Robert Moreau

Importance of melastatin-like transient receptor potential 7 and magnesium in the stimulation of osteoblast proliferation and migration by platelet-derived growth factor

Bone is a dynamic tissue that is continuously being remodeled throughout life. Specialized cells called osteoclasts transiently break down old bone (resorption process) at multiple sites as other cells known as osteoblasts are replacing it with new tissue (bone formation). Usually, both resorption and formation processes are in balance and thereby maintain skeletal strength and integrity. This equilibrium is assured by the coordination of proliferation, migration, differentiation, and secretory functions of the osteoblasts, which are essential for adequate formation and resorption processes. Disturbances of this equilibrium may lead to decreased bone mass (osteoporosis), increased bone fragility, and susceptibility to fractures. Epidemiological studies have linked insufficient dietary magnesium (Mg(2+)) intake in humans with low bone mass and osteoporosis. Here, we investigated the roles of Mg(2+) and melastatin-like transient receptor potential 7 (TRPM7), known as Mg(2+) channels, in human osteoblast cell proliferation and migration induced by platelet-derived growth factor (PDGF), which has been involved in the bone remodeling process. PDGF promoted an influx of Mg(2+), enhanced cell migration, and stimulated the gene expression of TRPM7 channels in human osteoblast MG-63 cells. The stimulation of osteoblast proliferation and migration by PDGF was significantly reduced under culture conditions of low extracellular Mg(2+) concentrations. Silencing TRPM7 expression in osteoblasts by specific small interfering RNA prevented the induction by PDGF of Mg(2+) influx, proliferation, and migration. Our results indicate that extracellular Mg(2+) and TRPM7 are important for PDGF-induced proliferation and migration of human osteoblasts. Thus Mg(2+) deficiency, a common condition among the general population, may be associated with altered osteoblast functions leading to inadequate bone formation and the development of osteoporosis.

Expression of transient receptor potential (TRP) channels in human and murine osteoblast-like cells

The preservation of bone mass relies on adequate proliferation, differentiation, secretion of matrix proteins and rate of apoptosis of the bone-forming osteoblasts. Although growing body of evidence indicates that the transient receptor potential (TRP) channels play important roles in numerous cellular functions, limited information is available about the TRP channels in osteoblasts. Here, we inventoried the gene expression and addressed some roles of the TRP channels in various osteoblast-like cells. The transcripts of canonical TRP (TRPC) channels were revealed for TRPC1, TRPC3, TRPC4 and TRPC6 in human MG-63, SaOS and U2 OS osteoblasts while transcripts for TRPC2, TRPC4, TRPC6 and TRPC7 were observed in the murine MC3T3 osteoblasts. PCR products were shown for the melastatin-related TRP (TRPM) channels TRPM4, TRPM6, TRPM7 and TRPM8 in all cell lines. The TRPM1 was specifically expressed by murine MC3T3 cells while the TRPM3 transcripts were revealed solely in human osteoblast-like cells. Transcripts for TRPV2 and TRPV4 were shown in osteoblastic cells. By interfering RNA approaches, the TRPC1 channels in osteoblasts were shown to be responsible for the capacitative calcium entry (CCE) and for the stimulation of cell proliferation by platelet-derived growth factor. On the other hand, interfering RNA-mediated abrogation of the expression of TRPM7, known as calcium and magnesium channels, resulted in the reduction of both basal and growth factor-stimulated osteoblastic cell proliferation. Our results provide the first complete reference for the gene expression of TRP channels in osteoblasts and point to their importance in cell proliferation.

Calcium uptake and calcium transporter expression by trophoblast cells from human term placenta

Placental transfer of maternal calcium (Ca(2+)) is a crucial step for fetal development although the biochemical mechanisms responsible for this process are largely unknown. This process is carried out in vivo by the placental syncytiotrophoblast layer. The aim of this study was to define the membrane gates responsible for the syncytiotrophoblast Ca(2+) entry, the first step in transplacental transfer. We have investigated the basal Ca(2+) uptake by primary culture of human term placenta syncytiotrophoblast. Kinetic studies revealed an active extracellular Ca(2+) uptake by cultured human syncytiotrophoblast. We demonstrated by Northern blot the presence of transcript for calcium transporter type 1 (CaT1) in cultured human syncytiotrophoblast and CaT1 expression was further confirmed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the expression of calcium transporter type 2 (CaT2) was revealed by RT-PCR in cultured human syncytiotrophoblast. It has been reported that the activity of this family of Ca(2+) channels is voltage-independent, and is not sensitive to L-type Ca(2+) channels agonist and antagonist. Interestingly, modulation of membrane potential by extracellular high potassium concentration and valinomycin had no effect on the basal Ca(2+) uptake of human syncytiotrophoblast. Moreover, the addition of L-type Ca(2+) channel modulators (Bay K 8644 and nitrendipine) to the incubation medium had also no effect on the basal Ca(2+) uptake, suggesting that the process is mainly voltage-independent and does not involved L-type Ca(2+) channels. On the other hand, we observed that two known blockers of CaT-mediated Ca(2+) transport, namely extracellular magnesium (Mg(2+)) and ruthenium red, dose-dependently inhibited Ca(2+) uptake by cultured human syncytiotrophoblast. Therefore, our results suggest that basal Ca(2+) uptake of human syncytiotrophoblast may be assured by CaT1 and CaT2.

Influence of oxidized low-density lipoproteins (LDL) on the viability of osteoblastic cells

Cardiovascular diseases have recently been noted as potential risk factors for osteoporosis development. Although it is poorly understood how these two pathologies are related, it is a known fact that oxidized low-density lipoproteins (OxLDL) constitute potential determinants for both of them. The current study investigated the metabolism of OxLDL by osteoblasts and its effect on osteoblastic viability. The results obtained show that OxLDL are internalized but not degraded by osteoblasts while they can selectively transfer their CE to these cells. It is also demonstrated that OxLDL induce proliferation at low concentrations but cell death at high concentrations. This reduction of osteoblast viability was associated with lysosomal membrane damage caused by OxLDL as demonstrated by acridine orange relocalization. Accordingly, chloroquine, an inhibitor of lysosomal activity, accentuated cell death induced by OxLDL. Finally, we demonstrate that osteoblasts have the capacity to oxidize LDL and thereby potentially increase the local concentration of OxLDL. Overall, the current study confirms the potential role of OxLDL in the development of osteoporosis given its influence on osteoblastic viability.

Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependent manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of (109)Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd(2+) species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive, suggesting that each ion inhibits the same uptake mechanism. Our results indicate that Cd uptake in human osteoblastic cells occurs, at least in part, through Ca- and Mg-inhibitable transport mechanisms, which may involve channels of the TRPM family. The effect of Cd on bone metabolism may be enhanced under low Ca and/or Mg levels.

Expression of Calcium Channels along the Differentiation of Cultured Trophoblast Cells from Human Term Placenta

Abstract Placental transfer of maternal calcium (Ca2+) is carried out in vivo by the syncytiotrophoblast layer. Although this process is crucial for fetal development, it remains poorly understood. Cytotrophoblasts isolated from human term placenta undergo spontaneous syncytiotrophoblast-like morphological and biochemical differentiation in vitro and are thought to reflect in vivo syncytiotrophoblast. In the present study, we characterized the Ca2+ uptake potential and the expression of several Ca2+ channels by human trophoblasts during differentiation in vitro for up to 6 days. Secretion of hCG (specific differentiation marker) and uptake of Ca2+ by trophoblasts increased gradually as a function of days in culture. Both hCG secretion and Ca2+ uptake were maximal on Day 4 and declined on Days 5–6. Expression of the Ca2+ transporter proteins CaT1 and CaT2 was revealed by reverse transcription-polymerase chain reaction in cytotrophoblasts freshly isolated from human term placenta. In addition, messengers for two L-type Ca2+ channel isoforms (α1C and α1D) were also detected. Levels of CaT1, CaT2, and L-type Ca2+ channel mRNA increased gradually during culture, reaching a maximum between Days 2 and 3. In contrast to CaT1 and CaT2 expression that declined thereafter to levels observed on Day 1, L-type channel expression decreased by 50% but remained above the expression level of Day 1. Our results indicate that the pattern of CaT1 and CaT2 expression correlates with the Ca2+ uptake potential along the differentiation of cultured human trophoblasts isolated from term placenta. This correlation provides circumstantial evidence for a role of this family of channels in basal Ca2+ uptake by the syncytiotrophoblast.

Role of Melastatin Transient Receptor Potential 7 Channels in the Osteoblastic Differentiation of Murine MC3T3 Cells

Adequate bone formation is assured by the coordinated proliferation, migration, differentiation, and secretory functions of osteoblasts. Epidemiological studies have linked insufficient dietary magnesium (Mg) intake to osteoporosis. Here, we investigated the role of melastatin-like transient receptor potential 7 (TRPM7), a calcium (Ca) and Mg channel, in osteoblastic differentiation of the murine MC3T3 cell line. Osteoblastic differentiation was monitored by alkaline phosphatase activity, osteocalcin gene expression, and extracellular matrix mineralization. Gene expression of TRPM7 increased with osteoblastic differentiation, suggesting the importance of intracellular Ca/Mg homeostasis to cell differentiation. Alteration of intracellular Ca/Mg homeostasis by culture conditions with low extracellular Ca or Mg significantly reduced the osteoblastic differentiation markers alkaline phosphatase activity and osteocalcin gene expression. In accordance, matrix mineralization was reduced under low extracellular Ca or Mg levels. Nevertheless, expression of collagen type I, the predominant matrix protein, was increased in low-Mg culture conditions, indicating that dysfunction of matrix protein production cannot account for the reduced mineralization. Silencing TRPM7 expression during the differentiation period also reduced osteoblastic differentiation and the extent of matrix mineralization. Gene expression of osteoblastic transcription factor Runx2 was reduced by conditions of culture under low extracellular Ca or Mg levels, as well as by TRPM7 silencing. Our results indicate that intracellular Ca and Mg homeostasis ensured by TRPM7 expression is important for the osteoblastic differentiation of MC3T3 cells. Thus, Mg deficiency, a common condition among the population, may be associated with altered osteoblastic differentiation leading to inadequate bone formation and the development of osteoporosis.

Characterization of oxidized low-density lipoprotein-induced hormesis-like effects in osteoblastic cells

Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Atherogenic determinants such as oxidized low-density lipoprotein (oxLDL) particles have been shown both to stimulate the proliferation and promote apoptosis of bone-forming osteoblasts. Given such opposite responses, we characterized the oxLDL-induced hormesis-like effects in osteoblasts. Biphasic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reductive activity responses were induced by oxLDL where low concentrations (10-50 microg/ml) increased and high concentrations (from 150 microg/ml) reduced the MTT activity. Cell proliferation stimulation by oxLDL partially accounted for the increased MTT activity. No alteration of mitochondria mass was noticed, whereas low concentrations of oxLDL induced mitochondria hyperpolarization and increased the cellular levels of reactive oxygen species (ROS). The oxLDL-induced MTT activity was not related to intracellular ROS levels. OxLDL increased NAD(P)H-associated cellular fluorescence and flavoenzyme inhibitor diphenyleneiodonium reduced basal and oxLDL-induced MTT activity, suggesting an enhancement of NAD(P)H-dependent cellular reduction potential. Low concentrations of oxLDL reduced cellular thiol content and increased metallothionein expression, suggesting the induction of compensatory mechanisms for the maintenance of cell redox state. These concentrations of oxLDL reduced osteoblast alkaline phosphatase activity and cell migration. Our results indicate that oxLDL particles cause hormesis-like response with the stimulation of both proliferation and cellular NAD(P)H-dependent reduction potential by low concentrations, whereas high concentrations lead to reduction of MTT activity associated with the cell death. Given the effects of low concentrations of oxLDL on osteoblast functions, oxLDL may contribute to the impairment of bone remodeling equilibrium.

The IL-12p70/IL-10 interplay is differentially regulated by free heme and hemozoin in murine bone-marrow-derived macrophages.

The outcome of malarial anemia is determined by a complex interplay between pro-inflammatory and anti-inflammatory cytokines, its severity associated with accumulation of hemozoin (Hz) in macrophages, elevated IL-10 responses and impaired IL-12 production. Although free heme contributes to malarial anemia by inducing oxidative damage of red blood cells (RBCs) and enhancing their clearance by phagocytes, its impact on IL-12/IL-10 interactions has not been fully characterized. Herein, the effect of hemin (HE) on IL-12 and IL-10 responses was studied in murine bone marrow-derived macrophages (BMDM) and compared with synthetic Hz. Our data reveal that HE induces modest inhibition of IL-12p70 responses to lipopolysaccharide (LPS) whereas Hz significantly impairs IL-12p70 responses to IFNgamma/LPS through down-regulation of IL-12p35 and p40 gene expression. Although reactive oxygen species (ROS) are generated after short-term exposure to HE and Hz, prolonged exposure to these iron protoporphyrins has opposite effects on the cellular redox status, HE being the only compound able to promote persistent ROS production. Accordingly, the inhibitory effect of HE on IL-12p70 seems sustained by redox-dependent induction of IL-10 and is partially controlled by the p38 mitogen-activated protein kinase (MAPK) signalling pathway. Indeed, treatment with n-acetylcysteine (NAC) or with the p38 MAPK inhibitor SB203580 inhibits IL-10 responses and significantly restores IL-12p70 responses to IFNgamma/LPS in HE-conditioned BMDM. Our results suggest that oxidant stress induced by free heme may potentially contribute to sustained production of IL-10 and down-regulation of IL-12 responses in malaria.

Alterations in bone and erythropoiesis in hemolytic anemia: comparative study in bled, phenylhydrazine-treated and Plasmodium-infected mice.

Sustained erythropoiesis and concurrent bone marrow hyperplasia are proposed to be responsible for low bone mass density (BMD) in chronic hemolytic pathologies. As impaired erythropoiesis is also frequent in these conditions, we hypothesized that free heme may alter marrow and bone physiology in these disorders. Bone status and bone marrow erythropoiesis were studied in mice with hemolytic anemia (HA) induced by phenylhydrazine (PHZ) or Plasmodium infection and in bled mice. All treatments resulted in lower hemoglobin concentrations, enhanced erythropoiesis in the spleen and reticulocytosis. The anemia was severe in mice with acute hemolysis, which also had elevated levels of free heme and ROS. No major changes in cellularity and erythroid cell numbers occurred in the bone marrow of bled mice, which generated higher numbers of erythroid blast forming units (BFU-E) in response to erythropoietin. In contrast, low numbers of bone marrow erythroid precursors and BFU-E and low concentrations of bone remodelling markers were measured in mice with HA, which also had blunted osteoclastogenesis, in opposition to its enhancement in bled mice. The alterations in bone metabolism were accompanied by reduced trabecular bone volume, enhanced trabecular spacing and lower trabecular numbers in mice with HA. Taken together our data suggests that hemolysis exerts distinct effects to bleeding in the marrow and bone and may contribute to osteoporosis through a mechanism independent of the erythropoietic stress.