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P Osdoby

Publications and source records attributed to P Osdoby.

At least 19 recordsLinked to original sources
PubMed2001

Receptor activator of NF-kappa B and osteoprotegerin expression by human microvascular endothelial cells, regulation by inflammatory cytokines, and role in human osteoclastogenesis.

The receptor activator of NF-kappaB (RANKL) is the essential signal required for full osteoclast (OC) development, activation, and survival. RANKL is highly expressed in areas of trabecular bone remodeling and inflammatory bone loss, is increased on marrow stromal cells or osteoblasts by osteotropic hormones or cytokines, and is neutralized by osteoprotegerin (OPG), a soluble decoy receptor also crucial for preventing arterial calcification. Vascular endothelial cells (VEC) are critically involved in bone development and remodeling and influence OC recruitment, formation, and activity. Although OCs develop and function in close association with bone VEC and sinusoids, signals mediating their interactions are not well known. Here, we show for the first time that human microvascular endothelial cells (HMVEC) express transcripts for both RANKL and OPG; inflammatory cytokines tumor necrosis factor-alpha and interleukin-1alpha elevate RANKL and OPG expression 5-40-fold in HMVEC (with an early OPG peak that declines as RANKL rises), and RANKL protein increases on the surface of tumor necrosis factor-alpha-activated HMVEC. Cytokine-activated HMVEC promoted the formation, fusion, and bone resorption of OCs formed in co-cultures with circulating human monocytic precursors via a RANKL-mediated mechanism fully antagonized by exogenous OPG. Furthermore, paraffin sections of human osteoporotic fractured bone exhibited increased RANKL immunostaining in vivo on VEC located near resorbing OCs in regions undergoing active bone turnover. Therefore, cytokine-activated VEC may contribute to inflammatory-mediated bone loss via regulated production of RANKL and OPG. VEC-derived OPG may also serve as an autocrine signal to inhibit blood vessel calcification.

Base Sequence↗
PubMed2000

Decreased nitric oxide levels stimulate osteoclastogenesis and bone resorption both in vitro and in vivo on the chick chorioallantoic membrane in association with neoangiogenesis.

High nitric oxide (NO) levels inhibit osteoclast (OC)-mediated bone resorption in vivo and in vitro, and nitrate donors protect against estrogen-deficient bone loss in postmenopausal women. Conversely, decreased NO production potentiates OC bone resorption in vitro and is associated with in vivo bone loss in rats and humans. Previously, we reported that bone sections from rats administered aminoguanidine (AG), a selective inhibitor of NO production via inducible NO synthase, exhibited both increased OC resorptive activity as well as greater numbers of OC. Here, we investigated further whether AG promoted osteoclastogenesis, in addition to stimulating mature OC function, using a modified in vivo chick chorioallantoic membrane (CAM) system and an in vitro chick bone marrow OC-like cell developmental model. AG, focally administered in small agarose plugs placed directly adjacent to a bone chip implanted on the CAM, dose-dependently elicited neoangiogenesis while stimulating the number, size, and bone pit resorptive activity of individual OC ectopically formed in vivo. In addition to enhancing OC precursor recruitment via neoangiogenesis, AG also exerted other vascular-independent effects on osteoclastogenesis. Thus, AG promoted the in vitro fusion and formation from bone marrow precursor cells of larger OC-like cells that contained more nuclei per cell and exhibited multiple OC differentiation markers. AG stimulated development was inversely correlated with declining medium nitrite levels. In contrast, three different NO donors each dose-dependently inhibited in vitro OC-like cell development while raising medium nitrite levels. Therefore, NO sensitively regulates OC-mediated bone resorption through affecting OC recruitment (angiogenesis), formation (fusion and differentiation), and bone resorptive activity in vitro and in vivo. Possibly, the stimulation of neoangiogenesis and OC-mediated bone remodeling via AG or other pro-angiogenic agents may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis.

Acid Phosphatase↗
PubMed1999

Estrogen's bone-protective effects may involve differential IL-1 receptor regulation in human osteoclast-like cells.

Declining estrogen levels during the first postmenopausal decade lead to rapid bone loss and increased fracture risk that can be reversed by estrogen replacement therapy. The bone-protective effects of estrogen may involve suppression of inflammatory cytokines that promote osteoclastogenesis and bone resorption, such as IL-1, TNF-alpha, and IL-6. We investigated whether estrogen modulates IL-1 actions on human osteoclasts (OCs) and other bone cell types. Isolated human OCs and primary bone marrow-derived OC-like cells expressed both the signaling (IL-1RI) and decoy (IL-1RII) IL-1 receptors, whereas only IL-1RI was detected in osteoblasts. IL-1RII/IL-1RI mRNA ratios and release of soluble IL-1RII (sIL-1RII) were lower in OC-like cells derived from women in the late postmenopausal period compared with younger women, but were unrelated to male donor age, suggesting that estrogen might play a role in regulating IL-1 receptor levels in vivo. Estrogen directly reduced in vitro OC-like cell IL-1RI mRNA levels while increasing IL-1RII mRNA levels and sIL-1RII release. These estrogenic events were associated with inhibited IL-1-mediated cytokine (IL-8) mRNA induction and cell survival, i.e., increased apoptosis. In contrast, estrogen did not alter IL-1R levels or IL-1 responsiveness in primary human osteoblasts or bone marrow stromal cells. We conclude that one novel mechanism by which estrogen exerts bone-protective effects may include a selective modulation of IL-1R isoform levels in OC or OC-like cells, thereby reducing their IL-1 responsiveness and cell survival. Conversely, this restraint on IL-1 actions may be lost as estrogen levels decline in aging women, contributing to an enhanced OC-mediated postmenopausal bone loss.

Aged↗
PubMed1998

Human osteoclasts and osteoclast-like cells synthesize and release high basal and inflammatory stimulated levels of the potent chemokine interleukin-8.

Chemokines, including interleukin-8 (IL-8), function as key mediators in diverse inflammatory disorders via promoting the recruitment, proliferation, and activation of vascular and immune cells. IL-8 levels are elevated in inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, osteomyelitis, and periodontal disease, that also exhibit progressive bone loss. Therefore, it is possible that IL-8 contributes to the osteopenia associated with these pathological conditions. Although macrophages, neutrophils, and endothelial cells are considered the primary sources of inflammation-induced IL-8 increases, we report here for the first time that human bone marrow-derived osteoclast-like cells (hOCL) as well as authentic bone-resorbing human osteoclasts (hOC) isolated from osteoporotic femoral heads express messenger RNA (mRNA) for IL-8 and secrete high levels of IL-8 during culture. Basal IL-8 release by cultured hOC or hOCL was orders of magnitude greater than the release of the proinflammatory cytokines IL-1beta, IL-6, and tumor necrosis factor-alpha. At a cellular level, in situ hybridization analysis revealed that IL-8 mRNA was expressed in resorbing hOC of rheumatoid arthritic pannus and was substantially greater than that expressed in hOC of noninflammatory giant cell tumor of bone tissue. Therefore, the potential inflammation-mediated induction of IL-8 was directly assessed using cultured hOCL. IL-8 release was stimulated by proinflammatory signals (IL-1alpha, tumor necrosis factor-alpha, lipopolysaccharide, or phorbol 12-myristate 13-acetate), unaffected by various other osteotropic modulators (transforming growth factor-beta1 and -beta3, IL-6, 17beta-estradiol, or calcitonin) and was decreased by interferon-gamma, vitamin D3, and the antiinflammatory glucocorticoid dexamethasone. Changes in IL-8 secretion were paralleled by corresponding changes in IL-8 mRNA steady state levels. We conclude that hOC and hOCL synthesize and secrete high constitutive and inflammation-stimulated levels of the chemokine IL-8. Consequently, hOC-derived IL-8 could act as an important regulatory signal for bone, vascular, and immune cell recruitment and activation during normal and pathological bone remodeling.

Arthritis, Rheumatoid↗
PubMed1998

Inhibition of avian osteoclast bone resorption by monoclonal antibody 121F: a mechanism involving the osteoclast free radical system.

Osteoclasts generate high levels of superoxide anions during bone resorption that contribute to the degradative process, although excessive levels of this free radical may be damaging. One mechanism for their removal is via superoxide dismutase (SOD), a protective superoxide scavenging enzyme. We have previously described a novel developmentally regulated 150 kDa plasma membrane glycoprotein of avian osteoclasts which is reactive with the osteoclast-specific monoclonal antibody (Mab) 121F and is related immunologically, biochemically, and in protein sequence to mitochondrial Mn2+/Fe2+ SOD. We hypothesized that this unusual osteoclast surface component may be involved in protection against superoxides generated during active bone resorption. Increasing concentrations of monovalent Fab fragments prepared from Mab 121F, but not those from another antiosteoclast Mab designated 29C, markedly inhibited both bone particle and bone pit resorption by avian osteoclasts, while reducing tartrate-resistant acid phosphatase activity and causing the morphological contraction of osteoclasts on bone. Thus, the SOD-related membrane antigen may be essential for osteoclast bone resorption. Osteoclast superoxide production, monitored kinetically by cytochrome c reduction and histochemically by nitroblue tetrazolium reduction staining, was significantly greater in the presence of 121F, but not 29C, Fab treatment. Furthermore, the release of another free radical known as nitric oxide, which is produced by osteoclasts, can scavenge superoxides, and acts to potently inhibit osteoclast bone resorption, was dose-dependently increased by 121F Fab in resorbing osteoclast cultures. Therefore, Mab 121F binding may block the potential protective function of the osteoclast plasma membrane SOD-related glycoprotein, leading to a rapid elevation of superoxide levels and a subsequent rise in osteoclast nitric oxide release, feedback messages which may be sensed by the osteoclast as signals to cease active bone resorption.

Acid Phosphatase↗
PubMed1997

A human homolog of the 150 kD avian osteoclast membrane antigen related to superoxide dismutase and essential for bone resorption is induced by developmental agents and opposed by estrogen in FLG 29.1 cells.

Osteoclast development from hematopoietic bone marrow precursors is associated with the expression of various enzymes, receptors, adhesion molecules, and other specialized components. Among these is a novel 150 kD superoxide dismutase-related membrane glycoprotein, originally identified by its reaction with the anti-osteoclast monoclonal antibody 121F. This antigen is uniquely restricted to osteoclasts in bone, universally present on osteoclasts from multiple species, induced during osteoclast differentiation in vitro and in ovo, and required at high levels for avian osteoclastic bone pit resorption. Expression of a comparable human antigen was investigated using human leukemic FLG 29.1 cells capable of differentiating towards an osteoclast-like phenotype. Phorbol ester, 1,25 (OH)2 vitamin D3, and osteoblast-derived soluble factors elicited dose and time-dependent inductions of this antigen as measured by enzyme-linked immunosorbent assay (ELISA) and immunocytochemical staining, coincident with their display of multiple other osteoclastic features. Synergistic interactions of these modulators led to further elevations in the ultimate expression levels of this antigen, although not to the full extent associated with in vivo-formed avian osteoclasts. The potent antiresorptive hormone 17beta-estradiol, but not its inactive alpha isomer, partially suppressed the phorbol ester-induced elevation of the 121F antibody-reactive antigen in FLG 29.1 cells as it does in avian osteoclast-like cells. Characterization of the human antigen isolated from FLG 29.1 cells by 121F immunoaffinity purification demonstrated that this regulated membrane component was synthesized by these human cells, more abundant following their differentiation into osteoclast-like cells, and similar biochemically and immunologically to the 150 kD integral membrane glycoprotein previously described from avian osteoclasts. Therefore, this report is the first documentation that human osteoclast-like FLG 29.1 cells express, in a developmentally regulated fashion, a homolog of the specific 150 kD avian osteoclast surface antigen that is related to superoxide dismutase, a protective free radical scavenging enzyme and is essential for osteoclastic bone resorption.

Acid Phosphatase↗
PubMed1997

Ca2+ or phorbol ester but not inflammatory stimuli elevate inducible nitric oxide synthase messenger ribonucleic acid and nitric oxide (NO) release in avian osteoclasts: autocrine NO mediates Ca2+-inhibited bone resorption.

Osteoclast bone resorption is essential for normal calcium homeostasis and is therefore tightly controlled by calciotropic hormones and local modulatory cytokines and factors. Among these is nitric oxide (NO), a multifunctional free radical that potently inhibits osteoclast bone resorption in vitro and in vivo. Previous findings led us to propose that NO might serve as an autocrine, as well as paracrine, regulator of osteoclast function. This premise was investigated using isolated bone-resorptive avian osteoclasts and focusing on the inducible isoform of NO synthase (iNOS) responsible for inflammatory stimulated high-level NO synthesis in other cells. Avian osteoclasts expressed both iNOS messenger RNA (mRNA) and protein. However, inflammatory cytokines that induce iNOS mRNA, protein, and NO in other cells did not do so in avian osteoclasts, consistent with the known role of inflammatory stimuli in promoting osteoclast resorption and localized bone loss. In searching for potential modulators of osteoclast iNOS, protein kinase C activation [by phorbol 12-myristate 13-acetate (PMA)] and intracellular Ca2+ rises (A23187) were each found to elevate osteoclast iNOS mRNA and protein levels, while increasing NO release and reducing osteoclast bone resorption. The iNOS selective inhibitor aminoguanidine suppressed stimulated osteoclast NO production elicited by either signal, but reversed only the resorption inhibition due to raised Ca2+. Thus, whereas additional inhibitory signals are presumably coproduced in osteoclasts treated with PMA, osteoclast iNOS-derived NO may act as an autocrine signal to mediate Ca2+-inhibited bone resorption. These findings document for the first time an iNOS whose mRNA levels are regulated by Ca2+ or PMA, but not inflammatory stimuli, and the autocrine production of NO as a Ca2+ sensing signal to suppress osteoclast bone resorption. The unusual regulation of osteoclast iNOS makes it a potentially attractive target for designing novel therapeutic agents to alleviate excessive bone loss.

Animals↗
PubMed1996

Proinflammatory agents, IL-8 and IL-10, upregulate inducible nitric oxide synthase expression and nitric oxide production in avian osteoclast-like cells.

Nitric oxide synthase (NOS) isoenzymes generate nitric oxide (NO), a sensitive multifunctional intercellular signal molecule. High NO levels are produced by an inducible NOS (iNOS) in activated macrophages in response to proinflammatory agents, many of which also regulate local bone metabolism. NO is a potent inhibitor of osteoclast bone resorption, whereas inhibitors of NOS promote bone resorption both in vitro and in vivo. The possibility that osteoclasts, like macrophages, express a regulated iNOS and produce NO as a potential autocrine signal following inflammatory stimulation was investigated in well-characterized avian marrow-derived osteoclast-like cells. NO production (reflected by medium nitrite levels) was markedly elevated in these cells by the proinflammatory agents lipopolysaccharide (LPS) and the synergistic action of IL-1 alpha, TNF alpha, and IFN gama. inhibitors of NOS activity (aminoguanidine, L-NAME) or iNOS induction (dexamethasone, TGF beta) reduced LPS-stimulated nitrite production. LPS also increased the NOS-associated diaphorase activity of these cells and their reactivity with anti-iNOS antibodies. RT-PCR cloning, using avian osteoclast-like cell RNA and human iNOS primers, yielded a novel 900 bp cDNA with high sequence homology (76%) to human, rat, and mouse iNOS genes. In probing osteoclast-like cell RNA with the PCR-derived iNOS cDNA, a 4.8 kb mRNA species was detected whose levels were greatly increased by LPS. Induction of iNOS mRNA by LPS, or by proinflammatory cytokines, occurred prior to the rise of medium nitrite in time course studies and was diminished by dexamethasone. Moreover, osteoclast-like cells demonstrated an upregulation of NO production and iNOS mRNA by IL-8 and IL-10, regulatory mechanism's not previously described. It is concluded that osteoclast-like cells express a novel iNOS that is upregulated by inflammatory mediators, leading to NO production. Therefore, NO may serve as both a paracrine and autocrine signal for modulating osteoclast bone resorption.

Animals↗
PubMed1995

Alternative splicing generates a second isoform of the catalytic A subunit of the vacuolar H(+)-ATPase.

We have identified a second isoform of the catalytic A subunit of the vacuolar H+ pump in chicken osteoclasts. In this isoform (A2) a 72-bp cassette replaces a 90-bp cassette present in the classical A1 isoform. The A1-specific cassette encodes a region of the protein that contains one of the three ATP-binding consensus sequences (the P-loop) identified in this polypeptide, as well as the pharmacologically relevant Cys254. In contrast, the A2-specific cassette does not contain any of these features. These two isoforms, which appear to be ubiquitously expressed, are encoded by a single gene and are generated by alternative splicing of two mutually exclusive exons. The alternative RNA processing involves the recognition of a single site, the boundary between the A2- and A1-specific exons, as either acceptor (in A1) or donor (in A2) splice site.

Adenosine Triphosphate↗
PubMed1995

Osteoclast 121F antigen expression during osteoblast conditioned medium induction of osteoclast-like cells in vitro: relationship to calcitonin responsiveness, tartrate resistant acid phosphatase levels, and bone resorptive activity.

Osteoclast differentiation from hematopoietic precursors into multinucleated cells uniquely capable of removing the organic and inorganic components of bone matrix occurs in multistep process, during which osteoclasts acquire the specialized characteristics necessary for bone resorptive activity and physiological regulation. Among those traits is a novel plasma membrane glycoprotein, reactive with the anti-osteoclast monoclonal antibody 121F, which is expressed during the course of osteoclast differentiation, shares structural and functional homologies with Mn2+/Fe2+ superoxide dismutase, and has been hypothesized to protect the osteoclast from the damaging effects of superoxide radicals generated during active bone resorption. We have reported previously that the expression of this membrane antigen is induced on multinucleated giant cells when the prefusion marrow mononuclear cells are cultured in conditioned medium from avian calvaria. The studies reported here were designed to investigate the relationship between expression of the 121F antibody-reactive osteoclast membrane antigen and tartrate resistant acid phosphatase levels, bone resorptive activity, calcitonin responsiveness, and ultrastructural features of avian bone marrow-derived multinucleated giant cells formed either in the presence or absence of diffusible osteoblast secreted factors. Parallel analyses of in vivo formed osteoclasts isolated from the same animals were performed for direct comparisons. In this report we demonstrate: (1) that the 121F monoclonal antibody-reactive osteoclast membrane antigen is stably induced in giant cells by soluble osteoblast-derived factors in a species nonrestricted but concentration- and temporal-dependent manner; (2) that osteoblast-mediated antigen induction is reflected in both increased numbers of cells and elevated expression of individual cells that are reactive with the 121F antibody, as determined by ELISA and histomorphometry; (3) that osteoblast conditioned medium, in addition to inducing this antigen in bone marrow cells, also elevates other defining osteoclast characteristics in these avian giant cells including their TRAP activity, cell retraction from the bone surface in response to calcitonin, bone resorptive function, and expression of a series of additional osteoclast antigenic markers; and (4) that secreted osteoblast products alone do not raise the levels of these traits for in vitro formed marrow giant cells to the extent associated with in vivo formed osteoclasts. Therefore, osteoblast soluble factors alone appear unable to promote the full differentiation of bone marrow cells in vitro into mature bone-resorbing osteoclasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗
PubMed1995

Bone cell function, regulation, and communication: a role for nitric oxide.

A large array of factors serve as vital communication links between cells and the characterization, regulation, and mechanisms of action of such factors are topics of intense research efforts. Most intercellular messenger molecules which have been described over the years are represented by proteins, small peptides, amino acids or their derivatives, ions, lipid metabolites, or steroids. However, a small uncharged free radical, nitric oxide, has recently garnered much attention as a potent multifunctional signal molecule with widespread actions within and between diverse tissues. Biochemical, molecular, and regulatory studies of the family of enzymes responsible for nitric oxide synthesis, nitric oxide synthases, have established that there are at least three distinct isoforms of this enzyme which are differentially expressed and regulated in various cells or tissues. Modulation of these isoenzyme levels or activities by diverse signals is mediated via transcriptional, translational, and/or post-translational mechanisms, and consequently, alterations in such control may influence normal or pathological processes. Nitric oxide appears to exert pronounced effects on skeletal physiology and its production by various bone cells, elicited target cell responses, modulation by other signalling molecules (e.g., cytokines, hormones, fatty acid derivatives), and chemical interactions with other free radicals (e.g., superoxide anions, hydroxyl radicals) may form one important facet of the many complicated communication pathways controlling bone cell physiology and remodeling. Further cell and molecular studies are needed to address the precise roles that nitric oxide plays in bone development and in the formation and degradation of bone during ordinary bone metabolism. In addition, alterations in the regulation and action of the bone nitric oxide system as a function of certain bone disorders may be manifested by perturbations in bone integrity or mineral homeostasis. In this article, we review the current evidence implicating nitric oxide as an important messenger molecule in bone intercellular communication, speculate on potential roles for this radical in bone biology, and discuss possible future directions for advanced research into the function of nitric oxide in skeletal physiology.

Amino Acid Oxidoreductases↗
PubMed1995

Enhanced expression of alpha v integrin subunit and osteopontin during differentiation of HL-60 cells along the monocytic pathway.

HL-60 cells, a promyelocytic leukemic cell line, provide a good model for studying the role of adhesion molecules and associated receptors involved in cell differentiation. When exposed to factors such as phorbol esters, these cells grown in suspension differentiate into monocytes and adhere to tissue culture dishes. In this study we showed that HL-60 cells exposed to phorbol esters express osteopontin (OPN), a cell adhesion molecule linked with osteoclast function. Moreover, the timed expression of OPN, in phorbol ester treated cells, was linked to increased cell adhesion. Subsequent to the expression of OPN, an increase in mRNA levels for alpha v integrin subunit was observed. The alpha v beta 3 integrin, a cell surface receptor found in high concentrations in osteoclasts, is considered to be a receptor for OPN. Furthermore, during differentiation we detected an increase in two cell surface markers specific for osteoclasts, 75B and 121F. This is the first report to demonstrate expression of OPN during differentiation of HL-60 cells, indicating that HL-60 cells can be used as a tool to enhance our understanding as to the role of OPN in cell differentiation.

Antigens, Surface↗
PubMed1994

Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase.

We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NO-generating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption.

Amino Acid Oxidoreductases↗
PubMed1993

Estrogen modulation of avian osteoclast lysosomal gene expression.

We have previously demonstrated that avian osteoclasts contain high levels of 17 beta-estradiol (17 beta E2) receptors and respond to 17 beta E2 treatment with a dose-dependent decrease in in vitro resorption of [3H] proline-labeled bone particles. To more accurately assess the influence of 17 beta E2 on osteoclastic activity, the specificity of estrogen modulation of resorption levels was determined using a quantitative pit resorption assay. Treatment with 17 beta E2 significantly decreased the number of osteoclast resorption pits formed compared with that after either vehicle or 17 alpha E2 treatment. Cotreatment with 17 beta E2 and hydroxytamoxifen (a complete 17 beta E2 antagonist in birds) abrogated the influence of 17 beta E2 on resorption activity. To elucidate the mechanism by which 17 beta E2 inhibits osteoclast activity, the effects of 17 beta E2 on the steady state mRNA levels of two avian osteoclast lysosomal proteins, lysozyme and a lysosomal membrane protein (LEP-100), were examined. Using highly purified avian osteoclasts, 17 beta E2 was shown to decrease lysosomal protein mRNA levels in a dose-dependent manner within 8 h of treatment in a process that required de novo protein synthesis. This response was specific for 17 beta E2, since the inactive stereoisomer 17 alpha E2 had no effect. Furthermore, coincubation of 17 beta E2 with hydroxytamoxifen eliminated the 17 beta E2 influence. After removal of 10(-8) M 17 beta E2, lysosomal gene mRNA levels returned to near-normal levels within 24 h. This is consistent with the previously reported ability of avian osteoclast-mediated resorption activity to recover from 17 beta E2 treatment. Lysozyme protein levels similarly decreased after 17 beta E2 treatment. These data suggest that avian osteoclasts are target cells for 17 beta E2 in vitro, that osteoclast activity in vivo is likely to be modulated by circulating levels of 17 beta E2, and that the 17 beta E2 inhibition of osteoclast resorption activity may be mediated at least in part via regulation of osteoclast lysosomal gene expression.

Animals↗
PubMed1992

Avian osteoblast conditioned media stimulate bone resorption by targeting multinucleating osteoclast precursors.

Osteoblasts are thought to secrete factors that regulate the rate of osteoclastic bone resorption. We studied the effect of osteoblast conditioned medium on bone degradation by multinucleated osteoclast-like cells generated in vitro from mononuclear precursors and found that the medium stimulates bone degradation primarily through interactions with osteoclast precursors. The conditioned medium also stimulates expression of the osteoclast-specific antigen 121F. The increased bone degradation, but not increased 121F expression, is due to the conditioned medium maintaining activity of the osteoclast precursors. Although the osteoclast precursors exhibit the DNA fragmentation characteristic of apoptosis, the osteoblast conditioned medium does not prevent such fragmentation. Chicken macrophage growth factor neither mimics nor augments the ability of the conditioned medium to stimulate bone degradation. Studies of osteoclast generation or function should carefully consider whether the effects are dependent on the viability of the resorbing cells.

Alkaline Phosphatase↗
PubMed1992

Osteoclast recruitment in response to human bone matrix is age related.

The effect of bone matrix age on the recruitment and differentiation of osteoclast precursors was studied using the chick chorioallantoic membrane (CAM) implant system. Devitalized mineralized bone particles (75-250 microns) were prepared from human femoral cortical bone obtained postmortem from 8 men (age range: 18-72 years). The particles were implanted onto the CAM and 8 days later implants were harvested and processed for light microscopic, morphometric or immunohistochemical analysis. Histomorphometric analysis was performed on samples representing each donor age. The analysis was grouped into three categories consisting of bone from young adults (18-20 years), adults (34-53 years) and aged individuals (67 years and older). Total osteoclast number, osteoclast number per bone particle, cell area, cell size, number of nuclei per cell profile, nucleocytoplasmic ratio, and the presence of a distinctive osteoclast antigen defined by monoclonal anti-body 121F were determined. Bone matrix from older individuals, and therefore the oldest age group (67 years and older), elicited significantly fewer multinucleated cells when compared to bone matrix from younger donors. The number of nuclei per cell profile was highest in the adult population (34-53 years), and there was a continuous increase in cell area with aging. As a consequence, the nucleocytoplasmic ratio decreased from the youngest to the oldest age group. These findings indicate that, relative to factors that affect the recruitment and differentiation of osteoclast precursor cells, bone matrix of older individuals is changed in quality and/or quantity compared to bone matrix from younger individuals. It is hypothesized that this decline in osteoclast formation in response to older bone matrix may contribute to the impaired bone remodeling associated with aging.

Adolescent↗
PubMed1991

Avian osteoclasts as estrogen target cells.

Although in vivo effects of 17 beta-estradiol (estrogen) on bone turnover have been shown to occur mainly through influences on osteoclast-mediated bone resorption, the mechanism by which estrogen reduces bone resorption is unclear. To approach this question, we have examined authentic osteoclasts for evidence of a direct osteoclast response to estrogen in vitro. Highly purified (greater than (90%) viable avian osteoclasts from birds maintained on a low calcium diet were obtained using an osteoclast-specific monoclonal antibody coupled to magnetic beads. Isolated cells were either analyzed directly for estrogen receptor (ER) levels or cultured to assess the biological effects of estrogen. Northern blot analysis revealed a 5.2-kilobase mRNA that hybridized with a cDNA to human ER mRNA in the osteoclasts. An anti-human ER antibody recognized proteins of 66 kDa and 140 kDa in osteoclast extracts by Western blot analysis. The 66-kDa size is in close agreement with the reported size of the human ER. Nuclear binding of estrogen to intact viable osteoclasts was steroid-specific and saturable, with 5662 +/- 1420 molecules bound per nucleus (mean +/- SEM). In vitro estrogen responses in osteoclasts included a dose-dependent decrease in resorption as well as an increase in nuclear protooncogene mRNA levels. These observations indicate that osteoclasts are capable of directly responding to estrogen in vivo.

Animals↗