Protease Activated Receptor-2 Expression and Function in Asthmatic Bronchial Smooth Muscle

<div><p>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.</p><p>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.</p><p>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.</p><p>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.</p></div

Increased PAR-2 expression in asthmatic bronchial smooth muscle cells.

<p>PAR-2 levels were assessed by flow cytometry (A), western blot (B) and quantitative RT-PCR (C). Normalized median fluorescence intensities were calculated by dividing median fluorescence intensity of PAR-2 by that of isotype control (A). Representative blots stained with anti–PAR-2 or anti–β-actin antibodies are shown (B). The RT-PCR expression of PAR-2 was presented as an arbitrary unit and normalized to endogenous references (geometric averaging of three internal control genes; <i>i.e.</i> YWHAZ, HPRT-1, and PO) according to GeNorm (C). Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 7) and control subjects (white bars, n = 7). Results are expressed as mean ± SD. *<i>P</i><0.05 using Mann & Whitney test.</p

Increased PAR-2 dependent calcium responses in control bronchial smooth muscle cells over-expressing PAR-2.

<p>The effects of lentivirus over-expressing PAR-2 (squared bars) in control bronchial smooth muscle cells were evaluated as compared to both control bronchial smooth muscle cells transduced by control lentivirus (hatched bars) and non transduced asthmatic bronchial smooth muscle cells (black bars). PAR-2 surface protein expression was assessed by flow cytometry (A). Relative calcium response ([Ca²⁺]_i peak, B) and area under the curve (AUC [Ca²⁺]_i, C) were assessed by microspectrofluorimetry from the cell response to 10⁻⁴ M SLIGKV-NH₂. Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 3) and control subjects (white bars, n = 3). Results are expressed as mean ± SEM. Calcium responses were obtained from a range of 9 to 35 cells per patient. * <i>P</i><0.05 using paired Wilcoxon-rank tests and Mann & Whitney test.</p

Clinical and functional characteristics of subjects.

<p>Data are mean ± SD. BMI: body mass index. OCS: oral corticosteroid. ICS: inhaled corticosteroid. LABA: long acting beta2 agonist. FEV1: forced expiratory volume in one second. FVC: forced vital capacity. FEF 25–75: forced expiratory flow between 25 and 75% of FVC.</p

Increased asthmatic bronchial smooth muscle cell phosphorylation of ERK and p38 following repeated PAR-2 stimulations.

<p>Phosphorylation of ERK (A) and p38 (B) was measured using western blot following stimulation for 0, 1 or 3 days by 10⁻⁴ M SLIGKV-NH₂. Representative blots stained with anti-Phospho-ERK (P-ERK), anti–ERK, anti-Phospho-p38 (P-p38) and anti-p38 antibodies are shown. Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 8) and control subjects (white bars, n = 7). Results are expressed as mean ± SD. *<i>P</i><0.05 using paired Wilcoxon-rank tests.</p

Increased PAR-2 dependent calcium response in asthmatic bronchial smooth muscle cells.

<p>Representative intracellular calcium responses following stimulation by 10⁻⁴ M SLIGKV-NH₂ for 30 sec are presented in bronchial smooth muscle cells from asthmatic (black line) or control subjects (grey line) (A). Basal calcium concentration (Basal [Ca²⁺]_i, B), relative calcium response ([Ca²⁺]_i peak, C) and area under the curve (AUC [Ca²⁺]_i, D) were assessed from cell response to 10⁻⁴ M SLIGKV-NH₂. Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 3) and control subjects (white bars, n = 3). Results are expressed as mean ± SEM from a range of 14 to 41 cells per patient. *<i>P</i><0.05 using Mann & Whitney test.</p

Increased asthmatic bronchial smooth muscle cell proliferation following repeated PAR-2 stimulations.

<p>Proliferation was measured using BrdU incorporation following stimulation for 1 or 3 days by 10⁻⁴ M SLIGKV-NH₂ or VKGILS-NH₂ (A). Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 5) and control subjects (white bars, n = 5). PAR-2 expression was assessed by flow cytometry following stimulation for 1 or 3 days by 10⁻⁴ M SLIGKV-NH₂ or VKGILS-NH₂ (B). Normalized median fluorescence intensities were calculated by dividing median fluorescence intensity of PAR-2 by that of isotype control. Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 6) and control subjects (white bars, n = 6). Relative calcium response was assessed by microspectrofluorimetry from the cell response to 10⁻⁴ M SLIGKV-NH₂ following stimulation for 1 or 3 days by 10⁻⁴ M SLIGKV-NH₂ (C). Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 3) and control subjects (white bars, n = 3). Calcium responses were obtained from a range of 16 to 35 cells per patient. Proliferation was measured using BrdU incorporation following stimulation for 1 or 3 days by 10⁻⁴ M SLIGKV-NH₂ or VKGILS-NH₂ (D). Bronchial smooth muscle cells obtained from control subjects were transduced with control lentivirus (hatched bars, n = 6) or lentivirus over-expressing PAR-2 (squared bars, n = 6). Results are expressed as mean ± SEM. *<i>P</i><0.05 using paired Wilcoxon-rank tests and Mann & Whitney tests.</p