The role of norepinephrine in adult human articular chondrocytes

Abstract

Norepinephrine (NE) belongs to the catecholamine family of tyrosine-derived neurotransmitters of the sympathetic nervous system. Tyrosine-hydroxylase positive sympathetic nerve fibers have been identified in bone marrow, in the periosteum, and in bone-adherent ligaments indicating that growth and metabolic activity of bone and joint tissues is regulated by sympathetic neurotransmitters. It is known that NE can regulate cell proliferation or apoptosis in several cell types, such as osteoblasts. It is further described that NE modulates inflammation during rheumatoid arthritis and gut inflammation. Here, we aim to understand the role of NE in human osteoarthritic chondrocytes with regard to inflammation and its impact on metabolic activity. Human chondrocytes were isolated from post-surgery discarded human osteoarthritic articular cartilage. Expression of adrenergic receptor on articular cartilage chondrocytes was tested with standard end point PCR and immunohistochemical analysis. Employing 3D cell cultures in fibrin gel, effects of NE on IL-1β induced gene expression of pro-inflammatory cytokines and matrix metalloproteinases (MMP) were analyzed with quantitative real-time PCR. The impact of NE on cell proliferation was determined in monolayer culture with BrdU ELISA and FACS analyses, the effect on apoptosis with Caspase 3/7 ELISA. To test the adrenergic receptor involved in apoptosis or proliferation, the α1-adrenergic receptor antagonist doxazosin, the α2-adrenergic receptor antagonist yohimbine, and the β1/2/3-adrenergic receptor antagonist nadolol were included in BrdU/caspase-3/7-ELISAs. Chondrocytes cultured in monolayer and in 3D under non- and inflammatory conditions expressed α1D-, α2A/B/C- and β2-adrenergic receptors. β2-Adrenergic receptors were detected on protein level in human osteoarthritic cartilage as well. Stimulation with NE has significantly reduced IL-1β induced gene expression of IL-8 and MMP-13 in human osteoarthritic chondrocytes cultured in 3D fibrin gel. Notably, we were unable to detect an impact on IL-1β induced gene expression of interleukin-6, MMP-2 and MMP-3. Furthermore, NE inhibits BrdU incorporation compared to the controls. This effect was reversed by nadolol. In cell cycle analyses, we showed that NE increased the G1/G0-phase population and decreased S-phase population. Additionally, we observed an increase of caspase-3/7 activity after NE treatment that was reversed after addition of α1-adrenergic receptor antagonist doxazosin. Neurotransmitters of the sympathetic nervous system like NE presumably mediate an anti-inflammatory/chondroprotective effect in human osteoarthritic chondrocytes via reducing IL-1β induced IL-8 and MMP-13. Furthermore, NE is able to modulate the metabolic activity of chondrocytes by a cell cycle slow-down via β2-adrenergic receptor signaling and by induction of apoptosis via α1D-adrenergic receptor signaling. We therefore assume a yet unknown function of catecholaminergic neurotransmitters in adult human cartilage that might have an impact on osteoarthritis pathology.