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Notch signaling critically controls cell fate decisions in mammals, both during embryogenesis and in adults. In the skeleton, Notch suppresses osteoblast differentiation and sustains bone marrow mesenchymal progenitors during postnatal life. Stabilizing mutations of Notch2 cause Hajdu-Cheney syndrome, which is characterized by early-onset osteoporosis in humans, but the mechanism whereby Notch inhibits bone accretion is not fully understood. Here, we report that activation of Notch signaling by either Jagged1 or the Notch2 intracellular domain suppresses glucose metabolism and osteoblast differentiation in primary cultures of bone marrow mesenchymal progenitors. Importantly, deletion of Notch2 in the limb mesenchyme increases both glycolysis and bone formation in the long bones of postnatal mice, whereas pharmacological reduction of glycolysis abrogates excessive bone formation. Mechanistically, Notch reduces the expression of glycolytic and mitochondrial complex I genes, resulting in a decrease in mitochondrial respiration, superoxide production, and AMPK activity. Forced activation of AMPK restores glycolysis in the face of Notch signaling. Thus, suppression of glucose metabolism contributes to the mechanism, whereby Notch restricts osteoblastogenesis from bone marrow mesenchymal progenitors., IntroductionNotch signaling between neighboring cells critically controls cell fate decisions in all metazoans (1, 2). In mammalian cells, engagement of the Notch receptors (Notch1-4) by the ligands (Jagged1 and -2 [...]

Although peptides are safe and useful as therapeutics, they are often easily degraded or metabolized. Dampening the clearance system for peptide ligands is a promising strategy for increasing the efficacy of peptide therapies. Natriuretic peptide receptor B (NPR-B) and its naturally occurring ligand, C-type natriuretic peptide (CNP), are potent stimulators of endochondral bone growth, and activating the CNP/NPR-B system is expected to be a powerful strategy for treating impaired skeletal growth. CNP is cleared by natriuretic peptide clearance receptor (NPR- C); therefore, we investigated the effect of reducing the rate of CNP clearance on skeletal growth by limiting the interaction between CNP and NPR-C. Specifically, we generated transgenic mice with increased circulating levels of osteocrin (OSTN) protein, a natural NPR-C ligand without natriuretic activity, and observed a dose-dependent skeletal overgrowth phenotype in these animals. Skeletal overgrowth in OSTN-transgenic mice was diminished in either CNP- or NPR-C-depleted backgrounds, confirming that CNP and NPR-C are indispensable for the bone growth-stimulating effect of OSTN. Interestingly, double-transgenic mice of CNP and OSTN had even higher levels of circulating CNP and additional increases in bone length, as compared with mice with elevated CNP alone. Together, these results support OSTN administration as an adjuvant agent for CNP therapy and provide a potential therapeutic approach for diseases with impaired skeletal growth., Introduction Most bones in mammals are formed through endochondral ossification, in that cartilaginous anlage is formed first and is subsequently replaced by calcified bone tissue (1). Although many signaling pathways, [...]

Osteocalcin (OCN) is an osteoblast-derived hormone that increases energy expenditure, insulin sensitivity, insulin secretion, and glucose tolerance. The cDNA sequence of OCN predicts that, like many other peptide hormones, OCN is first synthesized as a prohormone (pro-OCN). The importance of pro-OCN maturation in regulating OCN and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts are still unknown. Here, we show that the proprotein convertase furin is responsible for pro-OCN maturation in vitro and in vivo. Using pharmacological and genetic experiments, we also determined that furin-mediated pro-OCN cleavage occurred independently of its [gamma]- carboxylation, a posttranslational modification that is known to hamper OCN endocrine action. However, because pro-OCN is not efficiently decarboxylated and activated during bone resorption, inactivation of furin in osteoblasts in mice resulted in decreased circulating levels of undercarboxylated OCN, impaired glucose tolerance, and reduced energy expenditure. Furthermore, we show that Furin deletion in osteoblasts reduced appetite, a function not modulated by OCN, thus suggesting that osteoblasts may secrete additional hormones that regulate different aspects of energy metabolism. Accordingly, the metabolic defects of the mice lacking furin in osteoblasts became more apparent under pair-feeding conditions. These findings identify furin as an important regulator of bone endocrine function., Introduction It has been recently established that bone acts as an endocrine organ that secretes at least 3 hormones: fibroblast growth factor 23 (FGF23), lipocalin 2 (LCN2), and osteocalcin (OCN). [...]

Insulin signaling in osteoblasts has been shown recently to contribute to whole-body glucose homeostasis in animals fed a normal diet; however, it is unknown whether bone contributes to the insulin resistance that develops in animals challenged by a high-fat diet (HFD). Here, we evaluated the consequences of osteoblast-specific overexpression of or loss of insulin receptor in HFD-fed mice. We determined that the severity of glucose intolerance and insulin resistance that mice develop when fed a HFD is in part a consequence of osteoblast-dependent insulin resistance. Insulin resistance in osteoblasts led to a decrease in circulating levels of the active form of osteocalcin, thereby decreasing insulin sensitivity in skeletal muscle. Insulin resistance developed in osteoblasts as the result of increased levels of free saturated fatty acids, which promote insulin receptor ubiquitination and subsequent degradation. Together, these results underscore the involvement of bone, among other tissues, in the disruption of whole-body glucose homeostasis resulting from a HFD and the involvement of insulin and osteocalcin cross-talk in glucose intolerance. Furthermore, our data indicate that insulin resistance develops in bone as the result of lipotoxicity-associated loss of insulin receptors., Introduction The regulation of glucose metabolism is a complex physiological process that relies on the interplay of multiple hormones that themselves act in many target organs (1-3). Among all the [...]

A mouse model shows that osteoblast activating [beta]-catenin mutations alone are sufficient to initiate the development of acute myeloid leukaemia acting through increased Notch signalling. Notch involvement in leukaemia The tumour microenvironment has profound influences on tumorigenesis, and genetic alterations in stromal cells can contribute to the development of cancer. Stavroula Kousteni and colleagues show in a mouse model that activating mutations in [beta]-catenin in osteoblasts are sufficient to initiate the development of acute myeloid leukaemia (AML). These mutations trigger the release of ligands from osteoblasts that activate the Notch signalling pathway in haematopoietic cells; inhibition of the Notch pathway ameliorates the disease. The observation of increased [beta]-catenin signalling in osteoblasts in patients with myeloproliferative disease and AML suggests that a similar mechanism may contribute to leukaemia in humans. Cells of the osteoblast lineage affect the homing.sup.1,2 and the number of long-term repopulating haematopoietic stem cells.sup.3,4, haematopoietic stem cell mobilization and lineage determination and B cell lymphopoiesis.sup.5,6,7. Osteoblasts were recently implicated in pre-leukaemic conditions in mice.sup.8,9. However, a single genetic change in osteoblasts that can induce leukaemogenesis has not been shown. Here we show that an activating mutation of [beta]-catenin in mouse osteoblasts alters the differentiation potential of myeloid and lymphoid progenitors leading to development of acute myeloid leukaemia with common chromosomal aberrations and cell autonomous progression. Activated [beta]-catenin stimulates expression of the Notch ligand jagged 1 in osteoblasts. Subsequent activation of Notch signalling in haematopoietic stem cell progenitors induces the malignant changes. Genetic or pharmacological inhibition of Notch signalling ameliorates acute myeloid leukaemia and demonstrates the pathogenic role of the Notch pathway. In 38% of patients with myelodysplastic syndromes or acute myeloid leukaemia, increased [beta]-catenin signalling and nuclear accumulation was identified in osteoblasts and these patients showed increased Notch signalling in haematopoietic cells. These findings demonstrate that genetic alterations in osteoblasts can induce acute myeloid leukaemia, identify molecular signals leading to this transformation and suggest a potential novel pharmacotherapeutic approach to acute myeloid leukaemia., Author(s): Aruna Kode [sup.1] , John S. Manavalan [sup.1] , Ioanna Mosialou [sup.1] , Govind Bhagat [sup.2] , Chozha V. Rathinam [sup.3] , Na Luo [sup.1] , Hossein Khiabanian [sup.4] [...]

The gap junction protein connexin43 (Cx43) has been proposed to play key roles in bone differentiation and mineralization, but underlying cellular mechanisms are not totally understood. To further explore roles of Cx43 in these processes, we immortalized calvarial osteoblasts from wild-type and Cx43-null mice using human telomerase reverse transcriptase (hTERT). Osteoblastic (MOB) cell lines were generated from three individual wild-type and three individual Cx43-null mouse calvaria. Average population doubling times of the cell lines were higher than of the primary osteoblasts but did not greatly differ with regard to genotype. Modest to high level of Cx45 expression was detected in MOBs of both genotypes. Most of the cell lines expressed osteoblastic markers [Type I collagen, osteopontin, osteocalcin, parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP), periostin (OSF-2), osterix (Osx), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP)], and mineralization was comparable to that of primary osteoblasts. Two MOB cell lines from each genotype with most robust maintenance of osteoblast lineage markers were analyzed in greater detail, revealing that the Cx43-null cell lines showed a significant delay in early differentiation (up to 9 days in culture). Matrix mineralization was markedly delayed in one of the Cx43-null lines and slightly delayed in the other. These findings comparing new and very stable wild-type and Cx43-null osteoblastic cell lines define a role for Cx43 in early differentiation and mineralization stages of osteoblasts and further support the concept that Cx43 plays important role in the cellular processes associated with skeleton function. gap junction; alkaline phosphatase; differentiation; mineralization; human telomerase reverse transcriptase doi: 10.1152/ajpcell.00544.2009.

The potential of fibroblast growth factor-2 (FGF-2) to stimulate osteoprogenitors in aging bone was investigated. Previous work showed a decrease in bone formation in cell cultures derived from bone of elderly female patients, but not in cells from age-matched male or younger female patients, with transforming growth factor 13 increasing bone formation but not increasing osteoprogenitors. In the present study, FGF-2 was shown to significantly stimulate, in a dose-dependent manner, proliferation of mesenchyme-derived progenitor cells from bones of young and old mouse and humans. In proliferation assays, human cells were more responsive to lower concentrations (0.0016 ng/mL) of FGF-2 than mouse cells, but proliferation was less in cells from older bone. Immunofluorescence microscopy revealed that FGF-2 increased and prevented the decline in cells expressing activated leukocyte cell adhesion molecule, a novel marker for early lineage osteoblasts, but not a-smooth muscle actin. FGF-2 may have therapeutic potential for stimulating osteoblast progenitors in aging. Key Words: Aging--Bone--ell proliferation--Fibroblast growth factor-2--Osteoblasts. doi: 10.1093/gerona/glq114

Glutamatergic intercellular communication is involved in many aspects of metabolic homeostasis in normal bone. In bone metastasis, the balance between bone formation and degradation is disrupted. Although the responsible mechanisms are not clear, we have previously identified that cancer cell lines used in bone tumour models secrete glutamate, suggesting that tumour-derived glutamate may disrupt sensitive signalling systems in bone. This study examines the role of glutamate in mature osteoclastic bone resorption, osteoblast differentiation, and bone nodule formation. Glutamate was found to have no effect on the survival or activity of mature osteoclasts, although glutamate transporter inhibition and receptor blockade increased the number of bone resorption pits. Furthermore, transporter inhibition increased the area of resorbed bone while significantly decreasing the number of osteoclasts. Alkaline phosphatase activity and extracellular matrix mineralization were used as measurements of osteoblast differentiation. Glutamate significantly increased osteoblast differentiation and mineralization, but transport inhibitors had no effect. These studies support earlier findings suggesting that glutamate may be more important for osteoclastogenesis than for osteoclast proliferation or functions. Since glutamate is capable of changing the differentiation and activities of both osteoclast and osteoblast cell types in bone, it is reasonable to postulate that tumour-derived glutamate may impact bone homeostasis in bone metastasis. Key words: bone, cancer, glutamate, metastasis, osteoblast, osteoclast, transporter. La communication intercellulaire glutamatergique participe a de nombreux aspects de l'homeoslasie metabolique dans l'os normal. Dans la melastase osseuse, l'equilibre entre la formation et la degradation de l'os est rompu. Bien que les mecanismes a la base de ce desequilibre ne soient pas clairs, nous avons indique dans des travaux anterieurs que les lignees de cellules cancereuses utilisees dans les modeles de tumeurs osseuses secretent du glutamate, ce autorise a penser que le glutamate tumoral pourrait perturber les systemes de signalisation sensibles dans l'os. La presente etude examine le role du glutamate dans la resorption de l'osteoclaste mature, la differenciation osteoblastique et la formation de nodules osseux. Le glutamate n'a pas eu d'effet sur la survie ou l'activite des osteoclastes matures, quoique l'inhibition du transporteur de glutamate et le blocage du recepteur aient augmente les puits de resorption osseuse. De plus, l'inhibition du transporteur a augmente la surface de l'os resorbe tout en diminuant significativement le nombre d'osteoclastes. L'activite de la phosphatase alcaline et la mineralisation de la matrice extracellulaire ont servi de mesures de la differenciation osteoblastique. Le glutamate a augmente de maniere significative la differenciation et la mineralisation des osteoclastes, mais les inhibiteurs du transport n'ont eu aueira effet. Ces observations conforteni les resultats anterieurs suggerant que le glutamate pourrait etre plus important pour l'osteoclastogenese que les fonctions ou la proliferation des osteoclastes. Comme le glutamate est capable de modifier la differenciation et les activites des deux types de cellules dans l'os, il est raisonnable de postuler que le glutamate provenant des tumeurs pourrait avoir un impact sur l'homeostasie du tissu osseux dans la melastase osseuse. Mots-cles : os, cancer, glutamate, melastase, osteoblast, osteoclast, transporter. [Traduit par la Redaction], Introduction A variety of factors participate in the highly coordinated balancing of osteoclast (Oc) and osteoblast (Ob) cell functions in bone remodelling. Recent evidence suggests that the neurotransmitter L-glutamate is [...]

The objective of this study was to examine the effects of leptin on alpha (α) 1 (I) collagen gene expression in a human osteoblast-like MG63 cell line. MG63 cells were incubated with different doses of leptin ([10.sup.-8], [10.sup.-7], and [10.sup.-6] mol x [L.sup.-1]) for 24, 48, and 72 h. α1 (I) collagen gene expression in MG63 cells was detected by real-time fluorescence quantitative polymerase chain reaction (FQ-PCR), with 17β-estradiol (17β-[E.sup.2]) as the positive control. Expression of the al (I) collagen gene, regulated by leptin, was dose and time dependent, with maximal expression in the [10.sup.-7] mol x [L.sup.-1] group at 72 h of incubation. As a positive control, 17f5-E2 reached its maximal effect in the [10.sup.-7] mol x [L.sup.-1] group at 24 h. We conclude that leptin has the ability to up-regulate α1 (I) collagen gene expression in MG63 cells, with a more potent effect but a less rapid response than 17β-[E.sup.2]. Key words: leptin, MG63, real-time FQ-PCR, α1(I) collagen gene. L'objectif de cette etude etait d'examiner les effets de la leptine sur l'expression du gene du collagene alpha (a) 1 (I) par la lignee cellulaire osteoblastoide humaine MG63. Les cellules MG63 ont ete incubees en presence de differentes doses de leptine ([10.sup.-8], [10.sup.-7], [10.sup.-6] mol x [L.sup.-1]) pendant 24, 48 et 72 h. L'expression du gene du collagene α1 (I) par les cellules MG63 a ete detectee par une reaction en chaine par polymerase en temps reel couplee a une immunofluorescence quantitative (FQ-PCR), les cellules traitees a 17β-estradiol (17β-[E.sub.2]) servant de controle positif. L'expression du gene du collagene a1 (I) etait regulee par la leptine de facon dependante de la concentration et du temps, l'expression maximale etant obtenue avec [10.sup.-7] mol x [L.sup.-1] apres 72 h d'incubation. Comme controle positif, le 17β-[E.sub.2] atteignait son effet maximal a [10.sup.-7] mol x [L.sup.-1] apres 24 h. Nous concluons que la leptine a la capacite de stimuler l'expression du gene du collagene α1 (I) dans les cellules MG63, son effet etant plus puissant mais moins rapide que celui du 17β-[E.sub.2]. Mots-cles: leptine, MG63, FQ-PCR en temps reel, gene du collagene α1 (I). [Traduit par la Redaction], Introduction Obesity is associated with a lower risk for osteoporosis and higher serum leptin concentrations (Sinha et al. 1996; Reid 2002; Hadji et al. 2003; Thomas 2003). Leptin, the ob [...]

Henney NC, Li B, Elford C, Reviriego P, Campbell AK, Wann KT, Evans BA. A large-conductance (BK) potassium channel sub-type affects both growth and mineralization of human osteoblasts. Am J Physiol Cell Physiol 297: Cc1397-C1408, 2009. First published September 23, 2009; doi: 10.1152/ajpcell.00311.2009.--The pharmacology of the large-conductance [K.sup.+] (BK) channel in human osteoblasts is not well defined, and its role in bone is speculative. Here we assess BK channel properties in MG63 cells and primary human osteoblasts and determine whether pharmacological modulation affects cell function. We used RT-PCR and patch-clamp methods to determine the expression of BK channel subunits and cell number assays in the absence and presence of BK channel modulators. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3, and KCNMB4 subunits. The BK channel was voltage dependent, with a mean unitary conductance of 228.8 pS (n = 10) in cell-attached patches (140 mM [K.sup.+]/140 mM [K.sup.+]) and a conductance of 142.5 pS (n = 16) in excised outside-out and 155 pS (n = 6) in inside-out patches in 3 mM [K.sup.+]/140 mM [K.sup.+]. The selectivity ratio (ratio of [K.sup.+] to [Na.sup.+] permeability) was 15:1. The channel was blocked by tetraethylammonium (TEA, 0.3 mM), iberiotoxin (5-60 nM), tetrandrine (5-30 [micro]M), and paxilline (10 [micro]M) and activated by isopimaric acid (20 [micro]M). BK channel modulators affected MG63 cell numbers: TEA and tetrandrine significantly increased cell numbers at low concentrations (3 mM and 3 [micro]M, respectively) and reduced cell numbers at higher concentrations (>10 mM and >10 [micro]M, respectively). Neither iheriotoxin (20-300 nM) nor slotoxin (300 nM) affected cell numbers. The increase in cell numbers by TEA was blocked by isopimafic acid. TEA (0.1-3.0 mM) significantly increased mineralization in primary osteoblasts. In conclusion, the BK channel has a distinctive pharmacology and is thus a target for therapeutic strategies aimed at modulating osteoblast proliferation and function. MG63 cells; bone; potassium current; tetrandrine doi: 10.1152/ajpcell.00311.2009

Atkins GJ, Welldon KJ, Wijenayaka AR, Bonewald LF, Findlay DM. Vitamins K promotes mineralization, osteoblast-to-osteocyte transitionm, and an anticatabolic phenotype by [gamma]-carboxylation-dependent and-- independent mechanism. Am J Physiol Cell Physiol 297: C1358-C1367, 2009. First published August 12, 2009; doi: 10.1152/ajpcell.00216.2009.--The vitamin K family members phylloquinone (vitamin K1) and the menaquinones (vitamin K2) are under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologs, phytonadione (vitamin K1) and menatetrenone (vitamin K2), and those of the synthetic vitamin K, menadione (vitamin K3), on human primary osteoblasts. All homologs promoted in vitro mineralization by these cells. Vitamin K1-induced mineralization was highly sensitive to warfarin, whereas that induced by vitamins K2 and K3 was less sensitive, implying that [gamma]-carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor, are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of vitamin K2 or K3 in a three-dimensional type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K-treated cells revealed vitamin K-dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the osteoblast-to- osteocyte transition in humans. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K reduced receptor activator of NF-[kappa]B ligand expression relative to osteoprotegerin by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the osteoblast-to-osteocyte transition, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms by which vitamin K optimizes bone formation and integrity in vivo and may help explain the net positive effect of vitamin K on bone formation. phytonadione; vitamin K1; menatetrenone; vitamin K2; menadione; human primary osteoblast; osteocyte; MLO-Y4 doi: 10.1152/ajpcell.00216.2009

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.sup.2+]) intake in humans with low bone mass and osteoporosis. Here, we investigated the roles of and melastatin-like transient receptor potential 7 (TRPM7), known as [Mg.sup.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.sup.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.sup.2+] concentrations. Silencing TRPM7 expression in osteoblasts by specific small interfering RNA prevented the induction by PDGF of [Mg.sup.2+] influx, proliferation, and migration. Our results indicate that extracellular [Mg.sup.2+]and TRPM7 are important for PDGF-induced proliferation and migration of human osteoblasts. Thus [Mg.sup.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. TRPM7 channels; osteoblasts; adhesion

Decreased bone density and stature can occur in pediatric patients with inflammatory bowel disease (IBD). Little is known about how IBD broadly impacts the skeleton. To evaluate the influence of an acute episode of IBD on growing bone, 4-wk-old mice were administered 5% dextran sodium sulfate (DSS) for 5 days to induce colitis and their recovery was monitored. During active disease and early recovery, trabecular bone mineral density, bone volume, and thickness were decreased. Cortical bone thickness, outer perimeter, and density were also decreased, whereas inner perimeter and marrow area were increased. These changes appear to maintain bone strength since measures of moments of inertia were similar between DSS-treated and control mice. Histological (static and dynamic), serum, and RNA analyses indicate that a decrease in osteoblast maturation and function account for changes in bone density. Unlike some conditions of bone loss, marrow adiposity did not increase. Similar to reports in humans, bone length decreased and correlated with decreases in growth plate thickness and chondrocyte marker expression. During disease recovery, mice experienced a growth spurt that led to their achieving final body weights and bone length, density, and gene expression similar to healthy controls. Increased TNF-[alpha] and decreased IGF-I serum levels were observed with active disease and returned to normal with recovery. Changes in serum TNF-[alpha] (increased) and IGF-I (decreased) paralleled changes in bone parameters and returned to normal values with recovery, suggesting a potential role in the skeletal response. growth plate; cartilage; adipocyte; ulcerative colitis

Voltage-sensitive [Ca.sup.2+] channels (VSCCs) mediate [Ca.sup.2+] permeability in osteoblasts. Association between VSCC [[alpha].sub.1]- and [beta]-subunits targets channel complexes to the plasma membrane and modulates function. In mechanosensitive tissues, a 700-kDa ahnak protein anchors VSCCs to the actin cytoskeleton via the [[beta].sub.2]-subunit of the L-type [Ca.sub.v]1.2 ([[alpha].sub.1]c) VSCC complex. [Ca.sub.v]1.2 is the major [[alpha].sub.1]-subunit in osteoblasts, but the cytoskeletal complex and subunit composition are unknown. Among the four [beta]-subtypes, the [[beta].sub.2]-subunit and, to a lesser extent, the [[beta].sub.3]-subunit coimmunoprecipitated with the [Ca.sub.v]1.2 subunit in MC3T3-E1 preosteoblasts. Fluorescence resonance energy transfer revealed a complex between [Ca.sub.v]1.2 and [[beta].sub.2]-subunits and demonstrated their association in the plasma membrane and secretory pathway. Western blot and immunohistochemistry showed ahnak association with the channel complex in the plasma membrane via the [[beta].sub.]2-subunit. Cytochalasin D exposure disrupted the actin cytoskeleton but did not disassemble or disrupt the function of the complex of L-type VSCC [Ca.sub.v]1.2 and [[beta].sub.2]-subunits and ahnak. Similarly, small interfering RNA knockdown of ahnak did not disrupt the actin cytoskeleton but significantly impaired [Ca.sup.2+] influx. Collectively, we showed that [Ca.sub.v]1.2 and [[beta].sub.2]-subunits and ahnak form a stable complex in osteoblastic cells that permits [Ca.sup.2+] signaling independently of association with the actin cytoskeleton. osteoblasts; fluorescence resonance energy transfer; cytoskeleton

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

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 [micro]g/ml) increased and high concentrations (from 150 [micro]g/ml) reduced the MTF 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. osteoblasts; atherosclerosis; oxysterol

Patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a model of XLH characterized by a deletion in the Phex gene, manifest hypophosphatemia, renal phosphate wasting, and rickets/osteomalacia. Cloning of the PHEX/Phex gene and mutations in affected patients and hyp-mice established that alterations in PHEX/Phex expression underlie XLH. Although PHEX/Phex expression occurs primarily in osteoblast lineage cells, transgenic Phex expression in hyp-mouse osteoblasts fails to rescue the phenotype, suggesting that Phex expression at other sites underlies XLH. To establish whether abnormal Phex in osteoblasts and/or osteocytes alone generates the HYP phenotype, we created mice with a global Phex knockout ([Cre-Phex.sup.[Delta]flox/y] mice) and conditional osteocalcin-promoted (OC-promoted) Phex inactivation in osteoblasts and osteocytes ([OC-Cre-Phex.sup.[Delta]flox/y]). Serum phosphorus levels in [Cre-Phex.sup.[Delta]flox/y], [OC-Cre-Phex.sup.[Delta]flox/y], and hyp-mice were lower than those in normal mice. Kidney cell membrane phosphate transport in [Cre-Phex.sup.[Delta]flox/y], [OC-Cre-Phex.sup.[Delta]flox/y], and hyp-mice was likewise reduced compared with that in normal mice. Abnormal renal phosphate transport in [Cre-Phex.sup.[Delta]flox/y] and [OC-Cre-Phex.sup.[Delta]flox/y] mice was associated with increased bone production and serum FGF-23 levels and decreased kidney membrane type IIa sodium phosphate cotransporter protein, as was the case in hyp-mice. In addition, [Cre-Phex.sup.[Delta]flox/y], [OC-Cre-Phex.sup.[Delta]flox/y], and hyp-mice manifested comparable osteomalacia. These data provide evidence that aberrant Phex function in osteoblasts and/or osteocytes alone is sufficient to underlie the hyp-mouse phenotype., Introduction X-linked hypophosphatemia (XLH) is the archetypal vitamin D-resistant disease in humans and the most common form of inherited rickets, with an incidence of approximately 1 in 20,000 live births. [...]

The osteogenic Runt-related (Runx2) transcription factor negatively regulates proliferation and ribosomal gene expression in normal diploid osteoblasts, but is up-regulated in metastatic breast and prostate cancer cells. Thus, Runx2 may function as a tumor suppressor or an oncogene depending on the cellular context. Here we show that Runx2-deficient primary osteoblasts fail to undergo senescence as indicated by the absence of [beta]-gal activity and p[16.sup.INK4]a tumor suppressor expression. Primary Runx2-null osteoblasts have a growth advantage and exhibit loss of p[21.sup.wAF1/CIP1] and p[19.sup.ARF] expression. Reintroduction of WT Runx2, but not a subnuclear targeting-defective mutant, induces both p[21.sup.WAF/CIP1] and p[19.sup.ARF] mRNA and protein resulting in cell-cycle inhibition. Accumulation of spontaneous phospho-H2A.X foci, loss of telomere integrity and the Mre11/Rad50/Nbs1 DNA repair complex, and a delayed DNA repair response all indicate that Runx2 deficiency leads to genomic instability. We propose that Runx2 functions as a tumor suppressor in primary diploid osteoblasts and that subnuclear targeting contributes to Runx2-mediated tumor suppression. cancer | genomic instability | growth control | nuclear organization | osteoblast

Although dura mater tissue is believed to have an important role in calvarial reossification in many in vivo studies, few studies have shown the direct effect of dura mater cells on osteoblasts. In addition, no reports have yet identified the potential factor(s) responsible for various biological activities exerted by dura mater on calvarial reossification (e.g., cell proliferation). In this study, we tested the effect of dura mater on calvarial-derived osteoblasts by performing both heterotypic coculture and by culturing osteoblast cells with conditioned media harvested from dura mater cells of juvenile (3-day-old) and adult (30-day-old) mice. The results presented here demonstrate that cellular proliferation of juvenile osteoblast cells was significantly increased by juvenile dura mater either in the coculture system or when dura mater cell-conditioned medium was applied to the osteoblast cells. Moreover, high levels of FGF-2 protein were detected in juvenile dura mater cells and their conditioned medium. In contrast, low levels of FGF-2 protein were detected in adult dura mater cells, whereas FGF-2 protein was not detectable in their conditioned medium. Abrogation of the mitogenic effect induced by juvenile dura mater cell-conditioned medium was achieved by introducing a neutralizing anti-FGF-2 antibody, thus indicating that FGF-2 may be responsible for the mitogenic effect of the juvenile dura mater. Moreover, data obtained by exploring the three major FGF-2 signaling pathways further reinforced the idea that FGF-2 might be an important paracrine signaling factor in vivo supplied by the underlying dura mater to the overlying calvarial osteoblasts. mitogenic activity; fibroblast growth factor-2

Melatonin is produced not only by the pineal gland but by cells of the bone marrow. Moreover, melatonin is known to promote osteogenic differentiation in several cell line models and in multipotential bone marrow mesenchymal stem cells. Fatty acids have been independently shown to direct such cells to acquire the phenotype and molecular characteristics of adipocytes. To examine the effect of melatonin on intracellular triglyceride accumulation, an indicator of adipogenic differentiation in the rat osteoblastlike ROS17/2.8 cell line, cells were incubated with added oleic acid (100 [micro]M), fixed and stained with Oil Red O. Cellular lipid accumulation was quantitated by an Oil Red O method highly specific for triglycerides and expressed as a triglyceride accumulation index (TGAI, triglyceride per cell). Melatonin in nanomolar concentrations inhibited oleic acid-induced triglyceride accumulation. To identify the mechanism by which melatonin reduces triglyceride accumulation, cells were incubated with the two melatonin receptor antagonists, luzindole and $20928, or forskolin, a stimulator of adenylyl cyclase and cAMP production. These compounds prevented the inhibitory effect of melatonin on triglyceride accumulation, indicating that melatonin acts through known melatonin receptor-mediated mechanisms. In view of the previously demonstrated positive effects of melatonin in promoting osteoblastic differentiation in ROS17/2.8 cells and their reciprocal adipocytic differentiation induced by fatty acids, our observations may serve to relate the known age-related decreases of melatonin production, the shift in the bone marrow toward an adipocytic line of cell development, and the development of osteoporosis during aging. fatty acids; oleic acid; osteogenesis; luzindole: S20928 doi:10.1152/ajpregu.00013.2007

Fluid flow due to loading in bone is a potent mechanical signal that may play an important role in bone adaptation to its mechanical environment. Previous in vitro studies of osteoblastic cells revealed that the upregulation of cyclooxygenase-2 (COX-2) and c-fos induced by steady fluid flow depends on a change in actin polymerization dynamics and the formation of actin stress fibers. Exposing cells to dynamic oscillatory fluid flow, the temporal flow pattern that results from normal physical activity, is also known to result in increased COX-2 expression and PGE2 release. The purpose of this study was to determine whether dynamic fluid flow results in changes in actin dynamics similar to steady flow and to determine whether alterations in actin dynamics are required for PG[E.sub.2] release. We found that exposure to oscillatory fluid flow did not result in the development of F-actin stress fibers in MC3T3-E1 osteoblastic cells and that inhibition of actin polymerization with cytochalasin D did not inhibit intracellular calcium mobilization or PG[E.sub.2] release. In fact, PG[E.sub.2] release was increased threefold in the polymerization inhibited cells and this PG[E.sub.2] release was dependent on calcium release from the endoplasmic reticulum. This was in contrast to the PG[E.sub.2] release that occurs in normal cells, which is independent of calcium flux from endoplasmic reticulum stores. We suggest that this increased PG[E.sub.2] release involves a different molecular mechanism perhaps involving increased deformation due to the compromised cytoskeleton. mechanotransduction; cell mechanics doi:10.1152/ajpcell.00352.2005

Indian hedgehog (Ihh) is essential for chondrocyte and osteoblast proliferation/differentiation during prenatal endochondral bone formation. The early lethality of various Ihh-ablated mutant mice, however, prevented further analysis of its role in postnatal bone growth and development. In this study, we describe the generation and characterization of a mouse model in which the Ihh gene was successfully ablated from postnatal chondrocytes in a temporal/ spatial-specific manner; postnatal deletion of Ihh resulted in loss of columnar structure, premature vascular invasion, and formation of ectopic hypertrophic chondrocytes in the growth plate. Furthermore, destruction of the articular surface in long bones and premature fusion of growth plates of various endochondral bones was evident, resulting in dwarfism in mutant mice. More importantly, these mutant mice exhibited continuous loss of trabecular bone over time, which was accompanied by reduced Wnt signaling in the osteoblastic cells. These results demonstrate, for the first time, that postnatal chondrocyte-derived Ihh is essential for maintaining the growth plate and articular surface and is required for sustaining trabecular bone and skeletal growth. cre/IoxP | endochondral bone | tamoxifen-inducible | cartilage | osteoblast

Bone formation is carried out by the osteoblast, a mesenchymal cell whose lifespan and activity are regulated by growth factor signaling networks. Growth factors activate phosphatidylinositol 3-kinase (PI3K), which enhances cell survival and antagonizes apoptosis through activation of Akt/PKB. This process is negatively regulated by the Pten phosphatase, which inhibits the activity of PI3K. In this study, we investigated the effects of Akt activation in bone in vivo by conditionally disrupting the Pten gene in osteoblasts by using Cre-mediated recombination. Mice deficient in Pten in osteoblasts were of normal size but demonstrated a dramatic and progressively increasing bone mineral density throughout life. In vitro osteoblasts lacking Pten differentiated more rapidly than controls and exhibited greatly reduced apoptosis in association with markedly increased levels of phosphorylated Akt and activation of signaling pathways downstream of activated Akt. These findings support a critical role for this tumor-suppressor gene in regulating osteoblast lifespan and likely explain the skeletal abnormalities in patients carrying germ-line mutations of PTEN. Akt | bone acquisition | osteoblast survival | high bone mass

Several hormones that regulate nutritional status also impact on bone metabolism. Preptin is a recently isolated 34-amino acid peptide hormone that is cosecreted with insulin and amylin from the pancreatic [beta]-cells. Preptin corresponds to [Asp.sup.69]-[Leu.sup.102] of pro-IGF-II. Increased circulating levels of a pro-IGF-II peptide complexed with IGF-binding protein-2 have been implicated in the high bone mass phenotype observed in patients with chronic hepatitis C infection. We have assessed preptin's activities on bone. Preptin dose-dependently stimulated the proliferation (cell number and DNA synthesis) of primary fetal rat osteoblasts and osteoblast-like cell lines at periphysiological concentrations (>[10.sup.-11] M). In addition, thymidine incorporation was stimulated in murine neonatal calvarial organ culture, likely reflecting the proliferation of cells from the osteoblast lineage. Preptin did not affect bone resorption in this model. Preptin induced phosphorylation of p42/p44 MAP kinases in osteoblastic cells in a dose-dependent manner ([10.sup.-8]-[10.sup.-10] M), and its proliferative effects on primary osteoblasts were blocked by MAP kinase kinase inhibitors. Preptin also reduced osteoblast apoptosis induced by serum deprivation, reducing the number of apoptotic cells by >20%. In vivo administration of preptin increased bone area and mineralizing surface in adult mice. These data demonstrate that preptin, which is cosecreted from the pancreatic [beta]-cell with amylin and insulin, is anabolic to bone and may contribute to the preservation of bone mass observed in hyperinsulinemic states such as obesity. osteoblast; bone-active hormone; bone anabolic

Mesenchymal stem cell-derived osteochondroprogenitors express two master transcription factors, SOX9 and RUNX2, during condensation of the skeletal anlagen. They are essential for chondrogenesis and osteogenesis, respectively, and their haploinsufficiency causes human skeletal dysplasias. We show that SOX9 directly interacts with RUNX2 and represses its activity via their evolutionarily conserved high-mobility-group and runt domains. Ectopic expression of full-length SOX9 or its RUNX2-interacting domain in mouse osteoblasts results in an osteodysplasia characterized by severe osteopenia and down-regulation of osteoblast differentiation markers. Thus, SOX9 can inhibit RUNX2 function in vivo even in established osteoblastic lineage. Finally, we demonstrate that this dominant inhibitory function of SOX9 is physiologically relevant in human campomelic dysplasia. In campomelic dysplasia, haploinsufficiency of SOX9 results in up-regulation of the RUNX2 transcriptional target COL10A1 as well as all three members of RUNX gene family. In summary, SOX9 is dominant over RUNX2 function in mesenchymal precursors that are destined for a chondrogenic lineage during endochondral ossification. differentiation | mesenchymal | skeletal dysplasias | osteoblasts | transcriptional repressor

Declining estrogen production after menopause causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. We recently found that mice deficient in the [beta]-subunit of follicle-stimulating hormone (FSH[beta]) are protected from bone loss despite severe estrogen deficiency. Here we show that FSH[beta]-deficient mice have lowered TNF[alpha] levels. However, TNF[alpha]-deficient mice are resistant to hypogonadal bone loss despite having elevated FSH, suggesting that TNF[alpha] is critical to the effect of FSH on bone mass. We find that FSH directly stimulates TNF[alpha] production from bone marrow granulocytes and macrophages. We also explore how TNF[alpha] up-regulation induces bone loss. By modeling the known actions of TNF[alpha], we attribute the high-turnover bone loss to an expanded osteoclast precursor pool, together with enhanced osteoblast formation. TNF[alpha] inhibits osteoblastogenesis in the presence of ascorbic acid in culture medium, but in its absence this effect becomes stimulatory; thus, ascorbic acid reverses the true action of TNF[alpha]. Likewise, ascorbic acid blunts the effects of TNF[alpha] in stimulating osteoclast formation. We propose that hypogonadal bone loss is caused, at least in part, by enhanced FSH secretion, which in turn increases TNF[alpha] production to expand the number of bone marrow osteoclast precursors. Ascorbic acid may prevent FSH-induced hypogonadal bone loss by modulating the catabolic actions of TNF[alpha]. postmenopausal osteoporosis | TNF[alpha] | bone | ascorbic acid | hypogonadal

The osteoprotegerin (OPG)/receptor activator of nuclear factor-KB ligand (RANKL)/receptor activator of nuclear factor-[kappa]B (RANK) system was evaluated as a potential target of CGRP anabolic activity on bone. Primary cultures of human osteoblast-like cells (hOB) express calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1, and, because CGRP stimulates cAMP (one of the modulators of OPG production in osteoblasts), it was investigated whether it affects OPG secretion and expression in hOB. CGRP treatment of hOB ([10.sup.-11] M-[10.sup.-7] M) dose-dependently inhibited OPG secretion with an E[C.sub.50] of 1.08 x [10.sup.-10] M, and also decreased its expression. This action was blocked by the antagonist CGR[P.sub.8-37]. Forskolin, a stimulator of cAMP production, and dibutyryl cAMP also reduced the production of OPG. CGRP ([10.sup.-8] M) enhanced protein kinase A (PKA) activity in hOB, and hOB exposure to the PKA inhibitor, H89 (2 x [10.sup.-6] M), abolished the inhibitory effect of CGRP on OPG secretion. Conditioned media from CGRP-treated hOB increased the number of multinucleated tartrate-resistant acid phosphatase-positive cells and the secretion of cathepsin K in human peripheral blood mononuclear cells compared with the conditioned media of untreated hOB. These results show that the cAMP/PKA pathway is involved in the CGRP inhibition of OPG mRNA and protein secretion in hOB and that this effect favors osteoclastogenesis. CGRP could thus modulate the balance between osteoblast and osteoclast activity, participating in the fine tuning of all of the bone remodeling phases necessary for the subsequent anabolic effect. receptor-activity-modifying proteins; protein kinase A; osteoclast; cathepsin K doi:10.1152/ajpcell.00354.2005

The pathogenesis of glucocorticoid-induced (GC-induced) bone loss is unclear. For example, osteoblast apoptosis is enhanced by GCs in vivo, but they stimulate bone formation in vitro. This conundrum suggests that [...]

Abstract: Complexes of vanadyl(IV) with 4 monosaccharides and 5 disaccharides were tested in 2 osteoblast-like cell lines (MC3T3E1 and UMR106). Many complexes caused stimulation of UMR106 proliferation (120% basal) in [...]

Indication of the function of Hoxb8a in cell movement and in distinction of osteoblast during the formation of appendage by Medaka unextended-fin mutants is analyzed.

The stem cell niche is composed of a specialized population of cells that plays an essential role in regulating adult stem cell self-renewal and differentiation. In adults, osteoblasts, responsible for [...]

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx[43.sup.-]). hFOB/Cx[43.sup.-] cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/ plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx[43.sup.-] cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB I. 19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx[43.sup.-] cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteoalcin and core binding factor [alpha]1 (Cbfal) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx[43.sup.-]. Osteopontin mRNA levels were increased in hFOB/Cx[43.sup.-] relative to hFOB/pvc and decreased as a function of time in both hFOB/ Cx43 and hFOB/pvc. Transfection with Cx43 antisense did not affect express km of type I collagen in hFOB I. 19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfal expression in osteoblastic cells. gap junction communication; alkaline phosphatase activity; osteopontin; osteocalcin; hFOB 1.19

Insulin-like growth factor binding protein-5 (IGFBP5) is a multifunctional protein, which acts not only as a traditional binding protein, but also functions as a growth factor independent of IGFs to stimulate bone formation. It has been predicted that the intrinsic growth factor action of IGFBP5 involves binding of IGFBP5 to a putative receptor to induce downstream signaling pathways and/or nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell proliferation/ differentiation. Our study indentified proteins that bound to IGFBP5 using IGFBP5 as bait in a yeast two-hybrid screen of the U2 human osteosarcoma cell cDNA library. One of the clones that interacted strongly with the bait under high-stringency conditions corresponded to a novel IGFBP5 interacting protein (IGFBP5-IP) encoded by a gene that resides in mouse chromosome 10. The interaction between IGFBP5-IP and IGFBP5 is confirmed by in vitro coimmunoprecipitation studies that used pFlag and IGFBP5 polyclonal antibody, and cell lysates overexpressing both IGFBP5-IP and IGFBP5. Northern blot and RT-PCR analysis showed that the IGFBP-IP is expressed in both untransformed normal human osteoblasts and in osteosarcoma cell lines, which are known to produce IGFBP5. To determine the roles of IGFBP5-IP, we evaluated the effect of blocking the expression of IGFBP5-IP on osteoblast proliferation. We found that using a IGFBP5-IP-specific small interfering-hairpin plasmid resulted in a decrease in both basal and IGFBP5-induced osteoblast cell proliferation. On the basis of these findings, we predict that IGFBP5-IP may act as intracellular mediator of growth promoting actions of IGFBP5 and perhaps other osteoregulatory agents in bone cells. alternate splicing; yeast two-hybrid screen; small interfering RNA

The cardiac subtype of the L-type voltage-sensitive [Ca.sup.2+] channel (VSCC) Cav 1.2 ([[alpha].sub.1C]) is the primary voltage-sensitive channel responsible for [Ca.sup.2+] influx into actively proliferating osteoblasts. This channel also serves as the major transducer of [Ca.sup.2+] signals in growth-phase osteoblasts in response to hormone treatment. In this study, we have demonstrated that 24-h treatment of MC3T3-E1 preosteoblasts with 1,25-dihydroxyvitamin [D.sub.3][1,25[(OH).sub.2][D.sub.3]], a coupling factor for bone resorption, coordinately downregulates Cav 1.2 ([[alpha].sub.1C]) and uniquely upregulates T-type channel Cav3.2 ([[alpha].sub.1H]). No other voltage-sensitive channel [alpha]-subunit of the 10 that were surveyed was upregulated by 1,25[(OH).sub.2][D.sub.3]. The shift from predominantly L-type to T-type channel expression has been demonstrated to occur at both mRNA and protein levels detected using quantitative PCR and immunohistochemistry with antibodies specific for each channel type. Functional and pharmacological studies using specific inhibitors have revealed that treatment with 1,25[(OH).sub.2][D.sub.3] also alters the [Ca.sup.2+] permeability properties of the osteoblast membrane from a state of primarily L-current sensitivity to T-current sensitivity. We conclude that the L-type channel is likely to support proliferation of osteoblast cells, whereas T-type channels are more likely to be involved in supporting differentiated functions after 1,25[(OH).sub.2][D.sub.3]-mediated reversal of remodeling has occurred. This latter observation is consistent with the unique expression of the T-type VSCC Cav3.2 ([[alpha].sub.1H]) in terminally differentiated osteocytes as we recently reported. calcium influx; bone

The endogenous cannabinoids bind to and activate two G protein-coupled receptors, the predominantly central cannabinoid receptor type 1 (CB1) and peripheral cannabinoid receptor type 2 (CB2). Whereas CB1 mediates the cannabinoid psychotropic, analgesic, and orectic effects, CB2 has been implicated recently in the regulation of liver fibrosis and atherosclerosis. Here we show that CB2-deficient mice have a markedly accelerated age-related trabecular bone loss and cortical expansion, although cortical thickness remains unaltered. These changes are reminiscent of human osteoporosis and may result from differential regulation of trabecular and cortical bone remodeling. The CB[2.sup.-/-] phenotype is also characterized by increased activity of trabecular osteoblasts (bone-forming cells), increased osteoclast (the bone-resorbing cell) number, and a markedly decreased number of diaphyseal osteoblast precursors. CB2 is expressed in osteoblasts, osteocytes, and osteoclasts. A CB2-specific agonist that does not have any psychotropic effects enhances endocortical osteoblast number and activity and restrains trabecular osteoclastogenesis, apparently by inhibiting proliferation of osteoclast precursors and receptor activator of NF-[kappa]B ligand expression in bone marrow-derived osteoblasts/stromal cells. The same agonist attenuates ovariectomy-induced bone loss and markedly stimulates cortical thickness through the respective suppression of osteoclast number and stimulation of endocortical bone formation. These results demonstrate that the endocannabinoid system is essential for the maintenance of normal bone mass by osteoblastic and osteoclastic CB2 signaling. Hence, CB2 offers a molecular target for the diagnosis and treatment of osteoporosis, the most prevalent degenerative disease in developed countries. bone remodeling | HU-308 | osteoblast | osteoclast

p53 is a well known tumor suppressor. We show that p53 also regulates osteoblast differentiation, bone formation, and osteoblast-dependent osteoclast differentiation. Indeed, p[53.sup.-/-] mice display a high bone mass phenotype, and p[53.sup.-/-] osteoblasts show accelerated differentiation, secondary to an increase in expression of the osteoblast differentiation factor osterix, as a result. Reporter assays indicate that p53 represses osterix transcription by the minimal promoter in a DNA-binding--independent manner. In addition, p[53.sup.-/-] osteoblasts have an enhanced ability to favor osteoclast differentiation, in association with an increase in expression of macrophage-colony stimulating factor, which is under the control of osterix. Furthermore, inactivating p53 is sufficient to rescue the osteoblast differentiation defects observed in mice lacking c-Abl, a p53-interacting protein. Thus, these results identify p53 as a novel regulator of osteoblast differentiation, osteoblast-dependent osteo-clastogenesis, and bone remodeling.

The characteristic toughness and strength of bone result from the nature of bone matrix, the mineralized extracellular matrix produced by osteoblasts. The mechanical properties and composition of bone matrix, along with bone mass and architecture, are critical determinants of a bone's ability to resist fracture. Several regulators of bone mass and architecture have been identified, but factors that regulate the mechanical properties and composition of bone matrix are largely unknown. We used a combination of high-resolution approaches, including atomic-force microscopy, x-ray tomography, and Raman microspectroscopy, to assess the properties of bone matrix independently of bone mass and architecture. Properties were evaluated in genetically modified mice with differing levels of TGF-[beta] signaling. Bone matrix properties correlated with the level of TGF-[beta] signaling. Smad3+/- mice had increased bone mass and matrix properties, suggesting that the osteopenic Smad3-/- phenotype may be, in part, secondary to systemic effects of Smad3 deletion. Thus, a reduction in TGF-[beta] signaling, through its effector Smad3, enhanced the mechanical properties and mineral concentration of the bone matrix, as well as the bone mass, enabling the bone to better resist fracture. Our results provide evidence that bone matrix properties are controlled by growth factor signaling. osteoblast | Smad3 | atomic force microscopy

Bone remodeling involves the interplay of bone resorption and formation and is accurately controlled to maintain bone mass. Both processes require transcellular [Ca.sup.2+] transport, but the molecular mechanisms engaged remain largely elusive. The epithelial [Ca.sup.2+] channel TRPV5 is one of the most [Ca.sup.2+]-selective transient receptor potential (TRP) channels. In this study, the functional role of TRPV5 in bone was investigated. TRPV5 mRNA was expressed in human and murine bone samples and in osteoclasts along with other genes involved in transcellular [Ca.sup.2+] transport, including calbindin[D.sub.9K] and calbindin-[D.sub.28K], [Na.sup.+]/[Ca.sup.2+] exchanger 1, and plasma membrane [Ca.sup.2+]-ATPase 1b. TRPV5 expression in murine osteoclasts was confirmed by immunostaining and showed predominant localization to the ruffled border membrane. However, TRPV5 was absent in osteoblasts. Analyses of femoral bone sections from TRPV5 knockout (TRPV[5.sup.-/-]) mice revealed increased osteoclast numbers and osteoclast area, whereas the urinary bone resorption marker deoxypyridinoline was reduced compared with WT (TRPV[5.sup.+/+]) mice. In an in vitro bone marrow culture system, the amount of osteoclasts and number of nuclei per osteoclast were significantly elevated in TRPV[5.sup.-/-] compared with TRPV[5.sup.+/+] mice. However, using a functional resorption pit assay, we found that bone resorption was nearly absent in osteoclast cultures from TRP[V.sup.5-/-] mice, supporting the impaired resorption observed in vivo. In conclusion, TRPV5 deficiency leads to an increase in osteoclast size and number, in which [Ca.sup.2+] resorption is nonfunctional. This report identifies TRPV5 as an epithelial [Ca.sup.2+] channel that is essential for osteoclastic bone resorption and demonstrates the significance of transcellular [Ca.sup.2+] transport in osteoclastic function. tartrate-resistant acid phosphatase | 1,25[(OH).sub.2][D.sub.3] | osteoblast | Coomassie blue | laser scanning confocal microscopy

The transcription factor Sox9 is expressed in all chondroprogenitors and has an essential role in chondrogenesis. Sox9 is also expressed in other tissues, including central nervous system, neural crest, intestine, pancreas, testis, and endocardial cushions, and plays a crucial role in cell proliferation and differentiation in several of these tissues. To determine the cell fate of Sox9-expressing cells during mouse embryogenesis, we generated mice in which a Cre recombinase gene preceded by an internal ribosome entry site was inserted into the 3' untranslated region of the Sox9 gene (Sox9-Cre knock-in). In the developing skeleton, Sox9 was expressed before Runx2, an early osteoblast marker gene. Cell fate mapping by using Sox9-Cre;ROSA26 reporter (R26R) mice revealed that Sox9-expressing limb bud mesenchymal cells gave rise to both chondrocytes and osteoblasts. Furthermore, a mutant in which the Osterix gene was inactivated in Sox9-expressing cells exhibited a lack of endochondral and intra-membranous ossification and a lack of mature osteoblasts comparable with Osterix-null mutants. In addition, Sox9-expressing limb bud mesenchymal cells also contributed to tendon and synovium formation. By using Sox9-Cre;R26R mice, we also were able to systematically follow Sox9-expressing cells from embryonic day 8.0 to 17.0. Our results showed that Sox9-expressing cells contributed to the formation of all cell types of the spinal cord, epithelium of the intestine, pancreas, and mesenchyme of the testis. Thus, our results strongly suggest that all osteo-chondroprogenitor cells, as well as progenitors in a variety of tissues, are derived from Sox9-expressing precursors during mouse embryogenesis. chondrogenesis | Osterix | chondrocytes | osteoblasts

VEGF receptor 1 (VEGFR-1/FIt-1) is a high-affinity tyrosine kinase (TK) receptor for VEGF and regulates angiogenesis as well as monocyte/macrophage functions. We previously showed that the osteoclast deficiency in osteopetrotic [Csf1.sup.op]/[Csf1.sup.op] (op/op) mice is gradually restored in an endogenous, VEGF-dependent manner. However, the molecular basis of the recovery is still not clear. To examine which VEGFR is important and to clarify how colony-stimulating factor 1 (CSF-1) and VEGF signals interact in osteoclastogenesis, we introduced a VEGFR-1 signaling deficiency ([Flt1.sup.TK-/-]) into op/op mice. The original [Flt1.sup.TK-/-] mice showed mild osteoclast reduction without bone marrow suppression. The double mutant (op/op [FIt1.sup.TK-/-]) mice, however, exhibited very severe osteoclast deficiency and did not have numbers of osteoclasts sufficient to form the bone marrow cavity. The narrow bone marrow cavity in the op/op [FIt1.sup.TK-/-] mice was gradually replaced with fibrous tissue, resulting in severe marrow hypoplasia and extramedullary hematopoiesis. In addition to osteoclasts, osteoblasts also decreased in number in the op/op [FIt1.sup.TK-/-] mice. These results strongly suggest that the interaction of signals by means of VEGFR-1 and the CSF-1 receptor plays a predominant role not only in osteoclastogenesis but also in the maintenance of bone marrow functions. osteoblast | hematopoiesis | hematopoietic niche | osteopetrosis | op/op mouse

Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into several distinct Lineages. Two key transcription factors, Runx2 and peroxisome proliferator-activated receptor γ (PPARγ), drive MSCs to differentiate into either osteoblasts or adipocytes, respectively. How these two transcription factors are regulated in order to specify these alternate cell fates remains a pivotal question. Here we report that a 14-3-3-binding protein, TAZ (transcriptional coactivator with PDZ-binding motif), coactivates Runx2-dependent gene transcription while repressing PPARγ-dependent gene transcription. By modulating TAZ expression in model cell Lines, mouse embryonic fibroblasts, and primary MSCs in culture and in zebrafish in vivo, we observed alterations in osteogenic versus adipogenic potential. These results indicate that TAZ functions as a molecular rheostat that modulates MSC differentiation., Pluripotent MSCs can differentiate into several distinct cell types, including osteoblasts and adipocytes (1, 2). Two key transcription factors, Runx2 (also called Cbfal or Pebp2αA) and PPARγ, drive MSCs to [...]