Parathyroid Hormone Signaling Through Low-Density Lipoprotein-Related Protein 6

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Parathyroid hormone signaling through low-density lipoprotein-related protein 6

Mei Wan,1,6 Chaozhe Yang,1,2 Jun Li,1,2 Xiangwei Wu,1,2 Hongling Yuan,1,2 Hairong Ma,1,2 Xi He,3 Shuyi Nie,4 Chenbei Chang,4 and Xu Cao1,5 1Department of Pathology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA; 2Shihezi Medical College, Shihezi Univeristy, Xinjiang 832002, China; 3The Neurobiology Program, Children’s Hospital Boston and Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USA; 4Department of Cell Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA

Intermittent administration of PTH stimulates bone formation, but the precise mechanisms responsible for PTH responses in osteoblasts are only incompletely understood. Here we show that binding of PTH to its receptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternary complex containing PTH, PTH1R, and LRP6 promoted rapid phosphorylation of LRP6, which resulted in the recruitment of axin to LRP6, and stabilization of ␤-catenin. Activation of PKA is essential for PTH-induced ␤-catenin stabilization, but not for Wnt signaling. In vivo studies confirmed that PTH treatment led to phosphorylation of LRP6 and an increase in amount of ␤-catenin in osteoblasts with a concurrent increase in bone formation in rat. Thus, LRP6 coreceptor is a key element of the PTH signaling that regulates osteoblast activity. [Keywords: PTH signaling; LRP6; osteoblasts; ␤-catenin; PKA] Supplemental material is available at http://www.genesdev.org. Received June 6, 2008; revised version accepted September 4, 2008.

Parathyroid hormone is a circulating hormone that acts as al. 1997; Tintut et al. 1998; Siddappa et al. 2008); how- the central regulator of calcium metabolism by directly ever, the precise molecular mechanisms by which PKA targeting bone, kidney, and intestine. The classical concept mediates PTH responses in osteoblasts remain unresolved. of PTH action is that it regulates serum calcium levels Besides PKA and PKC activation, PTH also regulates by stimulating bone resorption; however, intermittent MAPKs (Gensure et al. 2005; Gesty-Palmer et al. 2006), administration of PTH selectively stimulates bone for- including p42/p44 ERKs, p38, and c-Jun N-terminal ki- mation (Jilka 2007; Potts and Gardella 2007). This latter nase subtypes. The direction of this regulation and its property has been exploited to develop PTH as the only mediation by more proximal effectors such as cAMP/ FDA-approved anabolic therapy for bone (Tam et al. PKA and PKC, especially in the case of p42/p44 ERKs, 1982; Hodsman et al. 2005). In the past decade, signifi- appears to depend on cell type and the concentration of cant progress has been made in determining PTH down- PTH. Such extensive signaling diversity suggested the stream signaling events. It is now known that PTH binds possibility that PTH might interact with more than one to its receptor PTH1R (Juppner et al. 1991; Abou-Samra type of receptor in these target tissues that coreceptors ␣ ␣ et al. 1992) and activates the G protein subunits G s may modify the signaling, and/or novel signaling path- ␣ Ј Ј and G q. This leads to production of 3 ,5 -cyclic adeno- ways are involved. sine-5Ј-monophosphate (cAMP) and activation of phos- Wnts are secreted growth factors that play essential pholipase C (PLC), which eventually results in the acti- roles in multiple developmental processes. The impor- vation of protein kinase A (PKA) and protein kinase C tance of this signaling pathway in skeletal biology and (PKC) (Pierce et al. 2002; Qin et al. 2004; McCudden et disease has been emphasized recently by the identifica- al. 2005). Activation of PKA is believed to mediate the tion of a link between bone mass in humans and gain- or anabolic effect of PTH on bone (Armamento-Villareal et loss-of-function mutations of the Wnt coreceptor LRP5 and the Wnt antagonist, sclerostin (Baron et al. 2006; Balemans and Van 2007; Glass and Karsenty 2007). Wnts Corresponding authors. 5 activate different downstream signaling pathways. Of E-MAIL [email protected]; FAX (205) 975-7414. ␤ 6E-MAIL [email protected]; FAX (205) 975-7414. these pathways, the canonical or -catenin pathway has Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1702708. been analyzed most extensively. At the cell membrane,

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LRP6 in PTH signaling

Wnts bind two different families of receptors to transduce (data not shown). Similarly, PTH enhanced the levels of the canonical signal: low-density lipoprotein-related pro- ␤-catenin in the cytosol in a concentration- and time- teins (LRP5 or LRP6) and the Frizzled (Fz) receptor family dependent manner in both mouse calvarial primary pre- members (Pinson et al. 2000; Tamai et al. 2000, 2004; osteoblasts (Fig. 1C) and HEK 293 cells (Supplemental Huelsken and Birchmeier 2001; J. Mao et al. 2001). Fig. 1). ␤-Catenin accumulation in the cytosol induced LRP5/6 acts synergistically with Fzs in the binding of by PTH is so rapid that the effect is unlikely to be me- Wnt and activatation of downstream signaling. In the diated through synthesis of Wnt ligands or sensitization absence of Wnt, ␤-catenin is found in a large cytoplasmic of Wnt-stimulated signaling. Indeed, Fz8CRD, a com- complex comprising other proteins that promote its in- petitive inhibitor of the Wnt receptor Fz (Hsieh et al. activation by phosphorylation and its proteasomal deg- 1999), inhibited Wnt3a-elevated, but not PTH-elevated, radation. This large protein complex includes ␤-catenin, ␤-catenin level (Fig. 1D), thus excluding the possibility adenomatous polyposis coli (APC), glycogen synthase ki- of the involvement of Wnts. To test whether PTH stim- nase (GSK)-3␤, and axin (Logan and Nusse 2004; Clevers ulates ␤-catenin in vivo, we analyzed the effects PTH 2006). Upon Wnt stimulation, an Fz–LRP6 complex for- (1–34) administered as a single dose to 5-mo-old rats. mation is induced, which causes LRP6 phosphorylation PTH (1–34) is a C-terminal-truncated synthetic analog of on PPPS/TP motifs and axin recruitment to the plasma PTH with an anabolic effect on bone formation in hu- membrane, resulting in the inhibition of ␤-catenin phos- mans (Potts et al. 1971; Tregear et al. 1973). Immunohis- phorylation/degradation (He et al. 2004). Stabilized ␤- tochemistry analysis of sections of the trabecular bone catenin protein accumulates in the nucleus and com- indicated that PTH induced expression of ␤-catenin in plexes with the T-cell factor/lymphoid enhancer factor preosteoblasts and osteoblasts on the bone surface with- (TCF/LEF) family of DNA-binding transcription factors in hours (Fig. 1E,F). At 8 h after injection, positive stain- to enhance gene expression (Staal and Clevers 2000; ing of ␤-catenin was observed in most osteoblasts Moon et al. 2002). (99.08 ± 0.57%) at the metaphysis subjacent to the Several recent reports that have linked PTH with the epiphyseal growth plates and ∼90.24 ± 0.68% of the os- downstream elements of the Wnt pathway provided the teoblasts at the diaphyseal bone marrow. Similar experi- framework for our analysis of the PTH-associated signal- ments were carried out using 2-mo-old male mice, and ing events. These reports indicated that PTH may regu- similar temporal ␤-catenin expression patterns were late the canonical Wnt pathway by mechanisms that had obtained in the mice injected with PTH (Supplemental not yet been identified. PTH regulates the levels of ex- Fig. 2). pression of key components of the canonical Wnt pathway (Qin et al. 2003; Keller and Kneissel 2005; Kulkarni LRP6 forms a complex with PTH/PTH1R et al. 2005; Tobimatsu et al. 2006), including the levels of ␤-catenin and the transcriptional activity of the transcrip- The rapid enhancement of ␤-catenin protein levels in tion factor TCF/LEF (Kulkarni et al. 2005; Tobimatsu et al. response to PTH treatment both in vitro and in vivo 2006). Here we found that PTH activates ␤-catenin sig- suggest that PTH may have a direct effect on the signal- naling in osteoblasts both in vitro and in vivo by sharing ing components that promote the stabilization of of a coreceptor with Wnt; however, PTH signal acts in a ␤-catenin. Both LRP5 and LRP6 are key components in distinct manner from that of the canonical Wnt signaling activating ␤-catenin signaling in canonical Wnt path- pathway in that LRP6 forms a complex with PTH/ way. We attempted to examine whether these two re- PTH1R. The ternary complex promoted phosphorylation ceptors are also important in PTH-stimulated effects in of LRP6, which recruits axin from the cytoplasma, and osteoblasts. Recent studies reported that PTH anabolic induces ␤-catenin stabilization. Activation of PKA was effect was not affected in LRP5 KO mice (Sawakami et necessary for the phosphorylation of LRP6 in response to al. 2006; Iwaniec et al. 2007), indicating that LRP5 is not PTH, but not to Wnt. These results reveal a novel sig- essential for the stimulatory effects of PTH on bone for- naling pathway of PTH and provide alternative interpre- mation. We therefore focused on the function of LRP6 in tations of the functions of LRP6 and ␤-catenin in osteo- PTH-activated signaling. We first tested whether inacti- blasts, which until now have been considered to affect vation of LRP6 would affect PTH-elevated ␤-catenin the canonical Wnt signaling pathway exclusively. level by introducing siRNA complementary to lrp6 mRNA to the cells. Reduction of LRP6 (Fig. 2A) attenu- ated PTH-stimulated accumulation of ␤-catenin in the Results cytosol (Fig. 2B) and TCF/LEF luciferase activity (Fig. 2C). PTH-stimulated mRNA expressions of osteocalcin PTH induces ␤-catenin stabilization in osteoblasts and RANKL, downstream target genes of PTH that are To determine whether PTH regulates expression of ␤- pertinent to osteoblast differentiation, were also inhib- catenin, the effects of PTH on ␤-catenin levels in rat ited by the siRNA (Fig. 2D,E). The results indicate that UMR-106 osteoblastic cells were examined. We found LRP6 is a critical mediator for PTH-induced ␤-catenin that PTH stimulated the transcription of a luciferase re- stabilization in osteoblasts. porter bearing TCF/LEF-binding elements (Fig. 1A), and We then examined the possibility that LRP6 may form enhanced the abundance of ␤-catenin in the cytosol (Fig. a ternary complex with PTH and PTH1R as it does with 1B), whereas the unrelated peptide had no such effects Wnt and Fz. Immunoprecipitation (IP) with antibodies to

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Wan et al.

Figure 1. Activation of ␤-catenin signaling by PTH in osteoblast-like cells. (A) PTH stimulation of a luciferase reporter with TCF/LEF-binding elements (TCF4-Luc) in UMR-106 cells. Cells were transfected with TCF4-Luc plasmid and treated with control condition medium (CM) collected from culture medium of cells transfected with empty vector, CM-containing Wnt3a, and CM with 10−8 M PTH (1–84). Luciferase activity was measured 8 h after transfection and normalized to internal controls as Renilla luciferase units (RLU). (*) P < 0.01, n =3.(B) PTH induced stabilization of ␤-catenin in UMR-106 cells. Cells were treated as described in A. Cytosolic and membrane fractions were prepared 1 h after treatment for detection of ␤-catenin levels by Western blotting analysis. (C) PTH-induced stabilization of ␤-catenin in mouse primary preosteoblasts. Mouse cavarial preosteoblasts were treated with vehicle (control), increasing dosages of PTH (1–84), or 50 ng/mL mouse recombinant Wnt3a. Cytosolic and membrane fractions were prepared 1 h after treatment for detection of ␤- catenin levels by Western blotting analysis. (D) PTH-elevated ␤-catenin level was not affected by Fz8CRD. Mouse cavarial preosteoblasts were treated with Wnt3a CM or 10−8 M PTH (1–34) together with control CM or Fz8CRD CM for 1 h. Cytosolic and membrane fractions were prepared 1 h after treatment for detection of ␤-catenin levels by Western blotting analysis. (E,F) Immu- nohistochemical analysis of ␤-catenin levels in femur sections from 5-mo-old male rats at the indicated time points after PTH (1–34) injection (40 µg/kg). Representative of sections immunohistochemically stained with antibody to ␤-catenin or control IgG and counterstained with hematoxylin viewed at lower power (top row) and higher power (middle and bottom rows). Metaphysis subjacent to the epiphyseal growth plates (middle row) or diaphyseal hematopoietic bone marrow (bottom row) were examined. (E) Red asterisks and green asterisks mark locations in the low-power images that are shown in the high-power fields below. ␤- Catenin-positive osteoblasts were counted in a blinded fashion using OsteoMeasure Software (OsteoMetrics, Inc.) from three random high- power fields per specimens at metaphysis subjacent to diaphyseal hematopoietic bone marrow, and a total of six specimens in each group were used. (F) The quantification of ␤-catenin-positive osteoblasts is presented as percentage of total osteoblasts. (*) P < 0.005; (**) P <0.001 (in comparison with control), n =6.

LRP6 from lysates of PTH-treated UMR-106 cells indi- indicated by co-IP. The PTH ligand was immunoprecipi- cated that PTH1R formed a complex with endogenous tated by LRP6 only when both LRP6 and PTH1R were LRP6 in response to PTH in a time-dependent manner present (Fig. 2H), suggesting that PTH forms a ternary (Fig. 2F). Unlike LRP6, PTH did not enhance the binding complex with LRP6 and PTH1R. Further evidence for of LRP5 to PTH1R, although there is detectable binding the PTH–PTH1R–LRP6 complex formation was ob- in the absence of PTH (Fig. 2G), indicating that LRP5 tained from PTH-induced close association of PTH1R may play a different role in PTH signaling. The presence with LRP6 in cells by photobleaching-based fluorescence of PTH ligand in the LRP6–PTH1R complex was also resonance energy transfer (FRET) (Fig. 2I–K). As shown in

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LRP6 in PTH signaling

Figure 2. Formation of complexes of LRP6 with PTH–PTH1R. (A) LRP6-specific siRNA reduced the amount of LRP6 protein in HEK 293 cells as determined by Western blotting. siRNA directed against GFP was used as an siRNA control. (B) LRP6-specific siRNA reduced PTH-induced ␤-catenin stabilization in HEK293 cells as determined by Western blotting analysis. (C) LRP6-specific siRNA reduced PTH-stimulated TCF/LEF activity in UMR-106 cells as determined using a luciferase assay. (*) P < 0.01 (in comparison with control), n = 3; (n.s.) not significant (in comparison with control), n =3.(D,E) Real-time PCR analysis of Osteocalcin (D) and RANKL (E) mRNA expression. C2C12 cells expressing siGFP (control) or siLRP6 together with PTH1R were treated with or without PTH (1–34) in osteogenic induction medium (100 nM ascorbic acid, 10 mM glycerophosphate, and 100 ng/mL BMP2) and harvested at day 3 for RNA extraction. (F) Co-IP of endogenous LRP6 with PTH1R in UMR-106 cells. Cells were serum deprived and treated with 10−8 M PTH (1–84). The LRP6-associated PTH1R was determined separately by Western blotting of the anti-LRP6 immunoprecipitates. (WCL) Whole-cell lysates. (G) PTH enhances binding of PTH1R to LRP6, but not LRP5. HEK 293 cells were transfected with VSVG-tagged LRP6 or HA-LRP5 together with PTH1R and treated with 10−8 M PTH (1–84). The PTH1R-associated LRP5 or LRP6 was determined by Western blotting analysis of the anti-PTH1R immunoprecipitates. (WCL) Whole cell lysates. (H) Ternary complex of LRP6, PTH, and PTH1R. HEK 293 cells were transfected with VSVG-tagged LRP6 and HA-PTH1R and treated with 10−8 M PTH (1–84). The LRP6-associated PTH ligand was determined by Western blotting analysis of the anti-VSVG immunoprecipitates. (WCL) Whole cell lysates. (I–K) PTH brings PTH1R and LRP6 into close proximity as demonstrated by FRET. (I) A photobleaching-based FRET (pbFRET) system was generated by transiently expressing two constructs in HEK293 cells in which CFP and YFP were fused at the C terminus of PTH1R and LRP6, respectively. The interactions of YFP-fused LRP6 with CFP-fused BMPRII or CFP-fused PTH1R with YFP-fused mLRP4T100 were also examined as controls. (J) Representative confocal imaging of the association of CFP-PTH1R with YFP-LRP6 at 5 min after PTH treatment in HEK293 cells by pbFRET. The total photobleached area (ROI_1) is marked with a green square. Quantification of fluorescent intensities of each chosen point within (ROI_2∼ROI_6) or outside of the marked bleached area (ROI_7∼ROI_9) by averaging fluorescence before and after the bleach was conducted. (K) Comparison of the FRET efficiencies (FRET Eff%) before and after photobleaching in the absence or presence of PTH. (*) P < 0.001, compare with unbleached, n = 6; (n.s.) not significant compare with unbleached. (L,M) Ventral injection of RNA for PTH (2 pg) plus PTH1R (50 pg) promotes LRP6 (200 pg)-induced axis duplication. (n) Numbers of embryos scored.

Figure 2J and K, PTH led to increased FRET efficiency hance the FRET efficiency in either YFP-LRP6 and CFP- between CFP-PTH1R and YFP-LRP6, but did not en- BMPRII, BMP type II receptor (Cao and Chen 2005), or

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Wan et al. between CFP-PTH1R and YFP-mLRP4T100, another Immunoprecipitated LRP6 from extracts of PTH-treated member of the low-density lipoprotein-related proteins UMR-106 cells were monitored for their phosphoryla- family (Li et al. 2000). Thus, LRP6 specifically interacts tion by Western blotting with an antibody that recog- with PTH1R upon PTH stimulation. The association of nizes phosphorylated PPPSP motifs (Ab1490) (Tamai et PTH1R with LRP6 is also supported by analysis of the al. 2004). PTH rapidly induced the phosphorylation of model of LRP6-mediated secondary axis induction in LRP6 at the PPPSP motifs (Fig. 4A). Phosphorylation of Xenopus, in which PTH enhanced LRP6-induced second- PPPSP motifs is required for axin recruitment from cy- ary axis induction (Fig. 2L,M). toplasm to LRP6 at cell membrane. PTH also increased axin1 level on cell membrane detected by cell fractionation assay in primary preosteoblasts (Fig. 4B). Consis- Extracellular domain of LRP6 interacts with PTH1R tently, PTH rapidly increased in the binding of axin to To confirm and extend the studies of the LRP6 and LRP6 by co-IP assays (Fig. 4C). Again, Fz8CRD, a com- PTH1R complex formation, we mapped the region of petitive inhibitor of the Wnt receptor Fz (Hsieh et al. LRP6 required for its interaction with PTH1R. PTH1R 1999), was used to exclude the possibility that these PTH was coexpressed in cells with LRP6, a truncated LRP6 effects are mediated through promotion of Wnts produc- containing the extracellular and transmembrane do- tion or sensitization of Wnt-stimulated signaling. Fz8CRD mains (LRP6N + T), or the transmembrane and intracel- inhibited Wnt3a-induced phosphorylation of LRP6 (Fig. lular domains (LRP6T + C) for IP assay. Binding of 4D, lane 8), but did not inhibit the effect of PTH (Fig. 4D, LRP6T + C to PTH1R could barely be detected, but the lane 4). In contrast, LRP6N blocked PTH-stimulated LRP6N + T associated with PTH1R as effectively as did LRP6 phosphorylation (Fig. 4E). The results indicate that full-length LRP6 (Fig. 3A). The presence of PTH in the PTH-induced formation of PTH1R–LRP6 complex LRP6N + T/PTH1R complex further suggested the for- through their extracellular domains is essential for phos- mation of a ternary complex. Moreover, PTH-induced phorylation of LRP6. direct interaction of LRP6N with PTH1R on the cell sur- Because the levels of ␤-catenin were increased in os- face was confirmed in an immunefluorescence colocal- teoblasts of rats with injection of a single dose of PTH ization assay. Immun-colocalization of LRP6N–IgG with (Fig. 1E,F), we tested whether the amounts of phosphor- PTH1R on the cell surface was increased from 22.8% to ylated LRP6 were enhanced in osteoblasts from the same 82.3% with addition of PTH ligand (Fig. 3B [top two tissue. Immunostaining with an antibody specific for the rows], C) whereas binding of IgG to PTH1R was barely phosphorylated PPPSP demonstrated that PTH-stimu- detected (Fig. 3B [bottom two rows], C). Collectively, the lated phosphorylation of LRP6 in preosteoblasts or os- results indicate that PTH induces formation of PTH1R/ teoblasts at the surface of trabecular bone (Fig. 4F, sec- LRP6 complex through LRP6 extracellular domain. ond and third rows), whereas the amount of total LRP6 We then examined whether LRP6N acts as a domi- protein remained unchanged (Fig. 4F, first row). The tem- nant-negative in PTH signaling through LRP6. As ex- poral pattern of phosphorylation of the PPPSP motifs pected, LRP6N blocked the PTH-induced association of was similar to that of ␤-catenin (cf Figs. 1E, 4F [second endogenous LRP6 with PTH1R (Fig. 3D). LRP6N inhib- and third rows] and Figs. 1F, 4G). Thus, PTH increases ited PTH-, but not LiCl-induced TCF transcriptional ac- the abundance of ␤-catenin in osteoblasts in vivo tivity (Fig. 3E,F). As LiCl directly inhibits GSK3 kinase through phosphorylation of LRP6. in the cytoplasm to stabilize of ␤-catenin (Stambolic et al. 1996), our results indicate that LRP6N acts upstream PKA is required in PTH-, but not in Wnt-activated of GSK3 and functions as a dominant-negative in the LRP6–␤-catenin signaling PTH-activated ␤-catenin signaling via binding to cell surface PTH1R. Furthermore, secreted proteins DKK1 As the activation of ␤-catenin signaling by PTH in os- and sclerostin, also binding to LRP6 at the extracellular teoblasts seems to be independent of Wnt, we attempted domain (Bafico et al. 2001; B. Mao et al. 2001; Semenov to investigate the mechanism responsible for the PTH et al. 2001, 2005; Li et al. 2005), disrupted PTH-induced effects. PTH activates cAMP-dependent PKA, which is ␤-catenin accumulation in the cytoplasm (Fig. 3G) and sufficient for initiation of signals mediating PTH action TCF/LEF luciferase activity (Fig. 3H). Thus, PTH-in- in osteoblasts. We assessed whether PKA participates in duced recruitment of LRP6 through its extracellular do- PTH-activated LRP6–␤-catenin signaling. Binding of main is essential in activation of ␤-catenin signaling intact PTH (1–84) or PTH (1–34) to PTH1R activates pathway. PKA. However, the native C-terminal fragments of PTH bind PTH1R but do not activate PKA (Kronenberg et al. 1998; Gensure et al. 2005; Murray et al. 2005). The C- PTH induces phosphorylation of LRP6 and axin terminal fragments PTH (7–84) and PTH (39–84) were recruitment in osteoblasts much less effective than PTH (1–84) in activating As phosphorylation of LRP6 at the PPPSP motifs plays a ␤-catenin signaling (Fig. 5A), altering the stability of ␤- crucial role in activating downstream ␤-catenin signal- catenin (Fig. 5B), and inducing axin–LRP6 binding (Fig. ing by Wnt (J. Mao et al. 2001; Tamai et al. 2004; Dav- 5C). These results suggest that cAMP–PKA activation is idson et al. 2005; Zeng et al. 2005), we examined whether involved in activation of LRP6. The minimum effects PTH induces phosphorylation of LRP6 at PPPSP motifs. induced by PTH (7–84) and PTH (39–84) (Fig. 5A–C) may

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LRP6 in PTH signaling

Figure 3. Extracellular domain of LRP6 interacts with PTH1R. (A) Interaction of the N-terminal domain of LRP6 with PTH1R. HEK 293 cells were transfected with indicated plasmids and treated with or without 10−8 M PTH (1–84). IP was done to identify the interaction. (B,C) Surface binding of LRP6N in the presence of PTH treatment. HEK 293 cells were transfected with HA-PTH1R and treated with CM containing human IgG or LRP6N-IgG for 1 h following PTH (1–84) treatment for 15 min. Cells were washed, fixed, and immunostained with either anti-HA (red) or anti-IgG (green). Nuclei were visualized using Hoechst 33342. (B) Representative images are shown. (C) IgG CM or LRP6N CM surface binding rates; i.e., the ratios of the number of cells showing in green to the number of cells showing in red were calculated. (D) Soluble LRP6N disrupts PTH1R binding to endogenous LRP6. UMR-106 cells were treated with control CM or LRP6N CM for 1 h followed by 10−8 M PTH (1–84) treatment for another 1 h. Cells were washed with PBS and cell lysates were collected. The endogenous PTH1R-associated LRP6 was determined by Western blotting of the anti-PTH1R immunoprecipitates. PI, preimmune serum control. (E) Inhibition of PTH-induced TCF4/LEF activation by soluble LRP6N. UMR-106 cells were transfected with TCF4-Luc plasmid and treated with control CM or LRP6N CM followed by PTH (1–84) treatment. Luciferase activity was measured and normalized to internal controls as Renilla luciferase units (RLU). (*) P < 0.01 (in comparison with control), n = 3; (n.s.) not significant (in comparison with control), n =3.(F) LRP6N does not affect LiCl-induced TCF4/LEF activation. UMR-106 cells were transfected with TCF4-Luc plasmid and treated with control CM or LRP6N CM followed by 20 mM LiCl treatment. Luciferase activity was measured and normalized to internal controls as Renilla luciferase units (RLU). (*) P < 0.001 (in comparison with control), n =3.(G) DKK1 reduced Wnt3a- or PTH-induced ␤-catenin stabilization in HEK293 cells as determined by Western blotting analysis. (H) Inhibition of PTH-induced TCF4/LEF activation by DKK1 and Sclerostin. UMR-106 cells were transfected with TCF4-Luc plasmid and treated with control CM, DKK1 CM, or Sclerostin CM followed by Wnt3a or PTH (1–84) treatment. Luciferase activity was measured and normalized to internal controls as Renilla luciferase units (RLU). (*) P < 0.01 (in comparison with Wnt3a or PTH treatment), n =3. be mediated by other signaling components than PKA as lane 3), and ␤-catenin-dependent transcription activity the unrelated peptide of the similar length did not exert (Fig. 5G), further indicating that PKA activity is essential such effect (data not shown). PKI-(14–22), a specific in- for PTH-activated LRP6–␤-catenin signaling. However, hibitor of PKA-directed phosphorylation, inhibited PTH- H89 did not affect Wnt3a-stimulated LRP6 phosphoryla- induced LRP6 phosphorylation (Fig. 5D). Moreover, the tion (Fig. 5H), ␤-catenin stabilization (Fig. 5F, lane 5), and PKA inhibitors, PKI-(14–22) and H89 reduced the binding ␤-catenin-dependent transcription activity (Fig. 5I). The of axin to LRP6 (Fig. 5E), ␤-catenin stabilization (Fig. 5F, results provide further evidence to support the concept

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Figure 4. PTH-induced phosphorylation of LRP6 and axin recruitment. (A) UMR-106 cells were serum deprived and treated with 10−8 M PTH (1–84). Phosphor- ylated endogenous LRP6 was identified by Western blotting analysis of the anti-LRP6 immunoprecipitates with Ab1490. (B) PTH induced the recruitment of axin1 to cell membrane in mouse primary preosteoblasts. Cells were treated with vehicle or 10−8 M PTH (1–84) for 30 min. Membrane fractions were prepared for detection of axin1 and LRP6 levels by Western blotting analysis. (C) LRP6–axin binding in HEK 293 cells. Cells were transfected with PTH1R, HA-tagged axin, and VSVG-tagged LRP6 and treated with 10−8 M PTH (1–84). The axin-associated LRP6 was determined by Western blotting of the anti-VSVG immunoprecipitates. (D) Fz8CRD does not inhibit PTH-induced LRP6 phosphorylation. HEK293 (for lanes 1–4) or MEF (for lanes 5–8) cells were transfected with VSVG-tagged LRP6, treated with control CM or Fz8CRD CM for 2 h, and then with 10−8 M PTH (1–84) or Wnt3a. The phosphorylated LRP6 was detected by Western blotting analysis of the anti-VSVG immunoprecipitates with Ab1490. (E) UMR-106 cells were serum deprived, treated with control CM or LRP6N CM for 1 h, and then with 10−8 M PTH (1–84) for another 15 min. Phos- phorylated endogenous LRP6 was identified by Western blotting analysis of the anti-LRP6 immunoprecipitates with Ab1490. (F,G) Immunohistochemical analysis of phosphorylated LRP6 levels in femur sections from 5-mo-old male rats at the indicated time points after PTH (1–34) injection (40 µg/kg). Representative of sections immunohistochemically stained with antibody to total LRP6 (top row) and phosphorylated LRP6 (Ab1490, middle and bottom rows), and counterstained with hematoxylin viewed at lower power (middle row) and higher power (bottom row). The metaphyseal area of distal femurs was examined. (F) Double asterisks mark locations in the low-power images that are shown in the high-power fields below. The phosphorylated LRP6- positive osteoblasts were counted in a blinded fashion using OsteoMeasure Software (OsteoMetrics, Inc.) from three random high-power fields per specimens at metaphysis subjacent to diaphyseal hematopoietic bone marrow, and a total of six specimens in each group were used. (G) The quantification of phosphorylated LRP6- positive osteoblasts is presented as percentage of total osteoblasts. (*) P < 0.005; (**) P < 0.001 (in comparison with control), n =6. that both PTH and Wnt activate LRP6–␤-catenin signal- ␤-catenin activation. In addition, DKK1 and sclerostin, ing in osteoblasts, but do so through distinct pathways. antagonists of LRP6 by direct binding to the extracellular domain of LRP6, inhibit PTH-induced axin–LRP6 binding (Supplemental Fig. 3). Thus, PTH-induced recruit- Discussion ment of LRP6 to PTH1R is essential in activation of The data reported here demonstrate that PTH activates ␤-catenin signaling. This PTH-activated LRP6–␤-catenin ␤-catenin signaling in osteoblasts in vitro and in vivo by pathway is likely a direct effect rather than via a Wnt direct recruitment of LRP6 to PTH/PTH1R complex. ligand-dependent process because the phosphorylation of PTH ligand induces direct interaction of the extracellu- LRP6 by PTH is rapid. In addition, Fz8CRD, the com- lar domain of LRP6 with PTH1R at the cell surface. As a petitive inhibitor of the Wnt receptor Fz, is not able to result, the PPPSP motifs of LRP6 are phosphorylated and block the action of PTH. axin is recruited to the phosphorylated PPPSP motifs, LRP6 is a well-recognized coreceptor for Wnt, and its leading to stabilization of ␤-catenin (Fig. 5J). The extra- function in osteoblasts has been considered primarily in cellular domain of LRP6, which inhibits LRP6–PTH1R terms of its effect on Wnt signaling (Baron et al. 2006; complex formation, blocks LRP6 phosphorylation and Balemans and Van 2007; Glass and Karsenty 2007). It

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Figure 5. PKA mediates PTH-, but not Wnt3a-activated LRP6–␤-catenin signaling. (A) Failure of PTH C-terminal ligands to stimulate TCF4/LEF activity. UMR-106 cells were transfected with TCF4-Luc plasmid, treated with 10−8 M PTH ligands and subject to a luciferase assay. (*) P < 0.05 (in comparison with control), n = 3; (**) P < 0.005 (in comparison with control), n =3.(B) Failure of PTH C-terminal ligands to stabilize ␤-catenin. UMR- 106 cells were treated with PTH ligands for 1 h. Cytosolic fractions were prepared for detection of ␤-catenin protein by West- ern blot analysis. (C) Effect of PTH C-terminal ligands on LRP6–axin binding. Cells were transfected with indicated plasmids and treated with PTH ligands. The LRP6- associated axin was determined by West- ern blotting of the anti-VSVG immunoprecipitates. (WCL) Whole-cell lysates. (D) Inhibition of PTH-induced LRP6 phosphorylation by PKA inhibitor. Cells were transfected with VSVG-LRP6 and PTH1R, metabolically labeled with [32P] phosphate, and treated with PKI-(14–22) for 1 h before adding PTH (1–34) for another 15 min. VSVG-LRP6 was immunoprecipitated from cell lysates. Proteins were re- solved by SDS-PAGE and visualized by au- toradiography. (E) Inhibition of the binding of axin with LRP6 by PKA inhibitors. Cells were transfected with indicated plasmids and pretreated with H89 or PKI-(14– 22) for 1 h before adding PTH (1–84) for another 30 min. The LRP6-associated axin was determined as in C.(F) Inhibition of PTH-induced, but not Wnt3a-induced ␤- catenin stabilization by PKA inhibitor. UMR-106 cells were treated with 10−8 M PTH (1–34) or 50 ng/mL recombinant mouse Wnt3a (rmWnt3a) together with vehicle (control) or H89. Cytosolic fractions were prepared for detection of ␤-catenin protein by Western blot analysis. (G) Inhibition of PTH-activated TCF4/ LEF activity by PKA inhibitors. UMR-106 cells were transfected with TCF4-Luc plasmid, treated with 10−8 M PTH together with vehicle (control), H89, or PKI-(14–22), and subject to a luciferase assay. (*) P < 0.005 (in comparison with PTH treatment group), I = 3. (H) Failure of PKA inhibitor to inhibit Wnt3a-induced LRP6 phosphorylation. MEFs were transfected with VSVG-LRP6 and pretreated with increasing doses of H89 for 1 h before adding control CM or Wnt3a CM for another 30 min. The phosphorylated LRP6 was detected by Western blotting analysis of the anti-VSVG immunoprecipitates with Ab1490. (I) Failure of PKA inhibitors to inhibit Wnt3a-stimulated TCF4/LEF activity. UMR-106 cells were transfected with TCF4-Luc plasmid, treated with control CM (Con) or Wnt3a CM (Wnt3a) together with vehicle, H89, or PKI-(14–22) and subject to a luciferase assay. (n.s.) No significance (in comparison with Wnt3a group), n =3.(J) Schematic model of LRP6–␤-catenin pathway activation in response to PTH. Upon PTH ligand binding to its receptor PTH1R, LRP6 are recruited and form complexes with PTH/PTH1R, thus positioning LRP6 in close proximity with PTH1R. In parallel, PKA is activated downstream from PTH1R and mediate the phosphorylation of LRP6, which leads to the recruitment of axin and stabilization of ␤-catenin. would be of considerable interest to further determine on the osteoblasts function. We found that knockdown the roles of LRP6 and ␤-catenin in PTH-mediated effects of LRP6 blocked PTH-stimulated phosphorylation of

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CREB at Ser133 and ERK (Supplemental Fig. 4), the 2000) tagged with HA and VSVG were subcloned into pCMV known direct PKA or PKC targets (Tyson et al. 1999; and pCS2+, respectively. LRP6N + T (LRP6 N-terminal plus the Gesty-Palmer et al. 2006), and importantly, inhibited transmembrane domain) and LRP6T + C (LRP6 transmembrane PTH-stimulated mRNA expression of osteocalcin and domain plus C-terminal) tagged with VSVG were subcloned into RANKL, osteoblast differentiation markers (Fig. 2D,E). pCS2 + . LRP6N + 1479m, LRP6N + 1490m, LRP6N + 1493m, and LRP6N + 1496m were generated by mutagenesis of either the Moreover, we analyzed mouse models of long-term ad- serine (at aa1490 or aa1496) or threonin (at aa1479 or aa1493) to ministration of PTH, which has paradoxical effects in alanine. LRP6N-IgG was generated by fusing the LRP6 extracel- that PTH stimulates bone formation when injected lular domain with IgG (Tamai et al. 2000). si-GFP (Wan et al. daily, but causes severe bone loss if continually infused 2005) and si-LRP6 plasmids were generated using a BS/U6 vec- (Supplemental Fig. 5A,B). Intermittent, but not continu- tor. Briefly, a 22-nucleotide (nt) oligo (oligo 1) corresponding to ous, administration of PTH causes phosphorylation of nucleotides 2981–3002 of the human LRP6 coding region was LRP6 and stabilization of ␤-catenin in osteoblasts first inserted into the BS/U6 vector digested with ApaI (blunted) (Supplemental Fig. 5C). In addition to stabilization of and HindIII. The inverted motif that contains the 6-nt spacer ␤-catenin by PTH and Wnts, TGF␤ also stimulates pro- and five Ts (oligo 2) was then subcloned into the HindIII and liferation of osteoprogenitors through stabilization of ␤- EcoRI sites of the intermediate plasmid to generate BS/U6/ LRP5/6. catenin (Jian et al. 2006). Thus, it appears that ␤-catenin is one of the common mediators of osteoblastic bone formation induced by different extracellular signals. Primary osteoblast isolation and culture PTH is also known to inhibit osteocyte expression Osteoblasts were isolated by digestion of calvaria of newborn of sclerostin (Bellido et al. 2005; Keller and Kneissel mice as decribed (Wang et al. 2007). Briefly, calvaria were incu- 2005; Leupin et al. 2007), which is an inhibitor of Wnt- bated with 10 mL of digestion solution containing 1.8 mg/mL of activated ␤-catenin signaling via directly binding to collagenase type I (Worthington Biochemical Corp.) for 15 min LRP6 and a negative regulator of bone formation (Win- at 37°C under constant agitation. The supernatant was then kler et al. 2003; van Bezooijen et al. 2004; Li et al. 2008). harvested, replaced with fresh collagenase, and the digestion Sclerostin also inhibits PTH-induced axin–LRP6 bind- repeated an additional four times. Digestion solutions containing, implying that suppression of sclerostin by PTH ing the osteoblasts were pooled together. After centrifugation, ␣ would increase the availability of LRP6 to facilitate PTH osteoblasts were obtained and cultured in -MEM containing signaling in a positive feedback fashion. The activation 10% FBS, and 1% penicillin/streptomycin at 37°C in a humidi- fied incubator supplied with 5% CO . of PKA by PTH may occur in parallel to mediate the 2 phosphorylation of LRP6 and axin recruitment for activation of ␤-catenin (Fig. 5J). Sequence analysis reveals Cell culture, conditioned media, transfection, and luciferase four PKA consensus sites in the cytoplasmic domain of reporter assays LRP6. Our results demonstrate that LRP6 can be directly HEK293, UMR-106, and mouse embryonic fibroblast (MEF) phosphorylated by PKA catalytic subunit (Supplemental cells were maintained in DMEM with 10% FCS. Mouse Wnt3a Fig. 6), and PKA inhibitor H89 inhibited PTH-induced conditioned medium (Wnt3a CM) was produced from mouse L phosphorylation of LRP6 as well as its binding to axin cells stably transfected with mouse Wnt3a (American Type Cul- (Fig. 5D,E). These data suggest that the functional bind- ture Collection) and control conditioned medium (Control CM) ing of LRP6 to axin is PKA phosphorylation-dependent. was from nontransfected L cells. IgG, LRP6N-IgG, DKK1, Therefore, both recruitment of LRP6 to PTH1R and Sclerostin, VSVG-LRP6N and Myc-Fz8CRD conditioned media phosphorylation of LRP6 by PKA are involved in PTH- were produced from HEK 293 cells transfected with the indi- induced stabilization of ␤-catenin. The fact that PTH- vidual plasmids. Transfections were carried out using lipofect- amine reagent (Invitrogen). Luciferase assays were carried out in stimulated bone formation can still occur in LRP5-defi- either UMR-106 or HEK 293 cells as described previously (Wan cient mice establishes that LRP5 alone is not essential et al. 2005), with 0.3 µg of TCF-Luc reporter plasmid plus 50 ng for the stimulatory effects of PTH on bone formation of Renilla luciferase plasmid (internal control) per well in the (Sawakami et al. 2006; Iwaniec et al. 2007). In our studies, 12-well plate. Experiments were repeated at least three times PTH directly phosphorylates the intracellular domain of with triplicate for each experiment. LRP6, but not LRP5 (Supplemental Fig. 6). Forskolin, a PKA agonist, induced binding of axin to LRP6, but not to LRP5 (Supplemental Fig. 7), further indicating that LRP6 Cell fractionation, co-IP, and Western blot analysis may act differently from LRP5 in PTH actions in osteo- Cells were harvested in cavitation buffer (5 mM HEPES at pH blasts. The distinct roles of these coreceptors in PTH 7.4, 3 mM MgCl2, 1 mM EGTA, 250 mM sucrose) containing function and whether their mutations cause bone defects protease and phosphatase inhibitors and homogenized by nitro- by altering PTH signaling remain to be investigated. gen cavitation (200 p.s.i., for 5 min) in a cell disruption bomb (Parr Instrument Co.). The cell homogenate was centrifuged twice at 700g for 10 min to pellet the nuclei. The supernatant was further centrifuged at 100,000g (Beckman SW50.1 rotor) for Materials and methods 1 h to separate the membrane and cytosol fractions, and the resulting membrane pellet was washed three times with cavi- cDNA constructs tation buffer before use in the assays (Zhang et al. 1999). IP and PTH1R tagged with HA was subcloned into pCDNA3.1. cDNAs Western blot analysis of cell lysates were performed as de- from human LRP5 (J. Mao et al. 2001) and LRP6 (Tamai et al. scribed previously (Wan et al. 2005).

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Cell suface binding by immunofluorescence colocalization acceptor fluorophores were within FRET range. HEK293 cells assay on coverslips in 35-mm dishes were cotransfected with 0.1 µg of each plasmid. Cells were observed using Leica TCS SP II AOBS Cells were transfected with HA-PTH1R and treated with IgG laser-scanning confocal microscope. An excitation wavelength conditioned medium or LRP6N-IgG conditioned medium for 1 of 405 nm and an emission range of 416–492 nm, and an exci- h followed by PTH (1–84) treatment for 15 min. Cells were then tation wavelength of 514 nm and an emission range of 525–600 washed with PBS, fixed with 4% paraformaldehyde, permeabi- nm were used to acquire images of CFP and YFP, respectively. lized with 0.1% Triton X-100, and incubated with primary an- YFP was photobleached by using full power of the 514 nm line tibody followed by incubation with chromophore-conjugated for 1–2 min. An image of CFP and YFP fluorescence after pho- secondary antibody. Digital pictures were taken using an Olym- tobleaching was obtained by using the respective filter sets. pus, IX TRINOC camera fitted to an Olympus, IX70 Inverted Images were representatives of three experiments. The FRET Research Microscope (Olympus) with objective lenses of Hoff- efficiencies were calculated according to man Modulation Contrast, HMC 10 LWD PL FL, 0.3NA ϱ/1, (OPTICS, Inc.) at room temperature, and processed using Mag- − Donorpost Donorpre FRET Eff% = naFire SP imaging software (Optronics). A Zeiss TCs SP2 sys- Donor tem was used for confocal imaging. The ratios of the number of post cells showing in green to the number of cells showing in red were calculated. For each treatment, 100 cells on each of three Xenopus embryo manipulation different slides were analyzed. RNAs for microinjection were synthesized using SP6 mMessage mMachine in vitro transcription kit (Ambion). RNAs were in- Metabolic 32P labeling and in vivo phosphorylation assays jected into the marginal zone region of two ventral blastomeres of four-cell stage embryos, and the phenotype of the embryos Cells were transfected with expression plasmids and were was observed at the tadpole stages. The doses of RNAs used washed twice with phosphate-free DMEM containing 2% dia- were 200 pg LRP6, 2 pg PTH, and 50 pg PTH1R. lyzed fetal calf serum, incubated in the same medium for 4 h, and then labeled with 1 mCi/mL [32P]orthophosphate (Perkin- Elmer) for an additional 2 h. The 32P-labeled cells were then Animals washed with ice-cold PBS and lysed with radioimmunoprecipi- The experimental protocol was reviewed and approved by the tation assay buffer. VSVG-LRP6 was immunoprecipitated with Institutional Animal Care and Use Committee (IACUC) of anti-VSVG, and the resultant precipitates were separated by University of Alabama at Birmingham. For the experiments 8.5% SDS-PAGE. Gels were dried and exposed to Biomax Mr or in which rats or mice were administered PTH as single-dose MS film (Eastman Kodak Co.). After autoradiographic analysis, injection, 5-mo-old male Sprague Dawley rats (Charles River dried gels were rehydrated with transfer buffer, and transferred Laboratories) or 2-mo-old male C57BL/J6 mice (The Jackson onto PVDF membranes. For equal loading confirmation, the Laboratory) (six per group) were administered a single dose of Plus transfected VSVG-LRP6 was visualized by the ECL Western either vehicle (1 mM acetic acid in sterile PBS) or PTH (1–34) blotting detection system (Amersham Biosciences). (Bachem, Inc.) at 40 µg/kg in a volume of 100 µl. In the mouse model, mouse recombinant Wnt3a (R&D Systems) was injected Quantitative real-time PCR at 25 µg/kg in a volume of 100 µL. All treatments were through bolus intravenous injection via the tail vein. Rats/mice were Cells were homogenized using Trizol reagent (Invitrogen), and sacrificed at 0.5, 2, 8, and 24 h after injection. total RNA was extracted according to the manufacturer’s protocol. cDNA was produced and quantitative real-time PCR were performed in an iCycler real-time PCR machine using iQ SYBR Immunohistochemical analysis of the bone tissue Green supermix (Bio-Rad). Primers are as follows: GAPDH (for- Formalin-fixed femur or tibia tissue sections of 5 µm thickness ward, 5Ј-GGGTGTGAACCACGAGAAAT-3Ј; reverse, 5Ј-CCT from rats or mice were processed with antigen retrieval and TCCACAATGCCAAAGTT-3Ј), Osteocalcin (forward, 5Ј-CT hydrogen peroxide treatment prior to incubation with primary TGGTGCACACCTAGCAGA-3Ј; reverse, 5Ј-CTCCCTCATGT monoclonal antibody specific for ␤-catenin (BD Biosciences), GTTGTCCCT-3Ј), and RANKL (forward, 5Ј- CCAAGATCTC goat polyclonal antibody sclerostin (R&D Systems), or rabbit TAACATGACG-3Ј; reverse, 5Ј-CACCATCAGCTGAAGATA polyclonal phosphorylated LRP6 (Ab1490) for 1 h at room tem- GT-3Ј). The quantity of RANKL and Osteocalcin mRNA in perature or overnight at 4°C. Negative controls were obtained each sample was normalized using the CT (threshold cycle) by replacing the primary antibodies with irrelevant control iso- value obtained for the GAPDH mRNA amplifications. type IgG. Antibody detection was accomplished using the bio- tin-streptavidin horseradish peroxidase (for ␤-catenin and sclerostin) or alkaline phosphatase (for Ab1490) (EnVision Sys- FRET procedure tem, Dako). ␤-Catenin and sclerostin staining was based on per- PTH1R and BMPRII cDNAs were cloned into ECFP-N1, and oxidase (HRP) using DAB as chromogen. Phospho-LRP6 stain- LRP6 and mLRP4T100 cDNAs were cloned into EYFP-N1 ing was based on alkaline phosphotase (AP) using Permanent (Clontech) expression vectors. These vectors were modified by Red as chromogen. The sections were then counterstained with site-directed mutagenesis that prevents the self-dimerization hematoxylin. Isotype-matched negative control antibodies (Bhatia et al. 2005). CFP and YFP were fused at the C termini of (R&D Systems) were used under the same conditions. Osteo- the receptors. Because CFP-PTH1R or CFP-BMPRII (the fluo- blasts/preosteoblasts were observed at the bone surface with rescent FRET donors) is quenched when in the proximity of large, spherical, and basal mononucleus. Only those specimens YFP-LRP6 or YFP-mLRP4T100 (the acceptors), FRET efficiency in which >10% of the cells were stained were considered as can be measured by comparing donor fluorescence pre- and post- positive. In the rat model, numbers of total osteoblasts and photobleaching of the acceptor. An increase in donor fluores- numbers of ␤-catenin- or p-LRP6-positive osteoblasts were cence after acceptor photobleaching indicates that donor and counted in three random high-power fields at metaphysis sub-

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Wan et al. jacent to the epiphyseal growth plates or the diaphyseal hema- disease. Cell 127: 469–480. topoietic bone marrow per specimen, and a total of six speci- Davidson, G., Wu, W., Shen, J., Bilic, J., Fenger, U., Stannek, P., mens in each group were used. In the mouse model, numbers of Glinka, A., and Niehrs, C. 2005. Casein kinase 1 ␥ couples total osteoblasts and numbers of ␤-catenin-positive osteoblasts Wnt receptor activation to cytoplasmic signal transduction. were counted in three random high-power fields in a 2-mm Nature 438: 867–872. square, 1 mm distal to the lowest point of the growth plate in Gensure, R.C., Gardella, T.J., and Juppner, H. 2005. Parathyroid the secondary spongiosa. Numbers of total osteocytes and num- hormone and parathyroid hormone-related peptide, and their bers of sclerostin-positive osteocytes were counted in three ran- receptors. Biochem. Biophys. Res. 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Parathyroid hormone signaling through low-density lipoprotein-related protein 6

Mei Wan, Chaozhe Yang, Jun Li, et al.

Genes Dev. 2008, 22: Access the most recent version at doi:10.1101/gad.1702708

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