Up-regulation of cyclin D1 expression in asthma serum-sensitized human airway smooth muscle promotes proliferation via protein kinase Cα

Abstract

ABSTRACTAbnormal hypertrophy and hyperplasia of airway smooth muscle cells play an important role in airway remodeling in chronic asthma. The authors’ previous studies have indicated that protein kinase Cα (PKCα) is involved in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). However, the underlying mechanisms remain unknown. Here, the authors examined the possible role of the α isoform of PKC in the control of cyclin D1 expression, using HASMCs passively sensitized on human atopic asthmatic serum as a model system. Cultured HASMCs were passively sensitized with serum from atopic asthmatic patients. Cell proliferation was measured by [3H]thymidine incorporation and an MTT assay. Cell cycle status was analyzed by flow cytometry. The mRNA and protein expression profiles of cyclin D1 were measured by reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blotting, respectively. Furthermore, the authors assessed the role of cyclin D1 in PKCα-induced HASMC proliferation by transfection with a recombinant cyclin D1 antisense construct. The activation of PKCα with phorbol myristate acetate (PMA), a PKC activator, up-regulated cyclin D1 expression and increased the proliferation of passively sensitized HASMCs. This effect was significantly decreased by specific inhibition of PKCα with Go6976. In addition, the authors showed that transfection with antisense cyclin D1 abolished PMA-induced G1/S progression and HASMC proliferation. These results demonstrate that PKCα promotes the proliferation of HASMCs sensitized with atopic asthmatic serum via up-regulation of cyclin D1 expression.