Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types,including osteoblast(OB)differentiation and function.Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive.A...Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types,including osteoblast(OB)differentiation and function.Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive.ATP6AP2,an accessory subunit of V-ATPase,plays important roles in multiple cell types/organs and multiple signaling pathways.However,little is known whether and how ATP6AP2 in OBs regulates Wnt/β-catenin signaling and bone formation.Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions.Conditionally knocking out(CKO)ATP6AP2 in the OB-lineage cells(Atp6ap2^(Ocn-Cre))reduced trabecular,but not cortical,bone formation and bone mass.Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs,but not osteocytes.Additional in vitro and in vivo studies revealed impairedβ-catenin signaling in ATP6AP2-KO BMSCs and OBs,but not osteocytes,under both basal and Wnt stimulated conditions,although LRP5 was decreased in ATP6AP2-KO osteocytes,but not BMSCs.Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression ofβ-catenin phosphorylation,but necessary for LRP6/β-catenin and N-cadherin/β-catenin protein complex distribution at the cell membrane,thus preventing their degradation.Expression of activeβ-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs.Taken together,these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability,and thus regulatingβ-catenin levels,demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/β-catenin signaling and trabecular bone formation.展开更多
The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogeneticallyconserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are...The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogeneticallyconserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are implicated in diseases of thekidney and brain. Here, we report high-resolution cryoelectron microscopy structures of LRP2 isolated from mouse kidney, atextracellular and endosomal pH. The structures reveal LRP2 to be a molecular machine that adopts a conformation for ligandbinding at the cell surface and for ligand shedding in the endosome. LRP2 forms a homodimer, the conformational transformationof which is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces. A subset of LRP2 deleteriousmissense variants in humans appears to impair homodimer assembly. These observations lay the foundation for furtherunderstanding the function and mechanism of LDL receptors and implicate homodimerization as a conserved feature of the LRPreceptor subfamily.展开更多
基金supported in part by grants from the National Institutes of Health(AG045781,AG051510,and AG066526)(to WCX).
文摘Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types,including osteoblast(OB)differentiation and function.Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive.ATP6AP2,an accessory subunit of V-ATPase,plays important roles in multiple cell types/organs and multiple signaling pathways.However,little is known whether and how ATP6AP2 in OBs regulates Wnt/β-catenin signaling and bone formation.Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions.Conditionally knocking out(CKO)ATP6AP2 in the OB-lineage cells(Atp6ap2^(Ocn-Cre))reduced trabecular,but not cortical,bone formation and bone mass.Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs,but not osteocytes.Additional in vitro and in vivo studies revealed impairedβ-catenin signaling in ATP6AP2-KO BMSCs and OBs,but not osteocytes,under both basal and Wnt stimulated conditions,although LRP5 was decreased in ATP6AP2-KO osteocytes,but not BMSCs.Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression ofβ-catenin phosphorylation,but necessary for LRP6/β-catenin and N-cadherin/β-catenin protein complex distribution at the cell membrane,thus preventing their degradation.Expression of activeβ-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs.Taken together,these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability,and thus regulatingβ-catenin levels,demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/β-catenin signaling and trabecular bone formation.
文摘The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogeneticallyconserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are implicated in diseases of thekidney and brain. Here, we report high-resolution cryoelectron microscopy structures of LRP2 isolated from mouse kidney, atextracellular and endosomal pH. The structures reveal LRP2 to be a molecular machine that adopts a conformation for ligandbinding at the cell surface and for ligand shedding in the endosome. LRP2 forms a homodimer, the conformational transformationof which is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces. A subset of LRP2 deleteriousmissense variants in humans appears to impair homodimer assembly. These observations lay the foundation for furtherunderstanding the function and mechanism of LDL receptors and implicate homodimerization as a conserved feature of the LRPreceptor subfamily.
