Prologation of c-Jun N-terminal kinase is associated with cell death induced by tumor necrosis factor alpha in human chondrocytes

Abstract

The aim of this study was to elucidate the role of JNK signaling pathway involved in tumor necrosis factor-alpha (TNF-alpha)-induced death of chondrocytes. Primary chondrocyte cultures were obtained from human knee osteoarthritis cartilages. First passage chondrocytes were treated with TNF-alpha and various potentiators, and cell death was measured with MTT assay. C-Jun N terminal kinase (JNK) activation was investigated with the solid phase kinase assay. Expression of apoptosis-related molecule was assayed with Western blot. Chondrocytes were resistant to TNF-alpha-induced cell death. In contrast, pretreatment with actinomycin D, the phosphatase inhibitor vanadate or MAP kinase phosphatase-1 (MKP-1) inhibitor Ro318220 invariably led to chondrocyte death. While TNF-alpha alone stimulated a single, brief JNK activity, a second JNK peak was observed when the cells were pretreated with actinomycin D. When the cells were pretreated with vanadate or Ro318220, TNF-alpha-induced JNK activation was greatly prolonged, which was associated with the induction of cell death. The expression of Bcl-2 and Mcl-1 decreased significantly in conditions of cell death. In conclusions, our data suggest that chondrocyte death induced by TNF-alpha is associated with sustained JNK activation. This effect may be due to downregulation of TNF-alpha induced phosphatase that inactivates JNK and of Bcl-2 family proteins.

Fig. 1

Patterns of c-Jun N terminal kinase (JNK) phosphorylation in human chondrocytes after treatment with TNF-alpha with or without vanadate, Ro318220, or actinomycin D. Both vanadate and Ro318220 increase the intensity and duration of JNK phosphorylation. Actinomycin D induces prolongation of SAPK/JNK activation, yielding second activation peak at 240 min of treatment. JNK activation was assessed by solid phase kinase assay. Data are representative of samples from 3 donors.

Fig. 2

(A) Kinetics of chondrocyte death induced by 10 ng/mL of TNF-α after 1 hr pretreatment with 500 µM vanadate or 10 µM Ro318220. Cell death increases significantly after 12 hr of treatment detected by MTT assay. Cell survival in control culture was set at 100%. Asterisks denote p<0.05. Data are means from samples from 6 donors. (B) Chondrocyte apoptosis is verified by quantitation of subdiploid cells analyzed with flow cytometry. Cells were fixed with 70% ethanol 24 hr after TNF-α treatment and stained with propidium iodide before analysis with flow cytometry. Numbers denote percent subdiploid cells. Data are representative of samples from 4 donors. (C) Annexin V-FITC/propidium iodide (PI) dual staining of chondrocytes analyzed by flow cytometry. Cells were stained 12 hr after TNF-α treatment. Numbers in the right lower quadrant denotes percentages of early apoptotic chondrocytes (annexin V positive, PI negative). Data are representative of 3 samples from different donors. (D) Processing of caspase-3 revealed by Western blot. Cells were treated with TNF-α for 6 hr and lysates were assayed for caspase-3. T: TNF-alpha, VT: vanadate pretreatment followed by TNF-alpha, RT: Ro318220 pretreatment followed by TNF-α . Arrow denotes processed caspase-3 band. Data are representative of samples from 3 donors. (E) Chondrocyte death induced by TNF-alpha and 500 µM vanadate or 10 µM Ro318220 is effectively inhibited by pan-caspase inhibitor, zVAD -FMK, implicating the involvement of caspase cascade. Cell death was measured by MTT assay, and the degree of cell death after 24 hr of incubation without zVAD-FMK was set at 100%. Asterisks denote p<0.05. Data are means from samples from 5 donors.

Fig. 3

(A) The influence of vanadate and Ro318220 on NF-κB activation induced by TNF-alpha in human articular chondrocyte. Nuclear extract was prepared 15 min after treatment with 10 ng/mL of TNF-alpha, and electrophoretic mobility shift assay was performed. Data are representative of samples from 3 donors. (B) Western blot for IκB of cytosolic extract of the same set of samples as in (A). (C) Western blot for phosphorylated IκB of cytosolic extract of the same set of samples as in (A).

Fig. 4

Patterns of Mcl-1 and Bcl-2 expression after TNF-alpha treatment with or without vanadate, or Ro318220. Twenty microgram of proteins were separated in SDS-PAGE gel and probed with respective antibodies. T, TNF-alpha; VT, vanadate pretreatment followed by TNF-alpha; RT, Ro318220 pretreatment followed by TNF-alpha. Data are representative of samples from 3 donors.