Yohann Wittrant

Oxidative stress in bone remodelling and disease

Oxidative stress is characterised by an increased level of reactive oxygen species (ROS) that disrupts the intracellular reduction-oxidation (redox) balance. Although initially shown to be involved in aging, physiological roles for ROS in regulating cell functions and mediating intracellular signals have emerged. In bone tissues, recent studies have demonstrated that ROS generation is a key modulator of bone cell function and that oxidative status influences the pathophysiology of mineralised tissues. Here, we review the crucial role of oxidative stress in bone pathophysiology, and discuss the possibility that ROS production might be a relevant therapeutic target under certain conditions. Further studies will be needed to investigate whether manipulation of the redox balance in bone cells represents a useful approach in the design of future therapies for bone diseases.

Therapeutic Relevance of Osteoprotegerin Gene Therapy in Osteosarcoma: Blockade of the Vicious Cycle between Tumor Cell Proliferation and Bone Resorption

Osteosarcoma is the most frequent primary bone tumor that develops mainly in the young, the median age of diagnosis being 18 years. Despite improvement in osteosarcoma treatment, survival rate is only 30% at 5 years for patients with pulmonary metastases at diagnosis. This warrants exploration of new therapeutic options, and among them, osteoprotegerin (OPG), a naturally occurring protein that inhibits bone resorption, is very promising in blocking the vicious cycle between bone resorption and tumor proliferation that takes place during tumor development in bone site. As OPG binds and inhibits the activity of tumor necrosis factor-related apoptosis-inducing ligand, the truncated form of murine OPG 1-194 was used. The cDNA encoding OPG was administered by gene transfer using replication-defective adenoviral vector or was associated with an amphiphilic polymer in two models of rodent osteosarcoma. In both models, OPG gene transfer was effective in preventing the formation of osteolytic lesions associated with osteosarcoma development, in reducing the tumor incidence and the local tumor growth, leading to a 4-fold augmentation of mice survival 28 days postimplantation. On the contrary, OPG did not prevent the development of pulmonary metastasis alone, suggesting that bone environment is necessary for OPG therapeutic efficacy. Because OPG has no direct activity on osteosarcoma cells in vitro (cell binding, cell proliferation, apoptosis, or cell cycle distribution), we show that OPG exerts indirect inhibitory effect on tumor progression through the inhibition of RANKL whose production is enhanced in bone tumor environment, leading to osteolysis inhibition as reflected by osteoclast number decrease.

Phosphate-Dependent Regulation of MGP in Osteoblasts: Role of ERK1/2 and Fra-1†‡

Inorganic phosphate (Pi) and the matrix Gla protein (MGP) are key regulators of bone formation. We have recently shown that Pi upregulates MGP in growth plate chondrocytes, which may represent a negative feedback loop for the control of mineralization. Osteoblasts from Fra‐1‐deleted mice express low levels of MGP, whereas the expression of MGP is elevated in Fra‐1 transgenic osteoblasts, suggesting a role for Fra‐1 in MGP expression and bone formation. In this study, we aimed at deciphering the relationships between Pi and MGP in osteoblasts to determine the molecular mechanisms involved in the Pi‐dependent regulation of MGP. In MC3T3‐E1 cells and primary calvaria‐derived osteoblasts, Pi increased MGP and Fra‐1 expression at both the mRNA and protein levels. We also found that Pi enhanced the phosphorylation of ERK1/2. U0126 (MEK1/2 inhibitor) suppressed Pi‐stimulated MGP and Fra‐1 expression, indicating that ERK1/2 is required for Pi‐dependent regulation of MGP and Fra‐1. In addition, using in vitro DNA binding and chromatin immunoprecipitation assays, we showed that Fra‐1 interacts with the MGP promoter in response to Pi in MC3T3‐E1 cells. Finally, we found that in fra‐1 knockdown MC3T3‐E1 osteoblasts, the level of MGP expression is no more significantly upregulated by Pi. We further showed that primary osteoblasts from Fra‐1‐deficient mice failed to exhibit a Pi‐dependent stimulation of MGP expression. These data show, for the first time, that Pi regulates MGP expression in osteoblasts through the ERK1/2‐Fra‐1 pathway.

Osteoprotegerin differentially regulates protease expression in osteoclast cultures

Cysteine proteases and matrix metalloproteinases (MMPs) are important factors in the degradation of organic matrix components of bone. Osteoprotegerin (OPG) is an osteoblast-secreted decoy receptor that inhibits osteoclast differentiation and activation. This study investigated the direct effects of human OPG on cathepsin K, MMP-9, MMP-2, and tissue inhibitors of metalloproteinases (TIMP1 and TIMP2) expressed by purified rabbit osteoclasts. The expression of two osteoclast markers, namely tartrate-resistant acid phosphatase (TRAP) and cathepsin K, was inhibited by 100 ng/mL hOPG, whereas MMP-9 expression was enhanced. Gelatinase activities were measured using a zymographic assay, and hOPG was shown to enhance both pro-MMP-9 and MMP-2 activities. Concomitantly, TIMP1 expression was greatly stimulated by hOPG, whereas TIMP2 mRNA levels were not modulated. Overall, these results show that hOPG regulates the proteases produced by purified osteoclasts differentially, producing a marked inhibitory effect on the expression of cathepsin K, the main enzyme involved in bone resorption.

Gallium modulates osteoclastic bone resorption in vitro without affecting osteoblasts

Background and purpose:  Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer‐related hypercalcemia and Pagets disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro.

Killer Dendritic Cells Link Innate and Adaptive Immunity against Established Osteosarcoma in Rats

We have previously reported that a distinct subset of splenic CD4(-) rat dendritic cells (DC) induces a rapid and caspase-independent apoptosis-like cell death in a large number of tumor cells in vitro. The killing activity of these killer DC (KDC) was restricted to their immature state and was immediately followed by their engulfment of the apoptotic target cells, suggesting that these KDC could directly link innate and adaptive immunity to tumors. Here, we addressed this question using a transplantable model of rat osteosarcoma. First, we showed that rat KDC have an MHC II(+)CD103(+)CD11b(+)NKp46(-) phenotype and are therefore distinct from natural killer cells, which are MHC II(-)CD103(-)CD11b(-)NKp46(+). KDC numbers could be specifically and strongly (up to 10-fold) enhanced by Flt3L in vivo. The OSRGa cell line derived from the osteosarcoma tumor was killed and phagocytosed in vitro by both normal and Flt3L-induced splenic KDC. Such tumor antigen-loaded KDC were used to s.c. vaccinate progressive tumor-bearing rats. Vaccination with OSRGa-loaded KDC but not KDC loaded with irrelevant tumor cells (Jurkat) delayed tumor progression or even induced tumor regression. This vaccine effect was not observed in CD8 T cell-depleted animals and protective against tumor rechallenge. These results suggest that KDC possess the intrinsic capability not only to kill and then engulf tumor cells but also to efficiently cross-present tumor cell-derived antigen in vivo and subsequently induce an adaptive antitumor immune response.

Inorganic phosphate regulates Glvr-1 and -2 expression: Role of calcium and ERK1/2

Sodium-dependent phosphate cotransporters are key regulators of phosphate homeostasis and play a major role in mineralized tissues remodelling. However, factors influencing their expression remain under consideration. In our study, modulation of type III sodium-dependent phosphate cotransporters expression by inorganic phosphate (Pi) was investigated in the murine odontoblast-like cell line MO6-G3. Experiments were designed to determine the effects of phosphate release on dental cells during tooth decay. By real-time RT-PCR we demonstrated that Glvr-1 and -2 expressions are up-regulated by Pi. The increase in Glvr-1 and -2 expressions was correlated with ERK1/2 phosphorylation and calcium/phosphate crystals formation in cultured wells. Using calcium-free culture conditions or the specific inhibitor of ERK phosphorylation (UO126), we demonstrated that Pi effects on Glvr-1 and -2 up-regulation require the presence of calcium and involve ERK signalling pathways. This study contributes to give new insights in the control of Pi transport during carious diseases.

Inorganic phosphate stimulates apoptosis in murine MO6-G3 odontoblast-like cells.

OBJECTIVE Dental pathologies such as caries are the most prevalent disease worldwide with infectious and social complications. During the process of caries formation, the tooth is degraded and demineralization of enamel and dentine leads to the release of large amounts of inorganic phosphate (Pi) within dental tubuli. As Pi has been shown to induce apoptosis in skeletal cells, including osteoblasts and chondrocytes, we questioned whether high concentrations of Pi could affect odontoblast viability, proliferation and apoptosis. DESIGN Using the odontoblast-like MO6-G3 cell line as a model, we used cell counting and MTS-based colorimetric assays to measure cell viability and proliferation. Apoptosis was assessed using Hoechst nuclei staining and detection of the early apoptotic markers annexin V and Apo2.7. RESULTS We show for the first time that a high Pi concentration (7 mM) induced a decrease in odontoblast viability and proliferation together with a large increase in apoptosis. These effects were blunted in calcium-free medium, possibly due to the formation of calcium-phosphate crystals in the presence of high Pi concentrations. CONCLUSION This study contributes to clarifying the effect of Pi on odontoblast viability and apoptosis, which may improve our understanding of the role of Pi during caries formation.

Olive and grape seed extract prevents post-traumatic osteoarthritis damages and exhibits in vitro anti IL-1β activities before and after oral consumption

Polyphenols exert a large range of beneficial effects in the prevention of age-related diseases. We sought to determine whether an extract of olive and grape seed standardized according to hydroxytyrosol (HT) and procyanidins (PCy) content, exerts preventive anti-osteoathritic effects. To this aim, we evaluated whether the HT/PCy mix could (i) have in vitro anti-inflammatory and chondroprotective actions, (ii) exert anti-osteoarthritis effects in two post-traumatic animal models and (iii) retain its bioactivity after oral administration. Anti-inflammatory and chondroprotective actions of HT/PCy were tested on primary cultured rabbit chondrocytes stimulated by interleukin-1 beta (IL-1β). The results showed that HT/PCy exerts anti-inflammatory and chondroprotective actions in vitro. The preventive effect of HT/PCy association was assessed in two animal models of post-traumatic OA in mice and rabbits. Diet supplementation with HT/PCy significantly decreased the severity of post-traumatic osteoarthritis in two complementary mice and rabbit models. The bioavailability and bioactivity was evaluated following gavage with HT/PCy in rabbits. Regular metabolites from HT/PCy extract were found in sera from rabbits following oral intake. Finally, sera from rabbits force-fed with HT/PCy conserved anti-IL-1β effect, suggesting the bioactivity of this extract. To conclude, HT/PCy extract may be of clinical significance for the preventive treatment of osteoarthritis.

Pomegranate Peel Extract Prevents Bone Loss in a Preclinical Model of Osteoporosis and Stimulates Osteoblastic Differentiation in Vitro

The nutritional benefits of pomegranate have attracted great scientific interest. The pomegranate, including the pomegranate peel, has been used worldwide for many years as a fruit with medicinal activity, mostly antioxidant properties. Among chronic diseases, osteoporosis, which is associated with bone remodelling impairment leading to progressive bone loss, could eventually benefit from antioxidant compounds because of the involvement of oxidative stress in the pathogenesis of osteopenia. In this study, with in vivo and ex vivo experiments, we investigated whether the consumption of pomegranate peel extract (PGPE) could limit the process of osteopenia. We demonstrated that in ovariectomized (OVX) C57BL/6J mice, PGPE consumption was able to significantly prevent the decrease in bone mineral density (−31.9%; p < 0.001 vs. OVX mice) and bone microarchitecture impairment. Moreover, the exposure of RAW264.7 cells to serum harvested from mice that had been given a PGPE-enriched diet elicited reduced osteoclast differentiation and bone resorption, as shown by the inhibition of the major osteoclast markers. In addition, PGPE appeared to substantially stimulate osteoblastic MC3T3-E1 alkaline phosphatase (ALP) activity at day 7, mineralization at day 21 and the transcription level of osteogenic markers. PGPE may be effective in preventing the bone loss associated with ovariectomy in mice, and offers a promising alternative for the nutritional management of this disease.