Marina Stolina

INTRODUCTION: Sclerosteosis is a rare high bone mass genetic disorder in humans caused by inactivating mutations in SOST, the gene encoding sclerostin. Based on these data, sclerostin has emerged as a key negative regulator of bone mass. We generated SOST knockout (KO) mice to gain a more detailed understanding of the effects of sclerostin deficiency on bone. MATERIALS AND METHODS: Gene targeting was used to inactivate SOST and generate a line of SOST KO mice. Radiography, densitometry, microCT, histomorphometry, and mechanical testing were used to characterize the impact of sclerostin deficiency on bone in male and female mice. Comparisons were made between same sex KO and wildtype (WT) mice. RESULTS: The results for male and female SOST KO mice were similar, with differences only in the magnitude of some effects. SOST KO mice had increased radiodensity throughout the skeleton, with general skeletal morphology being normal in appearance. DXA analysis of lumbar vertebrae and whole leg showed that there was a significant increase in BMD (>50%) at both sites. microCT analysis of femur showed that bone volume was significantly increased in both the trabecular and cortical compartments. Histomorphometry of trabecular bone revealed a significant increase in osteoblast surface and no significant change in osteoclast surface in SOST KO mice. The bone formation rate in SOST KO mice was significantly increased for trabecular bone (>9-fold) at the distal femur, as well as for the endocortical and periosteal surfaces of the femur midshaft. Mechanical testing of lumbar vertebrae and femur showed that bone strength was significantly increased at both sites in SOST KO mice. CONCLUSIONS: SOST KO mice have a high bone mass phenotype characterized by marked increases in BMD, bone volume, bone formation, and bone strength. These results show that sclerostin is a key negative regulator of a powerful, evolutionarily conserved bone formation pathway that acts on both trabecular and cortical bone.

Tumor-derived prostaglandin E2 (PGE2) modifies cytokine balance and inhibits host immunity. We hypothesized that a high level of PGE2 production by lung tumor cells is dependent on tumor cyclooxygenase (COX)-2 expression. We found that PGE2 production by A549 non-small cell lung cancer (NSCLC) cells was elevated up to 50-fold in response to interleukin (IL)-1beta. Reversal of IL-1beta-induced PGE2 production in A549 cells was achieved by specific pharmacological or antisense oligonucleotide inhibition of COX-2 activity or expression. In contrast, specific COX-1 inhibition was not effective. Consistent with these findings, IL-1beta induced COX-2 mRNA expression and protein production in A549 cells. Specific inhibition of COX-2 abrogated the capacity of IL-1beta-stimulated A549 cells to induce IL-10 in lymphocytes and macrophages. Furthermore, specific inhibition of A549 COX-2 reversed the tumor-derived PGE2-dependent inhibition of macrophage IL-12 production when whole blood was cultured in tumor supernatants. Our results indicate that lung tumor-derived PGE2 plays a pivotal role in promoting lymphocyte and macrophage IL-10 induction while simultaneously inhibiting macrophage IL-12 production. Immunohistochemistry of human NSCLC tissues obtained from lung cancer resection specimens revealed cytoplasmic staining for COX-2 within tumor cells. This is the first description of functional COX-2 expression by NSCLC cells and the definition of a pathway whereby tumor COX-2 expression and a high level of PGE2 production mediate profound alteration in cytokine balance in the lung cancer microenvironment.

RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG-Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG-Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock-in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting "huRANKL" mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG-Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.