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

Summary

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 μmol/L) was added to the HASMCs of group B₄ for 24 h. The cells from group B₅ were stimulated with Ro-31-8220 (5 μmol/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 and³H-TdR incorporation test. The expression of PKC-α 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 of³H-TdR incorporation, the ratios ofA value of PKC-α mRNA and theA value of PKC-α 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 ofA value of PKC-α mRNA and theA value of PKC-α 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.