Background/Purpose:

Multiple lines of evidence have established that osteoclasts are required for physiologic bone resorption and pathological bone loss in inflammatory disorders. Isolation of differentiated osteoclasts from bone is technically challenging, but with the discovery that M-CSF and RANKL are sufficient for osteoclast differentiation came the ability to generate multinucleated osteoclasts from myeloid precursor cells in vitro with high efficiency. In vivo, analysis of osteoclasts at sites of bone resorption reveals that functionally activated osteoclasts are almost exclusively localized to the immediate bone surface, indicating that cell-substrate interactions contribute to terminal osteoclast differentiation. In this study, we have employed a unique in vitro osteoclastogenesis system using authentic bone substrates and expression profiling and pathway analysis to identify critical bone substrate-mediated pathways of osteoclast formation and activation. We have further validated the sphingosine-1-phosphate (S1P) generation, transport and signaling pathway as a key component of the bone-substrate regulated osteoclast differentiation program in vitro and in vivo.

Methods:

Murine bone marrow derived macrophages were cultured in the presence or absence of RANKL on plastic, hydroxyapatite, or calvarial bone discs. RNA was isolated at different stages of osteoclast generation and subjected to microarray analysis. Pathway analysis was performed using GSEA and Ingenuity pathway analysis. SphK inhibitors were used to validate involvement of S1P signaling in osteoclastogenesis in vitro. Immunohistochemical analysis of the RA bone-pannus interface was used assess activation of this pathway in osteoclasts in vivo.

Results:

Microarray analysis revealed unique clusters of RANKL induced and bone substrate-modulated osteoclast genes. Of 1490 genes upregulated by RANKL in differentiated osteoclasts, 5.4% were further upregulated by culture on the bone substrate; an additional 8.5% were downregulated. Pathway analysis identified the S1P pathway as significantly regulated by both RANKL and bone substrate, and that this regulation was dependent upon the presence of integrin beta 3, a key mediator of osteoclast attachment to bone.  SphK inhibitors dose-dependently blocked RANKL-induced human and mouse osteoclastogenesis in vitro. Furthermore, SphK1 was highly induced in osteoclasts at sites of RA bone erosions.

Conclusion:

Interaction of osteoclasts with the bone surface regulates multiple critical pathways of osteoclast formation and activation. Pathway analysis and in vitro/in vivo validation identifies the S1P pathway as a critical regulator of RANKL-bone-dependent osteoclastogenesis.  This pathway represents a novel therapeutic target for preventing osteoclast-mediated bone destruction in inflammatory bone loss disorders.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-sphingosine-1-phosphate-pathway-is-a-key-regulator-of-bone-substrate-mediated-osteoclast-differentiation-in-inflammatory-arthritis/