Wayne R. Holloway

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.

Expression of functional RANK on mature rat and human osteoclasts

Although the important roles of RANK/RANKL in osteoclastogenesis have been established, their roles in the regulation of mature osteoclasts remain uncertain. Microisolation has been used to obtain pure populations of rat and human osteoclasts for RT‐PCR analysis. RANK and calcitonin receptor mRNA was detected in all the samples whereas OPG and ALP mRNA was not present in any. RANKL mRNA was detected in two of eight rat and one of four human samples. Treatment of osteoclasts with soluble RANKL resulted in translocation of NF‐κB to the nucleus and elevation of cytosolic and nuclear calcium levels. We have shown that RANK is highly expressed in mature osteoclasts and that its stimulation by RANKL results in activation of NF‐κB and calcium signalling.

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.