Rôle du facteur de transcription PITX1 dans les pathogenèses de l'ostéoporose et des maladies parodontales

L’ostéoporose est une maladie caractérisée par une faible masse osseuse et une détérioration du tissu osseux. Cette condition entraîne une plus grande fragilité osseuse et des risques de fractures. Plusieurs études ont associé l’ostéoporose à la faible densité osseuse des mandibules, à la perte d’attache parodontale, à l’augmentation de la hauteur de la crête alvéolaire et à la chute des dents. Cette étude vise à comprendre les mécanismes sous-jacents cette perte osseuse. En effet, au cours du développement des souris, PITX1 joue un rôle clé dans l'identité des membres postérieurs et dans le bon développement des mandibules et des dents. Son inactivation complète chez la souris mène à un phénotype squelettique sévère. Tandis que, son inactivation partielle provoque des symptômes apparentés à l'arthrose avec une augmentation de la masse osseuse au niveau de l’os cortical et de l’os trabéculaire. Inversement, une étude antérieure chez des jumelles monozygotiques discordantes pour l’ostéoporose, montrent une augmentation d’environ 8.6 fois du niveau d’expression du gène Pitx1 chez la jumelle ostéoporotique. Collectivement, ces données nous ont poussés à investiguer sur le rôle du facteur de transcription PITX1 dans le métabolisme osseux normal et pathologique. Dans ce contexte, des souris transgéniques Col1α1-Pitx1 sur-exprimant Pitx1 spécifiquement dans le tissu osseux sous le promoteur du collagène de type-I (fragment 2.1kpb) ont été générées et phénotypiquement caractérisées. Ces résultats ont révelé que les souris transgéniques Col1α1-Pitx1 présentaient un phénotype similaire à celui des patients ostéoporotiques accompagné d'une perte de dents et des problèmes dentaires et parodontaux. De plus, cette étude a révélé que la surexpression de Pitx1 induit une altération de l’homéostasie osseuse via l’inactivation de la voie de signalisation Wnt/β-caténine canonique. Cette hypothèse a été appuyée par le fait que le traitement des souris transgéniques Col1α1-Pitx1 avec du chlorure de lithium, un activateur de la voie Wnt canonique, prévient le phénotype ostéoporotique chez ces souris. Finalement, cette étude établit un rôle crucial de PITX1 dans la régulation de la masse osseuse et une implication possible dans l’ostéoporose et les maladies parodontales via l’inactivation de la voie de signalisation Wnt/β-caténine canonique.

Angiotensin receptor I stimulates osteoprogenitor proliferation through TGFβ-mediated signaling

Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti-hypertensive drugs that target angiotensin II (Ang II) activity may also improve loss of bone mineral density. Here we identified in a genetic screen the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20-17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20-17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20-17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)-Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor β (TGFβ) expression considerably and in an AT1R-dependent manner. Finally, exogenous TGFβ was able to restore high proliferative activity to W20-17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFβ and targets proliferation of osteoblast progenitors.

Anti-Metastatic Properties of a Marine Bacterial Exopolysaccharide-Based Derivative Designed to Mimic Glycosaminoglycans

Osteosarcoma is the most frequent malignant primary bone tumor characterized by a high potency to form lung metastases. In this study, the effect of three oversulfated low molecular weight marine bacterial exopolysaccharides (OS-EPS) with different molecular weights (4, 8 and 15 kDa) were first evaluated in vitro on human and murine osteosarcoma cell lines. Different biological activities were studied: cell proliferation, cell adhesion and migration, matrix metalloproteinase expression. This in vitro study showed that only the OS-EPS 15 kDa derivative could inhibit the invasiveness of osteosarcoma cells with an inhibition rate close to 90%. Moreover, this derivative was potent to inhibit both migration and invasiveness of osteosarcoma cell lines; had no significant effect on their cell cycle; and increased slightly the expression of MMP-9, and more highly the expression of its physiological specific tissue inhibitor TIMP-1. Then, the in vivo experiments showed that the OS-EPS 15 kDa derivative had no effect on the primary osteosarcoma tumor induced by osteosarcoma cell lines but was very efficient to inhibit the establishment of lung metastases in vivo. These results can help to better understand the mechanisms of GAGs and GAG-like derivatives in the biology of the tumor cells and their interactions with the bone environment to develop new therapeutic strategies.

Rôle du facteur de transcription PITX1 dans les pathogenèses de l'ostéoporose et des maladies parodontales

L’ostéoporose est une maladie caractérisée par une faible masse osseuse et une détérioration du tissu osseux. Cette condition entraîne une plus grande fragilité osseuse et des risques de fractures. Plusieurs études ont associé l’ostéoporose à la faible densité osseuse des mandibules, à la perte d’attache parodontale, à l’augmentation de la hauteur de la crête alvéolaire et à la chute des dents. Cette étude vise à comprendre les mécanismes sous-jacents cette perte osseuse. En effet, au cours du développement des souris, PITX1 joue un rôle clé dans l'identité des membres postérieurs et dans le bon développement des mandibules et des dents. Son inactivation complète chez la souris mène à un phénotype squelettique sévère. Tandis que, son inactivation partielle provoque des symptômes apparentés à l'arthrose avec une augmentation de la masse osseuse au niveau de l’os cortical et de l’os trabéculaire. Inversement, une étude antérieure chez des jumelles monozygotiques discordantes pour l’ostéoporose, montrent une augmentation d’environ 8.6 fois du niveau d’expression du gène Pitx1 chez la jumelle ostéoporotique. Collectivement, ces données nous ont poussés à investiguer sur le rôle du facteur de transcription PITX1 dans le métabolisme osseux normal et pathologique. Dans ce contexte, des souris transgéniques Col1α1-Pitx1 sur-exprimant Pitx1 spécifiquement dans le tissu osseux sous le promoteur du collagène de type-I (fragment 2.1kpb) ont été générées et phénotypiquement caractérisées. Ces résultats ont révelé que les souris transgéniques Col1α1-Pitx1 présentaient un phénotype similaire à celui des patients ostéoporotiques accompagné d'une perte de dents et des problèmes dentaires et parodontaux. De plus, cette étude a révélé que la surexpression de Pitx1 induit une altération de l’homéostasie osseuse via l’inactivation de la voie de signalisation Wnt/β-caténine canonique. Cette hypothèse a été appuyée par le fait que le traitement des souris transgéniques Col1α1-Pitx1 avec du chlorure de lithium, un activateur de la voie Wnt canonique, prévient le phénotype ostéoporotique chez ces souris. Finalement, cette étude établit un rôle crucial de PITX1 dans la régulation de la masse osseuse et une implication possible dans l’ostéoporose et les maladies parodontales via l’inactivation de la voie de signalisation Wnt/β-caténine canonique.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism

We have generated RANK (receptor activator of NF-κB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK−/− mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1−/− (recombinase activating gene 1) mice, indicating that RANK−/− mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK−/− mice without inducing hypercalcemia, although tumor necrosis factor α treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK−/− mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Is Nitric Oxide a Mediator of the Effects of Low-Intensity Electrical Stimulation on Bone in Ovariectomized Rats?

Low-intensity electrical stimulation (LIES) may counteract the effects of ovariectomy (OVX) on nitric oxide synthase (NOS) expression, osteocyte viability, bone structure, and microarchitecture in rats (Lirani-Galvo et al., Calcif Tissue Int 84:502-509, 2009). The aim of the present study was to investigate if these effects of LIES could be mediated by NO. We analyzed the effects of NO blockage (by l-NAME) in the response to LIES on osteocyte viability, bone structure, and microarchitecture in OVX rats. Sixty rats (200-220 g) were divided into six groups: sham, sham-l-NAME (6 mg/kg/day), OVX, OVX-l-NAME, OVX-LIES, and OVX-LIES-l-NAME. After 12 weeks, rats were killed and tibiae collected for histomorphometric analysis and immunohistochemical detection of endothelial NOS (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). In the presence of l-NAME, LIES did not counteract the OVX-induced effects on bone volume and trabecular number (as on OVX-LIES). l-NAME blocked the stimulatory effects of LIES on iNOS and eNOS expression of OVX rats. Both l-NAME and LIES decreased osteocyte apoptosis. Our results showed that in OVX rats l-NAME partially blocks the effects of LIES on bone structure, turnover, and expression of iNOS and eNOS, suggesting that NO may be a mediator of some positive effects of LIES on bone.

Low-Intensity Electrical Stimulation Counteracts the Effects of Ovariectomy on Bone Tissue of Rats: Effects on Bone Microarchitecture, Viability of Osteocytes, and Nitric Oxide Expression

Low Intensity Electrical Stimulation (LIES) has been used for bone repair, but little is known about its effects on bone after menopause. Osteocytes probably play a role in mediating this physical stimulus and they could act as transducers through the release of biochemical signals, such as nitric oxide (NO). The aim of the present study was to investigate the effects of LIES on bone structure and remodeling, NOS expression and osteocyte viability in ovariectomized (OVX) rats. Thirty rats (200-220 g) were divided into 3 groups: SHAM, OVX, and OVX subjected to LIES (OVX + LIES) for 12 weeks. Following the protocol, rats were sacrificed and tibias were collected for histomorphometric analysis and immunohistochemical detection of endothelial NO synthase (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). OVX rats showed significant (p < 0.05 vs. SHAM) decreased bone volume (10% vs. 25%) and trabecular number (1.7 vs. 3.9), and increased eroded surfaces (4.7% vs. 3.2%) and mineralization surfaces (15.9% vs. 7.7%). In contrast, after LIES, all these parameters were significantly different from OVX but not different from SHAM. eNOS and iNOS were similarly expressed in subperiosteal regions of tibiae cortices of SHAM, not expressed in OVX, and similarly expressed in OVX + LIES when compared to SHAM. In OVX, the percentage of apoptotic osteocytes (24%) was significantly increased when compared to SHAM (11%) and OVX + LIES (8%). Our results suggest that LIES counteracts some effects of OVX on bone tissue preserving bone structure and microarchitecture, iNOS and eNOS expression, and osteocyte viability.

Hemoglobin Regeneration Efficiency in Anemic Rats: Effects on Bone Mineral Composition and Biomechanical Properties

This study reports the effects of dietary iron (Fe) deficiency and recovery on bone mineral composition and strength in anemic rats submitted to a hemoglobin (Hb) repletion assay. Weanling male Wistar rats were fed a low-Fe diet (12 mg/kg) for 15 days followed by 2 weeks of Fe repletion with diets providing 35 mg Fe/kg as either ferrous sulfate (n = 8) or ferric pyrophosphate (FP; n = 12). At final day of each period (depletion and repletion), Fe-adequate animals were also euthanized. Iron status (blood Hb, Hb Fe pool, Hb regeneration efficiency), tibia mineral concentrations (Ca, Mg, Fe, Cu, and Zn) and biomechanical properties were evaluated. Iron-deficient rats had lower tibia Fe and Mg levels and bone strength when compared to controls. Yield load and resilience were positively related to tibia Mg levels (r = 0.47, P = 0.02 and r = 0.56, P = 0.004, respectively). Iron repletion did not recover tibia Mg concentrations impaired by Fe deficiency. Moreover, bone elastic properties were negatively affected by FP consumption. In conclusion, bone mineral composition and strength were affected by Fe deficiency, whereas dietary Fe source influenced tibia Mg and resistance in the period during which rats were recovering from anemia.

Methylenedioxymethamphetamine (Ecstasy) Decreases Neutrophil Activity and Alters Leukocyte Distribution in Bone Marrow, Spleen and Blood

Objective: Looking for possible neuroimmune relationships, we analyzed the effects of methylenedioxymethamphetamine (MDMA) administration on neuroendocrine, neutrophil activity and leukocyte distribution in mice. Methods: Five experiments were performed. In the first, mice were treated with MDMA (10 mg/kg) 30, 60 min and 24 h prior to blood sample collection for neutrophil activity analysis. In the second experiment, the blood of nave mice was collected and incubated with MDMA for neutrophil activity in vitro analysis. In the third and fourth experiments, mice were injected with MDMA (10 mg/kg) and 60 min later, blood and brain were collected to analyze corticosterone serum levels and hypothalamic noradrenaline (NA) levels and turnover. In the last experiment, mice were injected with MDMA 10 mg/kg and 60 min later, blood, bone marrow and spleen were collected for leukocyte distribution analysis. Results: Results showed an increase in hypothalamic NA turnover and corticosterone serum levels 60 min after MDMA (10 mg/kg) administration, a decrease in peripheral blood neutrophil oxidative burst and a decrease in the percentage and intensity of neutrophil phagocytosis. It was further found that MDMA (10 mg/kg) treatment also altered leukocyte distribution in blood, bone marrow and spleen. In addition, no effects were observed for MDMA after in vitro exposure both in neutrophil oxidative burst and phagocytosis. Conclusion: The effects of MDMA administration (10 mg/kg) on neutrophil activity and leukocyte distribution might have been induced indirectly through noradrenergic neurons and/or hypothalamic-pituitary-adrenal axis activations. Copyright (C) 2009 S. Karger AG, Basel

Modulation of doxorubicin-induced clastogenesis in Wistar rat bone marrow cells by vitamin B(6)

Vitamin B(6) has shown to be a potentially effective antioxidant agent, and dietary antioxidants are also frequently valuable inhibitors of clastogenesis and carcinogenesis. The purpose of the present work was to study the clastogenicity of different doses of vitamin B6 and to examine the possible modulating effect of this vitamin on chromosomal damage induced by the antitumor agent doxorubicin in Wistar rats. Experimental groups were set up for pre-and simultaneous treatment with vitamin B6 alone or in combination with DXR. The data obtained from administering diVerent doses of vitamin B(6) (12.5-100 mg/kg b. w.) showed no signigicant increase in total chromosomal aberrations when compared with the negative control. The administration of two doses of 25 mg/kg b. w. or one dose of 50 mg/kg b. w. of vitamin B6 before doxorubicin injection seemed equally effective in protecting cells against doxorubicin clastogenicity. The anticlastogenic effect of vitamin B(6) on DXR-induced chromosomal damage could be ascribed to its antioxidant properties. Vitamin B6 was not clastogenic or cytotoxic in rat bone marrow cells and it plays a role in inhibiting the clastogenicity induced by DXR.

Modification of the in vivo four-point loading model for studying mechanically induced bone adaptation

We modified the noninvasive, in vivo technique for strain application in the tibiae of rats (Turner et al,, Bone 12:73-79, 1991), The original model applies four-point bending to right tibiae via an open-loop, stepper-motor-driven spring linkage, Depending on the magnitude of applied load, the model produces new bone formation at periosteal (Ps) or endocortical surfaces (Ec.S). Due to the spring linkage, however, the range of frequencies at which loads can be applied is limited. The modified system replaces this design with an electromagnetic vibrator. A load transducer in series with the loading points allows calibration, the loaders' position to be adjusted, and cyclic loading completed under load central as a closed servo-loop. Two experiments were conducted to validate the modified system: (1) a strain gauge was applied to the lateral surface of the right tibia of 5 adult female rats and strains measured at applied loads from 10 to 60 N; and (2) the bone formation response was determined in 28 adult female Sprague-Dawley rats. Loading was applied as a haversine wave with a frequency of 2 Hz for 18 sec, every second day for 10 days. Peak bending loads mere applied at 33, 40, 52, and 64 N, and a sham-loading group tr as included at 64 N, Strains in the tibiae were linear between 10 and 60 N, and the average peak strain at the Ps.S at 60 N was 2664 +/- 250 microstrain, consistent with the results of Turner's group. Lamellar bone formation was stimulated at the Ec.S by applied bending, but not by sham loading. Bending strains above a loading threshold of 40 N increased Ec Lamellar hone formation rate, bone forming surface, and mineral apposition rate with a dose response similar to that reported by Turner et al, (J Bone Miner Res 9:87-97, 1994). We conclude that the modified loading system offers precision for applied loads of between 0 and 70 N, versatility in the selection of loading rates up to 20 Hz, and a reproducible bone formation response in the rat tibia, Adjustment of the loader also enables study of mechanical usage in murine tibia, an advantage with respect to the increasing variety of transgenic strains available in bone and mineral research. (Bone 23:307-310; 1998) (C) 1998 by Elsevier Science Inc. All rights reserved.

A six-year longitudinal study of the relationship of physical activity to bone mineral accrual in growing children: The University of Saskatchewan bone mineral accrual study

To investigate the influence of physical activity on bone mineral accrual during the adolescent years, we analyzed 6 years of data from 53 girls and 60 boys. Physical activity, dietary intakes, and anthropometry were measured every 6 months and dual-energy X-ray absorptiometry scans of the total body (TB), lumbar spine (LS), and proximal femur (Hologic 2000, array mode) were collected annually. Distance and velocity curves for height and bone mineral content (BMC) were fitted for each child at several skeletal sites using a cubic spline procedure, from which ages at peak height velocity (PHV) and peak BMC velocity (PBMCV) were identified. A mean age- and gender-specific standardized activity (Z) score was calculated for each subject based on multiple yearly activity assessments collected up until age of PHV. This score was used to identify active (top quartile), average (middle 2 quartiles), or inactive (bottom quartile) groups. Two-way analysis of covariance, with height and weight at PHV controlled for, demonstrated significant physical activity and gender main effects (but no interaction) for PBMCV, for BMC accrued for 2 years around peak velocity, and for BMC at 1 year post-PBMCV for the TB and femoral neck and for physical activity but not gender at the LS (all p < 0.05). Controlling for maturational and size differences between groups, we noted a 9% and 17% greater TB BMC for active boys and girls, respectively, over their inactive peers 1 year after the age of PBMCV. We also estimated that, on average, 26% of adult TB bone mineral was accrued during the 2 years around PBMCV.

Race and sex effects on the association between muscle strength, soft tissue, and bone mineral density in healthy elders: The Health, Aging, and Body Composition Study

Two factors generally reported to influence bone density are body composition and muscle strength. However, it is unclear if these relationships are consistent across race and sex, especially in older persons. If differences do exist by race and/or sex, then strategies to maintain bone mass or minimize bone loss in older adults may need to be modified accordingly. Therefore, we examined the independent effects of bone mineral-free lean mass (LM), fat mass (FM), and muscle strength on regional and whole body bone mineral density (BMD) in a cohort of 2619 well-functioning older adults participating in the Health, Aging, and Body Composition (Health ABC) Study with complete measures. Participants included 738 white women, 599 black women, 827 white men, and 455 black men aged 70-79 years. BMD (g/cm(2)) of the femoral neck, whole body, upper and lower limb, and whole body and upper limb bone mineral-free LM and FM was assessed by dual-energy X-ray absorptiometry (DXA). Handgrip strength and knee extensor torque were determined by dynamometry. In analyses stratified by race and sex and adjusted for a number of confounders, LM was a significant (p < 0.001) determinant of BMD, except in white women for the lower limb and whole body. In women, FM also was an independent contributor to BMD at the femoral neck, and both PM and muscle strength contributed to limb BMD. The following were the respective Beta-weights (regression coefficients for standardized data, Std beta) and percent difference in BMD per unit (7.5 kg) LM: femoral neck, 0.202-0.386 and 4.7-6.9 %; lower limb,.0.209-0.357 and 2.9-3.5%; whole body, 0.239-0.484 and 3.0-4.7 %; and upper limb (unit = 0.5 kg), 0.231-0.407 and 3.1-3.4%. Adjusting for bone size (bone mineral apparent density [BMAD]) or body size BMD/height) diminished the importance of LM, and the contributory effect of FM became more pronounced. These results indicate that LM and FM were associated with bone mineral depending on the bone site and bone index used. Where differences did occur, they were primarily by sex not race. To preserve BMD, maintaining or increasing LM in the elderly would appear to be an appropriate strategy, regardless of race or sex.

Gender differences in bone geometry during the adolescent growth spurt

To investigate whether there are gender differences in the bone geometry of the proximal femur during the adolescent years we used an interactive computer program ?Hip Strength Analysis? developed by Beck and associates (Beck et al., Invest Radiol. 1990,25:6-18.) to derive femoral neck geometry parameters from DXA bone scans (Hologic 2000, array mode). We analyzed a longitudinal data-set collected on 70 boys and 68 girls over a seven year period. Distance and velocity curves for height were fitted for each child utilizing a cubic spline procedure and the age of peak height velocity (PHV) was determined. To control for maturational differences between children of the same chronological age and between boys and girls, section modulus (Z) an index of bending strength, cross sectional area of bone (CSA), sub-periosteal width (SPW), and BMD values at the neck and shaft of the proximal femur were determined for points on each individual?s curve at the age of PHV and one and two years on either side of peak. To control for size differences, height and weight were introduced as co-variates in the two-way analyses of variance looking at gender over time measured at the maturational age points (-2, -1, age of PHV, +1, +2). The following figure presents the results of the analyses on two variables, BMD and Z at neck and shaft regions:After the age of peak linear growth (PHV), independent of body size, there was a gender difference in BMD at the shaft but not at the neck. Section modulus at both sites indicated that male bones became significantly stronger after PHV. Underlying these maturational changes, male bones became wider (SPW) after PHV in both the neck and shaft and enclosed more material (CSA) at all maturational age points at both regions. These results call into question the emphasis on using BMD as a measure of skeletal integrity in growing children