[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. CONCLUSION: 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.