Abstract
Asthmatic bronchial smooth muscle (BSM) is characterized by structural remodeling associated with mast cell infiltration displaying features of chronic degranulation. Mast cell-derived tryptase can activate protease activated receptor type-2 (PAR-2) of BSM cells. The aims of the present study were (i) to evaluate the expression of PAR-2 in both asthmatic and non asthmatic BSM cells and, (ii) to analyze the effect of prolonged stimulation of PAR-2 in asthmatic BSM cells on cell signaling and proliferation.BSM cells were obtained from both 33 control subjects and 22 asthmatic patients. PAR-2 expression was assessed by flow cytometry, western blot and quantitative RT-PCR. Calcium response, transduction pathways and proliferation were evaluated before and following PAR-2 stimulation by SLIGKV-NH2 or trypsin for 1 to 3 days.Asthmatic BSM cells expressed higher basal levels of functional PAR-2 compared to controls in terms of mRNA, protein expression and calcium response. When PAR-2 expression was increased by means of lentivirus in control BSM cells to a level similar to that of asthmatic cells, PAR-2-induced calcium response was then similar in both types of cell. However, repeated PAR-2 stimulations increased the proliferation of asthmatic BSM cells but not that of control BSM cells even following lentiviral over-expression of PAR-2. Such an increased proliferation was related to an increased phosphorylation of ERK in asthmatic BSM cells.In conclusion, we have demonstrated that asthmatic BSM cells express increased baseline levels of functional PAR-2. This higher basal level of PAR-2 accounts for the increased calcium response to PAR-2 stimulation, whereas the increased proliferation to repeated PAR-2 stimulation is related to increased ERK phosphorylation.
Highlights
Asthma is a chronic inflammatory disease characterized by bronchial hyperresponsiveness and airway remodeling [1]
protease activated receptor type-2 (PAR-2) surface expression was increased in asthmatic bronchial smooth muscle (BSM) cells compared to controls (Mean fluorescence intensity: 2.3610460.9 vs. 0.3610460.2, respectively) (Figure 1A)
The total protease activated receptors (PAR)-2 protein and mRNA expressions were increased in asthmatic BSM cells compared to controls using western blot (Figure 1B) and quantitative RT-PCR (Figure 1C), respectively
Summary
Asthma is a chronic inflammatory disease characterized by bronchial hyperresponsiveness and airway remodeling [1]. Tryptase has been shown to activate protease activated receptors (PAR) that are expressed at the site of the BSM [10] Among these receptors, the subtype 2 (PAR-2), plays a major role in bronchial hyperresponsiveness [11], and BSM cell calcium rise [12,13], as evidenced by pharmacological and RNA interference tools [13]. The subtype 2 (PAR-2), plays a major role in bronchial hyperresponsiveness [11], and BSM cell calcium rise [12,13], as evidenced by pharmacological and RNA interference tools [13] Taken together, all these findings suggest the presence of an auto-activation loop in asthma, involving mast cells and their mediators including tryptase. Mast cells chronically stimulate PAR-2 in BSM cell inducing bronchial hyperresponsiveness and chemotactic activity, which in turn recruits new mast cells [3]
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