Impact of Substance P and αCGRP on articular chondrocytes from osteoarthritic and non-osteoarthritic donors

Objectives Osteoarthritis (OA) is the most common joint disorder and the leading cause of disability and chronic pain in the elderly with a major socio-economic impact. The joints are innervated by calcitonin gene-related peptide (αCGRP) - 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 αCGRP on the metabolism of articular chondrocytes of OA and non-donors.

Methods Human chondrocytes from OA and non-OA donors were stimulated with SP or αCGRP (10-8M and 10-10M). Subsequently, proliferation was analyzed by determining doubling time, apoptosis via Caspase 3/7 activity assay and flow cytometric Annexin V measurement, 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 10-8M αCGRP for 7 days resulted in a significant decrease of proliferation in OA and non-OA chondrocytes. Stimulation with SP or αCGRP (10-8M) for 1 day increased apoptosis in OA chondrocytes, whereas in non-OA chondrocytes apoptosis was reduced (10-10M αCGRP). A significant increase in senescence was measured in OA-chondrocytes after SP and αCGRP stimulation (10-8 and 10-10M), but no changes for non-OA cells was observed. Moreover, SP stimulation decreased the expression of chondrogenic marker genes SOX9, COL2A1 and COL9A1 in OA-chondrocytes, but not in non-OA chondrocytes, and in contrary, increased the expression of inflammatory marker genes IL1, IL6 and TNFα in OA-chondrocytes, but not in non-OA chondrocytes. Phosphorylation of ERK1/2 was induced after stimulation with SP and αCGRP in both concentrations, after 15-30 minutes in OA and non-OA chondrocytes to the same extent, but phosphorylation of AKT was exclusively detected in non-OA chondrocytes after 5-15 minutes incubation with both neuropeptides. OA-chondrocytes revealed already a strong AKT phosphorylation without stimulation.

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 is already phosphorylated in OA chondrocytes without additional stimulation, which indicates different active signalling cascades presumably caused by the microenvironment from which the cells originate.

Stichwörter SP, CGRP, chondorcytes, osteoarthritis

Publication History

Article published online:
15 October 2020