Jason M. Hodge

Leptin inhibits osteoclast generation.

Originally, leptin was described as a product of adipocytes that acts on the hypothalamus to regulate appetite. However, subsequently, it has been shown that leptin receptors are distributed widely and that leptin has diverse functions, including promotion of hemopoietic and osteoblastic differentiation. It has been recognized for some time that both serum leptin and bone mass are correlated positively to body fat mass and, recently, we have shown a direct positive relationship between serum leptin and bone mass in nonobese women. We now report that leptin inhibits osteoclast generation in cultures of human peripheral blood mononuclear cells (PBMCs) and murine spleen cells incubated on bone in the presence of human macrophage colony‐stimulating factor (hM‐CSF) and human soluble receptor activator of NF‐κB ligand (sRANKL). The half‐maximal concentration inhibitory of leptin was approximately 20 nM in the presence of sRANKL at 40 ng/ml but decreased to approximately 2 nM when sRANKL was used at 5 ng/ml. The majority of the inhibitory effect occurred in the first week of the 3‐week cultures. Inhibition did not occur when the PBMC cultures were washed vigorously to remove nonadherent cells or when purified CD14+ monocytes were used to generate osteoclasts, indicating an indirect or permissive effect via CD14− PBMC. Leptin increased osteoprotegerin (OPG) messenger RNA (mRNA) and protein expression in PBMC but not in CD14+ cells, suggesting that the inhibitory effect may be mediated by the RANKL/RANK/OPG system. Leptin may act locally to increase bone mass and may contribute to linkage of bone formation and resorption.

Vitamin D action and regulation of bone remodeling : suppression of osteoclastogenesis by the mature osteoblast

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.

Osteoclastic Potential of Human CFU-GM: Biphasic Effect of GM-CSF

Human osteoclasts can be efficiently generated in vitro from cord blood mononuclear cells and derived CFU‐GM colonies. However, CFU‐M colonies are poorly osteoclastogenic. Short‐term (2–48 h) treatment with GM‐CSF stimulates osteoclast formation by proliferating precursors, whereas longer exposure favors dendritic cell formation.

Osteoblast-mediated effects of zinc on isolated rat osteoclasts: Inhibition of bone resorption and enhancement of osteoclast number

Zinc is an important element in biology yet little is understood of its role in bone cell metabolism and function. This study examined the effects of zinc on osteoclast (OC) function in cultures derived from neonatal rats and in cocultures of OC and UMR 106-01 osteoblast-like cells (UMR/OC cocultures). Treatment with zinc (10(-12)-10(-4) mol/L) had no effect on either bone resorption or the number of multinucleate cells positive for tartrate-resistant acid phosphatase (TRACP + ve MNC) in OC cultured for 24 h on bone slices. However, in UMR/OC cocultures, 10(-4) mol/L zinc (but not lower concentrations) decreased resorption pit formation by approximately 50% and increased TRACP + ve MNC number by approximately 40%. When osteoblast-like cells were pretreated with zinc prior to, but not during, coculture with OC, effects on TRACP + ve MNC and pit number persisted, although the effect was reduced. Zinc treatment also inhibited resorption and stimulated TRACP and calcitonin receptor (CTR) + ve MNC numbers in long-term (96-120 h) UMR/OC cocultures. Our results indicate that zinc increases TRACP + ve CTR + ve MNC numbers yet inhibits bone-resorbing activity, and that these effects are dependent on the presence of osteoblastic cells. Zinc is abundant in bone and may act as a local regulator of bone cells.

Differential utilization of eicosapentaenoic acid and docosahexaenoic acid in human plasma.

It has recently been shown that the ω3 fatty acid status in humans can be predicted by the concentration of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in plasma phospholipids [Bjerve, K.S., Brubakk, A.M., Fougner, K.J., Johnsen, H., Midjthell, K., and Vik, T. (1993)Am. J. Clin. Nutr., in press]. In countries with low intake of ω3 fatty acids, the level of EPA in plasma phospholipids is often only about one-fifth the concentration of DHA. The purpose of this study was to investigate whether this difference in the concentration of these two fatty acids was due to a selective loss of EPA relative to DHA or to a lower dietary intake of EPA. Seven female volunteers ingested four grams of MaxEPA daily for 2 wk and in the following 4 wk they ate a diet almost completely devoid of the long-chain ω3 fatty acids. The concentrations of the ω3 fatty acids in the plasma cholesteryl esters, triglycerides and phospholipids and the high density lipoprotein phospholipids were examined at weekly intervals throughout the study. There was a more rapid rise in the concentration of EPA than in DHA levels in the supplementation period in all lipid fractions, but there was a disproportionate rise in DHA relative to EPA in the plasma lipids compared with the ratio in the supplement. In the depletion phase there was a rapid disappearance of EPA from all fractions, such that pre-trial levels were reached by one week post-supplementation. The disappearance of DHA was slower, particularly for the plasma phospholipids: at 4 wk post-supplementation, the DHA concentration in this fraction was still 40% above the pre-trial value. It is suggested that the low plasma EPA values relative to DHA are the result of increased β-oxidation of EPA and/or low dietary intake, rather than a rapid conversion of EPA to DHA. One practical result of this experiment is that, compared with DHA, the maintenance of increased EPA levels in plasma (and therefore tissues) would require constant inputs of EPA due to its more rapid loss from the plasma.

Selective serotonin reuptake inhibitors inhibit human osteoclast and osteoblast formation and function.

BACKGROUND Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants and one of the most commonly used medications. There is growing concern that SSRIs, which sequester in bone marrow at higher concentrations than brain or blood, increase bone fragility and fracture risk. However, their mechanism of action on human osteoclasts (OC) and osteoblasts (OB) differentiation remains unclear. METHODS Expression of serotonin receptors (5-HTR), transporter (5-HTT), and tryptophan hydroxylase 1 (TPH1) was assessed in human OC (precursors and mature) and OB (nonmineralizing and mineralizing) by polymerase chain reaction. OC formation and resorption was measured in the presence of 5 SSRIs. OBs cultured with SSRIs for 28 days were assessed for alkaline phosphatase (ALP) and bone mineralization. Cell viability and apoptosis were determined by annexin V flow cytometry. RESULTS OCs and OB expressed TPH1, 5-HTT, and 5-HTR1B. The 5-HTR2A was expressed only in OB, whereas 5-HTR2B expression increased from precursor to mature OC. All SSRIs (except citalopram) dose-dependently inhibited OC formation and resorption between 1 μmol/L and 10 μmol/L; order of potency: sertraline > fluoxetine > paroxetine > fluvoxamine > citalopram. Similarly, SSRIs (except citalopram) inhibited ALP and bone mineralization by OB but only at 30 μmol/L. Apoptosis was induced by SSRIs in OC and OB in an identical pattern to inhibitory effects. Serotonin treatment had no effect on either OC or OB parameters. CONCLUSIONS These data demonstrate that SSRIs differentially inhibit bone cell function via apoptosis. This may explain the mechanisms of bone loss with chronic use and aid clinical choices.

M-CSF Potently Augments RANKL-Induced Resorption Activation in Mature Human Osteoclasts

Macrophage-CSF (M-CSF) is critical for osteoclast (OC) differentiation and is reported to enhance mature OC survival and motility. However, its role in the regulation of bone resorption, the main function of OCs, has not been well characterised. To address this we analysed short-term cultures of fully differentiated OCs derived from human colony forming unit-granulocyte macrophages (CFU-GM). When cultured on dentine, OC survival was enhanced by M-CSF but more effectively by receptor activator of NFκB ligand (RANKL). Resorption was entirely dependent on the presence of RANKL. Co-treatment with M-CSF augmented RANKL-induced resorption in a concentration-dependent manner with a (200–300%) stimulation at 25 ng/mL, an effect observed within 4–6 h. M-CSF co-treatment also increased number of resorption pits and F-actin sealing zones, but not the number of OCs or pit size, indicating stimulation of the proportion of OCs activated. M-CSF facilitated RANKL-induced activation of c-fos and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, but not NFκB nor nuclear factor of activated T-cells, cytoplasmic-1 (NFATc1). The mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 partially blocked augmentation of resorption by M-CSF. Our results reveal a previously unidentified role of M-CSF as a potent stimulator of mature OC resorbing activity, possibly mediated via ERK upstream of c-fos.

Interleukin-33, a Target of Parathyroid Hormone and Oncostatin M, Increases Osteoblastic Matrix Mineral Deposition and Inhibits Osteoclast Formation in Vitro

IL-33 is an important inflammatory mediator in allergy, asthma, and joint inflammation, acting via its receptor, ST2L, to elicit Th₂ cell cytokine secretion. IL-33 is related to IL-1 and IL-18, which both influence bone metabolism, IL-18 in particular inhibiting osteoclast formation and contributing to PTH bone anabolic actions. We found IL-33 immunostaining in osteoblasts in mouse bone and IL-33 mRNA expression in cultured calvarial osteoblasts, which was elevated by treatment with the bone anabolic factors oncostatin M and PTH. IL-33 treatment strongly inhibited osteoclast formation in bone marrow and spleen cell cultures but had no effect on osteoclast formation in receptor activator of nuclear factor-κB ligand/macrophage colony-stimulating factor-treated bone marrow macrophage (BMM) or RAW264.7 cultures, suggesting a lack of direct action on immature osteoclast progenitors. However, osteoclast formation from BMM was inhibited by IL-33 in the presence of osteoblasts, T cells, or mature macrophages, suggesting these cell types may mediate some actions of IL-33. In bone marrow cultures, IL-33 induced mRNA expression of granulocyte macrophage colony-stimulating factor, IL-4, IL-13, and IL-10; osteoclast inhibitory actions of IL-33 were rescued only by combined antibody ablation of these factors. In contrast to osteoclasts, IL-33 promoted matrix mineral deposition by long-term ascorbate treated primary osteoblasts and reduced sclerostin mRNA levels in such cultures after 6 and 24 h of treatment; sclerostin mRNA was also suppressed in IL-33-treated calvarial organ cultures. In summary, IL-33 stimulates osteoblastic function in vitro but inhibits osteoclast formation through at least three separate mechanisms. Autocrine and paracrine actions of osteoblast IL-33 may thus influence bone metabolism.

Cord Blood-Derived Macrophage-Lineage Cells Rapidly Stimulate Osteoblastic Maturation in Mesenchymal Stem Cells in a Glycoprotein-130 Dependent Manner

In bone, depletion of osteoclasts reduces bone formation in vivo, as does osteal macrophage depletion. How osteoclasts and macrophages promote the action of bone forming osteoblasts is, however, unclear. Since recruitment and differentiation of multi-potential stromal cells/mesenchymal stem cells (MSC) generates new active osteoblasts, we investigated whether human osteoclasts and macrophages (generated from cord blood-derived hematopoietic progenitors) induce osteoblastic maturation in adipose tissue-derived MSC. When treated with an osteogenic stimulus (ascorbate, dexamethasone and β-glycerophosphate) these MSC form matrix-mineralising, alkaline phosphatase-expressing osteoblastic cells. Cord blood-derived progenitors were treated with macrophage colony stimulating factor (M-CSF) to form immature proliferating macrophages, or with M-CSF plus receptor activator of NFκB ligand (RANKL) to form osteoclasts; culture medium was conditioned for 3 days by these cells to study their production of osteoblastic factors. Both osteoclast- and macrophage-conditioned medium (CM) greatly enhanced MSC osteoblastic differentiation in both the presence and absence of osteogenic medium, evident by increased alkaline phosphatase levels within 4 days and increased mineralisation within 14 days. These CM effects were completely ablated by antibodies blocking gp130 or oncostatin M (OSM), and OSM was detectable in both CM. Recombinant OSM very potently stimulated osteoblastic maturation of these MSC and enhanced bone morphogenetic protein-2 (BMP-2) actions on MSC. To determine the influence of macrophage activation on this OSM-dependent activity, CM was collected from macrophage populations treated with M-CSF plus IL-4 (to induce alternative activation) or with GM-CSF, IFNγ and LPS to cause classical activation. CM from IL-4 treated macrophages stimulated osteoblastic maturation in MSC, while CM from classically-activated macrophages did not. Thus, macrophage-lineage cells, including osteoclasts but not classically activated macrophages, can strongly drive MSC-osteoblastic commitment in OSM-dependent manner. This supports the notion that eliciting gp130-dependent signals in human MSC would be a useful approach to increase bone formation.

Multiple roles of M‐CSF in human osteoclastogenesis

Although the critical role of M‐CSF in osteoclastogenesis is well documented, there has been no detailed analysis of how it regulates human osteoclast formation and function in vitro. We used a human osteoclastogenesis model employing CFU‐GM osteoclast precursors cultured for 14 days on dentine with RANKL, with varying exposure to exogenous human M‐CSF. Short‐term treatment of precursors with M‐CSF (10–100 ng/mL) resulted in increased proliferation with or without RANKL. Treatment with M‐CSF (1–100 ng/mL) for 14 days caused a biphasic concentration‐dependent stimulation of formation, fusion, and resorption peaking at 10–50 ng/mL and almost complete abolition of resorption at 100 ng/mL. Time‐course studies using M‐CSF (25 ng/mL) showed that osteoclast size, nuclei/cell, and resorption increased with longer duration of M‐CSF treatment. When treatment was restricted to the first 4 days, M‐CSF (25–100 ng/mL) stimulated formation of normal numbers of osteoclasts that resorbed less. Blockade of endogenous M‐CSF signaling with neutralizing M‐CSF antibody during the first week of culture extensively inhibited osteoclastogenesis, whereas blockade during the second week produced only a small reduction in resorption. Treatment with M‐CSF during the second week of culture caused a small increase in osteoclast number and a concentration‐dependent increase in cytoplasmic spreading with inhibition of resorption. We have shown that M‐CSF modulates multiple steps of human osteoclastogenesis, including proliferation, differentiation and fusion of precursors. In the later stages of osteoclastogenesis, M‐CSF modulates osteoclast‐resorbing activity, but is not required for survival. Modulation of M‐CSF signaling is a potential therapeutic target for conditions associated with excess bone resorption. J. Cell. Biochem. 102: 759–768, 2007.