TNF-α stimulates alkaline phosphatase and mineralization through PPARγ inhibition in human osteoblasts.

The aims of the present study were to determine whether prostaglandins (PGs) and PPARγ are involved in the stimulation of tissue-non-specific alkaline phosphatase (TNAP) activity and mineralization by TNF-α in human osteoblasts. We used osteoblasts differentiated from MSCs from three different donors and MG-63 osteoblast-like cells. Inhibition of prostaglandin synthesis with the cyclooxygenase (COX) inhibitor indomethacin or the specific COX-2 blocker NS-398 abolished mineralization in the absence and presence of 1 ng/ml of TNF-α, suggesting that PGs were involved. The TNAP inhibitor levamisole abolished TNF-α effects on mineralization, suggesting that PGs were involved in TNAP expression and mineralization. TNF-α stimulated expression of COX-2 and PG E synthase before that of TNAP, but expression of PG D synthase later suggesting that PGE2 and PGF2α but not 15d-PGJ2 were involved in TNF-α effects. However, both PGE2 and PGF2α dose-dependently inhibited mineralization indicating that endogenous PG are required for mineralization but that TNF-α does not increase mineralization by increasing PG synthesis. Interestingly, TNF-α inhibited PPARγ expression and binding activity to PPRE consensus sequences independently of 15d-PGJ2. Inhibition of PPARγ activity with GW-9662 mimicked TNF-α effects in MG-63 cells, indicating that TNF-α stimulates mineralization by inhibiting PPARγ in osteoblasts. In MSC-derived osteoblast cultures, inhibition of PPARγ dropped TNAP expression and mineralization. Treatment of MG-63 cells with conditioned media from MSC-derived osteoblasts or MSC-derived adipocytes treated or not with GW-9662 revealed that TNF-α inhibition of PPARγ in undifferentiated MSCs and/or adipocytes was responsible for the decreased expression of TNAP in osteoblasts. In conclusion, TNF-α increases TNAP expression and stimulates mineralization by inhibiting PPARγ in osteoblasts, but PPARγ in adipocytes or undifferentiated MSCs controls the secretion of a factor leading to TNAP stimulation in osteoblasts.

IL-33 is expressed in human osteoblasts, but has no direct effect on bone remodeling.

The aim of the present study was to investigate the potential role of the recently discovered IL-1 family member IL-33 in bone remodeling. Our results indicate that IL-33 mRNA is expressed in osteocytes in non-inflammatory human bone. Moreover, IL-33 levels are increased by TNF-α and IL-1ß in human bone marrow stromal cells, osteoblasts and adipocytes obtained from three healthy donors. Experiments with the inhibitor GW-9662 suggested that expression of IL-33, in contrast to that of IL-1ß, is not repressed by PPARγ likely explaining why IL-33, but not IL-1ß, is expressed in adipocytes. The IL-33 receptor ST2L is not constitutively expressed in human bone marrow stromal cells, osteoblasts or CD14-positive monocytes, and IL-33 has no effect on these cells. In addition, although ST2L mRNA is induced by TNF-α and IL-1ß in bone marrow stromal cells, IL-33 has the same effects as TNF-α and IL-1ß, and, therefore, the biological activity of IL-33 may be redundant in this system. In agreement with this hypothesis, MC3T3-E1 osteoblast-like cells constitutively express ST2L mRNA, and IL-33 and TNF-α/IL-1ß similarly decrease osteocalcin RNA levels in these cells. In conclusion, our results suggest that IL-33 has no direct effects on normal bone remodeling.

Alteration of expression of muscle specific isoforms of the fragile X related protein 1 (FXR1P) in facioscapulohumeral muscular dystrophy patients.

BACKGROUND: The Fragile X Mental retardation-Related 1 (FXR1) gene belongs to the fragile X related family, that also includes the Fragile X Mental Retardation (FMR1) gene involved in fragile X syndrome, the most common form of inherited mental retardation. While the absence of FMRP impairs cognitive functions, inactivation of FXR1 has been reported to have drastic effects in mouse and xenopus myogenesis. Seven alternatively spliced FXR1 mRNA variants have been identified, three of them being muscle specific. Interestingly, they encode FXR1P isoforms displaying selective RNA binding properties. METHODS AND RESULTS: Since facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy characterised by altered splicing of mRNAs encoding muscle specific proteins, we have studied the splicing pattern of FXR1 mRNA in myoblasts and myotubes of FSHD patients. We show here that FSHD myoblasts display an abnormal pattern of expression of FXR1P isoforms. Moreover, we provide evidence that this altered pattern of expression is due to a specific reduced stability of muscle specific FXR1 mRNA variants, leading to a reduced expression of FXR1P muscle specific isoforms. CONCLUSION: Our data suggest that the molecular basis of FSHD not only involves splicing alterations, as previously proposed, but may also involve a deregulation of mRNA stability. In addition, since FXR1P is an RNA binding protein likely to regulate the metabolism of muscle specific mRNAs during myogenesis, its altered expression in FSHD myoblasts may contribute to the physiopathology of this disease.

[Experimental evaluation of microscan assessment of bone fusion]. / Etude expérimentale de la microtomographie X dans l'évaluation de la fusion osseuse.

PURPOSE OF THE STUDY: Certain confirmation of bone fusion remains difficult to obtain after arthrodesis despite progress in imaging techniques. Microscanning enables both qualitative and quantitative analysis of the bone microarchitecture. The purpose of this study was to evaluate this technique using a cervical arthrodesis with an intersomatic cage on an animal model and to validate results with histological analysis and electron scan microscopy (SEM). MATERIAL AND METHODS: C3-C4 discectomy was performed in 8 goats divided into two groups. In group 1 (3 animals), PEEK cages were inserted without bone graft. In group 2 (5 goats) the same cage was inserted and filled with an autologous iliac graft. The animals were sacrificed at six months. The instrumented levels were analyzed with a microscan. Histological slides were obtained and SEM performed. RESULTS: Nonunion was observed in the three animals with an empty cage (group 1) while only one animal in group 2 presented nonunion. Histology and SEM confirmed the diagnosis established with the microscan which also enabled a 3D analysis of the sample and study of the trabecular architecture of the intersomatic graft. DISCUSSION: The microscan enabled a micrometric analysis of the sample. This is the only technique enabling 3D analysis (slices can be obtained in the three planes for 3D reconstruction) for both qualitative and quantitative assessment. Analysis of the trabecular microstructure constitutes a major progress in evaluating the mechanical value of the fusion. The sample is not destroyed and can be studied further with other biomechanical techniques. CONCLUSION: Microscanning is an important technical advancement for the analysis of bone fusion. Future applications will undoubtedly be numerous (follow-up after arthrodesis, analysis of the mechanical quality of a graft). In vivo applications will probably be adapted soon.