Effect of cigarette smoke extract on proliferation of rat pulmonary artery smooth muscle cells and the relevant roles of protein kinase C.
BACKGROUND
Increased proliferation of pulmonary vascular cells and muscularisation of pulmonary vessels are frequently observed in human smokers and in animals exposed to cigarette smoke. To elucidate the molecular mechanisms leading to these changes, we studied the in vitro effect of cigarette smoke extract (CSE) on proliferation of pulmonary artery smooth muscle cells (PASMCs) and activation of protein kinase C (PKC), an important kinase implicated in cell proliferation.


METHODS
PASMCs cultured from 12 normal Wistar rats were studied in the following conditions: (1) PASMCs were exposed to different concentrations of CSE for 24 hours, then MTT colorimetric assay was used for detection of cell proliferation. Cell viability was assessed by trypan blue exclusion. (2) PASMCs were pre-incubated with phorbol 12-myristate 13-acetate (PMA) for 24 hours or Ro31-8220 for 30 minutes before exposure to 5% CSE for 24 hours. Cell proliferation was examined by MTT colorimetric assay, cell cycle analysis and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. (3) PASMCs were exposed to 5% CSE for 24 hours. Then PKC-alpha mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and protein expression by Western blotting, while PKC-alpha translocation was observed by immunofluorescence staining and confocal microscopy. (4) PASMCs were transfected with specific antisense oligodeoxynucleotides against PKC-alpha 6 hours before exposure to 5% CSE for 24 hours. PKC-alpha protein expression and cell proliferation were detected by methods described previously.


RESULTS
(1) Low concentration of CSE (5%) increased proliferation of PASMCs, whereas high concentrations (20%, 30%) were inhibitory as a result of cytotoxicity. (2) The value of absorbance (Value A), proliferation index (PI), S-phase cell fraction (SPF) and average optical density of PCNA staining in PASMCs from 5% CSE exposure group (0.306 +/- 0.033, 0.339 +/- 0.033, 0.175 +/- 0.021, 0.315 +/- 0.038, respectively) were significantly increased compared with those of control group (0.249 +/- 0.018, 0.177 +/- 0.055, 0.092 +/- 0.023, 0.187 +/- 0.022, respectively) (P < 0.05). PKC down-regulation by PMA pretreatment or PKC inhibition by Ro31-8220 pre-incubation abolished the effect of 5% CSE on PASMCs proliferation. (3) After exposure to 5% CSE for 24 hours, PKC-alpha mRNA and protein expression in PASMCs (1.054 +/- 0.078, 1.185 +/- 0.041, respectively) were much higher than in untreated cells (0.573 +/- 0.054, 0.671 +/- 0.055, respectively) (P < 0.01). Moreover, 5% CSE induced a translocation of PKC-alpha from cytoplasm toward the perinuclear area and into the nucleus. (4) Specific antisense oligodeoxynucleotides against PKC-alpha reduced 5% CSE-induced expression of PKC-alpha protein (0.713 +/- 0.047 vs 1.180 +/- 0.056), also abolished the effect of 5% CSE on PASMCs proliferation significantly.


CONCLUSIONS
CSE can be cytotoxic at high concentrations. But at low concentrations, it makes a mitogenic effect on cultured PASMCs. PKC, especially its alpha isozyme, seems to play an important role in CSE-induced proliferation of PASMC.
Hu
Jing
J
Department of Respiratory Medicine, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Xu
Yong-jian
YJ
Zhang
Zhen-xiang
ZX
Tian
Feng
F
D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D007211:Indoles / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000502:physiology*; D011651:Pulmonary Artery / Q000166:cytology*; D051381:Rats; D017208:Rats, Wistar; D012906:Smoke; D014026:Nicotiana
D007211:Indoles; D020319:Oligodeoxyribonucleotides, Antisense; D012906:Smoke; D051571:Protein Kinase C-alpha; C064758:Ro 31-8220