C. J. Aitken

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.

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.

Gene array identification of osteoclast genes: Differential inhibition of osteoclastogenesis by cyclosporin A and granulocyte macrophage colony stimulating factor

Treatment of adherent peripheral blood mononuclear cells (PBMCs) with macrophage colony stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) stimulates the formation of multinucleate osteoclast‐like cells. Treatment with M‐CSF alone results in the formation of macrophage‐like cells. Through the use of Atlas human cDNA expression arrays, genes regulated by RANKL were identified. Genes include numerous cytokines and cytokine receptors (RANTES and CSF2R∝), transcription factors (nuclear factor of activated T‐cells cytoplasmic 1 (NFATc1) and GA binding protein transcription factor alpha (GABPα)), and ribosomal proteins (60S L17 and 40S S20). Real‐time PCR analysis showed significant correlation (R2 of 0.98 P < 0.01) with array data for all genes tested. Time courses showed differential activation patterns of transcription factors with early induction of FUSE binding protein 1 (FBP) and c‐Jun, and later steady upregulation of NFATc1 and GABP by RANKL. Treatment with cyclosporin A, a known NFATc1 inhibitor, resulted in a blockade of osteoclast formation. The mononuclear cells resulting from high cyclosporin treatment (1,000 ng/ml) were cathepsin K (CTSK) and tartrate‐resistant acid phosphatase (TRAP) positive but expression of calcitonin receptor (CTR) was downregulated by more than 30‐fold. Constant exposure of M‐CSF‐ and RANKL‐treated cells to GM‐CSF resulted in inhibition of osteoclast formation and the downregulation of CTSK and TRAP implicating the upregulation of CSF2R in a possible feedback inhibition of osteoclastogenesis.

Adenoviral down-regulation of osteopontin inhibits human osteoclast differentiation in vitro

Although osteopontin (OPN) is highly expressed in osteoclasts, OPN‐deficient mice have a near‐normal bone phenotype and its role in osteoclast differentiation and function remains uncertain. We used an adenoviral OPN‐antisense vector (AdOPN‐AS) to down‐regulate OPN expression in a human in vitro osteoclastogenesis model employing CFU‐GM precursors treated with RANKL and M‐CSF. Cultures infected with AdOPN‐AS showed reduced secretion of OPN compared to cultures infected with a control adenoviral vector expressing β‐galactosidase. Infection with AdOPN‐AS co‐incident with exposure to RANKL was associated with substantial (approximately 50%) inhibition of osteoclast formation with a concomitant reduction in dentine resorption. There was also a small reduction in the size of generated osteoclasts but no significant effect on the size of resorption pits/tracks nor on the amount of resorption per osteoclast. When the cultures were infected with AdOPN‐AS after 4 days exposure to RANKL only minor effects on osteoclastogenesis were seen. Our data demonstrate that early down‐regulation of OPN in vitro inhibits human osteoclastogenesis. Since mice totally lacking OPN do not have reduced osteoclast numbers our results imply the existence in vivo of an alternative molecular pathway(s).

Regulation of human osteoclast function by redox and sRANKL-induced intracellular ROS production

S313 A bsracts aromatisable androgen 5α-dihydrotestosterone (DHT) was administered (45μg/day via subcutaneous implants) to orchidectomized male P6 and R1 mice at week 4 (start of puberty). Both bone resorption and formation markers (urinary deoxypyridinoline [DPD] and serum osteocalcin [oc]) and tibial bone mass (by in vivo pQCT) were measured every 4 weeks between 4-20 weeks of age. At baseline and endpoint serum osteoprotegerin (OPG) was evaluated. At the tibial diaphysis, DHT had similar bone anabolic effects in P6 and R1. DHT increased periosteal expansion more than endocortical expansion, resulting in thickening of the cortex (+7% in P6 and +4% in R1, respectively; P<0.001). At the tibial metaphysis, DHT also induced a sustained increase of trabecular bone mineral density during the entire experimental period; this increase was significantly greater in P6 (+150% in P6 versus +68% in R1, P<0.001), despite similar action of DHT on seminal vesicles in P6 and R1. In accordance with its bone sparing action on cancellous bone, DHT lowered oc and DPD in P6 (-47% and -59%, respectively, P<0.001) and R1 (-53% and -60%, respectively, P<0.001) during the entire experimental period. Moreover, DHT increased serum OPG (which was already higher in P6 than R1) in P6 (+63%, P<0.05) but not in R1. In conclusion, DHT action on the skeleton is not impaired in growing P6. DHT has similar anabolic action on cortical bone in P6 compared to controls. In cancellous bone, on the other hand, DHT shows a stronger bone sparing effect in P6; this effect is consistent with an increase of OPG in this animal model of senile osteoporosis. The longitudinal follow-up of bone markers in combination with in vivo pQCT appears an interesting tool for the combined evaluation of cortical and cancellous bone acquisition during growth in animal models.In animal models, reactive oxygen species (ROS) have been shown to activate osteoclasts and have been implicated as second messengers in RANKL-mediated osteoclastogenesis. The aim of this study was to investigate the role of ROS in the function of mature human osteoclasts. Osteoclasts were generated by culture of CFU-GM with human M-CSF and RANKL for 14 days. Modulation of resorbing capacity and survival were assessed in mature osteoclasts dissociated from the plastic substrate and re-cultured on dentine slices. Treatment with RANKL enhanced the survival of mature osteoclasts on dentine and was absolutely required for resorption. Resorption was further enhanced by the presence of both M-CSF and RANKL. Co-treatment with hydrogen peroxide (50μM) for 24 hours resulted in a 130% increase in osteoclast size and a 34% increase in resorption. In contrast, co-treatment with the thiol antioxidant N-acetylcysteine (NAC; 10 mM) reduced osteoclast size by 22% and number by 50%, and resorption was completely inhibited. When mature osteoclasts were RANKL- and serum-starved for 3 hours and then stimulated with RANKL (150 ng/mL) for 30 minutes, increased intracellular ROS-production was detected using fluorescence microscopy and the ROS-sensitive dye CM-H2DCFDA. Pre-treatment with NAC blocked RANKL-induced intracellular ROS. Treatment with RANKL also stimulated a timedependent increase in intracellular hydrogen peroxide, as measured using Amplex Red reagent, with peak accumulation at 10 minutes. Electrophoretic mobility shift assay demonstrated that pre-treatment of osteoclast precursors with hydrogen peroxide (10μM) for 16 hours resulted in increased RANKL-induced NF-κB activity, whereas pre-treatment with NAC (10mM) blocked this activity. Human osteoclast function is stimulated by ROS and inhibited by antioxidant quenching of ROS. Stimulation of osteoclast survival and resorbing activity by RANKL may be mediated by ligand-mediated intracellular ROS production.

Over-expression of TBP-2, a protein involved in redox regulation, inhibits osteoclastogenesis.

Using gene array analysis, we found that thioredoxin (TRX) binding protein-2 (TBP-2) was down-regulated during osteoclast (OC) differentiation. TBP-2 is a negative regulator of TRX, a small protein with a redox-active dithiol active site. TRX enhances DNA binding of redox-sensitve transcription factors such as NF?B and AP-1. OC were generated on dentine slices using human CFU-GM precursor cells treated with RANKL and M-CSF. At 4 days of culture, efficient (>80%) infection with adenovirus expressing galactosidase (AdLacZ) was achieved. Infection with adenovirus expressing TBP-2 (AdTBP-2) for 14 days resulted in 66% reduction in the total TRAP+ area and 50% reduction in OC numbers as compared to the AdLacZ control. The size of OC formed in the presence of AdTBP-2 was reduced by 66% and they contained fewer nuclei. Resorption of dentine was inhibited by 80%. In mature OC infected with AdTBP-2, RANKL-induced NF?B activation was reduced by 63% and Western analysis demonstrated markedly increased expression of TBP-2 protein. We have shown that the over-expression of TBP-2, a gene down-regulated during OC formation, inhibits OC differentiation and NF?B activation. These results are consistent with the known function of TBP-2 as a negative regulator of TRX and the importance of the redox-sensitive transcription factor NF?B in osteoclastogenesis.It has been postulated that muscle and bone form an operational unit; that is, factors that affect muscle will also influence bone. It also is thought that muscle action is paramount in affecting bone adaptation; if so, then it would be expected that muscle development (or lean tissue as a surrogate of muscle) during the growing years should precede bone mineral accrual. We have shown previously that this is the case for total body mass; however, in theory this relationship should be even stronger at the extremities where muscle action is more isolated. The purpose of this study was to investigate the relationship of the timing of bone-free peak lean mass accrual (PLM) to peak bone mineral mass accrual (PBM) at the arms and legs. Subjects (70 boys and 67 girls) were measured annually (DXA: Hologic 2000 QDR in array mode). Whole year velocity values were calculated for bone-free lean tissue and bone mineral content. The mean age of peak tissue accrual was then calculated as the mean of the peak. Dependent t-tests were done to test for differences between the age of PLM and PBM (p<.05). As shown in the following table, PLM occurred prior to PBM at both sites in both boys and girls; however, this difference was not statistically significant at the legs in girls. These preliminary data support the hypotheses that PLM (a surrogate measure for muscle mass) precedes PBM and that muscle and bone development are closely related.