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Bone and muscle are coupled through developmental, mechanical, paracrine, and autocrine signals. Genetic variants at the CPED1-WNT16 locus are dually associated with bone- and muscle-related traits. While Wnt16 is necessary for bone mass and strength, this fails to explain pleiotropy at this locus. Here, we show wnt16 is required for spine and muscle morphogenesis in zebrafish. In embryos, wnt16 is expressed in dermomyotome and developing notochord, and contributes to larval myotome morphology and notochord elongation. Later, wnt16 is expressed at the ventral midline of the notochord sheath, and contributes to spine mineralization and osteoblast recruitment. Morphological changes in wnt16 mutant larvae are mirrored in adults, indicating that wnt16 impacts bone and muscle morphology throughout the lifespan. Finally, we show that wnt16 is a gene of major effect on lean mass at the CPED1-WNT16 locus. Our findings indicate that Wnt16 is secreted in structures adjacent to developing bone (notochord) and muscle (dermomyotome) where it affects the morphogenesis of each tissue, thereby rendering wnt16 expression into dual effects on bone and muscle morphology. This work expands our understanding of wnt16 in musculoskeletal development and supports the potential for variants to act through WNT16 to influence bone and muscle via parallel morphogenetic processes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Watson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

RSV infection is typically associated with secondary bacterial infection. We hypothesise that the local airway immune response to RSV has incidental antibacterial effects. Using coordinated proteomics and metagenomics analysis we simultaneously analysed the microbiota and proteomes of the upper airway and determined direct antibacterial activity in airway secretions of RSV-infected children. Here, we report that the airway abundance of Streptococcus was higher in samples collected at the time of RSV infection compared with samples collected one month later. RSV infection is associated with neutrophil influx into the airway and degranulation and is marked by overexpression of proteins with known antibacterial activity including BPI, EPX, MPO and AZU1. Airway secretions of children infected with RSV, have significantly greater antibacterial activity compared to RSV-negative controls. This RSV-associated, neutrophil-mediated antibacterial response in the airway appears to act as a regulatory mechanism that modulates bacterial growth in the airways of RSV-infected children.

Objectives: To clarify the effects of neuromuscular dysfunction on hindlimb loading, muscle atrophy, and bone homeostasis., Methods: We quantified changes to hindlimb loading, muscle atrophy, and bone morphology following either Botulinum toxin A (BTxA) induced muscle paralysis or peripheral nerve injury (PNI) in mice; two in vivo models that we anticipated would differently alter gait and mechanical loading patterns due to their distinct effects on neuromuscular signaling. To confirm the expected behavioral effects of PNI, we assessed mechanical allodynia of the ipsilateral hindlimb using von Frey testing and activity (distance traveled and speed) was monitored in both groups using open field testing. Peak vertical ground reaction forces (GRF) and ankle and knee kinematics during normal locomotion were quantified and used to estimate peak mid-diaphyseal normal strains. Muscle atrophy and trabecular and cortical bone morphology were assessed via high-resolution microCT imaging., Results: BTxA-induced calf paralysis caused severe muscle atrophy and altered gait kinetics and kinematics and reduced gait-induced normal strains. PNI increased mechanical allodynia but did not alter gait, nor did it cause muscle atrophy. We observed that muscle paralysis and PNI both led to severe trabecular bone loss but only BTxA-induced paralysis increased cortical bone resorption., Conclusions: While mechanical stimuli clearly have essential functions in bone development and adaptation, these data emphasize that neuromuscular signaling, independent of load-induced mechanical strains, may modulate trabecular bone homeostasis in normal and disease states.

At sufficient dose, intramuscular injection of Botulinum toxin A causes muscle wasting that is physiologically consistent with surgical denervation and other types of neuromuscular dysfunction. The aim of this study was to clarify early molecular and micro-RNA alterations in skeletal muscle following Botulinum toxin A-induced muscle paralysis. Quadriceps were analyzed for changes in expression of micro- and messenger RNA and protein levels after a single injection of 0.4, 2 or 4U Botulinum toxin A (/100g body weight). After injection with 2.0U Botulinum toxin A, quadriceps exhibited significant reduction in muscle weight and increased levels of ubiquitin ligase proteins at 7, 14 and 28 days. Muscle miR-1 and miR-133a/b levels were decreased at these time points, whereas a dose-responsive increase in miR-206 expression at day 14 was observed. Expression of the miR-133a/b target genes RhoA, Tgfb1 and Ctfg, and the miR-1/206 target genes Igf-1 and Hdac4, were upregulated at 28 days after Botulinum toxin A injection. Increased levels of Hdac4 protein were observed after injection, consistent with anticipated expression changes in direct and indirect Hdac4 target genes, such as Myog. Our results suggest Botulinum toxin A-induced denervation of muscle shares molecular characteristics with surgical denervation and other types of neuromuscular dysfunction, and implicates miR-133/Tgf-β1/Ctfg and miR-1/Hdac4/Myog signaling during the resultant muscle atrophy., Competing Interests: The authors have declared that no competing interests exist.

Nearly all exogenous loading models of bone adaptation apply dynamic loading superimposed upon a time invariant static preload (SPL) in order to ensure stable, reproducible loading of bone. Given that SPL may alter aspects of bone mechanotransduction (eg, interstitial fluid flow), we hypothesized that SPL inhibits bone formation induced by dynamic loading. As a first test of this hypothesis, we utilized a newly developed device that enables stable dynamic loading of the murine tibia with SPLs ≥ -0.01 N. We subjected the right tibias of BALB/c mice (4-month-old females) to dynamic loading (-3.8 N, 1 Hz, 50 cycles/day, 10 s rest) superimposed upon one of three SPLs: -1.5 N, -0.5 N, or -0.03 N. Mice underwent exogenous loading 3 days/week for 3 weeks. Metaphyseal trabecular bone adaptation (μCT) and midshaft cortical bone formation (dynamic histomorphometry) were assessed following euthanasia (day 22). Ipsilateral tibias of mice loaded with a -1.5-N SPL demonstrated significantly less trabecular bone volume/total volume (BV/TV) than contralateral tibias (-12.9%). In contrast, the same dynamic loading superimposed on a -0.03-N SPL significantly elevated BV/TV versus contralateral tibias (12.3%) and versus the ipsilateral tibias of the other SPL groups (-0.5 N: 46.3%, -1.5 N: 37.2%). At the midshaft, the periosteal bone formation rate (p.BFR) induced when dynamic loading was superimposed on -1.5-N and -0.5-N SPLs was significantly amplified in the -0.03-N SPL group (>200%). These data demonstrate that bone anabolism induced by dynamic loading is markedly inhibited by SPL magnitudes commonly implemented in the literature (ie, -0.5 N, -1.5 N). The inhibitory impact of SPL has not been recognized in bone adaptation models and, as such, SPLs have been neither universally reported nor standardized. Our study therefore identifies a previously unrecognized, potent inhibitor of mechanoresponsiveness that has potentially confounded studies of bone adaptation and translation of insights from our field. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Transient muscle paralysis engendered by a single injection of botulinum toxin A (BTxA) rapidly induces profound focal bone resorption within the medullary cavity of adjacent bones. While initially conceived as a model of mechanical disuse, osteoclastic resorption in this model is disproportionately severe compared with the modest gait defect that is created. Preliminary studies of bone marrow following muscle paralysis suggested acute upregulation of inflammatory cytokines, including TNF-α and IL-1. We therefore hypothesized that BTxA-induced muscle paralysis would rapidly alter the inflammatory microenvironment and the osteoclastic potential of bone marrow. We tested this hypothesis by defining the time course of inflammatory cell infiltration, osteoinflammatory cytokine expression, and alteration in osteoclastogenic potential in the tibia bone marrow following transient muscle paralysis of the calf muscles. Our findings identified inflammatory cell infiltration within 24 h of muscle paralysis. By 72 h, osteoclast fusion and pro-osteoclastic inflammatory gene expression were upregulated in tibia bone marrow. These alterations coincided with bone marrow becoming permissive to the formation of osteoclasts of greater size and greater nuclei numbers. Taken together, our data are consistent with the thesis that transient calf muscle paralysis induces acute inflammation within the marrow of the adjacent tibia and that these alterations are temporally consistent with a role in mediating muscle paralysis-induced bone resorption., (Copyright © 2017 the American Physiological Society.)

The importance of Wnt pathway signaling in development of bone has been well established. Here we investigated the role of a known Wnt target, ENC1 (ectodermal-neural cortex 1; NRP/B), in osteoblast differentiation. Enc1 expression was detected in mouse osteoblasts, chondrocytes, and osteocytes by in situ hybridization, and osteoblastic expression was verified in differentiating primary cultures and MC3T3-E1 pre-osteoblast cells, with 57 kDa and 67 kDa ENC1 protein isoforms detected throughout differentiation. Induced knockdown of both ENC1 isoforms reduced alkaline phosphatase staining and virtually abolished MC3T3-E1 mineralization. At culture confluence, Alpl (alkaline phosphatase liver/bone/kidney) expression was markedly reduced compared with control cells, and there was significant and coordinated alteration of other genes involved in cellular phosphate biochemistry. In contrast, with 67 kDa-selective knockdown mineralized nodule formation was enhanced and there was a two-fold increase in Alpl expression at confluence. There was enhanced expression of Wnt/β-catenin target genes with knockdown of both isoforms at this time-point and a five-fold increase in Frzb (Frizzled related protein) with 67 kDa-selective knockdown at mineralization, indicating possible ENC1 interactions with Wnt signaling in osteoblasts. These results are the first to demonstrate a role for ENC1 in the control of osteoblast differentiation. Additionally, the contrasting mineralization phenotypes and transcriptional patterns seen with coordinate knockdown of both ENC1 isoforms vs selective knockdown of 67 kDa ENC1 suggest opposing roles for the isoforms in regulation of osteoblastic differentiation, through effects on Alpl expression and phosphate cellular biochemistry. This study is the first to report differential roles for the ENC1 isoforms in any cell lineage. J. Cell. Biochem. 118: 2141-2150, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Purpose: As our society becomes increasingly sedentary, compliance with exercise regimens that require numerous high-energy activities each week become less likely. Alternatively, given an osteogenic exercise intervention that required minimal effort, it is reasonable to presume that participation would be enhanced. Insertion of brief rest intervals between each cycle of mechanical loading holds potential to achieve this result because substantial osteoblast function is activated by many fewer loading repetitions within each loading bout. Here, we examined the complementary hypothesis that the number of bouts per week of rest-inserted loading could be reduced from three bouts per week without loss of osteogenic efficacy., Methods: We conducted a series of 3-wk in vivo experiments that noninvasively exposed the right tibiae of mice to either cyclic (1 Hz) or rest-inserted loading interventions and quantified osteoblast function via dynamic histomorphometry., Results: Although reducing loading bouts from three bouts per week (i.e., nine total bouts) to one bout per week (i.e., three total bouts) effectively mitigated the osteogenic benefit of cyclic loading, the same reduction did not significantly reduce periosteal bone formation parameters induced by rest-inserted loading. The osteogenic response was robust to the timing of the rest-inserted loading bouts (three bouts in the first week vs one bout per week for 3 wk). However, elimination of any single bout of the three one-bout-per-week bouts mitigated the osteogenic response to rest-inserted loading. Finally, periosteal osteoblast function assessed after the 3-wk intervention was not sensitive to the timing or number of rest-inserted loading bouts., Conclusions: We conclude that rest-inserted loading holds potential to retain the osteogenic benefits of mechanical loading with significantly reduced frequency of bouts of activity while also enabling greater flexibility in the timing of the activity.

Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (eg, development, growth, and healing), yet the mechanisms of neuromuscular-bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular-mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study, we developed a model of BTx-induced muscle paralysis in adult zebrafish, and we examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose-dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed late-stage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra- and/or inter-ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx-induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders., (© 2014 American Society for Bone and Mineral Research.)

Growing evidence suggests that aging compromises the ability of the skeleton to respond to anabolic mechanical stimuli. Recently, we reported that treating senescent mice with Cyclosporin A (CsA) rescued aging-related deficits in loading-induced bone formation. Given that the actions of CsA are often attributed to inhibition of the calcineurin/NFAT axis, we hypothesized that CsA enhances gene expression in bone cells exposed to fluid flow, by inhibiting nuclear NFATc1 accumulation. When exposed to flow, MC3T3-E1 osteoblastic cells exhibited rapid nuclear accumulation of NFATc1 that was abolished by CsA treatment. Under differentiation conditions, intermittent CsA treatment enhanced gene expression of late osteoblastic differentiation markers and activator protein 1 (AP-1) family members. Superimposing flow upon CsA further enhanced expression of the AP-1 members Fra-1 and c-Jun. To delineate the contribution of NFAT in this response, cells were treated with VIVIT, a specific inhibitor of the calcineurin/NFAT interaction. Treatment with VIVIT blocked flow-induced nuclear NFATc1 accumulation but did not recapitulate the CsA-mediated enhancement of flow-induced AP-1 component gene expression. Taken together, our study is the first to demonstrate that CsA enhances mechanically-induced gene expression of AP-1 components in bone cells, and suggests that this response requires calcineurin-dependent mechanisms that are independent of inhibiting NFATc1 nuclear accumulation.

The non-canonical Wnt pathway, a regulator of cellular motility and morphology, is increasingly implicated in cancer metastasis. In a quantitative PCR array analysis of 84 Wnt pathway associated genes, both non-canonical and canonical pathways were activated in primary and metastatic tumors relative to normal prostate. Expression of the Wnt target gene PITX2 in a prostate cancer (PCa) bone metastasis was strikingly elevated over normal prostate (over 2,000-fold) and primary prostate cancer (over 200-fold). The elevation of PITX2 protein was also evident on tissue microarrays, with strong PITX2 immunostaining in PCa skeletal and, to a lesser degree, soft tissue metastases. PITX2 is associated with cell migration during normal tissue morphogenesis. In our studies, overexpression of individual PITX2A/B/C isoforms stimulated PC-3 PCa cell motility, with the PITX2A isoform imparting a specific motility advantage in the presence of non-canonical Wnt5a stimulation. Furthermore, PITX2 specific shRNA inhibited PC-3 cell migration toward bone cell derived chemoattractant. These experimental results support a pivotal role of PITX2A and non-canonical Wnt signaling in enhancement of PCa cell motility, suggest PITX2 involvement in homing of PCa to the skeleton, and are consistent with a role for PITX2 in PCa metastasis to soft and bone tissues. Our findings, which significantly expand previous evidence that PITX2 is associated with risk of PCa biochemical recurrence, indicate that variation in PITX2 expression accompanies and may promote prostate tumor progression and metastasis.

Age-related decline in periosteal adaptation negatively impacts the ability to utilize exercise to enhance bone mass and strength in the elderly. We recently observed that in senescent animals subject to cyclically applied loading, supplementation with Cyclosporin A (CsA) substantially enhanced the periosteal bone formation rates to levels observed in young animals. We therefore speculated that if the CsA supplement could enhance bone response to a variety of types of mechanical stimuli, this approach could readily provide the means to expand the range of mild stimuli that are robustly osteogenic at senescence. Here, we specifically hypothesized that a given CsA supplement would enhance bone formation induced in the senescent skeleton by both cyclic (1-Hz) and rest-inserted loading (wherein a 10-s unloaded rest interval is inserted between each load cycle). To examine this hypothesis, the right tibiae of senescent female C57BL/6 mice (22 Mo) were subjected to cyclic or rest-inserted loading supplemented with CsA at 3.0 mg/kg. As previously, we initially found that while the periosteal bone formation rate (p.BFR) induced by cyclic loading was enhanced when supplemented with 3.0 mg/kg CsA (by 140%), the response to rest-inserted loading was not augmented at this CsA dosage. In follow-up experiments, we observed that while a 30-fold lower CsA dosage (0.1 mg/kg) significantly enhanced p.BFR induced by rest-inserted loading (by 102%), it was ineffective as a supplement with cyclic loading. Additional experiments and statistical analysis confirmed that the dose-response relations were significantly different for cyclic versus rest-inserted loading, only because the two stimuli required distinct CsA dosages for efficacy. While not anticipated a priori, clarifying the complexity underlying the observed interaction between CsA dosage and loading type holds potential for insight into how bone response to a broad range of mechanical stimuli may be substantially enhanced in the senescent skeleton.

Bone has long been established to be a highly mechanosensitive tissue. When subjected to mechanical loading, bone exhibits profoundly different anabolic responses depending on the temporal pattern in which the stimulus is applied. This phenomenon has been termed temporal processing, and involves complex signal amplification mechanisms that are largely unidentified. In this study, our goal was to characterize transcriptomic perturbations arising from the insertion of intermittent rest periods (a temporal variation with profound effects on bone anabolism) in osteoblastic cells subjected to fluid flow, and assess the utility of these perturbations to identify signaling pathways that are differentially activated by this temporal variation. At the level of the genome, we found that the common and differential alterations in gene expression arising from the two flow conditions were distributionally distinct, with the differential alterations characterized by many small changes in a large number of genes. Using bioinformatics analysis, we identified distinct up- and down-regulation transcriptomic signatures associated with the insertion of rest intervals, and found that the up-regulation signature was significantly associated with MAPK signaling. Confirming the involvement of the MAPK pathway, we found that the insertion of rest intervals significantly elevated flow-induced p-ERK1/2 levels by enabling a second spike in activity that was not observed in response to continuous flow. Collectively, these studies are the first to characterize distinct transcriptomic perturbations in bone cells subjected to continuous and intermittent stimulation, and directly demonstrate the utility of systems-based transcriptomic analysis to identify novel acute signaling pathways underlying temporal processing in bone cells.

We identified a new benzothiophene containing Rho kinase inhibitor scaffold in an ultra high-throughput enzymatic activity screen. SAR studies, driven by a novel label-free cellular impedance assay, were used to derive 39, which substantially reduced intraocular pressure in a monkey model of glaucoma-associated ocular hypertension., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Important and novel roles for neuropeptide Y (NPY) signaling in the control of bone homeostasis have recently been identified, with deletion of either the Y1 or Y2 receptors resulting in a generalized increase in bone formation. Whereas the Y2 receptor-mediated anabolic response is mediated by a hypothalamic relay, the Y1-mediated response is likely mediated by osteoblastic Y1 receptors. The presence of Y1 receptors on osteoblasts and various other peripheral tissues suggests that, in addition to neuronal input, circulating factors may also interact with the Y1-mediated pathways. The skeletal and adipose tissue (peripheral and marrow) responses to Y1 receptor deficiency were examined after (1) leptin deficiency, (2) gonadectomy, and (3) hypothalamic NPY overexpression. Bone formation was consistently increased in intact Y1(-/-) mice. However, the hypogonadism of gonadectomy or leptin deficiency blocked this anabolism in male Y1(-/-) mice, whereas females remained unchanged. The Y1-mediated bone anabolic pathway thus seems to be dependent on the presence of intact androgen signaling. Y1 deficiency also led to increased body weight and/or adiposity in all experimental models, with the exception of male ob/ob, showing a general adipogenic effect of Y1 deficiency that is not dependent on androgens. Interestingly, marrow adipocytes were regulated differently than general adipose depots in these models. Taken together, this interaction represents a novel mechanism for the integration of endocrine and neural signals initiated in the hypothalamus and provides further insight into the coordination of bone and energy homeostasis.

In mammals there are two ubiquitous, catalytically indistinguishable isoforms of inosine monophosphate dehydrogenase and mutations in the type I isoform, but not type II, cause retina-specific disorders. We have characterised the spatio-temporal expression of these proteins during development of the rat retina and performed functional investigations of the recently described retinal type I variants. Inosine monophosphate dehydrogenase was present in all immature cells throughout the retina during embryonic and neonatal development. Following eye opening and cell differentiation its distribution was restricted to the photoreceptors and bipolar cells, becoming prominent in Müller cells with aging. Type II was present in early, developing retinae whilst type I was undetectable. An isoform switch occurred around P10, after which the type I variants, type Ialpha and type Igamma, were the major forms. Functional investigations indicate type Igamma has greater catalytic activity compared with other variants and isoforms. Finally, all forms of type I show an increased propensity to form intracellular macrostructures compared to type II and these structures appear to be regulated in response to changing intracellular GTP levels. Collectively these data demonstrate that (i) type I does not play a role in early retinal development, (ii) type Igamma has greater activity and (iii) there are differences between type I and type II isoforms. These observations are consistent with the aetiology of retinitis pigmentosa and raise the possibility that programmed expression of specific inosine monophosphate dehydrogenase proteins may have arisen to meet the requirements of the cellular environment.

The Wnt pathway regulates cell proliferation and differentiation in development and disease, with a number of recent reports linking Wnt to control of osteoblast differentiation and bone mass. There is also accumulating evidence for interaction between the Wnt and nuclear receptor (NR)-mediated control pathways in non-osseous tissues. Calcitriol (1,25D(3)), which is the active hormonal ligand for the vitamin D receptor (VDR), a member of the NR superfamily, induces osteoblastic cell cycle arrest and expression of genes involved in matrix mineralization in vitro, with over-expression of VDR in mature osteoblasts increasing bone mass in mice. To determine whether the vitamin D and Wnt control pathways interact in osteoblastic regulation, we investigated the treatment effects of 1,25D(3) and/or lithium chloride (LiCl), which mimics canonical Wnt pathway activation, on osteoblast proliferation and differentiation. Treatments were initiated at various stages in differentiating cultures of the MC3T3-E1 osteoprogenitor cell line. Treatment of subconfluent cultures (day 1) with either agent transiently increased cell proliferation but decreased viable cell number, with additive inhibition after combined treatment. Interestingly, although early response patterns of alkaline phosphatase activity to 1,25D(3) and LiCl were opposite, mineralized nodule formation was virtually abolished by either treatment initiated at day 1 and remained very low after initiating treatments at matrix-formation stage (day 6). By contrast, mineralized nodule formation was substantial but reduced if 1,25D(3) and/or LiCl treatment was initiated at mineralization onset (day 13). Osteocalcin production was reduced by all treatments at all time points. Thus, vitamin D and/or canonical Wnt pathway activation markedly reduced mineralization, with additive inhibitory effects on viable cell number. The strength of the response was dependent on the stage of differentiation at treatment initiation. Importantly, the inhibitory effect of LiCl in this committed osteoblastic cell line contrasts with the stimulatory effects of genetic Wnt pathway activation in human and mouse bone tissue. This is consistent with the anabolic Wnt response occurring at a stage prior to the mature osteoprogenitor in the intact skeleton and suggests that prolonged or repeated activation of the canonical Wnt response in committed cells may have an inhibitory effect on osteoblast differentiation and function.

Germ line or hypothalamus-specific deletion of Y2 receptors in mice results in a doubling of trabecular bone volume. However, the specific mechanism by which deletion of Y2 receptors increases bone mass has not yet been identified. Here we show that cultured adherent bone marrow stromal cells from Y2(-/-) mice also demonstrate increased mineralization in vitro. Isolation of two populations of progenitor cell types, an immature mesenchymal stem cell population and a more highly differentiated population of progenitor cells, revealed a greater number of the progenitor cells within the bone of Y2(-/-) mice. Analysis of Y receptor transcripts in cultured stromal cells from wild-type mice revealed high levels of Y1 but not Y2, Y4, Y5, or y6 receptor mRNA. Interestingly, germ line Y2 receptor deletion causes Y1 receptor down-regulation in stromal cells and bone tissue possibly due to the lack of feedback inhibition of NPY release and subsequent overstimulation of Y1 receptors. Furthermore, deletion of Y1 receptors resulted in increased bone mineral density in mice. Together, these findings indicate that the greater number of mesenchymal progenitors and the altered Y1 receptor expression within bone cells in the absence of Y2 receptors are a likely mechanism for the greater bone mineralization in vivo and in vitro, opening up potential new treatment avenues for osteoporosis.

The importance of neuropeptide Y (NPY) and Y2 receptors in the regulation of bone and energy homeostasis has recently been demonstrated. However, the contributions of the other Y receptors are less clear. Here we show that Y1 receptors are expressed on osteoblastic cells. Moreover, bone and adipose tissue mass are elevated in Y1(-/-) mice with a generalized increase in bone formation on cortical and cancellous surfaces. Importantly, the inhibitory effects of NPY on bone marrow stromal cells in vitro are absent in cells derived from Y1(-/-) mice, indicating a direct action of NPY on bone cells via this Y receptor. Interestingly, in contrast to Y2 receptor or germ line Y1 receptor deletion, conditional deletion of hypothalamic Y1 receptors in adult mice did not alter bone homeostasis, food intake, or adiposity. Furthermore, deletion of both Y1 and Y2 receptors did not produce additive effects in bone or adiposity. Thus Y1 receptor pathways act powerfully to inhibit bone production and adiposity by nonhypothalamic pathways, with potentially direct effects on bone tissue through a single pathway with Y2 receptors.

The interplay in prostate cancer bone metastases between the 'seed' (the prostate cancer cells) and the 'soil' (the bone microenvironment) has been increasingly recognized as integral to the remarkable tropism for bone shown by prostate cancer. Increasing research into this area is elucidating the mechanisms involved in this complex 'cross-talk'. Recent developments, including the use of bisphosphonates in metastatic disease, highlight the important role of bone cells in the development and progression of metastatic prostate cancer. We review the current reports emphasising these possible mechanisms and indicating possible factors for future treatment directions.

Chemotactic responsiveness is crucial to neutrophil recruitment to sites of infection. During chemotaxis, highly divergent cytoskeletal programs are executed at the leading and trailing edge of motile neutrophils. The Rho family of small GTPases plays a critical role in cell migration, and recent work has focused on elucidating the specific roles played by Rac1, Rac2, Cdc42, and Rho during cellular chemotaxis. Rac GTPases regulate actin polymerization and extension of the leading edge, whereas Rho GTPases control myosin-based contraction of the trailing edge. Rac and Rho signaling are thought to crosstalk with one another, and previous research has focused on mutual inhibition of Rac and Rho signaling during chemotaxis. Indeed, polarization of neutrophils has been proposed to involve the activity of a negative feedback system where Rac activation at the front of the cell inhibits local Rho activation, and vice versa. Using primary human neutrophils and neutrophils derived from a Rac1/Rac2-null transgenic mouse model, we demonstrate here that Rac1 (and not Rac2) is essential for Rho and myosin activation at the trailing edge to regulate uropod function. We conclude that Rac plays both positive and negative roles in the organization of the Rhomyosin "backness" program, thereby promoting stable polarity in chemotaxing neutrophils.

Unlabelled: NeuropeptideY-, Y2 receptor (Y2)-, and leptin-deficient mice show similar anabolic action in cancellous bone but have not been assessed in cortical bone. Cortical bone mass is elevated in Y2(-/-) mice through greater osteoblast activity. In contrast, leptin deficiency results in reduced bone mass. We show opposing central regulation of cortical bone., Introduction: Treatment of osteoporosis is confounded by a lack of agents capable of stimulating the formation of bone by osteoblasts. Recently, the brain has been identified as a potent anabolic regulator of bone formation. Hypothalamic leptin or Y2 receptor signaling are known to regulate osteoblast activity in cancellous bone. However, assessment of these pathways in the structural cortical bone is critical to understanding their role in skeletal health and their potential clinical relevance to osteoporosis and its treatment., Materials and Methods: Long bones of 16-week male ob/ob and germline and hypothalamic Y2(-/-) mice were assessed by QCT. Cortical osteoblast activity was assessed histologically., Results: The femora of skeletally mature Y2(-/-) mice and of leptin-deficient ob/ob and Y2(-/-)ob/ob mice were assessed for changes in cortical osteoblast activity and bone mass. Ablation of Y2 receptors increased osteoblast activity on both endosteal and periosteal surfaces, independent of leptin, resulting in increased cortical bone mass and density in Y2(-/-) mice along the entire femur. Importantly, these changes were evident after deletion of hypothalamic Y2 receptors in adult mice, with a 5-fold elevation in periosteal bone formation. This is in marked contrast to leptin-deficient models that displayed reduced cortical mass and density. These changes were associated with substantial differences in calculated strength between the Y2(-/-) and leptin-deficient mice., Conclusions: These results indicate that the Y2-mediated anabolic pathway stimulates cortical and cancellous bone formation, whereas the leptin-mediated pathway has opposing effects in cortical and cancellous bone, diminishing the production of cortical bone. The findings from conditional hypothalamic Y2 knockout show a novel, inducible control mechanism for cortical bone formation and a potential new pathway for anabolic treatment of osteoporosis.

Unlabelled: Vitamin D acts through the immature osteoblast to stimulate osteoclastogenesis. Transgenic elevation of VDR in mature osteoblasts was found to inhibit osteoclastogenesis associated with an altered OPG response. This inhibition was confined to cancellous bone. This study indicates that vitamin D-mediated osteoclastogenesis is regulated locally by OPG production in the mature osteoblast., Introduction: Vitamin D stimulates osteoclastogenesis acting through its nuclear receptor (VDR) in immature osteoblast/stromal cells. This mobilization of calcium stores does not occur in a random manner, with bone preferentially removed from cancellous bone. The process whereby the systemic, humoral regulator is targeted to a particular region of the skeleton is unclear., Materials and Methods: Bone resorption was assessed in mice with vitamin D receptor transgenically elevated in mature osteoblasts (OSVDR). Vitamin D-mediated osteoclastogenesis was examined in vitro using OSVDR osteoblasts and osteoblastic RANKL: osteoprotegerin (OPG) examined in vivo and in vitro after vitamin D treatment., Results: Vitamin D-mediated osteoclastogenesis was reduced in OSVDR mice on chow and calcium-restricted diets, with effects confined to cancellous bone. OSVDR osteoblasts had a reduced capacity to support osteoclastogenesis in culture. The vitamin D-mediated reduction in OPG expression was reduced in OSVDR osteoblasts in vivo and in vitro, resulting in a reduced RANKL/OPG ratio in OSVDR compared with wildtype, after exposure to vitamin D., Conclusions: Mature osteoblasts play an inhibitory role in bone resorption, with active vitamin D metabolites acting through the VDR to increase OPG. This inhibition is less active in cancellous bone, effectively targeting this region for resorption after the systemic release of activated vitamin D metabolites.

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.

Context and Objective: Hip fracture is partially genetically determined. The present study was designed to examine the contributions of vitamin D receptor (VDR) and collagen I alpha1 (COLIA1) genotypes to the liability to hip fracture in postmenopausal women., Design: The study was designed as a prospective population-based cohort investigation., Subjects: Six hundred seventy-seven postmenopausal women of Caucasian background, aged 70 +/- 7 yr (mean +/- SD), have been followed for up to 14 yr. Sixty-nine women had sustained a hip fracture during the period., Main Outcome: Atraumatic hip fractures were prospectively identified through radiologists' reports. Bone mineral density (BMD) at the hip and lumbar spine was measured by dual-energy x-ray absorptiometry. GENOTYPES: The TaqI and SpI COLIA1 polymorphisms of the VDR and COLIA1 genes were determined. Using the Single Nucleotide Polymorphism database, VDR TT, Tt, and tt genotypes were coded as TT, TC, and CC, whereas COLIA1 SS, Ss, and ss were coded as GG, GT, and TT., Results: Women with VDR CC genotype (16% prevalence) and COLIA1 TT genotype (5% prevalence) had an increased risk of hip fracture [odds ratio (OR) associated with CC, 2.6; 95% confidence interval (CI), 1.2-5.3; OR associated with TT, 3.8; 95% CI, 1.3-10.8] after adjustment for femoral neck BMD (OR, 3.4 per SD; 95% CI, 2.3-5.0) and age (OR, 1.4 per 5 yr; 95% CI, 1.1-1.7). Approximately 20 and 12% of the liability to hip fracture was attributable to the presence of the CC genotype and TT genotype, respectively., Conclusion: The VDR CC genotype and COLIA1 TT genotype were associated with increased hip fracture risk in Caucasian women, and this association was independent of BMD and age.

Unlabelled: Leptin and Y2 receptors on hypothalamic NPY neurons mediate leptin effects on energy homeostasis; however, their interaction in modulating osteoblast activity is not established. Here, direct testing of this possibility indicates distinct mechanisms of action for leptin anti-osteogenic and Y2-/- anabolic pathways in modulating bone formation., Introduction: Central enhancement of bone formation by hypothalamic neurons is observed in leptin-deficient ob/ob and Y2 receptor null mice. Similar elevation in central neuropeptide Y (NPY) expression and effects on osteoblast activity in these two models suggest a shared pathway between leptin and Y2 receptors in the central control of bone physiology. The aim of this study was to test whether the leptin and Y2 receptor pathways regulate bone by the same or distinct mechanisms., Materials and Methods: The interaction of concomitant leptin and Y2 receptor deficiency in controlling bone was examined in Y2-/- ob/ob double mutant mice, to determine whether leptin and Y2 receptor deficiency have additive effects. Interaction between leptin excess and Y2 receptor deletion was examined using recombinant adeno-associated viral vector overproduction of NPY (AAV-NPY) to produce weight gain and thus leptin excess in adult Y2-/- mice. Cancellous bone volume and bone cell function were assessed., Results: Osteoblast activity was comparably elevated in ob/ob, Y2-/-, and Y2-/- ob/ob mice. However, greater bone resorption in ob/ob and Y2-/- ob/ob mice reduced cancellous bone volume compared with Y2-/-. Both wildtype and Y2-/- AAV-NPY mice exhibited marked elevation of white adipose tissue accumulation and hence leptin expression, thereby reducing osteoblast activity. Despite this anti-osteogenic leptin effect in the obese AAV-NPY model, osteoblast activity in Y2-/- AAV-NPY mice remained significantly greater than in wildtype AAV-NPY mice., Conclusions: This study suggests that NPY is not a key regulator of the leptin-dependent osteoblast activity, because both the leptin-deficient stimulation of bone formation and the excess leptin inhibition of bone formation can occur in the presence of high hypothalamic NPY. The Y2-/- pathway acts consistently to stimulate bone formation; in contrast, leptin continues to suppress bone formation as circulating levels increase. As a result, they act increasingly in opposition as obesity becomes more marked. Thus, in the absence of leptin, the cancellous bone response to loss of Y2 receptor and leptin activity can not be distinguished. However, as leptin levels increase to physiological levels, distinct signaling pathways are revealed.

A 3.9 kb DNA fragment of human osteocalcin promoter and 3.6 kb DNA fragment of the rat collagen type1a1 promoter linked with visually distinguishable GFP isomers, topaz and cyan, were used for multiplex analysis of osteoblast lineage progression. Three patterns of dual transgene expression can be appreciated in primary bone cell cultures derived from the transgenic mice and by histology of their corresponding bones. Our data support the interpretation that strong pOBCol3.6GFPcyan alone is found in newly formed osteoblasts, while strong pOBCol3.6GFPcyan and hOC-GFPtpz are present in osteoblasts actively making a new matrix. Osteoblasts expressing strong hOC-GFPtpz and weak pOBCol3.6GFPcyan are also present and may or may not be producing mineralized matrix. This multiplex approach reveals the heterogeneity within the mature osteoblast population that cannot be appreciated by current histological methods. It should be useful to identify and isolate populations of cells within an osteoblast lineage as they progress through stages of differentiation., (genesis 43:87-98, 2005. (c) 2005 Wiley-Liss, Inc.)

Unlabelled: Bisphosphonates have clinical benefit in children with severe osteogenesis imperfecta or osteoporosis and potential benefit in children with Perthes disease or undergoing distraction osteogenesis. However, there is concern about the effects of bisphosphonates on the physis and bone length. In 44 growing rabbits, zoledronic acid caused a transient disruption of physeal morphology, retention of cartilaginous matrix in trabeculae and cortical bone of the metaphysis, and a minor decrement in tibial bone length at maturity., Introduction: Data from growing animal models suggest that bisphosphonates cause retention of longitudinal cartilaginous septa at the chondro-osseous junction, extension of trabeculae to the metaphyseal-diaphyseal junction, and varying dose-dependent effects on longitudinal growth. However, there is a lack of data regarding effects of intermittent use of nitrogen-containing bisphosphonates on the physis and on tibial length in models reaching maturity., Materials and Methods: Contralateral tibias of juvenile rabbits were examined after right tibial distraction osteogenesis from two previous studies. Animals were randomized to receive 0.1 mg/kg zoledronic acid (ZA) IV at 8 weeks of age (ZA*1) or 8 and 10 weeks of age (ZA*2) or saline. Body mass was analyzed from 5 to 44 weeks of age; tibial length and proximal physeal-metaphyseal histology and histomorphometry were analyzed at 8-52 weeks of age., Results: Tibial length was 3% less at 14 weeks of age in the ZA*2-treated versus saline group (p<0.05) in both studies, and this difference persisted at maturity in the long-term study group (26 weeks of age, p<0.05). Total body mass gain from 5 to 26 weeks of age was 14% less in ZA*2-treated than saline animals (p<0.05). Rate of weight gain from 8 to 10 weeks of age was 76% less in ZA*2 compared with saline animals (p<0.05). Radiographs showed radiodense lines in the metaphyses of ZA-treated bones, corresponding to the number of doses. Histologically, lines resulting from the first dose of ZA contained longitudinal cartilaginous matrix cores surrounded by bone, whereas those from the second dose contained spherical cores of matrix caused by transient disruption of physeal morphology after the first dose of ZA. Resorption of these lines at later times was radiographically and histologically evident, but remnants of cartilaginous matrix remained in the cortical bone of ZA-treated animals., Conclusions: ZA treatment within the final 13.5% of the rabbit tibial growth period caused a transient disruption in physeal morphology and resorption associated with retention of cartilaginous matrix and coinciding with a persistent 3% decrement in tibial length. Disruption of physeal morphology and potential loss of bone length should be considered when administering nitrogen-containing bisphosphonates to children before closure of the major physes.

The vitamin D receptor (VDR) mediates the effects of 1,25(OH)(2)D(3), the active form of vitamin D. The human VDRB1 isoform differs from the originally described VDR by an N-terminal extension of 50 amino acids. Here we investigate cell-, promoter-, and ligand-specific transactivation by the VDRB1 isoform. Transactivation by these isoforms of the cytochrome P450 CYP24 promoter was compared in kidney (HEK293 and COS1), tumor-derived colon (Caco-2, LS174T, and HCT15), and mammary (HS578T and MCF7) cell lines. VDRB1 transactivation in response to 1,25(OH)(2)D(3) was greater in COS1 and HCT15 cells (145%), lower in HEK293 and Caco-2 cells (70-85%) and similar in other cell lines tested. By contrast, on the cytochrome P450 CYP3A4 promoter, 1,25(OH)(2)D(3)-induced VDRB1 transactivation was significantly lower than VDRA in Caco-2 (68%), but comparable to VDRA in HEK293 and COS1 cells. Ligand-dependence of VDRB1 differential transactivation was investigated using the secondary bile acid lithocholic acid (LCA). On the CYP24 promoter LCA-induced transactivation was similar for both isoforms in COS1, whereas in Caco-2 and HEK293 cells VDRB1 was less active. On the CYP3A4 promoter, LCA activation of VDRB1 was comparable to VDRA in all the cell lines tested. Mutational analysis indicated that both the 1,25(OH)(2)D(3) and LCA-regulated activities of both VDR isoforms required a functional ligand-dependent activation function (AF-2) domain. In gel shift assays VDR:DNA complex formation was stronger in the presence of 1,25(OH)(2)D(3) than with LCA. These results indicate that regulation of VDRB1 transactivation activity is dependent on cellular context, promoter, and the nature of the ligand.

Although the key components of the cellular nuclear transport machinery have largely been characterized through extensive efforts in recent years, in vivo measurements of the kinetics of nuclear protein import/export are patently few. The present study applies the approach of FRAP (fluorescence recovery after photobleaching) to examine the nucleocytoplasmic flux of a novel human VDRB1 (vitamin D receptor B1) isoform in living cells. Through an N-terminal extension containing a consensus nuclear targeting sequence, VDRB1 is capable of localizing in nuclear speckles adjacent to SC-35 (35 kDa splicing component)-containing speckles as well as in the nucleoplasm, dependent on ligand. Investigation of VDRB1 nucleocytoplasmic transport using FRAP indicates for the first time that the VDRB1 has a serum-modulated, active nuclear import mechanism. There is no evidence of an efficient, active export mechanism for VDRB1, probably as a result of nuclear retention. VDRB1 nuclear import in the absence of serum occurred more rapidly and to a greater extent to nuclear speckles compared with import to other nuclear sites. This preferential transport from the cytoplasm to and accumulation within nuclear speckles is consistent with the idea that the latter represent dynamic centres of VDRB1 interaction with other nuclear proteins. The results are consistent with the existence of specialized pathways to target proteins to nuclear subdomains.

Unlabelled: Bisphosphonates induce major increases in strength of callus in distraction osteogenesis in the short term. Poor understanding of the underlying mechanism, however, raises concerns about long-term consequences. In this long-term study in 32 rabbits, zoledronic acid transiently increased trabeculae by delayed temporal progression of endochondral bone remodeling but did not prevent radiographic completion of bone repair., Introduction: We hypothesized that bisphosphonate inhibition of osteoclast-mediated resorption would retain bone during repair, producing a larger callus in the short term. However, if remodeling was not restored, completion of the bone repair process in the long term could be jeopardized., Materials and Methods: Juvenile rabbits underwent right tibial osteotomy and 2 weeks of distraction, followed by a period of consolidation. Animals received saline (controls) or zoledronic acid (ZA; 0.1 mg/kg at surgery and again 2 weeks later), and distracted tibias were examined by radiograph, DXA, histology, and histomorphometry at 2, 4, 6, 18, and 44 weeks after surgery., Results: Regenerated bone in ZA-treated animals was denser than controls on radiographs at 6 weeks and had more distinct radiodense trabeculae and retention of original cortices at 18 weeks. By 44 weeks, controls and ZA-treated animals were radiographically healed and indistinguishable. Regenerate BMD and BMC increased between 2 and 4 weeks in all animals, with a greater effect in ZA. At 6 weeks, BMD and BMC in ZA-treated animals were 1.6- and 2-fold greater, respectively, than controls (p < 0.01). From 6 to 44 weeks, the control values gradually increased and approached the ZA-treated values. Regenerate bone volume and trabecular number by histomorphometry were from 1.6- to 2-fold greater in ZA-treated animals at 6 and 18 weeks (p < 0.05). Endochondral cartilaginous matrix volume was up to 2.4-fold greater in ZA-treated animals at 2 and 4 weeks (p < 0.05). TRACP+ cells in ZA-treated animals were larger with more nuclei. Mineral apposition rate and osteoblast number and surface were lower in ZA-treated animals at 6 weeks (p < 0.01) but not at later times., Conclusions: Disruption of TRACP+ cell function by ZA during bone regeneration seems to lead to an accretion of cancellous bone built on a larger endochondral cartilaginous matrix and increased bone mass, consistent with reported increases in short-term callus strength. This increase in bone mass, caused by a delay in remodeling, provided a transient advantage without preventing radiographic completion of the bone repair process in the long term. Noncontinuous treatment with nitrogen-containing bisphosphonates thus can have short-term beneficial effects without preventing long-term bone repair.

Cytoskeletal remodeling is critical for cell adhesion, spreading, and motility. p21-activated kinase (PAK), an effector molecule of the Rho GTPases Rac and Cdc42, has been implicated in cytoskeletal remodeling and cell motility. PAK kinase activity and subcellular distribution are tightly regulated by rapid and transient localized Rac and Cdc42 activation, and by interactions mediated by adapter proteins. Here, we show that endogenous PAK is constitutively activated in certain breast cancer cell lines and that this active PAK is mislocalized to atypical focal adhesions in the absence of high levels of activated Rho GTPases. PAK localization to focal adhesions in these cells is independent of PAK kinase activity, NCK binding, or GTPase binding, but requires the association of PAK with PIX. Disruption of the PAK-PIX interaction with competitive peptides displaces PAK from focal adhesions and results in a substantial reduction in PAK hyperactivity. Moreover, disruption of the PAK-PIX interaction is associated with a dramatic decrease of PIX and paxillin in focal adhesions, indicating that PAK localization to these structures via PIX is required for the maintenance of paxillin- and PIX-containing focal adhesions. Abnormal regulation of PAK localization and activity may contribute to the tumorigenic properties of certain breast cancer cells.

The vitamin D receptor (VDR) shares a conserved structural and functional organization with other nuclear receptor (NR) superfamily members. For many NRs, N-terminal variant isoforms that display distinct cell-, stage- and promoter-specific actions have been identified. The novel VDR isoform VDRB1, with a 50 amino acid N-terminal extension, is produced from low abundance transcripts that contain exon 1d of the human VDR locus. There is evidence for the conservation of this exon in other mammalian and avian species. The transactivation differences between VDRB1 and the original VDR, clarified here, provide insights into mechanisms that may contribute to functional differences and potentially distinct physiological roles for these two VDR isoforms.

Ski-interacting protein (SKIP), a vitamin D receptor (VDR) coactivator, also functions as a repressor in Notch signalling in association with the corepressor SMRT. Here we show that SKIP bifunctionally modulates (activates or represses) Retinoid-X receptor (RXR)- and VDR-dependent gene transcription in a cell line-specific manner, with activation in CV-1 and repression in P19 cells. The coactivator function of SKIP in these cells appeared to correlate with the relative level and ratio of expression of N-CoR and p300, with greater SKIP activation in higher p300-expressing and lower N-CoR-expressing cell-lines. C-terminal deletion of SKIP (delta334-536 aa) was associated with strong activation in both CV-1 and P19 cells. The corepressors N-CoR and SMRT and the coregulator p300 interacted with SKIP through the same N-terminal region (1-200 aa). Overall these results suggest that transcriptional action of SKIP may depend on distinct functional domains and cell line-specific interactions with both corepressors and coactivators.

Increased cross-sectional area and strength of long bones has been observed in transgenic mice with 2-fold (OSV9) and 3-fold (OSV3) elevation of osteoblast vitamin D receptor (VDR) levels. In the present study, mineralization density distributions, including typical calcium content (Ca(Peak)) and homogeneity of mineralization (Ca(Width)) of femoral bone and growth plate cartilage, were determined by quantitative backscattered electron imaging (qBEI). Fourier-transform infrared (FTIR) microspectroscopy was used to examine mineral content, collagen and crystal maturation, and scanning small angle X-ray scattering (scanning-SAXS) for studying mineral particle thickness and alignment. In addition, X-ray diffraction (XRD) of distal tibiae revealed mineral particle c-axis size. In trabecular bone, the increase in Ca(Peak) was significant for both OSV9 (+ 3.14%, P = 0.03) and OSV3 (+ 3.43%, P = 0.02) versus controls with 23.61 +/- 0.45 S.D. wt% Ca baseline values. In cortical bone, Ca(Peak) was enhanced for the OSV3 mice (+ 1.84%, P = 0.02) versus controls with 26.61 +/- 0.28 S.D. wt% Ca, and OSV9 having intermediate values. Additionally, there was significantly increased homogeneity of mineralization as denoted by a reduction of Ca(Width) (-8.4%, P = 0.01) in primary spongiosa. FTIR microspectroscopy, with the exception of an increased collagen maturity in OSV3 trabecular bone (+ 9.9%, P = 0.02), XRD, and scanning-SAXS indicated no alterations in the nanostructure of transgenic bone. These findings indicate that elevation of osteoblastic vitamin D response led to formation of normal bone with higher calcium content. These material properties, together with indications of decreased bone resorption in secondary spongiosa and increased cortical periosteal bone formation, appear to contribute to the improved mechanical properties of their long bones and suggest an important physiological role of the vitamin D-endocrine system in normal bone mineralization.

Neuropeptide Y regulates numerous physiological processes via at least five different Y receptors, but the specific roles of each receptor are still unclear. We previously demonstrated that Y2 receptor knockout results in a lean phenotype, increased cancellous bone volume, and an increase in plasma pancreatic polypeptide (PP), a ligand for Y4 receptors. PP-overexpressing mice are also known to have a lean phenotype. Deletion of the Y4 receptor also produced a lean phenotype and increased plasma PP levels. We therefore hypothesized that part of the Y2 phenotype results from increased PP action on Y4 receptors and tested this in PP transgenic Y4(-/-) and Y2(-/-) Y4(-/-) double knockout mice. Bone mass was not altered in Y4 knockout mice. Surprisingly, despite significant hyperphagia, Y2(-/-) Y4(-/-) mice retained a markedly lean phenotype, with reduced body weight, white adipose tissue mass, leptinemia, and insulinemia. Furthermore, bone volume was also increased threefold in Y2(-/-) Y4(-/-) mice, and this was associated with enhanced osteoblastic activity. These changes were more pronounced than those observed in Y2(-/-) mice, suggesting synergy between Y2 and Y4 receptor pathways. The lack of bone changes in PP transgenic mice suggests that PP alone is not responsible for the bone mass increases but might play a major role in the lean phenotype. However, a synergistic interaction between Y2 and Y4 pathways seems to regulate bone volume and adiposity and could have important implications for possible interventions in obesity and for anabolic treatment of osteoporotic bone loss.

Unlabelled: Prolonged healing times and stress-shielding osteopenia remain problematic in distraction osteogenesis. In this study of 30 rabbits, zoledronic acid increased regenerate volume, mineralization, and tibial strength and prevented osteopenia over a 6-week period. Translation to the clinical setting, if safe, could improve outcomes in distraction osteogenesis in children., Introduction: Because the external fixators for limb lengthening and reconstruction are designed to control the positions of bone fragments accurately, they also produce stress-shielding effects on the forming regenerate and surrounding bone. Osteopenia, leading to refracture and limitations on rehabilitation, are common consequences, potentially increasing morbidity and detracting from final clinical outcome., Materials and Methods: We examined the effect of zoledronic acid on distraction osteogenesis in 42 immature male NZW rabbits. The model chosen results in reliable regenerate formation and stress-shielding osteopenia. Fourteen animals received either Saline, zoledronic acid 0.1 mg/kg at surgery (ZOL), or another dose 2 weeks postoperatively (Redosed ZOL). Rabbits underwent DXA for bone mineral content and bone mineral density in regenerate and surrounding segments of operated and contralateral tibias. After death at 6 weeks, 30 pairs of tibias underwent quantitative computerized tomography (QCT) and four-point bend testing, and 12 were examined by histomorphometry. The study was powered at 0.8 to show differences of 1.3 SDs for mineral and mechanical parameters., Results: Osteopenia observed in tibias of the Saline group was absent in ZOL and Redosed ZOL tibias, the latter exhibiting higher bone mineral density and bone mineral content over contralateral regions (p < 0.01). Regenerate bone mineral content was higher in ZOL and Redosed ZOL versus Saline groups at 4 and 6 weeks (p < 0.01). Cross-sectional area was 49% and 59% greater at 6 weeks in ZOL and Redosed ZOL regenerates compared with the Saline group (p < 0.01). ZOL and Redosed ZOL tibias were 29% and 89% stronger by four-point bending than the Saline group (p < 0.01). Histomorphometry in the regenerate of ZOL and Redosed ZOL groups revealed higher trabecular bone volume and trabecular number compared with the Saline group (p < 0.001)., Conclusions: Zoledronic acid administration led to significantly greater bone area, mineral content, strength, and trabecular number with reduced stress-shielding osteopenia in this model of distraction osteogenesis. These data suggest that intraoperative and postoperative zoledronic acid administration could improve outcomes in children undergoing limb lengthening.

The vitamin D receptor (VDR) is a ligand-dependent transcription factor that heterodimerizes with retinoid X receptor (RXR) and interacts with the basal transcription machinery and transcriptional cofactors to regulate target gene activity. The p160 coactivator GRIP1 and the distinct coregulator Ski-interacting protein (SKIP)/NCoA-62 synergistically enhance ligand-dependent VDR transcriptional activity. Both coregulators bind directly to and form a ternary complex with VDR, with GRIP1 contacting the activation function-2 (AF-2) domain and SKIP/NCoA-62 interacting through an AF-2 independent interface. It was previously reported that SKIP/NCoA-62 interaction with VDR was independent of the heterodimerization interface (specifically, helices H10/H11). In contrast, the present study defines specific residues within a conserved and surface-exposed region of VDR helix H10 that are required for interaction with SKIP/NCoA-62 and for full ligand-dependent transactivation activity. SKIP/NCoA-62, the basal transcription factor TFIIB, and RXR all interacted with VDR helix H10 mutants at reduced levels compared with wild type in the absence of ligand and exhibited different degrees of increased interaction upon ligand addition. Thus, SKIP/NCoA-62 interacts with VDR at a highly conserved region not previously associated with coregulator binding to regulate transactivation by a molecular mechanism distinct from that of p160 coactivators.

The ability of cells to recognize and respond with directed motility to chemoattractant agents is critical to normal physiological function. Neutrophils represent the prototypic chemotactic cell in that they respond to signals initiated through the binding of bacterial peptides and other chemokines to G protein-coupled receptors with speeds of up to 30 microm/min. It has been hypothesized that localized regulation of cytoskeletal dynamics by Rho GTPases is critical to orchestrating cell movement. Using a FRET-based biosensor approach, we investigated the dynamics of Rac GTPase activation during chemotaxis of live primary human neutrophils. Rac has been implicated in establishing and maintaining the leading edge of motile cells, and we show that Rac is dynamically activated at specific locations in the extending leading edge. However, we also demonstrate activated Rac in the retracting tail of motile neutrophils. Rac activation is both stimulus and adhesion dependent. Expression of a dominant-negative Rac mutant confirms that Rac is functionally required both for tail retraction and for formation of the leading edge during chemotaxis. These data establish that Rac GTPase is spatially and temporally regulated to coordinate leading-edge extension and tail retraction during a complex motile response, the chemotaxis of human neutrophils.

Previously, we have reported that nitric oxide synthases (NOSs), which generate NO, modulate fracture healing. However, the cellular sources of the NOS isoforms during the course of fracture healing have not been studied systematically. The purpose of this study was to localize the cellular distribution of NOS isoforms (inducible NOS [iNOS], endothelial NOS [eNOS], and neuronal NOS [bNOS]) by in situ hybridization and immunohistology after femoral fractures in rats. The iNOS signal was detected during the initial stages (on day 4 and day 7) of fracture healing in 52 +/- 2% (mean +/- SE, n = 7) of cells within the intramembranous region, along the edge of the periosteal callus. The iNOS signal in callus cells declined to an undetectable level on day 14. eNOS was detected during the middle stages (on day 7 and day 14) of fracture healing in cells lining the blood vessels and also in 49 +/- 3% of cells in the chondral region. The bNOS signal was found to be increased at the later stages (day 14 and day 21) of fracture healing in 51 +/- 3% of cells at the junction between fibrous tissue and cartilage within the fibrochondral region. In summary, the,expression of NOS isoforms during fracture healing was time dependent and cellular distinctive.

Neuropeptide Y (NPY) is a downstream modulator of leptin action, possibly at the level of the arcuate nucleus where NPY neurons are known to express both leptin receptors and Y2 receptors. In addition to the well-described role of NPY and leptin in energy balance and obesity, intracerebroventricular administration of NPY or leptin also causes bone loss. Here we show that Y2 receptor-deficient mice have a twofold increase in trabecular bone volume as well as greater trabecular number and thickness compared with control mice. We also demonstrate that central Y2 receptors are crucial for this process, since selective deletion of hypothalamic Y2 receptors in mature conditional Y2 knockout mice results in an identical increase in trabecular bone volume within 5 weeks. This hypothalamus-specific Y2 receptor deletion stimulates osteoblast activity and increases the rate of bone mineralization and formation, with no effect on osteoblast or osteoclast surface measurements. The lack of any changes in plasma total calcium, leptinemia, or hypothalamo-pituitary-corticotropic, -thyrotropic, -somatotropic, or -gonadotropic output suggests that Y2 receptors do not modulate bone formation by humoral mechanisms, and that alteration of autonomic function through hypothalamic Y2 receptors may play a key role in a major central regulatory circuit of bone formation.

The importance of N-terminal regions of nuclear hormone receptors in transcriptional regulation is increasingly recognized. As variant VDR gene transcripts indicated possible N-terminally extended receptors, we investigated their natural occurrence, transactivation capacity, and subcellular localization. A novel 54-kDa VDRB1 protein, in addition to the previously recognized 48-kDa VDRA form, was detected in human kidney tissue as well as in osteoblastic (MG63), intestinal (Int-407, DLD-1, and COLO 206F), and kidney epithelial (786) human cell lines by Western blots using isoform-specific and nonselective anti-VDR antibodies. VDRB1 was present at approximately one-third the level of VDRA. Isoform-specific VDRB1 expression constructs produced lower ligand-dependent transactivation than VDRA when transiently transfected with a vitamin D-responsive promoter into cell lines with low endogenous VDR. Intracellular localization patterns of the green fluorescent protein-tagged VDR isoforms differed. VDRB1 appeared as discrete intranuclear foci in the absence of 1,25-dihydroxyvitamin D3, whereas VDRA produced diffuse nuclear fluorescence. After 1,25-dihydroxyvitamin D3 treatment, both VDR isoforms exhibited similar diffuse nuclear signal. In the absence of 1,25-dihydroxyvitamin D3, the VDRB1 foci partially colocalized with SC-35 speckles and a subset of promyelocytic leukemia nuclear bodies. These data provide the first evidence of VDRB1, a novel N-terminally variant human VDR that is expressed at a level comparable to VDRA in human tissue and cell lines. It is characterized by reduced transactivation activity and a ligand-responsive speckled intranuclear localization. The intranuclear compartmentalization and altered functional activity of VDRB1 may mediate a specialized physiological role for this receptor isoform.

In this study, we show that phosphorylated 3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates p21-activated kinase 1 (PAK1) in the presence of sphingosine. We identify threonine 423, a conserved threonine in the activation loop of kinase subdomain VIII, as the PDK1 phosphorylation site on PAK1. Threonine 423 is a previously identified PAK1 autophosphorylation site that lies within a PAK consensus phosphorylation sequence. After pretreatment with phosphatases, autophosphorylation of PAK1 occurred at all major sites except threonine 423. A phosphothreonine 423-specific antibody detected phosphorylation of recombinant, catalytically inactive PAK1 after incubation with wild-type PAK1, indicating phosphorylation of threonine 423 occurs by an intermolecular mechanism. The biological significance of PDK1 phosphorylation of PAK1 at threonine 423 in vitro is supported by the observation that these two proteins interact in vivo and that PDK1-phosphorylated PAK1 has an increased activity toward substrate. An increase of phosphorylation of catalytically inactive PAK1 was observed in COS-7 cells expressing wild-type, but not catalytically inactive, PDK1 upon elevation of intracellular sphingosine levels. PDK1 phosphorylation of PAK1 was not blocked by pretreatment with wortmannin or when PDK1 was mutated to prevent phosphatidylinositol binding, indicating this process is independent of phosphatidylinositol 3-kinase activity. The data presented here provide evidence for a novel mechanism for PAK1 regulation and activation.

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the alpha Tm(fast) and beta-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and alpha(f)9d (detects specific Tms from the alpha Tm(fast) and beta-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by alpha(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and alpha(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the alpha Tm(fast) and beta-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to alpha Tm(fast) and beta-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo., (Copyright 2000 Wiley-Liss, Inc.)