Background Spondyloarthritis (SpA) is an inflammatory and chronic joint disease belonging to a group of diseases called spondyloarthropathies. SpA is one of the most common rheumathological pathologies, in second position after rheumatoid arthritis (RA). Pain can be relieved by non-steroidal anti-inflammatory drugs or anti-TNFα, but not ectopic ossification of the enthesis (fibrocartilaginous insertion of tendons in bones). Because SpA physiopathology remains controversial, we aim to better understand the mechanisms involved in the ossification of the enthesis.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that regulates crucial biological processes such as proliferation, survival, cell cycle, apoptosis or inflammatory response. S1P can act intra- or extra-cellularly, triggering signal transduction through G protein-coupled receptors (S1P_1-5). Sphingosine kinases (SphK_1-2), which convert sphingosine into S1P, control the fine balance between the central molecules of sphingolipid metabolism.

Objectives S1P metabolic pathway has been attributed an important role in bone homeostasis [1], but also in osteoarticular pathologies, such as RA and osteoarthritis. However, its implication in SpA has not been investigated yet and we aim to determine if S1P metabolism has a role in enthesis ossification.

Methods We used primary cultures from newborn mice of the three cell types located at the enthesis: osteoblasts (from calvaria), chondrocytes (from posterior joints) and tenocytes (from tail tendons). Gene expression of SphK_1-2 and S1P_1-5 receptors was determined by real-time PCR. The enzymatic activity of SphK_1-2 was also analyzed. The expression of SphK_1-2 in adult mouse enthesis was examined by immunohistochemistry.

Results In cell culture, SphK₁ expression appeared 6-fold higher in osteoblasts than in chondrocytes and tenocytes. SphK₂ expression and SphK₁/SphK₂ ratio were also more elevated in osteoblasts. We confirmed that SphK₁ expression and activity increased during osteogenic differentiation in murine osteoblasts [2] and showed that SphK₂ was also increased. Of interest, SphK regulation seemed different during the differentiation of hypertrophic chondrocytes. S1P_1-3 receptors were the most represented in enthesis cells, whereas S1P_4-5 were nearly undetected. Moreover, gene expression of S1P_1-3 differed among the three cell types; in particular, S1P₁ appeared overrepresented in osteoblasts. Thus, S1P metabolism may be differentially regulated in osteoblasts, chondrocytes and tenocytes.

By immunohistochemistry, we located SphKs expression in borderline connective tissues surrounding the tendon (epitenon) and the bone (periosteum), a tissue rich in osteoprogenitor cells. This result reinforces the assumption of a link between SphKs and osteogenic differentiation.

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Conclusions These preliminary results highlight the possible implication of S1P metabolism in enthesis ossification. Strategies with SphK inhibitors and S1P receptor antagonists will be used in cell culture to confirm our hypothesis. In addition, S1P levels will be determined in SpA patients to validate that S1P metabolism could be a therapeutic target in SpA.

References

1. Biochim Biophys Acta. 2013. Ishii, Kikuta.

2. Mol Oncol. 2014. Brizuela, Martin, et al.

Acknowledgements Grant from the French Society of Rheumatology (SFR)

Disclosure of Interest None declared