Effect of cigarette smoke extract on the role of protein kinase C in the proliferation of passively sensitized human airway smooth muscle cells

Abstract

To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A1, A2 and A3 and the group B was sub-divided into the group of B1, B2, B3, B4 and B5. No other agents were added to the group A1 and B1. The cells of group A2 and B2 were stimulated with 5% CSE for 24 h. HASMCs from group A3 and B3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5%) for 24 h. PKC inhibitor Ro-31-8220 (5 micromol/L) was added to the HASMCs of group B4 for 24 h. The cells from group B5 were stimulated with Ro-31-8220 (5 micromol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-a in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-alpha mRNA and the A value of PKC-alpha protein in HASMCs from group B1, B2 and B3 were significantly increased compared to those of group A1, A2 and A3 correspondingly and respectively (P< 0.01). The proliferation of HASMCs of group A2 and B2 stimulated with CSE and group A3 and B3 stimulated with CSE and PMA were also significantly enhanced when group A1, A2 and A3 and group B1, B2 and B3 compared to each other (P<0.05, P<0.01, respectively). The percentage of S phase, absorbency (A) value, 3H-TdR incorporation rate, the ratios of A value of PKC-alpha mRNA and the A value of PKC-alpha protein in HASMCs from group B4 treated with Ro-31-8220 and group B5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B1 and B2 correspondingly and respectively (P<0.05, P<0.01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.