[PKCalpha-ERK1/2 cascade is involved in up-regulation of cyclinD1 and P21(cip1) in human airway smooth muscle cells sensitized by sera from atopic asthmatics].
OBJECTIVE
To explore the role of PKCalpha-ERK1/2 cascade in PMA induced up-regulation of cyclinD1 and P21(cip1) in human airway smooth muscle cells (HASMCs) sensitized by sera from atopic asthmatics.


METHODS
HASMCs in cultures were passively sensitized by 10% serum from asthmatic patients and were randomly divided into five groups: the control group, PMA treated group, PMA and PKCalpha mismatched Oligodeoxynucleotides (PKCalpha-mmODN) treated group, PMA and PKCalpha antisense Oligodeoxynucleotides (PKCalpha-asODN) treated group, PMA and U0126 (MAP Kinase Kinase inhibitor)treated group. The expression of p-PKCalpha, ERK1/2, p-ERK1/2, cyclinD1 and P21(cip1) protein were determined by western blotting. The proliferation of HASMC was examined by cell cycle analysis and MTT colorimetric assay.


RESULTS
Compared with the control group, the expression of p-PKCalpha and ERK1/2, p-ERK1/2 protein increased, the expression of cyclinD1, P21(cip1) protein increased correspondingly (the A value % control was 2.10 +/- 0.29, 1.67 +/- 0.19, 2.20 +/- 0.27, 1.99 +/- 0.22 and 3.11 +/- 0.29 respectively; q value was 9.87, 7.06, 10.57, 11.10 and 20.33 respectively; all P < 0.05) in PMA treated group, and cells proliferation [the percentage of cells in S phase was (30.3 +/- 2.4)%, A(490) value was 0.80 +/- 0.06] enhanced significantly compared with those [the percentage of cells in S phase was (13.9 +/- 2.6)%, A(490) value was 0.41 +/- 0.04] of the control group (q = 6.07, 12.63; all P < 0.05). In PMA and PKCalpha-asODN treated group, the level of p-PKCalpha decreased, the expression of ERK1/2, p-ERK1/2 and the expression of cyclinD1, P21(cip1) decreased correspondingly (the A value % control was 1.23 +/- 0.19, 1.34 +/- 0.18, 1.52 +/- 0.20, 1.45 +/- 0.18 and 1.49 +/- 0.18 respectively; q value was 7.49, 3.58, 5.97, 6.06 and 15.65 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (21.2 +/- 2.8)%, A(490) value was 0.51 +/- 0.04; q = 6.07, 12.63; all P < 0.05], as compared with those of the PMA treated group. In PMA and U0126 treated group, the level of p-PKCalpha had no significant change (A value was1.99 +/- 0.18, q = 0.94, P > 0.05), but the levels of ERK1/2, p-ERK1/2 decreased, the expression of cyclinD1, P21(cip1) reduced (the A value % control was 0.95 +/- 0.21, 1.15 +/- 0.19, 1.37 +/- 0.15 and 1.96 +/- 0.21 respectively; q value was 7.79, 9.16, 6.92 and 11.16 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (22.0 +/- 3.2)%, A(490) value was 0.49 +/- 0.03; q = 5.51, 13.45; all P < 0.05], as compared with those of the PMA treated group.


CONCLUSIONS
ERK1/2 is one of the downstream regulators of PKCalpha, and PKCalpha-ERK1/2 cascade is involved in PMA induced up-regulation of cyclinD1 and P21(cip1) and proliferation in HASMC sensitized by sera from atopic asthmatics.
DU
Chun-Ling
CL
Pulmonary Disease Laboratory of Ministry of Health of China, Department of Respiratory Medicine, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Xu
Yong-Jian
YJ
Liu
Xian-Sheng
XS
Xie
Jun-Gang
JG
Zhang
Zhen-Xiang
ZX
Zhang
Jian
J
Qiao
Li-Fen
LF
Ni
Wang
W
Chen
Shi-Xin
SX
D000328:Adult; D001249:Asthma / Q000097:blood / Q000378:metabolism*; D002453:Cell Cycle; D049109:Cell Proliferation; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008875:Middle Aged; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D015398:Signal Transduction; D015854:Up-Regulation; D055815:Young Adult
D050759:Cyclin-Dependent Kinase Inhibitor p21; D019938:Cyclin D1; D051571:Protein Kinase C-alpha; D048049:Extracellular Signal-Regulated MAP Kinases; D048052:Mitogen-Activated Protein Kinase 3