Several lines of evidence implicate

Several lines of evidence implicate VPS35/retromer in bone remodeling or bone homeostasis. First, VPS35 is highly expressed in both OB- and OC-lineage hypoxia inducible factor (Xia et al., 2013b). Second, VPS35/retromer\'s cargos, such as PTH1R, Wntless, and RANK, are critical for bone remodeling or bone homeostasis (Cheloha et al., 2015; Coudreuse et al., 2006; Eaton, 2008; Feinstein et al., 2011; Franch-Marro et al., 2008; Xia et al., 2013b; Zhong et al., 2012). Third, young adult Vps35-heterozygote (Vps35+/m) mice display lower bone-mass with reduced bone formation and increased bone resorption (Xia et al., 2013b). RANKL (receptor activator of nuclear factor kappa-B ligand) signaling is increased and sustained in Vps35-deficient bone marrow macrophages (BMMs), resulting in an increased OC formation and bone resorption (Xia et al., 2013b). While this study has pointed to the importance of hyper-resorptive OCs for the osteoporotic deficit in Vps35+/m mice, the reduced bone formation may also have a critical role in this deficit. However, it remains unclear the exact role of VPS35/retromer and the functional significance of VPS35/retromer regulation of PTH1R in OB-lineage cells. PTH1R, a receptor of PTH, is an essential regulator of not only calcium–phosphorus metabolism, but also bone remodeling (Cheloha et al., 2015; Vilardaga et al., 2012). Intermittent treatment with human recombinant PTH(1–34) promotes recruitments of both OB and OC and a net bone-gain; but continued treatment leads to more OC activation with a net bone-loss (Cheloha et al., 2015; Vilardaga et al., 2012). It is of considerable interest to investigate how PTH(1–34) activation of PTH1R results in such complex metabolic effects. PTH(1–34) activation of PTH1R stimulates adenylate cyclase (AC)-mediated cAMP production by Gαs and increases PLC-mediated [Ca]i (cytosolic free Ca concentration) by Gαq (Cheloha et al., 2015; Vilardaga et al., 2012). These G-protein mediated signaling events, so called cell surface or canonical GPCR (G-protein coupled receptor) signaling, contribute to the complex metabolic effects induced by PTH (Vilardaga et al., 2012; Whalen et al., 2011). However, recent studies have found that β-arrestin serves as a multifunctional scaffolding protein linking the PTH1R to signaling endosomes independent of the cell surface or canonical GPCR pathway, and thus called endosomal or non-canonical GPCR signaling, which are also important for the complex metabolic effects (Vilardaga et al., 2012; Whalen et al., 2011). Exactly how both PTH-induced cell surface and endosomal signaling events are involved in the complex metabolic functions, and how both pathways are regulated and/or terminated, remain poorly understood. Here, we provide evidence that VPS35 in OB-lineage cells is necessary for maintaining bone mass. Mice that selectively knock out VPS35 in mature OBs, Vps35Ocn-Cre, as that of Vps35+/−, displayed reduced bone-mass. However, PTH(1–34) treatments diminished such an osteoporotic deficit in both Vps35Ocn-Cre and Vps35+/− mutant alleles. In addition, a more dramatic trabecular bone-gain response to PTH(1–34) was detected in both Vps35 mutant alleles, as compared with that of control mice. The increased bone-gain response might be due to an impaired PTH(1–34)-driven catabolic response or bone resorption. Further mechanistic studies showed that VPS35 in OB-lineage cells is required to turn off PTH(1–34)-signaling. Such a negative regulation of PTH(1–34) signaling (in particular, the endosomal signaling) is likely due to VPS35 promotion of PTH(1–34)-induced PTH1R translocation to the Golgi apparatus as well as VPS35 interaction with an inhibitor of PP1 phosphatase, PPP1R14C. This negative regulation of PTH(1–34)-driven endosomal signaling appeared to be crucial for PTH(1–34)-induction of catabolic response. Taken together, these results demonstrate a critical role for osteoblastic VPS35 to de-regulate PTH1R signaling, reveal a mechanism underlying VPS35 suppression of PTH1R-driven endosomal signaling, and provide insights into PTH(1–34)-induced catabolic response and adequate bone remodeling.