P38 Kinase Regulates Nitric Oxide-induced Apoptosis of Articular Chondrocytes by Accumulating p53 via NFκB-dependent Transcription and Stabilization by Serine 15 Phosphorylation

Song-Ja Kim,Sang-Gu Hwang,Deug Y Shin,Shin-Sung Kang,Jang-Soo Chun

doi:10.1074/jbc.m202862200

Abstract

Nitric oxide (NO) during primary culture of articular chondrocytes causes apoptosis via p38 mitogen-activated protein kinase in association with elevation of p53 protein level, caspase-3 activation, and differentiation status. In this study, we characterized the molecular mechanism by which p38 kinase induces apoptosis through activation of p53. We report here that NO-induced activation of p38 kinase leads to activation of NFkappaB, which in turn induces transcription of the p53 gene. Activated p38 kinase also physically associates and phosphorylates the serine 15 residue of p53, which results in accumulation of p53 protein during NO-induced apoptosis. Ectopic expression of wild-type p53 enhanced NO-induced apoptosis, whereas expression of a dominant negative p53 blocked it, indicating that p53 plays an essential role in NO-induced apoptosis of chondrocytes. The increased accumulation of p53 caused expression of Bax, a pro-apoptotic member of the Bcl-2 family that is known to cause apoptosis via release of cytochrome c and caspase activation. These results suggest that NO-activated p38 kinase activates p53 function in two different ways, transcriptional activation by NFkappaB and direct phosphorylation of p53 protein, leading to apoptosis of articular chondrocytes.

Highlights

Nitric oxide (NO) during primary culture of articular chondrocytes causes apoptosis via p38 mitogen-activated protein kinase in association with elevation of p53 protein level, caspase-3 activation, and differentiation status
The increased accumulation of p53 caused expression of Bax, a pro-apoptotic member of the Bcl-2 family that is known to cause apoptosis via release of cytochrome c and caspase activation. These results suggest that NO-activated p38 kinase activates p53 function in two different ways, transcriptional activation by nuclear factor ␬B (NF␬B) and direct phosphorylation of p53 protein, leading to apoptosis of articular chondrocytes
Our recent study demonstrated that apoptosis of chondrocyte apoptosis caused by direct production of NO with the NO donor sodium nitroprusside (SNP)1 is regulated by opposite functions of two mitogen-activated protein kinase subtypes, extracellular signal-regulated kinase 1/2 and p38 kinase, in association with the elevation of p53 protein level, caspase-3 activation, and differentiation status [9]

Summary

EXPERIMENTAL PROCEDURES

Culture of Rabbit Articular Chondrocytes and Experimental Condition—Rabbit articular chondrocytes were released from cartilage slices by enzymatic digestion, as previously described [9]. For p38 kinase assay, the beads were re-suspended in 20 ␮l of kinase reaction buffer containing 25 mM Tris-HCl, pH 7.5, 5 mM ␤-glycerol phosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, 10 mM MgCl2, [␥-32P]ATP, and 1 ␮g of ATF-2 fusion protein as a substrate (New England Biolabs). After electrophoresis and transfer of proteins to a nitrocellulose membrane, phosphorylation of p53 was determined by Western blot analysis using phosphorylation site-specific anti-p53 monoclonal antibody from Cell Signaling Technology (Beverly, MA). Cell pellets were suspended in hypotonic buffer containing 10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, and inhibitors of proteases and phosphatases as described above and lysed by the addition of 0.07% Nonidet P-40. The blots were developed using a peroxidase-conjugated secondary antibody and enhanced chemiluminescence system

RESULTS

DISCUSSION