Oral squamous cell carcinomas (OSCC) are malignant tumors with a potent activity of local bone invasion; however, the molecular mechanisms of tumor osteolysis are unclear. In this study, we identified high level expression of chemokine ligand, CXCL13 and RANK ligand (RANKL) in OSCC cells (SCC1, SCC12 and SCC14a). OSCC cell‐conditioned media (20%) induced osteoclast differentiation which was inhibited by OPG in peripheral blood monocyte cultures indicating that OSCC cells produce soluble RANKL. Recombinant hCXCL13 (10 ng/ml) significantly enhanced RANKL‐stimulated osteoclast differentiation in these cultures. Trans‐well migration assay identified that CXCL13 induces chemotaxis of peripheral blood monocytes in vitro which was inhibited by addition of anti‐CXCR5 receptor antibody. Zymogram analysis of conditioned media from OSCC cells revealed matrix metalloproteinase‐9 (MMP‐9) activity. Interestingly, CXCL13 treatment to OSCC cells induced CXCR5 and MMP‐9 expression suggesting an autocrine regulatory function in OSCC cells. To examine the OSCC tumor cell bone invasion/osteolysis, we established an in vivo model for OSCC by subcutaneous injection of OSCC cells onto the surface of calvaria in NCr‐nu/nu athymic mice, which developed tumors in 4–5 weeks. μCT analysis revealed numerous osteolytic lesions in calvaria from OSCC tumor‐bearing mice. Histochemical staining of calvarial sections from these mice revealed a significant increase in the numbers of TRAP‐positive osteoclasts at the tumor‐bone interface. Immunohistochemical analysis confirmed CXCL13 and MMP‐9 expression in tumor cells. Thus, our data implicate a functional role for CXCL13 in bone invasion and may be a potential therapeutic target to prevent osteolysis associated with OSCC tumors in vivo.

Loss of Ostm1 leads to the most severe form of osteopetrosis in mice and humans. Because functional rescue of the osteopetrotic defect in these mice extended their lifespan from ∼3 weeks to 6 weeks, this unraveled a second essential role of Ostm1. We discovered that Ostm1 is highly expressed in the mouse brain in neurons, microglia, and astrocytes. At 3-4 weeks of age, mice with Ostm1 loss showed 3-10-fold stimulation of reactive gliosis, with an increased astrocyte cell population and microglia activation. This inflammatory response was associated with marked retinal photoreceptor degeneration and massive neuronal loss in the brain. Intracellular characterization of neurons revealed abnormal storage of carbohydrates, lipids, and ubiquitinated proteins, combined with marked accumulation of autophagosomes that causes frequent axonal swelling. Stimulation of autophagy was provided by specific markers and by significant down-regulation of the mammalian target of rapamycin signaling, identifying a cellular pathologic mechanism. A series of transgenic mouse lines specifically targeted to distinct central nervous system cell subpopulations determined that Ostm1 has a primary and autonomous role in neuronal homeostasis. Complete functional complementation demonstrated that the development of severe and rapid neurodegeneration in these mice is independent of the hematopoietic lineage and has clinical implications for treatment of osteopetrosis. Importantly, this study establishes a novel neurodegenerative mouse model critical for understanding the multistep pathogenic cascade of cellular autophagy disorders toward therapeutic strategy design.

In osteoclasts, Src controls podosome organization and bone degradation, which leads to an osteopetrotic phenotype in src ^–/– mice. Since this phenotype was even more severe in src ^–/– hck ^–/– mice, we examined the individual contribution of Hck in bone homeostasis. Compared to wt mice, hck ^–/– mice exhibited an osteopetrotic phenotype characterized by an increased density of trabecular bone and decreased bone degradation, although osteoclastogenesis was not impaired. Podosome organization and matrix degradation were found to be defective in hck ^–/– osteoclast precursors (preosteoclast) but were normal in mature hck ^–/– osteoclasts, probably through compensation by Src, which was specifically overexpressed in mature osteoclasts. As a consequence of podosome defects, the 3‐dimensional migration of hck ^–/– preosteoclasts was strongly affected in vitro. In vivo , this translated by altered bone homing of preosteoclasts in hck ^–/– mice: in metatarsals of 1‐wk‐old mice, when bone formation strongly depends on the recruitment of these cells, reduced numbers of osteoclasts and abnormal developing trabecular bone were observed. This phenotype was still detectable in adults. In summmary, Hck is one of the very few effectors of preosteoclast recruitment described to date and thereby plays a critical role in bone remodeling.—Vérollet, C., Gallois, A., Dacquin, R., Lastrucci, C., Pandruvada, S. M. N., Ortega, N., Poincloux, R., Behar, A., Cougoule, C., Lowell, C., Al Saati, T., Jurdic, P., Maridonneau‐Parini, I., Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors. FASEB J. 27, 3608–3618 (2013). www.fasebj.org