Abstract
Many positive signalling pathways of osteoclastogenesis have been characterized, but negative signalling pathways are less well studied. Here we show by microarray and RNAi that guanine nucleotide-binding protein subunit α13 (Gα13) is a negative regulator of osteoclastogenesis. Osteoclast-lineage-specific Gna13 conditional knockout mice have a severe osteoporosis phenotype. Gna13-deficiency triggers a drastic increase in both osteoclast number and activity (hyper-activation), mechanistically through decreased RhoA activity and enhanced Akt/GSK3β/NFATc1 signalling. Consistently, Akt inhibition or RhoA activation rescues hyper-activation of Gna13-deficient osteoclasts, and RhoA inhibition mimics the osteoclast hyperactivation resulting from Gna13-deficiency. Notably, Gα13 gain-of-function inhibits Akt activation and osteoclastogenesis, and protects mice from pathological bone loss in disease models. Collectively, we reveal that Gα13 is a master endogenous negative switch for osteoclastogenesis through regulation of the RhoA/Akt/GSK3β/NFATc1 signalling pathway, and that manipulating Gα13 activity might be a therapeutic strategy for bone diseases.
Highlights
Many positive signalling pathways of osteoclastogenesis have been characterized, but negative signalling pathways are less well studied
Using murine bone marrow monocytes (BMMs), which are widely used as primary osteoclast precursors, we demonstrated that whereas guanine nucleotide-binding protein subunit a13 (Ga13) expression was only mildly induced by macrophage colony-stimulating factor (M-CSF) (B2-fold at both mRNA and protein level), its expression was strongly induced by combined stimulation with M-CSF and RANKL (B6-fold at mRNA level and 8-fold at protein level) (Fig. 1d,e)
These results indicated that Ga13 might have important roles in osteoclasts
Summary
Gna[13] silencing promotes osteoclastogenesis. Given the limited attention attributed to the investigation of negative regulators of osteoclastogenesis, we performed a genome-wide screening to identify factors that negatively regulate osteoclast formation (Fig. 1). Using murine bone marrow monocytes (BMMs), which are widely used as primary osteoclast precursors, we demonstrated that whereas Ga13 expression was only mildly induced by M-CSF (B2-fold at both mRNA and protein level), its expression was strongly induced by combined stimulation with M-CSF and RANKL (B6-fold at mRNA level and 8-fold at protein level) (Fig. 1d,e) These results indicated that Ga13 might have important roles in osteoclasts. Histomorphometry based on Trichrome staining showed that trabecular bone number was reduced without much change of osteoblast number and osteoblast surface per bone surface in the Gna13f/f Ctsk-Cre mice, confirming that the reduced bone density was not caused by decreased bone formation (Supplementary Fig. 2a,b). Unlike WT BMMs, Gna13-deficient cells formed osteoclasts when attended by permissive RANKL dose (one tenth of the optimum 10 ng ml À 1 RANKL) (Fig. 3a) and formed 25% more a
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