52 – INCREASED PHOSPHORYLATION OF ERK AND P38 SIGNAL TRANSDUCTION PROTEINS IN MESENCHYMAL STEM CELLS FROM OSTEOARTHRITIC PATIENTS

Abstract

Purpose: Mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients are not well characterized and little is known of how they are regulated. Recent evidence indicates that a major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human MSCs from OA patients express type X collagen (COL10), a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification). However, the intracellular pathways for transducing signals that regulate hypertrophy in MSCs remain unclear. In chondrocytes, this pathway is mediated by mitogen activated protein kinase (MAPK) p38. The aim of this study was to determine the phosphorylation levels of ERK/p38 MAPK signaling molecules in MSCs from OA patients compared to those from normal patients.

Method: MSCs were obtained from aspirates from the intramedullary canal of donors (60–80 years of age) undergoing total hip replacement for OA. Cells were cultured in DMEM high glucose supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin for 2–3 passages. Cells were then lysed and proteins were separated on 10% acrylamide gels and transferred to nitrocellulose membranes. Protein expression was determined by Western blot using specific antibodies directed against type X collagen, ERK, phosphorylated-ERK, p38, phosphorylated-p38, JNK, phosphorylated-JNK, AKT, and phosphorylated-AKT. GAPDH was used as a housekeeping gene. Proteins were detected using the West Pico Chemiluminescence substrates and analyzed using the Bio-Rad VersaDoc equipped with a cooled CCD 12 bit camera. Normal mesenchymal stem cells from a 22 years old woman were purchased from Lonza (Switzerland).

Results: Results show that the expression of COL10 was markedly increased in MSCs of OA patients compared to control patient. Results also shows that the phosphorylation of all the signal transduction proteins studied was induced in MSCs of patients with OA. Indeed, the phosphorylation of ERK (3.4±0.9 times the control), p38 (1.7±0.3 times the control), JNK (5.40±1.14 times the control), and AKT (4.3±0.8 times the control) was higher in MSCs of OA patients compared to control normal patients.

Conclusion: In the normal donor, MSCs continue to exhibit their in situ behavior in that they expressed very little or no COL10. This may relate to the fact that normal MSCs being multipotent in nature like to maintain an undifferentiated state. In contrast, MSCs from OA patients expressed COL10: this suggests that they are in a situation were they can be preprogrammed not only to replace the degraded articular cartilage but also the damaged subchondral bone. Since the phosphorylation of ERK/p38 MAPK signaling molecules is also lower in normal MSCs, our results also suggest that this signaling pathway is implicated in the control of COL10 expression. This finding is of great importance for the understanding of COL10 regulation in general and may lead to important advances in the comprehension of COL10 related diseases.