Enhanced BMP-2/BMP-4 ratio in patients with peripheral spondyloarthritis and in cytokine- and stretch-stimulated mouse chondrocytes.

BACKGROUND: Excessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process. METHODS: BMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4. RESULTS: In SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes. CONCLUSIONS: Our results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.

The role of sphingosine 1-phosphate metabolism in bone and joint pathologies and ectopic calcification.

Sphingolipids display important functions in various pathologies such as cancer, obesity, diabetes, cardiovascular or neurodegenerative diseases. Sphingosine, sphingosine 1-phosphate (S1P), and ceramide are the central molecules of sphingolipid metabolism. Sphingosine kinases 1 and 2 (SK1 and SK2) catalyze the conversion of the sphingolipid metabolite sphingosine into S1P. The balance between the levels of S1P and its metabolic precursors ceramide and sphingosine has been considered as a switch that could determine whether a cell proliferates or dies. This balance, also called « sphingolipid rheostat ¼, is mainly under the control of SKs. Several studies have recently pointed out the contribution of SK/S1P metabolic pathway in skeletal development, mineralization and bone homeostasis. Indeed, SK/S1P metabolism participates in different diseases including rheumatoid arthritis, spondyloarthritis, osteoarthritis, osteoporosis, cancer-derived bone metastasis or calcification disorders as vascular calcification. In this review, we will summarize the most important data regarding the implication of SK/S1P axis in bone and joint diseases and ectopic calcification, and discuss the therapeutic potential of targeting SK/S1P metabolism for the treatment of these pathologies.

Cytokine-Induced and Stretch-Induced Sphingosine 1-Phosphate Production by Enthesis Cells Could Favor Abnormal Ossification in Spondyloarthritis.

Spondyloarthritis (SpA) is a common rheumatic disease characterized by enthesis inflammation (enthesitis) and ectopic ossification (enthesophytes). The current pathogenesis model suggests that inflammation and mechanical stress are both strongly involved in SpA pathophysiology. We have previously observed that the levels of sphingosine 1-phosphate (S1P), a bone anabolic molecule, were particularly high in SpA patients' serum compared to healthy donors. Therefore, we wondered how this deregulation was related to SpA molecular mechanisms. Mouse primary osteoblasts, chondrocytes, and tenocytes were used as cell culture models. The sphingosine kinase 1 (Sphk1) gene expression and S1P secretion were significantly enhanced by cyclic stretch in osteoblasts and chondrocytes. Further, TNF-α and IL-17, cytokines implicated in enthesitis, increased Sphk1 mRNA in chondrocytes in an additive manner when combined to stretch. The immunochemistry on mouse ankles showed that sphingosine kinase 1 (SK1) was localized in some chondrocytes; the addition of a pro-inflammatory cocktail augmented Sphk1 expression in cultured ankles. Subsequently, fingolimod was used to block S1P metabolism in cell cultures. It inhibited S1P receptors (S1PRs) signaling and SK1 and SK2 activity in both osteoblasts and chondrocytes. Fingolimod also reduced S1PR-induced activation by SpA patients' synovial fluid (SF), demonstrating that the stimulation of chondrocytes by SFs from SpA patients involves S1P. In addition, when the osteogenic culture medium was supplemented with fingolimod, alkaline phosphatase activity, matrix mineralization, and bone formation markers were significantly reduced in osteoblasts and hypertrophic chondrocytes. Osteogenic differentiation was accompanied by an increase in S1prs mRNA, especially S1P1/3 , but their contribution to S1P-impact on mineralization seemed limited. Our results suggest that S1P might be overproduced in SpA enthesis in response to cytokines and mechanical stress, most likely by chondrocytes. Moreover, S1P could locally favor the abnormal ossification of the enthesis; therefore, blocking the S1P metabolic pathway could be a potential therapeutic approach for the treatment of SpA. © 2019 American Society for Bone and Mineral Research.

Effects of phospholipase D during cultured osteoblast mineralization and bone formation.

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.

Involvement of sphingosine kinase/sphingosine 1-phosphate metabolic pathway in spondyloarthritis.

Spondyloarthritis (SpA) is a relatively common chronic inflammatory joint disorder, with a prevalence of about 0.2-0.5% worldwide. The primary target of the pathological process is the enthesis, where tendons and ligaments attach to underlying bone. These insertion sites are hotspots of bone formation (enthesophytes), which can lead to ankylosis. Unfortunately, the mechanisms causing the onset and progression of entheseal ossification remain largely unknown. Sphingosine 1-phosphate (S1P), a lipid generated after sphingosine phosphorylation by sphingosine kinases 1 and 2 (SK1/2), plays important roles in cell proliferation, differentiation and survival. S1P regulates fundamental biological processes such as cell cycle, inflammatory response or bone homeostasis. Indeed, S1P has been involved in some of most-spread skeletal diseases such as rheumatoid arthritis or osteoarthritis. On the other hand, the implication of S1P in SpA has not been explored yet. In the present work, we observed by ELISA that S1P content was significantly increased in the serum of SpA patients (6.1±4.2µM, n=21) compared to healthy donors (1.6±0.9µM, n=12). In vitro, gene expression of SK1 and SK2 as well as their activity were increased during differentiation of primary murine chondrocytes and osteoblasts into mineralizing cells. In addition, mRNA of the S1P-specific transporter Spns2 and S1P secretion were augmented. Using the pharmacological drugs SKi (SK pan-inhibitor), PF-543 (SK1 specific inhibitor) or K-145 (SK2 specific inhibitor), we showed that the inhibition of SK1 and/or SK2 decreased matrix mineralization, alkaline phosphatase activity and the mRNA expression of Runx2 and Bglap in chondrocytes and osteoblasts. To our knowledge, this is the first study indicating that S1P levels are significantly increased in serum from SpA patients. Moreover, we showed in vitro that SK activity was involved in the mineralization capacity of osteoblasts and chondrocytes. S1P metabolic pathway may represent an ingenious therapeutic target for SpA in the future.