Sfrp4 Repression Of The Ror2/Jnk Cascade In Osteoclasts Ensures Proper Cortical Bone Thickness

BackgroundPyle's disease?OMIM 265900?is a rare skeletal disorder characterized by wide metaphyses,significant thinning of cortical bone and fragility fractures despite the increase in trabecular bone.We have recently reported that this disease is caused by loss of function mutations in the Wnt inhibitor Secreted Frizzled Receptor Protein 4?SFRP4?and shown that in cortical bone,Sfrp4 is a key regulator of periosteal bone formation and endosteal bone remodeling,highly coordinated processes that define shape during growth and cortical homeostasis in the adult.However,whether Sfrp4 has a cell autonomous effect on osteoclasts?OCs?and whether the increased OCs number in endocortical bone leads to the thinning of the cortical bone in Pyle's desease patients is till not known.Objective1.To detect the effects of Sfrp4 on osteoclast's differentiation and functions.2.To evaluate the underlying mechanism of Sfrp4's effects on osteoclast.3.To investigate the reasons that lead to the significant thinning of cortical bone and fragility fractures in Sfrp4^-/-mice and Pyle's desease patients.Method1.The bone marrow macrophages?BMMs?were isolated from wide type?Wt?and Sfrp4^-/-mice,stimulated with M-CSF and RANKL to induce osteoclasts.Trap staining,RT-PCR and bone resorption pit assays were used to compare the osteoclasteogenesis abilities between Wt and Sfrp4^-/-BMMs;Wt BMMs were isolated,treated with different doses of Sfrp4?0?g/ml,5?g/ml,10?g/ml,20?g/ml?during osteoclasteogenesis to evaluate the effects of Sfrp4 treatment on osteocalsteogenesis;Calvarial osteoclasts?cOBs?and BMMs were isolated from Wt and Sfrp4^-/-mice,mix and match co-culture were did between cOBs and BMMs to distinguish the paracrine and cell-autonomous effects of Sfrp4 on osteocalsts.2.BMMs were isolated from Wt and Sfrp4^-/-mice,treated with M-CSF and RANKL to induce osteoclasteogenesis.The protein and RNA were isolated to run WB and RT-PCR in order to test the expression of Wnt/?-Catenin canonical Wnt casecade and Ror2/Jnk non-canonical Wnt signaling;Different doses of canonical Wnt/?-Catenin signaling inhibitor XAV939?0?M,1?M,5?M?or non-canonical Ror2/Jnk casecade inhibitor Sp600?0?M,2.5?M,5?M?were used during the osteoclast differentiation of Sfrp4^-/-BMMs to test wheather the inhibition of canonical or non-canonical wnt signaling could rescue the Sfrp4^-/-BMMs'phenotype on osteoclast differentiation;BMMs from Cre-ER^T2;Lrp5/6^fl/fll/fl mice were isolated,treated with Tamoxifen to delete the canonical Wnt/?-Catenin receptor Lrp5 and Lrp6,then Sfrp4?10?g/ml?was used during the osteoclast differentiation of these BMMs to evaluate wheather Sfrp4 can still function normally when canonical Wnt/?-Catenin signaling patheway was inhibited;Cre-ER^T2;Ror2^fl/fll/fl BMMs were isolated,treated with Tamoxifen to delete the non-canonical Ror2/Jnk casecade receptor Ror2,then Sfrp4?10?g/ml?was added during the osteoclast differentiation of the BMMs to test wheather Sfrp4's function on osteoclastogenesis could be affected when non-canonical Ror2/Jnk signaling was blocked.3.Ctsk_crere Ror2^fl/fll/fl mice were constructed to delete the non-canonical Ror2/Jnk casecade receptor Ror2 in osteoclasts in vivo specifically.Then these mice were mated with Sfrp4^-/-mice to get Ctsk_crere Ror2^fl/fSfrp4^+/-and Ror2^fl/fSfrp4^+/-mice.Micro-CT and histomorphology analysis were used to evaluate wheather the deletion of Ror2 receptor in osteoclasts in vivo could rescue Sfrp4^+/-cortical bone phenotype.Results1.OC differentiation and bone resorption activity were significantly enhanced in Sfrp4^-/-BMMs,as indicated by the increase in tartrate resistant acid phosphatase multinucleated cells?TRAP⁺MNCs?,pit assay and OC-specific gene expression.Confirming these findings,sFrp4-treated wt BMMs were significantly inhibited in their ability to respond to Rankl-induced OC differentiation.Together with mix-and-matched co-culture assays our data demonstrate that Sfrp4 deletion in osteoblasts or BMMs favors OCgenesis.2.Sfrp4 deletion resulted in activation of canonical Wnt and non-canonical Wnt-Ror2-Jnk signaling in Rankl-induced OCs.However,while pharmacological inhibition of canonical Wnt signaling had no effect on the Sfrp4 deficiency-dependent increase in OCgenesis in vitro,blocking the Ror2/Jnk cascade markedly inhibited its effect on OC differentiation of BMMs.Similarly,while in vitro excision of Lrp5/6 did not alter the effect of Sfrp4 on OCgenesis,in vitro excision of Ror2 significantly impaired it.3.The deletion of Ror2 exclusively in OCs(Ctsk _Crere Ror2^fl/fl)in Sfrp4 mutant mice,significantly suppressed the number of endosteal OCs and corrected their cortical thinning.Conclusion1.Sfrp4 inhibits osteoclasteogenesis in both cell-autonomous and paracrine manner.2.Sfrp4 inhibits osteoclasteogenesis via inhibiting non-canonical Ror2/Jnk cascade rather than canonical wnt signaling.3.Sfrp4-dependent suppression of the non-canonical Wnt/Ror2/Jnk cascade in osteoclasts regulates endocortical resorption ensuring proper cortical bone thickness...