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Altered expression and production of leptin is responsible for abnormal cell markers in subchondral osteoblasts from osteoarthritic patients
Arthritis Research & Therapy volume 9, Article number: P36 (2007)
Leptin is a peptide hormone with a role in body weight regulation, immune response, bone metabolism and possibly in rheumatic diseases. Osteoarthritis (OA) is characterized by cartilage damage and loss, synovial membrane inflammation and bone sclerosis and the formation of osteophytes. It is now evident that the subchondral bone tissue plays a prominent role in the pathophysiology of OA, a situation that is related to abnormal osteoblast (Ob) differentiation. Leptin can promote the differentiation of Obs; however, a direct role for leptin in human OA has yet to be demonstrated.
Materials and methods
We prepared primary cultures of normal and OA Obs from subchondral bone and OA chondrocytes from articular cartilage from tibial plateaus removed for knee replacement surgery of OA patients or at autopsy. We determined the expression and production of leptin using RT-PCR and ELISA.
Alkaline phosphatase activity was determined by p-nitrophenyl phosphate hydrolysis, osteocalcin release by enzyme immunoassay and collagen production by the release of the CICP propeptide. TGFβ1 production was determined by ELISA. Inactivating antibodies raised against leptin receptors and inhibitors of leptin signaling, typhostin (Tyr) and piceatannol (Pce) were used to determine their effect on Ob cell markers. Cellular proliferation was assessed using the BrdU cell proliferation assay whereas activation of the Erk1/2 pathway was determined by western blot analysis.
Our results indicated using two different sets of primers for RT-PCR experiments that leptin was expressed only in Obs not in chondrocytes. The expression of leptin was also higher in OA Obs compared with normal using real-time PCR and was responsible for the increase in leptin levels noted in conditioned-media from OA Obs. Although leptin was not expressed by chondrocytes, it was present in articular cartilage – suggesting that leptin produced in bone tissue reached the overlaying cartilage. The long-form leptin receptor mRNA levels were slightly reduced in OA Obs compared with normal Obs. Since leptin can promote the differentiation of Obs, we next questioned whether the observed increase in alkaline phosphatase activity (ALP) and osteocalcin release (OC) observed in OA Obs was linked with their endogenous leptin production. Inactivating antibodies against the leptin receptor reduced both ALP and OC in OA Obs about 35%, a situation reproduced with Tyr and Pce. Likewise, Tyr and Pce also reduced ALP, CICP release, and TGFβ1 production in OA Obs. Last, leptin dose-dependently (1 ng/ml to 10 mg/ml) stimulated cellular proliferation in OA Obs and this was reflected by an increase in phosphorylation of Erk1/2 signaling.
These results indicate that OA Obs produce more leptin than normal, and suggest that leptin found in articular cartilage is derived from bone tissue. The increase in leptin in OA bone tissue could also be responsible for increased ALP, OC and TGFβ1 in Obs. Since leptin can promote inflammation and cartilage loss in combination with cytokines, this suggests that subchondral bone production of leptin may be responsible, at least in part, for cartilage loss in OA.
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Lajeunesse, D., Mutabaruka, M., Aoulab-Aissa, M. et al. Altered expression and production of leptin is responsible for abnormal cell markers in subchondral osteoblasts from osteoarthritic patients. Arthritis Res Ther 9, P36 (2007). https://doi.org/10.1186/ar2262
- Articular Cartilage
- Subchondral Bone
- Cartilage Loss
- Receptor mRNA Level