gms | German Medical Science

Objectives: Osteoarthritis (OA) is the most common joint disorder with a major adverse socio-economic impact. Thus, there is a need for identification of novel targets for diagnostic and therapeutic approaches. 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 the effects of SP and α CGRP on the metabolism of articular chondrocytes from OA patients.

Methods: Human chondrocytes obtained from OA patients after joint replacement, were expanded for 1 passage and stimulated with SP or α CGRP (10-8M and 10-10M) in 2D- and 3D-cell culture systems. Subsequently, proliferation was analyzed via BrdU assay, apoptosis via Caspase 3/7 activity assay and Annexin V FACS measurement, senescence via ß-galactosidase assay, adhesion ability with crystal violet staining, gene expression of marker genes with qPCR and activation of signaling pathways with western blot and ELISA. GAG concentration was evaluated via DMMB assay.

Results and conclusion: Stimulation with SP (10-8M and 10-10M) for 1 day resulted in a significant increase in apoptosis, and long term stimulation with SP (10-8M) for 7 days increased gene expression of the pro-inflammatory genes TNF α and IL-6 significantly and IL-1ß by trend. Cells cultured in fibringels for 21 days showed decreased GAG production after 7 and 21 days, when treated with α CGRP (10-10M). Moreover, α CGRP (10-8M and 10-10M) treatment decreased adhesion and cell spreading ability on cell culture plastic, increased senescence and the expression of the senescence-related gene p21. Chondrocytes contained enhanced intracellular cAMP-levels after stimulation with α CGRP, but not with SP. Phosphorylation of ERK1/2 was induced after stimulation with both neuropeptides, SP and α CGRP, in both concentrations after 15-30 min.

We conclude for both neurotransmitters dose-dependent effects on OA-chondrocyte metabolism. SP exerted a rather catabolic influence on the cells by inducing the expression of pro-inflammatory genes and apoptosis, presumably mediated at least in part by activation of the ERK pathway. In contrast, α CGRP affects the cells in an ambivalent manner, suggested by the fact that the pro-inflammatory ERK pathway together with the anti-inflammatory cAMP pathway was activated. We hypothesize therefore that SP signals via the α q-subunit, whereas α CGRP probably mediates its signalling cascade via the α q- and α s-subunits of their receptors in OA-chondrocytes. Analysing and comparing the signalling of SP and α CGRP in non-OA articular chondrocytes will contribute to understanding the role of sensory neurotransmitters in OA-pathology and is therefore focus in upcoming experiments.