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Apoptosis in Vascular Smooth Muscle Cells is induced by Protein Kinase C Delta Cleavage but not Phosphorylation
Author(s) -
Kato Kaori,
Kamiya Kentaro,
Wang Chunjie,
Kent K. Craig,
Liu Bo
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.610
Subject(s) - apoptosis , phosphorylation , protein kinase c , western blot , dna fragmentation , kinase , microbiology and biotechnology , vascular smooth muscle , cleavage (geology) , chemistry , biology , programmed cell death , biochemistry , endocrinology , smooth muscle , paleontology , fracture (geology) , gene
Oxidative stress and apoptosis are considered critical to many vascular diseases. We have previously shown that protein kinase C delta (PKCδ) plays a critical role in early stage apoptosis. In this study, we examined various activation of PKCδ and its relationship with apoptosis and necrosis in vascular smooth muscle cells (SMCs). ELISA for DNA fragmentation first showed that in rat A10 SMC line, 100–400 μM hydrogen peroxide (H 2 O 2 ) results in a 1.21–1.59 fold increase in apoptosis, but a decrease after 800 μM. This was confirmed by flow cytometry where we found increasing apoptosis at low doses of H 2 O 2 and increasing necrosis at higher doses. Western blot for PKCδ's constitutively active catalytic fragment revealed that it was significantly increased after treatment with 100–400 μM H 2 O 2. To test the impact of these results on apoptosis, we used a competitive inhibitor (z‐DIPD‐fmk) that mimics the caspase‐3 cleavage site on PKCδ thus inhibiting its cleavage, and found it to inhibit apoptosis. We also examined the role of PKCδ phosphorylation at Tyr‐311 and Tyr‐332. Western Blot showed increased phosphorylation only at high doses of H 2 O 2. Inhibition of phosphorylation by Src Family Kinase inhibitors PP2 (10 μM) and SU6656 (5μM) increased apoptosis by 1.72 and 2.5 fold. Our results suggest that the cleavage of PKCδ induces apoptosis but its phosphorylation is anti‐apoptotic and may be driving SMCs toward necrosis.