Mechanical loading highly increases IL-6 production and decreases OPG expression by osteoblasts

Abstract

In osteoarthritis (OA), mechanical factors play a key role, not only in cartilage degradation, but also in subchondral bone sclerosis. The aim of this study was to develop on original compression model for studying the effect of mechanical stress on osteoblasts. We investigate the effects of compression on primary calvaria osteoblasts isolated from newborn mice and cultured for 28 days in monolayer. At the end of this period, osteoblasts were embedded in a newly synthesized extracellular matrix which formed a three-dimensional membrane. This membrane was then submitted to compression in Biopress Flexercell plates (1-1.7 MPa compressions at 1 Hz frequency) during 1-8h. The expression of 20 genes was investigated by real time reverse transcriptase polymerase chain reaction. Interleukin (IL)-6, matrix metalloproteinase (MMP)-3 and prostaglandin (PG)E(2) were assayed in the culture medium by specific immunoassays. The compression highly increased IL-6 and cyclooxygenase (COX)-2 mRNA levels in osteoblasts. In parallel, increased amount of IL-6 and PGE(2) was found in the supernatant of loaded osteoblasts. This stimulation reached a maximum after 4h of 10% compression. MMP-2, MMP-3, and MMP-13 mRNA levels were also increased by compressive stress, while 15-hydroxyprostaglandin-dehydrogenase and osteoprotegerin (OPG) start to decrease at hour 4. COX-1, microsomial PG E synthase-1 (mPGES1), mPGES2 and cytosolic PGES and receptor activator of nuclear factor ligand (RANKL) were unmodified. Finally, we observed that alpha 5 beta 1 integrin, intracellular Ca(++), nuclear factor-kappaB and extracellular signal-regulated kinase 1/2 pathways were involved in the compression-induced IL-6 and PGE(2) production. IL-6 neutralizing antibodies and piroxicam inhibited the decrease OPG expression, but did not modify RANKL mRNA level, indicating that IL-6 and PGE(2) induce a decrease of the OPG/RANKL ratio. This work demonstrates that IL-6 is mechano-sensitive cytokine and probably a key factor in the biomechanical control of bone remodeling in OA.