Cigarette smoke exposure inhibits extracellular MMP-2 (gelatinase A) activity in human lung fibroblasts.
BACKGROUND
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. 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.


METHODS
In 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.


RESULTS
CSE 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).


CONCLUSIONS
Present 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.
La Rocca
Giampiero
G
Sezione di Anatomia Umana, Dipartimento di Medicina Sperimentale, Università degli Studi di Palermo, Via del Vespro 129, 90127 Palermo, Italy. giampylr@hotmail.com
Anzalone
Rita
R
Magno
Francesca
F
Farina
Felicia
F
Cappello
Francesco
F
Zummo
Giovanni
G
D016923:Cell Death; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004781:Environmental Exposure / Q000032:analysis; D004784:Environmental Monitoring; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D008168:Lung / Q000201:enzymology*; D020778:Matrix Metalloproteinase 2 / Q000187:drug effects* / Q000378:metabolism*; D010936:Plant Extracts / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism; D014028:Tobacco Smoke Pollution / Q000032:analysis*
D017077:Culture Media, Conditioned; D010936:Plant Extracts; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D014028:Tobacco Smoke Pollution; D020778:Matrix Metalloproteinase 2