Possible Role of Extracellular Nucleotides in Ectopic Ossification of Human Spinal Ligaments

Abstract

Abstract. To reveal the involvement of extracellular nucleotides in the ossification process in ossification of the posterior longitudinal ligament of the spine (OPLL), the mRNA expression profiles of P2 purinoceptors, mechanical stress-induced ATP release, and ATP-stimulated expression of osteogenic genes were analyzed in ligament cells derived from the spinal ligament of OPLL patients (OPLL cells) and non-OPLL cells derived from the spinal ligaments of cervical spondylotic myelopathy patients as a control. The extracellular ATP concentrations of OPLL cells in static culture were significantly higher than those of non-OPLL cells, and this difference was diminished in the presence of ARL67156, an ecto-nuclease inhibitor. Cyclic stretch markedly increased the extracellular ATP concentrations of both cell types to almost the same level. P2Y1 purinoceptor subtypes were intensively expressed in OPLL cells, but only weakly expressed in non-OPLL cells. Not only ATP addition but also cyclic stretch raised the mRNA levels of alkaline phosphatase and osteopontin in OPLL cells, which were blocked by MRS2179, a selective P2Y1 antagonist. These increases in the expression of osteogenic genes were not observed in non-OPLL cells. These results suggest an important role of P2Y1 and extracellular ATP in the progression of OPLL stimulated by mechanical stress.

Effect of Combined Action of Extracellular ATP and Elevated Calcium on Osteogenic Differentiation of Primary Cultures From Rat Calvaria

“…The observation that OPN was significantly up‐regulated by ATPγ‐S in the presence of 5.35 mM Ca 2+ suggests that signaling pathways associated to purinergic receptors and the calcium messenger system regulate bone matrix synthesis and mineralization. Interestingly, up‐regulation of OPN expression in osteoblastic cell monolayer cultures has also been shown by mechanical stress as compressive forces, which also stimulates ATP release from these cells [Rath et al, ; Sawada et al, ]. Our findings show increased ALP activity after cell stimulation with low concentration of UTP (10 µM) or high concentration of UDP (100 µM) in osteogenic medium containing 5.35 mM Ca 2+ , suggesting that these conditions promote osteogenic differentiation.…”

The genetics of ossification of the posterior longitudinal ligament

“…Additionally, it seems clear that multiple growth factors, 4,[9][10][11]35,40,41 cytokines, 23,42 and transcription factors 1,29 are involved in the pathogenesis, inheritance, and development of OPLL. Multiple polymorphisms in multiple genes of multiple bone modulating proteins have been linked to OPLL.…”

“…40 Horikoshi et al 4 demonstrated that an alternative TGF gene, TGFB3, was found to have a significant association to OPLL (while many other previously implicated candidate genes, such as COL11A2, did not show significant association). Other growth factors, cytokines, and genes that have been shown to have increased expression or different polymorphisms between patients with OPLL and those without included purinoceptors, 29 osteopontin, 1 CTGF/Hcs24, 42 the estrogen receptor, and interleukin-1.…”

Ossification of the posterior longitudinal ligament: a review

“…47 The P2Y1 purinoceptor subtypes, intensively expressed in OPLL cells, responded to mechanical stress-induced extracellular adenosine triphosphate, which stimulated OPLL progression. 110 Frequent consumption of pickles, nondaily consumers of rice, 100 family history of myocardial infarction, high body mass index at age 40, long working hours, and working night shifts 58 were associated with increased risk of OPLL. On the other hand, frequent consumption of chicken and soy products 100 and good sleeping habits (6-8 hours/night) in the prime of life may decrease the risk of OPLL.…”