PubMed Health⌕ Search

Biomedical subjects

Harry C Blair

Publications and source records attributed to Harry C Blair.

At least 19 recordsLinked to original sources
PubMed2006

Serum phosphate and lactate vary with FSH in an early postmenopausal population.

OBJECTIVE: We examined serum in recent postmenopausal women to determine the relationship of menopausal status, as FSH level, to serum acid-base balance. DESIGN, SETTING, AND PATIENTS: Serum electrolytes of 58 women, aged 53-58, were measured relative to serum FSH. The subjects were over one year since the last menstrual period and were from an academic practice setting. RESULTS: In women with FSH <35 IU/L (n=20, mean 16.6 IU/L, SD 6.8), phosphate and lactate were reduced relative to women with FSH >35 IU/L (n=38, mean 84.8 IU/L, SD 34.5). No other major anions showed significant differences. Both groups were analyzed by mass spectroscopy for fatty acids and anionic metabolic intermediates. Lactate was the predominant anion in the organic group but accounted for only about 10% of the FSH-responsive anion change. This change was mainly due to a 0.1-mM increase in phosphate in the high FSH group. CONCLUSIONS: There is a direct correlation of early postmenopausal FSH to increasing serum phosphate. Changes in phosphorus may reflect differences in the rate of bone loss.

Calcium↗
PubMed2006

Follicle-stimulating hormone stimulates TNF production from immune cells to enhance osteoblast and osteoclast formation.

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

Animals↗
PubMed2006

FSH directly regulates bone mass.

Postmenopausal osteoporosis, a global public health problem, has for decades been attributed solely to declining estrogen levels. Although FSH levels rise sharply in parallel, a direct effect of FSH on the skeleton has never been explored. We show that FSH is required for hypogonadal bone loss. Neither FSHbeta nor FSH receptor (FSHR) null mice have bone loss despite severe hypogonadism. Bone mass is increased and osteoclastic resorption is decreased in haploinsufficient FSHbeta+/- mice with normal ovarian function, suggesting that the skeletal action of FSH is estrogen independent. Osteoclasts and their precursors possess G(i2alpha)-coupled FSHRs that activate MEK/Erk, NF-kappaB, and Akt to result in enhanced osteoclast formation and function. We suggest that high circulating FSH causes hypogonadal bone loss.

Animals↗
PubMed2006

Death receptor-3 mediates apoptosis in human osteoblasts under narrowly regulated conditions.

We previously reported that a soluble form of the TNF-family receptor death receptor-3 (DR3) is expressed in osteoblasts. DR3 regulates death or differentiation in other tissues, and DR3 ligands occur in bone, but the function of DR3 in the osteoblast was unknown. We studied the expression of DR3 and the effects crosslinking antibodies to DR3 or of natural DR3 ligands in human osteoblasts. Western analysis showed that nontransformed osteoblasts and the MG63 osteosarcoma cell line produce both soluble decoy receptor and transmembrane isoforms of DR3. Cell surface labeling showed that low and high DR3-expressing osteoblast populations occur. Verification of by cloning showed a point mutation in DR3 from MG63 cells. Activation of DR3 by antibody crosslinking or with DR3 ligands caused apoptosis in osteoblasts and in MG63 cells, but only in low-density cell cultures. In dense cultures apoptosis did not occur, but nuclear factor-kappaB nuclear translocation was observed under some conditions. Crosslinking of DR3 in high-density MG63 cultures blocked expression of bone matrix elements. DR3 activation in high-density nontransformed osteoblasts had only minor effects on cell maturation. We conclude that DR3 activation can mediate apoptosis in osteoblasts. Its activity is, however, highly restricted by its soluble ligand-binding isoform and possibly also by alternate survival signals. In the presence of survival signals, DR3 may affect cell maturation although effects on differentiation were clearly seen only in the MG63 transformed cell line.

Animals↗
PubMed2006

Osteoclastic differentiation and function regulated by old and new pathways.

The osteoclast is a specialized multinucleated variant of the macrophage family. It degrades mineralized tissue, and is required for modeling and remodeling of bone. The osteoclast has long been known to require vitamin D for its differentiation and to be regulated by parathyroid hormone via circulating Ca(2+) levels. Two local signals important in osteoclast survival and differentiation, CSF-1 and RANKL, were characterized by the mid-1990 s. A basic framework of specialized cell attachment and resorption molecules was also clear by that time, including the alpha(v)beta(3) integrin, the key adhesion molecule of the mature osteoclast, the highly expressed vacuolar-type H(+)-ATPase that drives acid secretion to dissolve mineral, and cathepsin K, the predominant acid proteinase for collagenolysis. Recently, additional detail has been added to this framework, showing that the osteoclast has more complex regulation than was previously believed. These include the findings that one component of the V-H(+)-ATPase is unique to the osteoclast, that chloride transport and probably Cl(-)/H(+) exchange are also required for mineral degradation, and that additional receptors besides RANK and Fms regulate osteoclast formation and survival. Additional receptors include estrogen receptor-alpha, TNF-family receptors other than RANK, and, at least in some cases, glycoprotein hormone receptors including the TSH-R and the FSH-R. Challenges in understanding osteoclast biology include how the signalling mechanisms function cooperatively. Recent findings suggest that there is a network of cytoplasmic adapters, including Gab-2 and BCAR1, which are modified by multiple signalling mechanisms and which serve to integrate the signalling pathways.

Animals↗
PubMed2006

Comparison of fetal and adult marrow stromal cells in osteogenesis with and without glucocorticoids.

To better understand the potential use of fetal marrow stromal cells (MSCs) in bone tissue engineering, we compared the ability of these cells with those of adult MSCs with respect to osteoblasts differentiation in the presence or absence of glucocorticoids. Cells were grown for 3-4 weeks in basal medium or supplemented with 100 nM dexamethasone (DEX, a synthetic glucocorticoid analog) or with 50 microM L-ascorbate and 10 mM glycerol-2-phosphate (AS+GP) or with AS+GP+DEX. At various time points in culture, the following parameters were compared between fetal and adult MSCs: cell morphology, cell proliferation, alkaline phosphatase activity, calcium (45Ca) uptake, von Kossa staining, and glucocorticoids receptor expression were analyzed. Compared with adult MSCs, fetal cells showed a less dramatic change to cuboidal morphology in DEX-containing media. Fetal MSCs in all media conditions showed higher proliferation rates and lower alkaline phosphatase activities (p < 0.001) than adult cells. Both fetal and adult MSCs responded similarly in DEX-containing media with respect to suppressing cell proliferation, stimulating alkaline phosphatase activity, and consistently accumulating calcium (usually higher in fetal cells) with subsequent formation of mineralized matrix when compared with cells cultured in AS+GP. Our findings further implicate the requirement of glucocorticoids in osteogenesis. In conclusion, compared with adult MSCs, fetal cells showed greater ability in sustaining cell proliferation and calcium uptake suggesting that they may be useful for bone tissue repair.

Adolescent↗
PubMed2006

Bone cell precursors and the pathophysiology of bone loss.

In health, changes in bone formation and degradation rates are coupled and adequate cellular resources are available in the bone so that a change in bone formation rate occurs with an opposing change in resorption. On the other hand, the regulation of bone volume, particularly in pathological conditions, is dependent not only on the pathways that mediate terminal pathways of bone cell differentiation, but also on the availability of stem cells for allowing the differentiation to occur. Regulation of cell numbers in stem cell compartments and release of stem cells for differentiation of osteoblast or osteoclast precursors are not well understood, although it is clear that changes in stem cell numbers underlie pathological changes in bone mass. This may include effects of aging, fracture, metastatic disease, and autoimmune diseases on the precursor cell pools available for bone formation and degradation. Increases in osteoclast precursors or decreases in osteoblast precursors are common features of bone-losing states; increases in precursors may conversely occur during growth or repair processes. Rational therapy based on modifying stem cell populations may, when the processes are better understood, help prevent chronic bone-losing states and may also be of use in preventing or treating aplastic anemia and related conditions.

Bone Density↗
PubMed2006

TSH and bone loss.

We have recently challenged the view that the bone loss associated with hyperthyroidism is solely due to elevated thyroid hormone levels. We find that thyroid-stimulating hormone (TSH), derived from the anterior pituitary gland, inhibits bone resorption by the osteoclast. Mice haploinsufficient in the TSH receptor show reduced bone density and evidence of enhanced bone resorption in the face of normal thyroid function. In humans, TSH inhibits markers of bone resorption with a single administration, and low TSH levels correlate with increased fracture risk. The evidence that low TSH levels predispose to osteoporosis in hyperthyroidism is discussed in view of the emerging role of pituitary hormones in bone biology.

Animals↗
PubMed2006

Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.

UNLABELLED: We identified a previously unknown integrin, alpha(9)beta(1), on OCLs and their precursors. Antibody to alpha(9) inhibited OCL formation in human marrow cultures, and OCLs from alpha(9) knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity. INTRODUCTION: Integrins play important roles in osteoclast (OCL) formation and function. Mature OCLs mainly express alpha(v)beta(3) integrin, a heterodimer adhesion receptor that has been implicated in osteoclastic bone resorption. We identified ADAM8, a disintegrin and metalloproteinase, as a novel stimulator of OCL differentiation and showed that the disintegrin domain of ADAM8 mediated its effects on OCL formation. Because the disintegrin domain of ADAM8 does not bind Arg-Gly-Asp (RGD) sequences, we determined which integrin bound ADAM8 and characterized its role in OCL formation and activity. MATERIALS AND METHODS: Chinese hamster ovary cells (CHO) expressing different integrin subunits were tested for their capacity to bind the disintegrin domain of ADAM8. Mouse or human bone marrow cells and purified OCL precursors were tested for alpha(9)beta(1) integrin expression by Western blot, immunocytochemistry, and real-time RT-PCR. A monoclonal antibody to human alpha(9) was used to block alpha(9)beta(1) on OCL precursors stimulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or RANKL. Vertebrae of 7-day-old alpha(9)(-/-) mice and wildtype (WT) littermates were compared using bone histomorphometry and 3D microCT analysis. RESULTS: Alpha(9) integrin was expressed by mouse and human bone marrow-derived OCLs and their precursors. Importantly, the anti-alpha(9) antibody inhibited human OCL formation stimulated by 1alpha,25(OH)(2)D(3) or RANKL dose-dependently. Furthermore, analysis of OCLs formed in marrow cultures from alpha(9)(-/-) mice showed that the OCLs formed were more contracted and formed significantly less bone resorption pits on dentin slices. Histologic analysis of alpha(9)(-/-) vertebrae showed thickened trabecular regions and retained cartilage within vertebral bodies of alpha(9)(-/-) mice. 3D microCT analysis of alpha(9)(-/-) vertebrae also showed a significant increase in trabecular bone volume/total tissue volume and a tendency for decreased trabecular separation compared with WT mice. CONCLUSIONS: These results support a previously unknown role for alpha(9)beta(1) integrin in OCL formation and function.

ADAM Proteins↗
PubMed2005

NO-dependent osteoclast motility: reliance on cGMP-dependent protein kinase I and VASP.

The osteoclast degrades bone in cycles; between cycles, the cell is motile. Resorption occurs by acid transport into an extracellular compartment defined by an alphavbeta3 integrin ring. NO has been implicated in the regulation of bone turnover due to stretch or via estrogen signals, but a specific mechanism linking NO to osteoclastic activity has not been described. NO stimulates osteoclast motility, and at high concentrations NO causes detachment and terminates resorption. Here we demonstrate that NO regulates attachment through the cGMP-dependent protein kinase I (PKG I) via phosphorylation of the intermediate protein VASP. VASP colocalized with the alphavbeta3 ring in stationary cells, but alternating bands of VASP and alphavbeta3 occurred when motility was induced by NO donors or cGMP. Redistribution of VASP correlated with its phosphorylation. Dependency of NO-induced motility on PKG I and on VASP was shown by siRNA knockdown of each protein. VASP knockdown also altered distribution of alphavbeta3 at the attachment site. We conclude that PKG I and VASP are essential for reorganization of attachment and cytoplasmic proteins in motility induced by NO or by cGMP.

Cell Adhesion Molecules↗
PubMed2005

Calcineurin regulates bone formation by the osteoblast.

Two of the most commonly used immunosuppressants, cyclosporine A and tacrolimus (FK506), inhibit the activity of a ubiquitously expressed Ca(2+)/calmodulin-sensitive phosphatase, calcineurin. Because both drugs also cause profound bone loss in humans and in animal models, we explored whether calcineurin played a role in regulating skeletal remodeling. We found that osteoblasts contained mRNA and protein for all isoforms of calcineurin A and B. TAT-assisted transduction of fusion protein TAT-calcineurin Aalpha into osteoblasts resulted in the enhanced expression of the osteoblast differentiation markers Runx-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. This expression was associated with a dramatic enhancement of bone formation in intact calvarial cultures. Calcineurin Aalpha(-/-) mice displayed severe osteoporosis, markedly reduced mineral apposition rates, and attenuated colony formation in 10-day ex vivo stromal cell cultures. The latter was associated with significant reductions in Runx2, bone sialoprotein, and osteocalcin expression, paralleled by similar decreases in response to FK506. Together, the gain- and loss-of-function experiments indicate that calcineurin regulates bone formation through an effect on osteoblast differentiation.

Animals↗
PubMed2005

CHDL: a cadherin-like domain in Proteobacteria and Cyanobacteria.

We identified a cadherin-like domain (CHDL) using computational analysis. The CHDL domain is mostly distributed in Proteobacteria and Cyanobacteria, although it is also found in some eukaryotic proteins. Prediction of three-dimensional protein folding indicated that the CHDL domain has an immunoglobulin beta-sandwich fold and belongs to the cadherin superfamily. The CHDL domain does not have LDRE and DxNDN motifs, which are conserved in the cadherin domain, but has three other motifs: PxAxxD, DxDxD and YT-V/I-S/T-D, which might contribute to forming a calcium-binding site. The identification of this cadherin-like domain indicates that the cadherin superfamily may exhibit wider sequence and structural diversity than previously appreciated. Domain architecture analysis revealed that the CHDL domain is also associated with other adhesion domains as well as enzyme domains. Based on computational analysis and previous experimental data, we predict that the CHDL domain has calcium-binding and also carbohydrate-binding activity.

Amino Acid Motifs↗
PubMed2005

Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.

Autosomal recessive osteopetrosis (ARO) is a paradigm for genetic diseases that cause severe, often irreversible, defects before birth. In ARO, osteoclasts cannot remove mineralized cartilage, bone marrow is severely reduced, and bone cannot be remodeled for growth. More than 50% of the patients show defects in the osteoclastic vacuolar-proton-pump subunit, ATP6a3. We treated ATP6a3-deficient mice by in utero heterologous hematopoietic stem cell (HSC) transplant from outbred GFP transgenic mice. Dramatic phenotype rescue by GFP osteoclasts was obtained with engraftment, which was observed in most cases. Engraftment survived for variable periods. Recipients were not immunosuppressed, and graft-versus-host disease was not observed in all pups born after in utero treatment. Thus, differentiation of unmatched HSC transplanted in utero is sufficient to prevent fatal defects in ARO and may prevent complications of ARO unresponsive to conventional bone marrow transplantation. The presence of defective cells is not a barrier to the rescue of the phenotype by donor HSC.

Animals↗
PubMed2005

Epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMMSCs) are pluripotent cells capable of differentiating into several cell types and are thus an attractive cell source for connective tissue engineering. A challenge in such a use is expansion and directed seeding in vitro, requiring proliferation and survival, and directed migration, respectively, prior to functional differentiation. The epidermal growth factor (EGF) receptor (EGFR) is the prototypal growth factor receptor and elicits these responses from a wide variety of stromal, epithelial, and endothelial cells. Ligands for this receptor are appealing for use in tissue engineering because they are relatively resistant to biological extremes and amenable to high-volume production. Therefore, we determined whether an EGFR ligand, EGF, could be used for ex vivo expansion of BMMSCs. EGF stimulated motility in rat and immortalized human BMMSCs. EGF-induced proliferation was observed in immortalized human BMMSCs but was not apparent in rat BMMSCs under our experimental conditions. EGF did not, however, rescue either type of BMMSC from apoptosis due to lack of serum. During our examination of key signaling intermediaries, EGF caused robust phosphorylation of extracellular signal-regulated protein kinase (ERK) and protein kinase B/akt (AKT) but only minimal phosphorylation of EGFR and phospholipase C-gamma in rat BMMSCs, whereas in the human BMMSCs these intermediaries were all strongly activated. EGF also induced robust ERK activation in primary porcine mesenchymal stem cells. EGF pretreatment or cotreatment did not interfere with secondarily induced differentiation of either type of BMMSC into adipogenic or osteogenic lineages. Platelet-derived growth factor (PDGF) effects were similar to but not additive with those elicited by EGF, with some quantitative differences; however, PDGF did interfere with the differentiation of these BMMSCs. These findings suggest that EGFR ligands could be used for ex vivo expansion and direction of BMMSCs.

Animals↗
PubMed2005

Osteoclast signalling pathways.

The osteoclast is a monocyte-derived cell with complex regulatory control due to its role, balancing calcium homeostasis with skeletal modelling and repair. Normal differentiation requires tyrosine kinase- and tumor necrosis-family receptors, normally fms and RANK. Ligands for these receptors plus unidentified serum or cell-presented factor(s) are needed for in vitro differentiation, possibly signalling via an immune-like tyrosine kinase acceptor molecule. Osteoclast development and activity are increased by cytokines signalling through GP130, such as IL-6, by TGF-beta, and by IL-1, although these cannot replace serum. Other tyrosine kinase receptors including kit and met can augment fms signalling, and TNFs other than RANKL, including TNFalpha and TRAIL, modify RANK signalling, which is also susceptible to interference by interferons. The situation is further complicated by G-protein coupled receptors including the calcitonin receptor, by integrin or calcium-mediated signals, and by estrogen receptors, which operate in bone largely via NO downstream signals. Differentiation, activity, and survival signals merge in intracellular second messengers. These include cytoplasmic kinases of several families; differentiation pathways often terminate in Erk/Jun kinases or NF-kappaB. Key regulatory intermediates include TRAF6, src, Smad3, phosphatidylinositol-3-kinase, Jak/Stat, and the cGMP-dependent protein kinase I. There are substantial uncertainties regarding how intracellular agents connect to primary signals. The frontier includes characterization of how scaffolding/adapter proteins, such as cbl, gab, grb, p130Cas, and shc, as well as itam-containing proteins and nonreceptor tyrosine kinase adapters of the src and syk families, delimit and integrate signals of multiple receptors to bring about specific outcomes.

Animals↗
PubMed2005

Comparative modeling of TNFRSF25 (DR3) predicts receptor destabilization by a mutation linked to rheumatoid arthritis.

We constructed a three-dimensional model of TNFRSF25 (death receptor-3; DR3), a tumor necrosis-receptor family member that is expressed on immune cells and on osteoblasts, to determine whether mutations that are linked to rheumatoid arthritis are likely to have effects on receptor function. Since the crystal structure of DR3 is not known, comparative modeling was used, aligning structural elements of the primary sequences of DR3 with TNFs which have been determined by crystallography, substituting the amino acids of the target protein for those in the known structure, introducing necessary deletions or insertions, followed by energy minimization to yield a putative structure. This approach has been validated by studies of other TNF-family receptors. The results show that the DR3 extracellular domain is comprised of four homologous cysteine-rich domains (CRDs), and that a mutation linked to rheumatoid arthritis is in a region critical for structural integrity of ligand-receptor complexes at the end of CRD3. Specifically, the D158G mutation eliminates two hydrogen bonds normally present in a N/D-T-V/D-C consensus motif typically found flanking the last cysteine of each CRD. This may cause aberrations in either T cell function or in response of bone cells to DR3 ligands, which may contribute to pathology in rheumatoid arthritis. Comparison of RA mutants to mutants in other TNFRSF receptors shows that these occur in homologous positions in CRDs, so that this site is proposed to be a 'hot spot' for mutations in TNFRSF family proteins.

Amino Acid Sequence↗
PubMed2005

Negative regulation of RANKL-induced osteoclastic differentiation in RAW264.7 Cells by estrogen and phytoestrogens.

We studied estrogen effects on osteoclastic differentiation using RAW264.7, a murine monocytic cell line. Differentiation, in response to RANKL and colony-stimulating factor 1, was evaluated while varying estrogen receptor (ER) stimulation by estradiol or nonsteroidal ER agonists was performed. The RAW264.7 cells were found to express ERalpha but not ERbeta. In contrast to RANKL, which decreased ERalpha expression and induced osteoclast differentiation, 10 nm estradiol, 3 microm genistein, or 3 microm daidzein all increased ERalpha expression, stimulated cell proliferation, and decreased multinucleation, with the effects of estrogen > or = daidzein > genistein. However, no estrogen agonist reduced RANKL stimulation of osteoclast differentiation markers or its down-regulation of ERalpha expression by more than approximately 50%. Genistein is also an Src kinase antagonist in vitro, but it did not decrease Src phosphorylation in RAW264.7 cells relative to other estrogen agonists. However, both phytoestrogens and estrogen inhibited RANKL-induced IkappaB degradation and NF-kappaB nuclear localization with the same relative potency as seen in proliferation and differentiation assays. This study demonstrates, for the first time, the direct effects of estrogen on osteoclast precursor differentiation and shows that, in addition to effecting osteoblasts, estrogen may protect bone by reducing osteoclast production. Genistein, which activates ERs selectively, inhibited osteoclastogenesis less effectively than the nonselective phytoestrogen daidzein, which effectively reproduced effects of estrogen.

Animals↗