lung diseases
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