Fournier, C.

Layer, P. G.

Thangaraj, G.

GSI Repository

Does Radon act via the cholinergic anti-inflammatory signaling pathway  in arthritic mice and mesenchymal stem cells? Gopenath Thangaraj1, Claudia Fournier2, Paul G. Layer1 1 Technische Universität Darmstadt, Germany; 2 GSI, Darmstadt, Germany  Introduction Treatment with Radon is believed to suppress the severity of the autoimmune disorder Rheumatoid Arthritis (RA). Thereby, associated with excess pain, cartilage undergoes severe destruction and synovial fluids accumulate in the joints. One of the mechanisms supposedly involved in RA is the cholinergic anti-inflammatory pathway (CAIP). Roles of the cholinergic system in CNS functioning are well established. But also, non-neural, e.g. developmental functions of the cholinergic system are now well accept-ed. Recently, we have shown improper formation of carti-lage in an AChE/BChE double knockout mouse, thus providing evidence for the involvement of cholinesterases in cartilage development (J. Klaczinski, Dissertation 2012). In AP6 of the GREWIS project, we analyse the effects of Radon treatment on the expression of choliner-gic components (ACh receptors, particularly the  7-nAChRs, a main player of CAIP, and AChE, ChAT) dur-ing the formation of cartilage, both in vivo and in vitro.  Materials and Methods 11 day-old pregnant mice and chicken embryos up to stages HH23 are used. Mesenchymal cells were isolated from limb buds of both mouse and chick embryos, plated as high density micro-mass cultures and incubated for 2 weeks at 37°C. Also, human primary osteoblast cells were cultured until passage 4 and collected for mRNA isola-tion. Alcian blue staining marks cartilage development, while Alizarin red and alkaline phosphatase stainings in-dicates differentiation of osteoblasts. Cholinesterase en-zyme activity was visualized by Karnovsky-Roots stain-ing. cDNA was synthesized and used for PCR analysis.  Results Human primary osteoblast cells express an entire set of cholinergic components First results showed that human osteoblast cells expressed cholinesterases (AChE and BChE); they also synthesize ACh and express both muscarinic and nicotinic ACh re-ceptors (Fig. 1), suggesting the involvement of an entire cholinergic system in the differentiation of osteoblasts.    Fig. 1: PCR analyses of human osteoblast cells.  They express all tested cholinergic components.   Micromass cultures as 3D in vitro-systems for cartilage formation     Fig. 2: Light microscopy of micromass cultures. Chon-drocytes condense to form nodules which later differenti-ate to osteoblasts, both for chick (a) and mouse mesen-chymal cells(b). Such cultures will be exposed to radon and to analyze the effects on cartilage formation. Formation of cartilage in vitro In mouse micromass cultures, cartilage formation starts as early as div 2, as visualized by Alcian blue staining. It rises until 6 days and drops from 8 days, when osteoblast differentiation takes over.           Fig. 3: Cartilage nodules from mouse mesenchymal cells from div2-8, stained with Alcian blue. Cholinesterase activity during cartilage formation            Fig. 4: Note different expression patterns of AChE and BChE in mouse cartilage nodules after 4 div. AChE sup-posedly supports further osteoblast differentiation.  Future work Treatment of osteoblasts, MSC-derived chondrogenic cells (to be established) and micromass cultures with cho-linesterase and nAChR inhibitors, +/- radon exposure. Correlate changes of cholinergic components with results of other APs (cytokines, NF-kB, TRP channels).  Work is funded by BMBF (GREWIS, 02NUK017A). a b a b c d div 2 div 6 div 4 div 8 a b AChE BChE HEALTH-14 GSI SCIENTIFIC REPORT 2012438

Does Radon act via the cholinergic anti-inflammatory signaling pathway  in arthritic mice and mesenchymal stem cells?

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Does Radon act via the cholinergic anti-inflammatory signaling pathway in arthritic mice and mesenchymal stem cells?

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