gms | German Medical Science

Objectives: Osteoarthritis (OA) is the most prevalent reason for chronic joint pain and is characterized by the progressive loss of cartilage. The joints are innervated by calcitonin gene-related peptide (α CGRP)- and substance P (SP) positive sensory nerve fibers which are a potential source of tibial-femoral pain during OA pathogenesis. Alteration of sensory joint innervation might be partly responsible for degenerative changes which contribute to development of OA. We, therefore, aim to analyze and compare the molecular effects of SP and α CGRP on the metabolism of articular chondrocytes of OA and non-OA donors.

Methods: Human chondrocytes from OA and non-OA donors were stimulated with SP or αCGRP (10^-8 M and 10^-10 M). Subsequently, proliferation was analyzed by determining doubling time, apoptosis via Caspase 3/7 activity assay, senescence via SA-ß-galactosidase assay, gene expression with qPCR and activation of signaling pathways with western blot and ELISA.

Results and Conclusion: Stimulation with 10^-8 M αCGRP for 7 days resulted in a significant decrease of proliferation in OA and non-OA chondrocytes. Stimulation with SP or αCGRP (10^-8 M) for 1 day increased apoptosis in OA chondrocytes, whereas in non-OA chondrocytes apoptosis was reduced (10^-10 M αCGRP). A significant increase in senescence was measured in OA-chondrocytes after SP andαCGRP stimulation, but not in non-OA cells. Moreover, SP stimulation decreased the expression of chondrogenic marker genes SOX9, COL2A1 and COL9A1 in OA chondrocytes and, in contrary, increased the expression of inflammatory marker genes IL1, IL6 and TNFα in OA chondrocytes. Phosphorylation of ERK1/2 was induced after stimulation with SP and αCGRP in OA and non-OA chondrocytes to the same extent, but phosphorylation of AKT was exclusively detected in non-OA chondrocytes after incubation with both neuropeptides. We examined in addition the level of the second messenger cyclic AMP (cAMP). In non-OA chondrocytes, both neuropeptides provoked an increase in the cAMP-level after stimulation with a high concentration (10^-8 M), compared to the unstimulated control. However, in OA chondrocytes, only αCGRP (10^-10 M) increased cAMP level significantly. We conclude for SP and αCGRP dose-dependent effects on chondrocytes. Especially SP provokes catabolic and inflammatory responses in OA-chondrocytes, by inducing the expression of pro-inflammatory genes and the decrease of chondrogenic genes. In addition, SP enhances apoptosis and senescence, presumably mediated by activation of the ERK pathway. In contrast, αCGRP affects chondrocytes in an ambivalent manner, suggested by the fact that apoptosis was reduced in non-OA chondrocytes, but also proliferation was impaired in non-OA and OA chondrocytes. In non-OA chondrocytes, SP and αCGRP induce the pro-inflammatory AKT pathway, whereas AKT was not phosphorylated in OA chondrocytes, which indicates different active signaling cascades presumably caused by the microenvironment from which the cells originate.