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We studied hypoxia-induced dynamic changes in the balance between PKA and PKA-counteracting phosphatases in the microfluidic environment in single cells using picosecond fluorescence spectroscopy and intramolecular fluorescence resonance energy transfer (FRET)-based sensors of PKA activity. First, we found that the apparent PKA activity in bone cells (MC3T3-E1 cells) and endothelial cells (bovine aortic endothelial cells) is rapidly and sensitively modulated by the level of O2 in the media. When the O2 concentration in the glucose-containing media was lowered due to O2 consumption by the cells in the microfluidic chamber, the apparent PKA activity increases; the reoxygenation of cells under hypoxia leads to a rapid (~2 min) decrease of the apparent PKA activity. Second, lack of glucose in the media led to a lower apparent PKA activity and to a reversal of the response of the apparent PKA activity to hypoxia and reoxygenation. Third, the apparent PKA activity in cells under hypoxia was predominantly regulated via a cAMP-independent pathway since 1) changes in the cAMP level in the cells were not detected using a cAMP FRET sensor, 2) the decay of cAMP levels was too slow to account for the fast decrease in PKA activity levels in response to reoxygenation, and 3) the response of the apparent PKA activity due to hypoxia/reoxygenation was not affected by an adenylate cyclase inhibitior (MDL-12,330A) at 1 mM concentration. Fourth, the immediate onset of ROS accumulation in MC3T3-E1 cells subjected to hypoxia and the sensitivity of the apparent PKA acitivity to redox levels suggest that the apparent PKA activity change during hypoxia and reoxygenation in this study can be linked to a redox potential change in response to intermittent hypoxia through the regulation of activities of PKA-counteracting phosphatases such as protein phosphatase 1. Finally, our results suggest that the detection of PKA activity could be used to monitor responses of cells to hypoxia in real time. oxidative stress; phosphatase; ischemia; reperfusion; cAMP; protein kinase A doi: 10.1152/ajpheart.01073.2009.

Ageing in multicellular organisms typically involves a progressive decline in cell replacement and repair processes, resulting in several physiological deficiencies, including inefficient muscle repair, reduced bone mass, and dysregulation of blood formation (haematopoiesis). Although defects in tissue-resident stem cells clearly contribute to these phenotypes, it is unclear to what extent they reflect stem cell intrinsic alterations or age-related changes in the stem cell supportive microenvironment, or niche. Here, using complementary in vivo and in vitro heterochronic models, we show that age-associated changes in stem cell supportive niche cells deregulate normal haematopoiesis by causing haematopoietic stem cell dysfunction. Furthermore, we find that age-dependent defects in niche cells are systemically regulated and can be reversed by exposure to a young circulation or by neutralization of the conserved longevity regulator, insulin-like growth factor-1, in the marrow microenvironment. Together, these results show a new and critical role for local and systemic factors in signalling age-related haematopoietic decline, and highlight a new model in which blood-borne factors in aged animals act through local niche cells to induce age-dependent disruption of stem cell function., Age-associated pathologies represent a significant and growing global health care concern, particularly as demographic trends predict a doubling in the number of individuals over 65 years of age in the [...]

The molecular mechanisms by which bone cells transduce mechanical stimuli into intracellular biochemical responses have yet to be established. There is evidence that mechanical stimulation acts synergistically with parathyroid hormone PTH(1-34) in mediating bone growth. Using picosecond time-resolved fluorescence microscopy and G protein-coupled receptor conformation-sensitive fluorescence resonance energy transfer (FRET), we investigated conformational transitions in parathyroid hormone type 1 receptor (PTH1R). 1) A genetically engineered PTH1R sensor containing an intramolecular FRET pair was constructed that enabled detection of conformational activity of PTH1R in single cells. 2) The nature of ligand-dependent conformational change of PTH1R depends on the type of ligand: stimulation with the PTH(1-34) leads to conformational transitions characterized by decrease in FRET efficiency while [NH.sub.2]-terminal truncated ligand PTH(3-34) stimulates conformational transitions characterized by higher FRET efficiencies. 3) Stimulation of murine preosteoblastic cells (MC3T3-E1) with fluid shear stress (FSS) leads to significant changes in conformational equilibrium of the PTH1R in MC3T3-E1 cells, suggesting that mechanical perturbation of the plasma membrane leads to ligand-independent response of the PTH1R. Conformational transitions induced by mechanical stress were characterized by an increase in FRET efficiency, similar to those induced by the [NH.sub.2]-terminal truncated ligand PTH(3-34). The response to the FSS stimulation was inhibited in the presence of PTH(1-34) in the flow medium. These results indicate that the FSS can modulate the action of the PTH(1-34) ligand. 4) Plasma membrane fluidization using benzyl alcohol or cholesterol extraction also leads to conformational transitions characterized by increased FRET levels. We therefore suggest that PTH1R is involved in mediating primary mechanochemical signal transduction in MC3T3-E1 cells. mechanosensor; fluorescence resonance energy transfer; fluid shear stress; G protein-coupled receptor; mechanotransduction

Byline: Ram. Chander, Taru. Garg, Sarita. Sanke, Kiran. Agarwal, Aruna. Chhikara A 55-year-old woman, recently diagnosed with dermatomyositis, presented with multiple, tiny, firm papular lesions on cheeks for many years. [...]

X-linked hypophosphatemia (XLH) is characterized by hypophosphatemia and impaired mineralization caused by mutations of the PHEX endopeptidase (phosphate-regulating gene with homologies to endopeptidases on the X chromosome), which leads to the overproduction of the phosphaturic fibroblast growth factor 23 (FGF23) in osteocytes. The mechanism whereby PHEX mutations increase FGF23 expression and impair mineralization is uncertain. Either an intrinsic osteocyte abnormality or unidentified PHEX substrates could stimulate FGF23 in XLH. Similarly, impaired mineralization in XLH could result solely from hypophosphatemia or from a concomitant PHEX-dependent intrinsic osteocyte abnormality. To distinguish between these possibilities, we assessed FGF23 expression and mineralization after reciprocal bone cross-transplantations between wild-type (WT) mice and the Hyp mouse model of XLH. We found that increased FGF23 expression in Hyp bone results from a local effect of PHEX deficiency, since FGF23 was increased in Hyp osteocytes before and after explantation into WT mice but was not increased in WT osteocytes after explantation into Hyp mice. WT bone explanted into Hyp mice developed rickets and osteomalacia, but Hyp bone explanted into WT mice displayed persistent osteomalacia and abnormalities in the primary spongiosa, indicating that both phosphate and PHEX independently regulate extracellular matrix mineralization. Unexpectedly, we observed a paradoxical suppression of FGF23 in juvenile Hyp bone explanted into adult Hyp mice, indicating the presence of an age-dependent systemic inhibitor of FGF23. Thus PHEX functions in bone to coordinate bone mineralization and systemic phosphate homeostasis by directly regulating the mineralization process and producing FGF23. In addition, systemic counter-regulatory factors that attenuate the upregulation of FGF23 expression in Hyp mouse osteocytes are present in older mice. PHEX endopeptidase; X-linked hypophosphatemia; fibroblast growth factor 23: rickets; osteomalacia

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

The strong correlation between a bone's architectural properties and the mechanical forces that it experiences has long been attributed to the existence of a cell that not only detects mechanical load but also structurally adapts the bone matrix to counter it. One of the most likely cellular candidates for such a 'mechanostat' is the osteocyte, which resides within the mineralized bone matrix and is perfectly situated to detect mechanically induced signals. However, as osteocytes can neither form nor resorb bone, it has been hypothesized that they orchestrate mechanically induced bone remodeling by coordinating the actions of cells residing on the bone surface, such as osteoblasts. To investigate this hypothesis, we developed a novel osteocyte-osteoblast coculture model that mimics in vivo systems by permitting us to expose osteocytes to physiological levels of fluid shear while shielding osteoblasts from it. Our results show that osteocytes exposed to a fluid shear rate of 4.4 dyn/[cm.sup.2] rapidly increase the alkaline phosphatase activity of the shielded osteoblasts and that osteocytic-osteoblastic physical contact is a prerequisite. Furthermore, both functional gap junctional intercellular communication and the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 signaling pathway are essential components in the osteoblastic response to osteocyte communicated mechanical signals. By utilizing other nonosteocytic coculture models, we also show that the ability to mediate osteoblastic alkaline phosphatase levels in response to the application of fluid shear is a phenomena unique to osteocytes and is not reproduced by other mesenchymal cell types. osteocyte; osteoblast; fluid-flow; coculture; mechanical stimulation; gap junction; intercellular communication

Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major systemic regulator of calcium homeostasis that activates PTH/PTHrP receptors (PTH1Rs) on target cells. Carboxyl fragments of PTH (CPTH), secreted by the parathyroids or generated by PTH proteolysis in the liver, circulate in blood at concentrations much higher than intact PTH-(1-84) but cannot activate PTH1Rs. Receptors specific for CPTH fragments (CPTHRs), distinct from PTH1Rs, are expressed by bone cells, especially osteocytes. Activation of CPTHRs was previously reported to modify intracellular calcium within chondrocytes. To further investigate the mechanism of action of CPTHRs in osteocytes, cytosolic free calcium concentration ([[[Ca.sup.2+]].sub.i]) was measured in the PTH1R-null osteocytic cell line OC59, which expresses abundant CPTHRs but no PTH1Rs. [[[Ca.sup.2+]].sub.i] was assessed by single-cell ratiometric microfluorimetry in fura-2-loaded OC59 cells. A rapid and transient increase in [[[Ca.sup.2+]].sub.i] was observed in OC59 cells in response to the CPTH fragment hPTH-(53-84) (250 nM). No [[[Ca.sup.2+]].sub.i] signal was observed in COS-7 cells, in which CPTHR binding also cannot be detected. Neither hPTH-(1-34) nor a mutant CPTH analog, [[Ala.sup.55-57]]hPTH-(53-84), that does not to bind to CPTHRs, increased [[[Ca.sup.2+]].sub.i] in OC59 cells. The [[[Ca.sup.2+]].sub.i] response to hPTH-(53-84) required the presence of extracellular calcium and was blocked by inhibitors of voltage-dependent calcium channels (VDCCs), including nifedipine (100 nM), [omega]-agatoxin IVA (10 nM), and [omega]-conotoxin GVIA (100 nM). We conclude that activation of CPTHRs in OC59 osteocytic cells leads to a rapid increase in influx of extracellular calcium, most likely through the opening of VDCCs. calcium influx; osteocytes

Osteocytes, former osteoblasts entombed in the bone matrix, form an extensive cell communication network that is thought to detect microdamage and mechanical strains and to transmit signals leading to repair and compensatory bone augmentation or reduction. Bone active hormones and drugs control the integrity of this network by regulating osteocyte apoptosis, which might be a determinant of bone strength. Herein we demonstrate that mechanical stimulation by stretching activates the ERKs, which in turn are responsible for the attenuation of osteocyte apoptosis. The effect of osteocyte stretching is transmitted by integrins and cytoskeletal and catalytic molecules, such as Src kinases. Stretch-induced antiapoptosis also requires nuclear translocation of ERKs and new gene transcription. The evidence linking mechanical stimulation, activation of an integrin/cytoskeleton/Src/ERK signaling pathway, and osteocyte survival provides a mechanistic basis for the profound role of mechanical forces, or lack thereof, on skeletal health and disease. bone; mechanotransduction; osteoblastic cells; caveolae; stretching

Solute transport through the bone lacunar-canalicular system is believed to be essential for osteocyte survival and function but has proved difficult to measure. We report an approach that permits direct measurement of real-time solute movement in intact bones. By using fluorescence recovery after photobleaching, the movement of a vitally injected fluorescent dye (sodium fluorescein) among individual osteocytic lacunae was visualized in situ beneath the periosteal surface of mouse cortical bone at depths up to 50 [micro]m with laser scanning confocal microscopy. Transport was analyzed by using a two-compartment mathematical model of solute diffusion that accounted for the characteristic anatomical features of the lacunar-canalicular system. The diffusion coefficient of fluorescein (376 Da) was determined to be 3.3 [+ or -] 0.6 x [10.sup.-6] [cm.sup.2]/sec, which is 62% of its diffusion coefficient in water and is similar to diffusion coefficients measured for comparably sized molecules in cartilage. The diffusion of fluorescein in bone is also consistent with the presence of an osteocyte pericellular matrix whose structure resembles that proposed for the endothelial glycocalyx [Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001) J. Struct. Biol. 136, 239-255]. To our knowledge, this is the first instance where the dynamics of molecular movement has been measured directly in the bone lacunar-canalicular system. This in situ imaging approach should also facilitate the analysis of convection-based transport mechanisms in bones of living animals. diffusion coefficient I fiber matrix theory I fluorescence recovery after photobleaching | osteocyte | confocal microscopy

Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(L-lactide-coglycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions. gene expression; osteogenesis; angiogenesis

We studied the effect of the 5[alpha]-reductase inhibitor MK-434 on responses to testosterone (T) in orchiectomized (ORX) male Brown Norway (BN) rats aged 13 too. At 4 wk after ORX or sham surgery, a second surgery was performed to implant pellets delivering 1 mg T/day or placebo pellets. During the second 4 wk of the study, rats received injections of MK-434 (0.75 mg/day) or vehicle injections. Treatment with T elevated serum T to 75% above that for sham animals (P = 0.002) and did not affect serum dihydrotestosterone (DHT) or serum estradiol. T treatment also caused an elevation of prostate T and a marked elevation of prostate DHT. During the second half of the study, ORX rats lost an average of 18.86 [+ or -] 4.62 g body wt. T completely prevented weight loss, and the effect was not inhibited by MK-434 (P < 0.001). ORX produced a nonsignificant trend toward a small (5%) decrease in the mass of the gastrocnemius muscle (P = 0.0819). This trend was also reversed by T, and the effect of T was not blocked by MK-434. T caused a significant 16% decrease in subcutaneous fat that was not blocked by MK-434 (P < 0.05). Finally, T caused a 65% decrease in urine excretion of deoxypyridinoline, a marker of bone resorption, and again the effect was not blocked by MK-434 (P < 0.0001). In contrast, T caused a greater than fivefold increase in prostate mass, and the effect was almost completely blocked by MK-434 (P < 0.0001). This study demonstrates that 5[alpha]-reductase inhibitors may block the undesirable effects oft on the prostate, without blocking the desirable anabolic effects of T on muscle, bone, and fat. Dihydrotestosterone; body composition; bone resorption

We examined changes in electrical and morphological properties of rat osteoclasts in response to prostaglandin (PG)[E.sub.2]. PG[E.sub.2] (> 10 nM) stimulated an outwardly rectifying [Cl.sup.-] current in a concentration-dependent manner and caused a long-lasting depolarization of cell membrane. This PG[E.sub.2]-induced [Cl.sup.-] current was reversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and tamoxifen. The anion permeability sequence of this current was [I.sup.-] > [Br.sup.-][approximately equal to] [Cl.sup.-] > [gluconate.sup.-]. When outwardly rectifying [Cl.sup.-] current was induced by hyposmotic extracellular solution, no further stimulatory effect of PG[E.sub.2] was seen. Forsknlin and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) mimicked the effect of PG[E.sub.2]. The PG[E.sub.2]-induced [Cl.sup-] current was inhibited by pretreatment with guanosine 5'-O-2-(thiodiphosphate) (GDP[beta]S), Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS), N-(2-[p-bromocinnmnylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89), and protein kinase A inhibitors. Even in the absence of nonosteoclastic cells, PG[E.sub.2] (1 [micro]M) reduced cell surface area and suppressed motility of osteoclasts, and these effects were abolished by Rp-cAMPS or H-89. PG[E.sub.2] is known to exert its effects through four subtypes of PGE receptors (EP1-EP4). EP2 and EP4 agonists (ONO-AE1-259 and ONO-AE1-329, respectively), but not EP1 and EP3 agonists (ONO-DI-004 and ONO-AE-248, respectively), mimicked the electrical and morphological actions of PG[E.sub.2] on osteoclasts. Our results show that PG[E.sub.2] stimulates rat osteoclast [Cl.sup.-] current by activation of a cAMP-dependent pathway through EP2 and, to a lesser degree, EP4 receptors and reduces osteoclast motility. This effect is likely to reduce bone resorption. prostanoid receptor agonists; electrophysiology; motile activity; bone resorption

During development of the skeleton, osteoclast (OC) recruitment and migration are required for the vascular invasion of the cartilaginous anlage and the ossification of long bones. c-Cbl lies downstream of the vitronectin receptor and forms a complex with c-Src and Pyk2 in a signaling pathway that is required for normal osteoclast motility. To determine whether the decreased motility we observed in vitro in c-[Cbl.sup.-/-] OCs translated into decreased cell migration in vivo, we analyzed the long bones of c-[Cbl.sup.-/-] mice during development. Initiation of vascularization and replacement of cartilage by bone were delayed in c-[Cbl.sup.-/-] mice, due to decreased osteoclast invasion of the hypertrophic cartilage through the bone collar. Furthermore, [c-Cbl.sup.-/-] mice show a delay in the formation of secondary centers of ossification, a thicker hypertrophic zone of the growth plate, and a prolonged presence of cartilaginous remnants in the spongiosa, confirming a decrease in resorption of the calcified cartilage. Thus, the decrease in motility of c-[Cbl.sup.-/-] osteoclasts observed in vitro results in a decreased ability of osteoclasts to invade and resorb bone and mineralized cartilage in vivo. These results confirm that c-Cbl plays an important role in osteoclast motility and resorbing activity.

Bone is removed or replaced in defined locations by targeting osteoclasts and osteoblasts in response to its local history of mechanical loading. There is increasing evidence that osteocytes modulate this targeting by their apoptosis, which is associated with locally increased bone resorption. To investigate the role of osteocytes in the control of loading-related modeling or remodeling, we studied the effects on osteocyte viability of short periods of mechanical loading applied to the ulnae of rats. Loading, which produced peak compressive strains of -0.003 or -0.004, was associated with a 78% reduction in the resorption surface at the midshaft. The same loading regimen resulted in a 40% relative reduction in osteocyte apoptosis at the same site 3 days after loading compared with the contralateral side (P = 0.01). The proportion of osteocytes that were apoptotic was inversely related to the estimated local strain (P < 0.02). In contrast, a single short period of loading resulting in strains of -0.008 engendered both tissue microdamage and subsequent bone remodeling and was associated with an eightfold increase in the proportion of apoptotic osteocytes (P = 0.02) at 7 days. This increase in osteocyte apoptosis was transient and preceded both intracortical remodeling and death of half of the osteocytes (P < 0.01). The data suggest that osteocytes might use their U-shaped survival response to strain as a mechanism to influence bone remodeling. We hypothesize that this relationship reflects a causal mechanism by which osteocyte apoptosis regulates bone's structural architecture. in vivo; rat ulnae; osteocytes; cell death

Research indicates that human bone cell phenotypic markers exhibit distinct age-related alterations, with some elderly people demonstrating significantly lower quantities of bone proteins. Immunologically purified bone preosteoblast-like cell populations were used in the research, with the findings facilitating a better understanding of the early stages of bone cell development.

Research was conducted to understand bone growth and formation using two models. The data indicate that a heparin-binding growth associated molecule mediates bone growth and formation by gathering and depositing precursor cells on to a substrate.

Research was conducted to determine whether bone cells obtained from osteoporotic patients respond differently to mechanical stress than cells obtained from age-matched controls. Bone-derived cell cultures were monitored for production of osteocalcin and alkaline phosphatase activity. Then the response of the bone cells to mechanical stress was tested by analyzing the changes in the release of prostaglandin E2, nitric oxide and transforming growth factor-beta. Results suggest that their release is a normal response of human bone cells to fluid shear stress.

A study was conducted on the wall-shear stress induction of prostaglandin E2 and nitric oxide in osteoblasts. Strain and fluid flow responsiveness of bone cells in vivo were determined using nitric oxide and prostaglandin production. Results indicate that increases in wall-shear stress is associated with fluid flow. Results also suggest that osteoblastic cells use fluid flow-mediated wall-shear stress to sense bone mechanical loading.

The quadratojugal (QJ) is a neural crest-derived membrane bone in the maxillary region of the avian head. In vivo its periosteum undergoes both osteogenesis to form membrane bone and chondrogenesis to form secondary cartilage. This bipotential property, which also exists in some other membrane bones, is poorly understood. The present study used cell culture to investigate the differentiation potential of QJ periosteal cells. Three cell populations were enzymatically released from QJ periostea and plated at different densities. Cell density greatly affected phenotypic expression and differentiation pathways. We found two culture conditions that favored osteogenesis and chondrogenesis, respectively. In micromass culture, the periosteal cells produced a layer of osteogenic cells that expressed alkaline phosphatase (APase) and secreted bony extracellular matrix (ECM). In contrast, low-density monolayer culture elicited chondrogenesis. Cells with pericellular refractile ECM and round shape appeared at 7 to 8 days and formed colonies later. The chondrogenic phenotype of these cells was confirmed by immunolocalization of type II collagen and Alcian blue staining of ECM. This result demonstrated that a fully expressed chondrogenic phenotype can be achieved from membrane bone periosteal cells in primary monolayer culture. Chondrogenesis requires a cell density lower than confluence and cannot be initiated in confluent cultures. Among the three cell populations, those cells from the outer layer have the highest growth rate and require the lowest initial plating density (below 5 x [10.sup.3] cells/ml) to achieve chondrogenesis. Cells from the inner layer have the slowest growth rate and chondrify at the highest initial density (below 5 x [10.sup.4] cells/ml). Chondrocytes from all populations express distinct phenotypic markers - APase and type I collagen - from initial chondrogenesis, but are not hypertrophic morphologically. Furthermore, the fact that chondrocytes arise within the same colony as APase-positive polygonal cells suggests that chondrocytes may differentiate from precursors related to the osteogenic cell lineage. This cell culture approach mimics secondary cartilage and membrane bone formation in vivo.

The signals released by the amphibian gastrula's Spemann organizer were found to be capable of directly inducing neural tissue from ectoderm and dorsalizing ventral mesoderm to form muscle. The secreted polypeptide noggin duplicates these activities. It is expressed at the right time and place to participate in the organizer signal. Noggin protein was observed to bind bone morphogenetic protein 4 (BMP4) with high affinity. It can also abolish BMP4 activity by inhibiting binding to cognate cell-surface receptors. Results suggest that the organizer-secreted noggin interrupts BMP signaling to pattern the embryo.

The Xenopus protein was expressed in the baculovirus system and a peptide antibody was raised against its NH2-terminus to study the biochemical function of chordin (Chd). A myc epitope was also introduced at the COOH-terminus. Radio-receptor binding assay results showed that Chd protein is capable of inhibiting the binding of BMP-4 protein to its cognate receptor at subnanomolar concentrations. The execution of noncell-autonomous effects of Spemann's organizer on dorsoventral patterning is partly caused by diffusible signals that directly bind to and neutralize ventral bone morphogenetic proteins during gastrulation. This creates pattern in the ectodermal and mesodermal germ layers.

Although apparently phenotypically normal at birth, mice heterozygous for inactivation of the gene encoding parathyroid hormone-related peptide (PTHrP) develop haplotype insufficiency by 3 months of age. In addition to histologic and morphologic abnormalities similar to those seen in homozygous mutants, heterozygous animals demonstrated alterations in trabecular bone and bone marrow. These included metaphyseal bone spicules which were diminished in volume, irregularly distributed, and less well developed than those seen in age-matched controls as well as bone marrow, which contained an inordinate number of adipocytes. A substantial reduction in PTHrP mRNA was detected in heterozygous tissue, while circulating parathyroid hormone (PTH) and calcium concentrations were normal. Thus, while a physiologic concentration of PTH was capable of maintaining calcium homeostasis, it was incapable of compensating for PTHrP haploinsufficiency in developing bone. In normal animals, both PTHrP and the PTH/PTHrP receptor were expressed predominantly in chondrocytes situated throughout the proliferative zone of the tibial growth plate. In the metaphysis, the PTH/PTHrP receptor was identified on osteoblasts and preosteoblastic cells situated in the bone marrow, while PTHrP was expressed only by osteoblasts. These observations indicate that postnatal bone development involves susceptible pathways that display exquisite sensitivity to critical levels of PTHrP and imply that the skeletal effects of PTH are influenced by locally produced PTHrP. Moreover, identification of both the ligand and its N-terminal receptor in metaphyseal osteoblasts and their progenitors suggests an autocrine/paracrine role for the protein in osteoblast differentiation and/or function. Impairment in this function as a consequence of PTHrP haploinsufficiency may critically influence the course of bone formation, resulting in altered trabecular architecture and perhaps low bone mass and increased bone fragility.

Both, the transport rate and steady-state concentration of ascorbate in bone cells, are controlled by transforming growth factor-beta. The increase in collagen synthesis and bone formation and collagen synthesis is sudden. The osteoblastic cells have a saturable Na+ dependent ascorbate transport system which mediates concentrative uptake of reduced vitamin C. The Na+ ascorbate co-transport activity regulates ascorbate concentration in osteoblasts.

The role of pp60 (super c-src) was found to be critical in osteoclast activity. Excision of the c-src gene in transgenic mice resulted in the development of osteoporosis that indicated the possible specific functions of this proto-oncogene in bone cells.Measurement in osteoclasts and immunolocalization in bones of pp60 (super c-src) revealed the high expression of this protein the bone cells similar to those found in brain and platelets. The association of this protein with membranes of intracellular organelles indicated its possible role in recognition or fusion of membrane vesicles.

Ahlqvist and Damsten's (1969) modification of the Kerley (1965) method for histological age estimation uses percent osteonal bone, rather than actual osteon counts, in order to eliminate the difficulty of distinguishing between intact and fragmentary osteons. Since their method has been developed for the femur only, and several more recent methods have been proposed that utilize percent osteonal bone, a study was undertaken to ascertain the relative value of percent osteonal bone compared with osteon counts to estimate age at death fr the radius, tibia, and fibula. First the question of how much of the cross-section of a bone should be sampled was addressed by comparing the results of regression against age for percent osteonal bone derived from sampling only four fields with those derived from the entire cross-section of the radius. A significant age association was found only when the entire cross-section was sampled. In order to evaluate the relative merit of using either percent osteonal bone, or osteon counts to estimate age, each variable was regressed against age. Significant correlation coefficients were found for all three bones when the independent variable was osteon counts. When percent osteonal bone was employed, a significant correlation was found only for the radius. Stepwise linear regression found osteon counts for the fibula alone to the best age predictor. Finally, a repeated measures analysis of variance revealed that percent osteonal bone and osteon counts both differ among the three bones within an individual. Based upon these results, osteon counts, rather than percent osteonal bone, should be the variable of choice when developing histological age predicting methods.

A few years ago, researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging, at Tufts University in Boston, Massachusetts, showed that healthy men and women over 65 [...]

Osteoclasts are unique cells that utilize podosomes instead of focal adhesions for matrix attachment and cytoskeletal remodeling during motility. We have shown that osteopontin (OP) binding to the [[Alpha].sub.v][[Beta].sub.3] integrin of osteoclast podosomes stimulated cytoskeletal reorganization and bone resorption by activating a heteromultimeric signaling complex that includes gelsolin, [pp.sup.60c-scr] and phosphatidylinositol 3'-kinase. Here we demonstrate that gelsolin deficiency blocks podosome assembly and [[Alpha].sub.v][[Beta].sub.3]-stimulated signaling related to motility in gelsolin-null mice. Gelsolin-deficient osteoclasts were hypomotile due to retarded remodeling of the actin cytoskeleton. They failed to respond to the autocrine factor, OP, with stimulation of motility and bone resorption. Gelsolin deficiency was associated with normal skeletal development and endochondral bone growth. However, gelsolin-null mice had mildly abnormal epiphyseal structure, retained cartilage proteoglycans in metaphyseal trabeculae, and increased trabecular thickness. With age, the gelsolin-deficient mice expressed increased trabecular and cortical bone thickness producing mechanically stronger bones. These observations demonstrate the critical role of gelsolin in podosome assembly, rapid cell movements, and signal transduction through the [[Alpha].sub.v][[Beta].sub.3] integrin. Key words: gelsolin * phosphatidylinositol 3'-kinase * actin * podosomes * osteoclasts

Bozorg Grayeli, Alexis, Olivier Sterkers, Pierre Roulleau, Pierre Elbaz, Evelyne Ferrary, and Caroline Silve. Parathyroid hormone-parathyroid hormone-related peptide receptor expression and function in otosclerosis. Am. J. Physiol. 277 (Endocrinol. Metab. 40): E1005-E1012, 1999.--The aim of this study was to investigate the possibility that an abnormality related to parathyroid hormone (PTH) action is involved in the increased bone turnover observed in otosclerosis. To do so, expression and function of the PTH-PTH-related peptide (PTHrP) receptor were studied in the involved tissue (stapes) and compared with that in control bone sample obtained from the external auditory canal (EAC) in the same patient in 10 cases of otosclerosis and in 1 case of osteogenesis imperfecta. PTH-PTHrP receptor expression was studied by RT-PCR of RNA prepared from cultured cells in three patients and RNA directly extracted from bone samples in four patients. PTH-PTHrP receptor function was assessed by measuring the stimulation of cAMP production by 0.8, 8, and 80 nM PTH in bone cell cultures in seven cases. Results showed that PTH-PTHrP receptor mRNA expression in the otosclerotic stapes was lower than that in EAC samples (P [is less than] 0.05), whereas it was higher in stapes than that in EAC in the case of osteogenesis imperfecta, cAMP production after PTH stimulation was lower in bone cells cultured from otosclerotic stapes compared with that in cells cultured from EAC (range of increase in stimulation: 0.8-4.5 and 1.5-7 in stapes and EAC bone cells, respectively, P [is less than] 0.05). In contrast, the stimulation of cAMP production by forskolin was not significantly different in otosclerotic stapes and EAC bone cells (range of increase in stimulation: 20.7-83.1 and 4.9-99.8 in stapes and EAC, respectively, P [is greater than]0.05). These results show a lower stimulation of cAMP production in response to PTH associated with a lower PTH-PTHrP receptor mRNA expression in pathological stapes from patients with otosclerosis compared with that in control EAC samples. This difference supports the hypothesis that an abnormal cellular response to PTH contributes to the abnormal bone turnover in otosclerosis. bone turnover; middle ear; stapes

Saadeh, Pierre B., Babak J. Mehrara, Douglas S. Steinbrech, Matthew E. Dudziak, Joshua A. Greenwald, Jonathan S. Luchs, Jason A. Spector, Hikaru Ueno, George K. Gittes, and Michael T. Longaker. Transforming growth factor-[Beta] 1 modulates the expression of vascular endothelial growth factor by osteoblasts. Am. J. Physiol. 277 (Cell Physiol. 46): C628-C637, 1999.--Angiogenesis is essential to both normal and pathological bone physiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-[Beta]1 (TGF[Beta] 1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-[Beta] 1 and VEGF expression in osteoblasts and osteoblast-like cells. Northern blot analysis revealed an early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cells and MC3T3-E1 osteoblast-like cells after stimulation with TGF-[Beta] 1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-[Beta] 1 treatment. Actinomycin D inhibited the TGF-[Beta]1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-[Beta] 1 signal transduction via a dominantnegative receptor II adenovirus significantly decreased TGF-[Beta] 1 induction of VEGF mRNA. Additionally, TGF-[Beta]1-induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells (P [is less than] 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-[Beta]1 mRNA and VEGF mRNA were concurrently present in rat membranous bone, and both followed similar patterns of expression during rat mandibular fracture healing (mRNA and protein). In summary, TGF [Beta]1-induced VEGF expression by osteoblasts and osteoblast-like cells is a dose-dependent event that may be intimately related to bone development and fracture healing. angiogenesis; bone; fracture

Researchers at the University of Bern in Switzerland have identified a compound in the popular vegetable that appears to decrease bone loss in laboratory studies using rat bone cells. It is suggested that eating onions might help prevent bone loss and osteoporosis, a disease, which predominantly affects older women.

Abstract. Fluoride is one of the most potent but least well understood stimulators of bone formation in vivo. Bone formation was shown to arise from direct effects on bone cells. […]

Research was conducted to examine the mechanism by which osteoprotegerin (OPGL) increases bone resorption that is independent of its role in osteoclast (OC) differentiation in cultures of mature rat OCs and in mice. OPGL was found to bind to individual mature OC-inducing cytoskeletal changes indicative of OC activation and stimulates multiple spatially associated cycles of robust bone resorption in vitro. Results demonstrate that in addition to its role in OC differentiation, OPGL is a potent and direct regulator of OC activity in vivo and in vitro.

A study was conducted to examine the function of the actin cytoskeleton and actin-membrane interactions in the mechanical signal transmission that leads to altered gene expression in culture MC3T3-E1 osteoblasts. Results indicate that fluid shear-induced mechanical signaling in osteoblasts increases expression of cyclooxygenase-2 and c-Fos via a mechanism that involves the rearrangement of the actin cytoskeleton. Moreover, the development of stress fibers and their anchorage to integrins in focal adhesion lead to fluid shear-induced changes in bone cells.