Introduction: 

Osteoclasts are the unique bone resorbing cells of hematopoietic origin. The phosphoinositide 3-kinase (PI3K) family consists of various PI3K isoforms which are thought to mediate different and isoform-specific biological functions. However their importance in osteoclast biology is poorly understood. The aim of this study was to test the role of the PI3Kb isoform in in vitro osteoclast development and in vivo bone homeostasis.

Methods: 

The trabecular architecture of the distal femoral metaphysis of wild-type and PI3Kb^–/– mice was tested by micro-CT analysis. Bone marrow cells were isolated from long bones of wild-type and PI3Kb^–/– mice and differentiated into osteoclasts in vitro in the presence of murine M-CSF and RANKL. Human monocytes were purified over the Ficoll-Paque gradient from whole blood of healthy volunteers and differentiated into osteoclasts in vitro in the presence of human M-CSF and RANKL.

Osteoclast differentiation and function were examined by TRAP-staining and resorption of bone slices or artificial hydroxyapatite surface. TGX221 was used as selective inhibitor of PI3Kb. For survival assays cells were incubated in binding buffer containing annexinV-phycoerythrin and 7-aminoactinomycinD then analysed by flow cytometry. For actin ring formation assay cells were fixed, permeabilized and stained with Alexa488-Phalloidin. For gene expression analysis total cellular RNA was extracted, cDNA was synthesized and multiplex PCR was performed with probes specific for the gene of interest.

Results. 

PI3Kb^–/– mice had significantly higher trabecular bone mass under basal conditions than wild-type mice. In vitro differentiation of murine bone marrow progenitors to multinucleated osteoclasts was strongly reduced by the genetic deficiency of PI3Kb. Human osteoclast development was reduced by the presence of the PI3Kb inhibitor TGX221. Pharmacological inhibition or genetic disruption of PI3Kb inhibited the in vitro resorptive capacity of osteoclasts. While PI3Kb was not required for the upregulation of osteoclast-specific gene expression or for the survival of preosteoclasts, it was highly involved in actin ring formation of mature osteoclasts.

Conclusion. 

Our results indicate that PI3Kb participates in bone resorption under basal conditions and plays a critical role in osteoclast development likely by regulating the organization of the osteoclast cytoskeleton.

This research was supported by Wellcome Trust, ERC, EMBO, TÁMOP (4.2.1B-09/1/KMR-2010-0001).