Inhibition of Mitogen-activated Protein Kinase Kinase Induces Apoptosis of Human Chondrocytes

Abstract

We previously have reported that the mitogen-activated protein kinase (MAPK) pathway is stimulated by adhesion of human chondrocytes to anti-␤ 1 -integrin antibodies or collagen type II in vitro. These mechanisms most likely prevent chondrocyte dedifferentiation to fibroblast-like cells and chondrocyte death. To investigate whether this pathway plays an essential role for the differentiation, phenotype, and survival of chondrocytes, we blocked mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) (MEK), a kinase upstream of the kinase Erk by using U0126. Exposure of chondrocytes to U0126 caused activation of caspase-3 in a dose-dependent manner. Western blot analysis with an antibody specific for dually phosphorylated Erk shows that collagen type II induced phosphorylation of Erk1/2 was specifically blocked by U0126 in a dose-dependent manner. Immunohistochemical analysis showed that treated chondrocytes were caspase-3 positive. In treated chondrocytes, the cleavage of 116-kDa poly(ADP-ribose)polymerase resulted in the 85-kDa apoptosis-related cleavage fragment and was associated with caspase-3 activity. Analysis by electron microscopy showed typical morphological signs of apoptosis, such as crescent-shaped clumps of heterochromatin, and a degraded pericellular matrix. Thus, these results indicate that the MEK/Erk signal transduction pathway is involved in the maintenance of chondrocytes differentiation and survival. These data stimulate further investigations on the role of mitogen-activated protein kinase pathways in human chondrocytes.Apoptosis, or programmed cell death, plays a key role in embryogenesis, immunological competence and tissue homeostasis for cell removal and can distinguished biochemically and morphologically from cell necrosis, which is a passive, energyindependent form of cell death. Chondrocyte degradation and death occurs in enchondral ossification as well as in age-associated athropathies such as osteoarthritis (1, 2). Chondrocyte apoptosis, can be induced in vitro by a variety of agents, such as nitric oxide, oxygen radical scavengers (3), tumor necrosis factor (4), and interleukin-1␤ (5).It is known that many cell types including chondrocytes require integrin mediated interactions with the extracellular matrix to survive, differentiate, and proliferate (6, 7). Cells undergo a specific cell death or apoptosis in the absence of specific matrix components (8). Interaction between chondrocytes and cartilage matrix components or anti-␤ 1 -integrin antibodies leads to a rearrangement of cytoskeletal and signaling proteins localized at focal adhesions and focal adhesion kinase (6 -9). These stimulate docking proteins such as Src-homology collagen. Src-homology collagen then associates with growth factor receptor-bound protein 2, and extracellular signal-regulated kinase (Erk) 1 (7). These mechanisms most likely prevent chondrocyte dedifferentiation to fibroblast-like cells and chondrocytes death (7)(8)10).It is well known that one of the early reactions occurring in the c...

IGF-I stimulation of proteoglycan synthesis by chondrocytes requires activation of the PI 3-kinase pathway but not ERK MAPK

“…In other cell types, IGF-I survival signalling involves the activation of the PI3K/Akt pathway, although additional pathways appear to be capable of promoting survival [33,34]. In chondrocytes plated on to collagen, IGF-I stimulation of the ERK/MAPK signalling pathway was shown to prevent apoptosis [35]. Under the conditions used in the present study, significant cell death was not observed with inhibition of either the PI3K pathway or the ERK/ MAPK signalling pathway.…”

Synergistic effects of dexamethasone and quinolones on human-derived tendon cells

“…Western blot analysis was performed as described previously in detail [21]. Briefly, tenocyte proteins were extracted with lysis buffer [50 mM Tris/HCl (pH 7.2)], 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% (v/v) aprotinin, 4 g/mL pepstatin A, 10 g/mL leupeptin and 1 mM PMSF for 30 min on ice.…”

“…They activate different signal transduction pathways, such as the mitogen-activated protein (MAP) kinase pathway, and thus control proliferation, differentiation and survival of tenocytes [18,19]. Undisturbed cell-matrix interactions are essential for cell viability, and inhibition of the MAP kinase pathway can induce programmed cell death [20,21].…”

Borrelia burgdorferi-Induced Expression of Matrix Metalloproteinases from Human Chondrocytes Requires Mitogen-Activated Protein Kinase and Janus Kinase/Signal Transducer and Activator of Transcription Signaling Pathways

“…To confirm the role of p38 MAPK, ERK1/2 and JNK on MMP-1 and MMP-3 induction by B. burgdorferi, we examined expression of MMP-1 and MMP-3 in the presence of specific inhibitors for each pathway. Concentrations of inhibitors were selected based on prior studies of these inhibitors with chondrocytes (9,15,16,21,25,28,29,31,33,34). Cells were in- (Fig.…”