Molecular characterization of rabbit CPP32 and its function in vascular smooth muscle cell apoptosis.

Vascular remodeling in atherogenesis is marked not only by cellular proliferation and migration but is also impacted by apoptotic cell death. Extensive studies have focused on the signal transduction events leading to apoptosis. CPP32, a member of the caspase/interleukin-1 beta-converting enzyme (ICE) protease family, has emerged as a central player in several reports of apoptosis pathways. Vascular smooth muscle cells (SMC) undergo apoptosis after treatment with various stimuli, including nitric oxide (NO) donors, such as sodium nitroprusside (SNP, 0.1-1 mM). The aim of the present study was to evaluate the role of CPP32 in SNP-induced apoptosis of SMC. We isolated a rabbit CPP32 cDNA by using degenerate primers and polymerase chain reaction technique. The predicted protein encoded by this cDNA contains the conserved sequence (QACRG) necessary for covalent linkage to poly(ADP-ribose) polymerase as well as the three amino acids responsible for substrate recognition and catalysis reported in other caspase members. Using a segment of this cDNA as a probe, we found no change of CPP32 mRNA in cultured arterial SMC before and after SNP treatment. We also measured the protease activity of CPP32 against a chromophore p-nitroaniline (pNA)-labeled substrate, DEVD-pNA. Our results showed a dose-dependent increase of CPP32 activity in SMC, with a maximal 10-fold increase after SNP treatment. Addition of a competitive CPP32 inhibitor, DEVD-CHO, produced a 50% reduction in maximal stimulation. Immunoblot analysis illustrated that SNP treatment induced proteolytic cleavage of CPP32 into its enzymatically active subunit p17 as well as the degradation of into a 85-kDa fragment. We further demonstrated that incubation of cultured SMC with DEVD-CHO significantly reduced SNP-induced DNA fragmentation. DNA fragmentation analysis was carried out using several methods including a cell death detection enzyme-linked immunosorbent assay kit, in situ end labeling, and DNA electrophoresis in agarose gel. Our data indicate that CPP32 mRNA is constitutively expressed in rabbit SMC and activation of CPP32 protein has a pivotal role in SNP-induced SMC apoptosis.

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