F. P. Ross

Estrogen Deficiency Increases the Ability of Stromal Cells to Support Murine Osteoclastogenesis via an Interleukin-1and Tumor Necrosis Factor-mediated Stimulation of Macrophage Colony-stimulating Factor Production

To analyze how estrogen blocks osteoclastogenesis, we investigated the effects of ovariectomy on osteoclast (OC) formation in co-cultures of purified OC precursors and purified stromal cells (SC). OC formation was higher in co-cultures containing SC from ovariectomized mice than in those containing SC from sham-operated mice, thus suggesting that estrogen regulates osteoclastogenesis by targeting SC. Ovariectomy also increased the mononuclear cell secretion of interleukin (IL)-1) and tumor necrosis factor (TNF) and the SC production of macrophage colony-stimulating factor (MCSF). Osteoclastogenesis and SC production of M-CSF were not blocked by in vitro estrogen treatment but were decreased by in vivo treatment of donor mice with either estrogen or a combination of the IL-1 inhibitor, IL-1 receptor antagonist, and the TNF inhibitor, TNF binding protein. IL-1 and TNF production were also blocked by in vivo estrogen treatment, demonstrating that the increased bone marrow levels of IL-1 and TNF characteristic of ovariectomized mice induce the formation of a SC population characterized by a high production of M-CSF and increased pro-osteoclastogenic activity. Since in co-cultures of SC and OC precursors M-CSF levels correlated with OC production (r = 0.7, p < 0.0001), the data also indicate that the pro-osteoclastogenic activity of SC is proportional to their secretion of M-CSF. The ability of estrogen to decrease SC production of M-CSF and the pro-osteoclastogenic activity of these cells by regulating IL-1 and TNF production is a previously undescribed mechanism by which estrogen down-regulates osteoclastogenesis.

Tumor Necrosis Factor-α Activation of Nuclear Transcription Factor-κB in Marrow Macrophages Is Mediated by c-Src Tyrosine Phosphorylation of IκBα

Tumor necrosis factor-α (TNF) exerts its transcriptional effects via activation of nuclear transcription factor-κ B (NF-κB). NF-κB is sequestered in the cytosol by IκBα and, in most cells, released upon serine phosphorylation of this inhibitory protein which then undergoes rapid, ubiquitin-dependent degradation. In contrast, we find TNF induction of NF-κB in murine bone marrow macrophages (BMMs), is mediated, by c-Src, in a cell, and cytokine specific manner. The non-receptor tyrosine kinase is rapidly mobilized and activated upon TNF exposure. Within the same time frame, TNF induced c-Src associates with IκBα in a long lived complex. The proto-oncogene, when associated with IκBα phosphorylates the inhibitory protein on tyrosine 42. Consistent with the pivotal role played by c-Src in TNF-induced IκBα tyrosine phosphorylation, NF-κB activation, by the cytokine, is markedly delayed and reduced in c-src−/− BMMs. Underscoring the physiological significance of c-Src activation of NF-κB, TNF induction of IL-6, which is an NF-κB mediated event, is substantially diminished in c-src−/− BMMs.

Enrichment of generated murine osteoclasts

Biochemical and molecular studies of osteoclasts generally require cells in a reasonable degree of purity. The chicken has been extremely useful in this regard, as abundant avian osteoclasts can be generated in vitro entirely from pure populations of marrow macrophage precursors. Propagation of murine osteoclasts is, in contrast, far less efficients, demanding the presence of stromal cells. The aims of this study were to develop a method by which murine osteoclasts generated in culture, can be effectively enriched while maintaining viability and, to explore the mechanisms by which stromal cells promote murine osteoclast generation and survival. We find that 106 fractionated murine marrow cells enriched, for marrow-residing colony-forming units (CFU-cs), yield 3000–4000 tartrate-resistant acid phosphatase (TRAP)-expressing multinucleated giant cells when cultured for 12 days with ST-2 stromal cells. These cells are osteoclasts as evidenced by their ability to “pit” bone slices, resorb radiolabeled bone particles, and generate cyclic AMP in response to calcitonin. Treatment of these generated osteoclast cultures with bacterial collagenase for 2 hours at 37° selectively removes virtually all ST-2 cells, yielding a >60% pure population of TRAP and calcitonin receptor-expressing cells, 90% of which are viable. These cells continue to respond to calcitonin and survive for 24 hours in the absence of ST-2 cells. We also found that murine osteoclast generation depends upon contact of osteoclast precursors with viable ST-2 cells. Furthermore, the stromal cells secrete macrophage colony-stimulating factor (CSF-1), and the anti-CSF-1 antibody 5A1 inhibits murine osteoclastogenesis. Exogenous CSF-1, in turn, partially overrides the anti-osteoclastogenic effect of 5A1. We conclude that (1) the purity of murine osteoclasts generated from bone marrow cells enriched for CFU-cs can be greatly enhanced by selective removal of associated stromal cells, and (2) both soluble and membraneresiding stromal cell factors are necessary for generation of murine osteoclasts.

Species differences in the immunophenotype of osteoclasts and mononuclear phagocytes.

SummaryHuman osteoclasts, in contrast to mononuclear phagocytes, are known to express a well-defined restricted range of myeloid antigens. To determine whether these antigenic differences are present in other species, we examined the immunophenotype of chicken and rabbit ostoeclasts, macrophages, macrophage polykaryons, and monocytes and compared them with similarly derived and cultured human cells. Human, rabbit, and avian osteoclasts reacted with monoclonal antibodies against human β1 integrins (CD29, CD49b, CD49d), β3 integrins (CD51, CD61), as well as human macrophage-associated antigen CD68. Avian osteoclasts also reacted for CD11a/18 and CD14 which are not present on human osteoclasts. Avian and mammalian monocytes, macrophages, and macrophage polykaryons expressed all the above antigens. Both avian and human macrophage polykaryons produced by culture of peritoneal macrophages reacted with anti-CD51 antibodies indicating that expression of the vitronectin receptor alone does not distinguish between these cells in vitro.

Binding of osteopontin to the osteoclast integrin αvβ3

Occupancy of the chicken osteoclast αvβ3 integrin stimulates immediate cell signals. Peptides from osteopontin containing Arg-Gly-Asp and peptides from the osteopontin and bone sialoprotein sequences containing Arg-Gly-Asp stimulated immediate reductions in osteoclast cytosolic Ca2+. The changes in cytosolic Ca2+ required the Arg-Gly-Asp sequence, and were blocked by LM609, a monoclonal antibody to the αvβ3 integrin. Osteoclast stimulation by the proteins through the integrin did not require immobilization since soluble peptides produced changes in cytosolic Ca2+ and inhibited osteoclast binding to bone particles and bone resorption. The decrease in cytosolic Ca2+ stimulated by osteopontin and related peptides was due to activation of a plasma membrane Ca2+-ATPase. Thus, the data suggest that ligand binding to the osteoclast αvβ3 integrin results in a reduction in cytosolic Ca2+ which participates in regulation of osteoclast function.

Immunocytochemical localization of vacuolar H+-ATPase and Cl−-HCO 3 − anion exchanger (erythrocyte band-3 protein) in avian osteoclasts: Effect of calcium-deficient diet on polar expression of the H+-ATPase pump

Osteoclasts attach to the bone surface and resorb bone by secreting protons into an isolated subosteoclastic compartment. Previous studies have shown the presence of a vacuolar type H+-ATPase, and a functional Cl−-HCO3− anion exchanger in the osteoclast. In the present studies, using a monoclonal antibody to the 31-kDa subunit of H+-ATPase and a rabbit antiserum to the erythrocyte band-3 protein (Cl−-HCO3− anion exchanger) we have immunocytochemically localized the respective pumps in bone sections obtained from chickens fed a normal or a calcium-deficient diet for 4 weeks. Our results indicate that although H+-ATPase is either evenly distributed throughout the osteoclast or is more polarized at its ruffled membrane juxtaposed to the bone surface, the band-3 protein immunoreactivity is always localized to the plasma membrane which is not attached to the bone surface (basolateral membrane). Four weeks of a calcium-deficient diet resulted in a significant increase in the percentage of osteoclasts that were polarized for the H+-ATPase pump at their ruffled membrane, and a trend toward increased total number of osteoclasts, although the latter did not reach statistical significance (P =0.09). These changes were not accompanied by a significant increase in the intensity of staining for H+-ATPase. Band-3 protein immunoreactivity was always prominent, limited to the basolateral membrane, and did not alter with calcium-deficient diet or with changes in the degree of H+-ATPase polarization.

Cloning of the Murine β5 Integrin Subunit Promoter IDENTIFICATION OF A NOVEL SEQUENCE MEDIATING GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR-DEPENDENT REPRESSION OF β5 INTEGRIN GENE TRANSCRIPTION

We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin αvβ5 and that granulocyte-macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the β5 gene. We herein report cloning of the β5 integrin gene promoter and identification of a GM-CSF-responsive sequence. A 13-kilobase (kb) genomic fragment containing part of the β5 gene was isolated by screening a mouse genomic library with a probe derived from the most 5′-end of a murine β5 cDNA. A combination of primer extension and S1 nuclease studies identifies two transcriptional start sites, with the major one designated +1. A 1-kb subclone containing sequence −875 to + 110 is transcriptionally active in a murine myeloid cell line. This 1-kb fragment contains consensus binding sequences for basal (Sp1), lineage-specific (PU.1), and regulatable (signal transducer and activator of transcription) transcription factors. Reflecting our earlier findings, promoter activity is repressed in transfected myeloid cells treated with GM-CSF. Using deletion mutants, we localized a 109-base pair (bp) promoter region responsible for GM-CSF-inhibited β5 transcription. We further identified a 19-bp sequence within the 109-bp region that binds GM-CSF-induced nuclear proteins by gel shift/competition assays. Mutation of the 19-bp sequence not only ablates its capacity to bind nuclear proteins from GM-CSF-treated cells, in vitro, but the same mutation, when introduced in the 1-kb promoter, abolishes its ability to respond to GM-CSF treatment. Northern analysis demonstrates that cycloheximide treatment abrogates the capacity of GM-CSF to decrease β5 mRNA levels. In summary, we have identified a 19-bp cis-element mediating GM-CSF-induced down-regulation of β5 by a mechanism requiring protein synthesis.