Parathyroid hormone signaling through low-density ...genesdev.cshlp.org/content/22/21/2968.full.pdfParathyroid hormone signaling through low-density lipoprotein-related protein 6 Mei

Parathyroid hormone signalingthrough low-densitylipoprotein-related protein 6Mei 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 HospitalBoston and Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USA; 4Department of CellBiology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA

Intermittent administration of PTH stimulates bone formation, but the precise mechanisms responsible forPTH responses in osteoblasts are only incompletely understood. Here we show that binding of PTH to itsreceptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternarycomplex containing PTH, PTH1R, and LRP6 promoted rapid phosphorylation of LRP6, which resulted in therecruitment 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 tophosphorylation of LRP6 and an increase in amount of �-catenin in osteoblasts with a concurrent increase inbone formation in rat. Thus, LRP6 coreceptor is a key element of the PTH signaling that regulates osteoblastactivity.

[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 asthe central regulator of calcium metabolism by directlytargeting bone, kidney, and intestine. The classical conceptof PTH action is that it regulates serum calcium levelsby stimulating bone resorption; however, intermittentadministration of PTH selectively stimulates bone for-mation (Jilka 2007; Potts and Gardella 2007). This latterproperty has been exploited to develop PTH as the onlyFDA-approved anabolic therapy for bone (Tam et al.1982; Hodsman et al. 2005). In the past decade, signifi-cant progress has been made in determining PTH down-stream signaling events. It is now known that PTH bindsto its receptor PTH1R (Juppner et al. 1991; Abou-Samraet al. 1992) and activates the G protein � subunits G�s

and G�q. This leads to production of 3�,5�-cyclic adeno-sine-5�-monophosphate (cAMP) and activation of phos-pholipase C (PLC), which eventually results in the acti-vation of protein kinase A (PKA) and protein kinase C(PKC) (Pierce et al. 2002; Qin et al. 2004; McCudden etal. 2005). Activation of PKA is believed to mediate theanabolic effect of PTH on bone (Armamento-Villareal et

al. 1997; Tintut et al. 1998; Siddappa et al. 2008); how-ever, the precise molecular mechanisms by which PKAmediates PTH responses in osteoblasts remain unresolved.Besides PKA and PKC activation, PTH also regulatesMAPKs (Gensure et al. 2005; Gesty-Palmer et al. 2006),including p42/p44 ERKs, p38, and c-Jun N-terminal ki-nase subtypes. The direction of this regulation and itsmediation by more proximal effectors such as cAMP/PKA and PKC, especially in the case of p42/p44 ERKs,appears to depend on cell type and the concentration ofPTH. Such extensive signaling diversity suggested thepossibility that PTH might interact with more than onetype of receptor in these target tissues that coreceptorsmay modify the signaling, and/or novel signaling path-ways are involved.

Wnts are secreted growth factors that play essentialroles in multiple developmental processes. The impor-tance of this signaling pathway in skeletal biology anddisease has been emphasized recently by the identifica-tion of a link between bone mass in humans and gain- orloss-of-function mutations of the Wnt coreceptor LRP5and the Wnt antagonist, sclerostin (Baron et al. 2006;Balemans and Van 2007; Glass and Karsenty 2007). Wntsactivate different downstream signaling pathways. Ofthese pathways, the canonical or �-catenin pathway hasbeen analyzed most extensively. At the cell membrane,

Corresponding authors.5E-MAIL [email protected]; FAX (205) 975-7414.6E-MAIL [email protected]; FAX (205) 975-7414.Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1702708.

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Wnts bind two different families of receptors to transducethe canonical signal: low-density lipoprotein-related pro-teins (LRP5 or LRP6) and the Frizzled (Fz) receptor familymembers (Pinson et al. 2000; Tamai et al. 2000, 2004;Huelsken and Birchmeier 2001; J. Mao et al. 2001).LRP5/6 acts synergistically with Fzs in the binding ofWnt and activatation of downstream signaling. In theabsence of Wnt, �-catenin is found in a large cytoplasmiccomplex comprising other proteins that promote its in-activation by phosphorylation and its proteasomal deg-radation. This large protein complex includes �-catenin,adenomatous polyposis coli (APC), glycogen synthase ki-nase (GSK)-3�, and axin (Logan and Nusse 2004; Clevers2006). Upon Wnt stimulation, an Fz–LRP6 complex for-mation is induced, which causes LRP6 phosphorylationon PPPS/TP motifs and axin recruitment to the plasmamembrane, resulting in the inhibition of �-catenin phos-phorylation/degradation (He et al. 2004). Stabilized �-catenin protein accumulates in the nucleus and com-plexes with the T-cell factor/lymphoid enhancer factor(TCF/LEF) family of DNA-binding transcription factorsto enhance gene expression (Staal and Clevers 2000;Moon et al. 2002).

Several recent reports that have linked PTH with thedownstream elements of the Wnt pathway provided theframework for our analysis of the PTH-associated signal-ing events. These reports indicated that PTH may regu-late the canonical Wnt pathway by mechanisms that hadnot yet been identified. PTH regulates the levels of ex-pression of key components of the canonical Wnt path-way (Qin et al. 2003; Keller and Kneissel 2005; Kulkarniet al. 2005; Tobimatsu et al. 2006), including the levels of�-catenin and the transcriptional activity of the transcrip-tion factor TCF/LEF (Kulkarni et al. 2005; Tobimatsu et al.2006). Here we found that PTH activates �-catenin sig-naling in osteoblasts both in vitro and in vivo by sharingof a coreceptor with Wnt; however, PTH signal acts in adistinct manner from that of the canonical Wnt signalingpathway in that LRP6 forms a complex with PTH/PTH1R. The ternary complex promoted phosphorylationof LRP6, which recruits axin from the cytoplasma, andinduces �-catenin stabilization. Activation of PKA wasnecessary for the phosphorylation of LRP6 in response toPTH, but not to Wnt. These results reveal a novel sig-naling pathway of PTH and provide alternative interpre-tations of the functions of LRP6 and �-catenin in osteo-blasts, which until now have been considered to affectthe canonical Wnt signaling pathway exclusively.

Results

PTH induces �-catenin stabilization in osteoblasts

To determine whether PTH regulates expression of �-catenin, the effects of PTH on �-catenin levels in ratUMR-106 osteoblastic cells were examined. We foundthat PTH stimulated the transcription of a luciferase re-porter bearing TCF/LEF-binding elements (Fig. 1A), andenhanced the abundance of �-catenin in the cytosol (Fig.1B), whereas the unrelated peptide had no such effects

(data not shown). Similarly, PTH enhanced the levels of�-catenin in the cytosol in a concentration- and time-dependent manner in both mouse calvarial primary pre-osteoblasts (Fig. 1C) and HEK 293 cells (SupplementalFig. 1). �-Catenin accumulation in the cytosol inducedby PTH is so rapid that the effect is unlikely to be me-diated through synthesis of Wnt ligands or sensitizationof Wnt-stimulated signaling. Indeed, Fz8CRD, a com-petitive inhibitor of the Wnt receptor Fz (Hsieh et al.1999), inhibited Wnt3a-elevated, but not PTH-elevated,�-catenin level (Fig. 1D), thus excluding the possibilityof the involvement of Wnts. To test whether PTH stim-ulates �-catenin in vivo, we analyzed the effects PTH(1–34) administered as a single dose to 5-mo-old rats.PTH (1–34) is a C-terminal-truncated synthetic analog ofPTH with an anabolic effect on bone formation in hu-mans (Potts et al. 1971; Tregear et al. 1973). Immunohis-tochemistry analysis of sections of the trabecular boneindicated that PTH induced expression of �-catenin inpreosteoblasts and osteoblasts on the bone surface with-in hours (Fig. 1E,F). At 8 h after injection, positive stain-ing of �-catenin was observed in most osteoblasts(99.08 ± 0.57%) at the metaphysis subjacent to theepiphyseal growth plates and ∼90.24 ± 0.68% of the os-teoblasts at the diaphyseal bone marrow. Similar experi-ments were carried out using 2-mo-old male mice, andsimilar temporal �-catenin expression patterns wereobtained in the mice injected with PTH (SupplementalFig. 2).

LRP6 forms a complex with PTH/PTH1R

The rapid enhancement of �-catenin protein levels inresponse to PTH treatment both in vitro and in vivosuggest that PTH may have a direct effect on the signal-ing components that promote the stabilization of�-catenin. Both LRP5 and LRP6 are key components inactivating �-catenin signaling in canonical Wnt path-way. We attempted to examine whether these two re-ceptors are also important in PTH-stimulated effects inosteoblasts. Recent studies reported that PTH anaboliceffect was not affected in LRP5 KO mice (Sawakami etal. 2006; Iwaniec et al. 2007), indicating that LRP5 is notessential for the stimulatory effects of PTH on bone for-mation. We therefore focused on the function of LRP6 inPTH-activated signaling. We first tested whether inacti-vation of LRP6 would affect PTH-elevated �-cateninlevel by introducing siRNA complementary to lrp6mRNA to the cells. Reduction of LRP6 (Fig. 2A) attenu-ated PTH-stimulated accumulation of �-catenin in thecytosol (Fig. 2B) and TCF/LEF luciferase activity (Fig.2C). PTH-stimulated mRNA expressions of osteocalcinand RANKL, downstream target genes of PTH that arepertinent to osteoblast differentiation, were also inhib-ited by the siRNA (Fig. 2D,E). The results indicate thatLRP6 is a critical mediator for PTH-induced �-cateninstabilization in osteoblasts.

We then examined the possibility that LRP6 may forma ternary complex with PTH and PTH1R as it does withWnt and Fz. Immunoprecipitation (IP) with antibodies to

LRP6 in PTH signaling

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LRP6 from lysates of PTH-treated UMR-106 cells indi-cated that PTH1R formed a complex with endogenousLRP6 in response to PTH in a time-dependent manner(Fig. 2F). Unlike LRP6, PTH did not enhance the bindingof LRP5 to PTH1R, although there is detectable bindingin the absence of PTH (Fig. 2G), indicating that LRP5may play a different role in PTH signaling. The presenceof PTH ligand in the LRP6–PTH1R complex was also

indicated by co-IP. The PTH ligand was immunoprecipi-tated by LRP6 only when both LRP6 and PTH1R werepresent (Fig. 2H), suggesting that PTH forms a ternarycomplex with LRP6 and PTH1R. Further evidence forthe PTH–PTH1R–LRP6 complex formation was ob-tained from PTH-induced close association of PTH1Rwith LRP6 in cells by photobleaching-based fluorescenceresonance energy transfer (FRET) (Fig. 2I–K). As shown in

Figure 1. Activation of �-catenin signaling byPTH in osteoblast-like cells. (A) PTH stimula-tion of a luciferase reporter with TCF/LEF-bind-ing elements (TCF4-Luc) in UMR-106 cells. Cellswere transfected with TCF4-Luc plasmid andtreated with control condition medium (CM) col-lected from culture medium of cells transfectedwith empty vector, CM-containing Wnt3a, andCM with 10−8 M PTH (1–84). Luciferase activitywas measured 8 h after transfection and normal-ized to internal controls as Renilla luciferaseunits (RLU). (*) P < 0.01, n = 3. (B) PTH inducedstabilization of �-catenin in UMR-106 cells.Cells were treated as described in A. Cytosolicand membrane fractions were prepared 1 h aftertreatment for detection of �-catenin levels byWestern blotting analysis. (C) PTH-induced sta-bilization of �-catenin in mouse primary preos-teoblasts. Mouse cavarial preosteoblasts weretreated with vehicle (control), increasing dosagesof PTH (1–84), or 50 ng/mL mouse recombinantWnt3a. Cytosolic and membrane fractions wereprepared 1 h after treatment for detection of �-catenin levels by Western blotting analysis. (D)PTH-elevated �-catenin level was not affectedby Fz8CRD. Mouse cavarial preosteoblasts weretreated 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 prepared1 h after treatment for detection of �-cateninlevels by Western blotting analysis. (E,F) Immu-nohistochemical analysis of �-catenin levels infemur sections from 5-mo-old male rats at theindicated time points after PTH (1–34) injection(40 µg/kg). Representative of sectionsimmunohistochemically stained with antibodyto �-catenin or control IgG and counterstainedwith hematoxylin viewed at lower power (toprow) and higher power (middle and bottomrows). Metaphysis subjacent to the epiphysealgrowth plates (middle row) or diaphyseal hema-topoietic bone marrow (bottom row) were exam-ined. (E) Red asterisks and green asterisks marklocations in the low-power images that areshown in the high-power fields below. �-Catenin-positive osteoblasts were counted in ablinded fashion using OsteoMeasure Software(OsteoMetrics, Inc.) from three random high-power fields per specimens at metaphysis subja-cent to diaphyseal hematopoietic bone marrow,and a total of six specimens in each group wereused. (F) The quantification of �-catenin-positiveosteoblasts is presented as percentage of total os-teoblasts. (*) P < 0.005; (**) P <0.001 (in compari-son with control), n = 6.

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Figure 2J and K, PTH led to increased FRET efficiencybetween CFP-PTH1R and YFP-LRP6, but did not en-

hance the FRET efficiency in either YFP-LRP6 and CFP-BMPRII, BMP type II receptor (Cao and Chen 2005), or

Figure 2. Formation of complexes of LRP6 with PTH–PTH1R. (A) LRP6-specific siRNA reduced the amount of LRP6 protein in HEK293 cells as determined by Western blotting. siRNA directed against GFP was used as an siRNA control. (B) LRP6-specific siRNAreduced PTH-induced �-catenin stabilization in HEK293 cells as determined by Western blotting analysis. (C) LRP6-specific siRNAreduced PTH-stimulated TCF/LEF activity in UMR-106 cells as determined using a luciferase assay. (*) P < 0.01 (in comparison withcontrol), 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 forRNA extraction. (F) Co-IP of endogenous LRP6 with PTH1R in UMR-106 cells. Cells were serum deprived and treated with 10−8 MPTH (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-taggedLRP6 or HA-LRP5 together with PTH1R and treated with 10−8 M PTH (1–84). The PTH1R-associated LRP5 or LRP6 was determinedby 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). TheLRP6-associated PTH ligand was determined by Western blotting analysis of the anti-VSVG immunoprecipitates. (WCL) Whole celllysates. (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 terminusof PTH1R and LRP6, respectively. The interactions of YFP-fused LRP6 with CFP-fused BMPRII or CFP-fused PTH1R with YFP-fusedmLRP4T100 were also examined as controls. (J) Representative confocal imaging of the association of CFP-PTH1R with YFP-LRP6 at5 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 (FRETEff%) before and after photobleaching in the absence or presence of PTH. (*) P < 0.001, compare with unbleached, n = 6; (n.s.) notsignificant compare with unbleached. (L,M) Ventral injection of RNA for PTH (2 pg) plus PTH1R (50 pg) promotes LRP6 (200pg)-induced axis duplication. (n) Numbers of embryos scored.






between CFP-PTH1R and YFP-mLRP4T100, anothermember of the low-density lipoprotein-related proteinsfamily (Li et al. 2000). Thus, LRP6 specifically interactswith PTH1R upon PTH stimulation. The association ofPTH1R with LRP6 is also supported by analysis of themodel of LRP6-mediated secondary axis induction inXenopus, in which PTH enhanced LRP6-induced second-ary axis induction (Fig. 2L,M).

Extracellular domain of LRP6 interacts with PTH1R

To confirm and extend the studies of the LRP6 andPTH1R complex formation, we mapped the region ofLRP6 required for its interaction with PTH1R. PTH1Rwas coexpressed in cells with LRP6, a truncated LRP6containing the extracellular and transmembrane do-mains (LRP6N + T), or the transmembrane and intracel-lular domains (LRP6T + C) for IP assay. Binding ofLRP6T + C to PTH1R could barely be detected, but theLRP6N + T associated with PTH1R as effectively as didfull-length LRP6 (Fig. 3A). The presence of PTH in theLRP6N + T/PTH1R complex further suggested the for-mation of a ternary complex. Moreover, PTH-induceddirect interaction of LRP6N with PTH1R on the cell sur-face was confirmed in an immunefluorescence colocal-ization assay. Immun-colocalization of LRP6N–IgG withPTH1R on the cell surface was increased from 22.8% to82.3% with addition of PTH ligand (Fig. 3B [top tworows], C) whereas binding of IgG to PTH1R was barelydetected (Fig. 3B [bottom two rows], C). Collectively, theresults indicate that PTH induces formation of PTH1R/LRP6 complex through LRP6 extracellular domain.

We then examined whether LRP6N acts as a domi-nant-negative in PTH signaling through LRP6. As ex-pected, LRP6N blocked the PTH-induced association ofendogenous LRP6 with PTH1R (Fig. 3D). LRP6N inhib-ited PTH-, but not LiCl-induced TCF transcriptional ac-tivity (Fig. 3E,F). As LiCl directly inhibits GSK3 kinasein the cytoplasm to stabilize of �-catenin (Stambolic etal. 1996), our results indicate that LRP6N acts upstreamof GSK3 and functions as a dominant-negative in thePTH-activated �-catenin signaling via binding to cellsurface PTH1R. Furthermore, secreted proteins DKK1and sclerostin, also binding to LRP6 at the extracellulardomain (Bafico et al. 2001; B. Mao et al. 2001; Semenovet al. 2001, 2005; Li et al. 2005), disrupted PTH-induced�-catenin accumulation in the cytoplasm (Fig. 3G) andTCF/LEF luciferase activity (Fig. 3H). Thus, PTH-in-duced recruitment of LRP6 through its extracellular do-main is essential in activation of �-catenin signalingpathway.

PTH induces phosphorylation of LRP6 and axinrecruitment in osteoblasts

As phosphorylation of LRP6 at the PPPSP motifs plays acrucial role in activating downstream �-catenin signal-ing by Wnt (J. Mao et al. 2001; Tamai et al. 2004; Dav-idson et al. 2005; Zeng et al. 2005), we examined whetherPTH induces phosphorylation of LRP6 at PPPSP motifs.

Immunoprecipitated LRP6 from extracts of PTH-treatedUMR-106 cells were monitored for their phosphoryla-tion by Western blotting with an antibody that recog-nizes phosphorylated PPPSP motifs (Ab1490) (Tamai etal. 2004). PTH rapidly induced the phosphorylation ofLRP6 at the PPPSP motifs (Fig. 4A). Phosphorylation ofPPPSP motifs is required for axin recruitment from cy-toplasm to LRP6 at cell membrane. PTH also increasedaxin1 level on cell membrane detected by cell fraction-ation assay in primary preosteoblasts (Fig. 4B). Consis-tently, PTH rapidly increased in the binding of axin toLRP6 by co-IP assays (Fig. 4C). Again, Fz8CRD, a com-petitive inhibitor of the Wnt receptor Fz (Hsieh et al.1999), was used to exclude the possibility that these PTHeffects are mediated through promotion of Wnts produc-tion or sensitization of Wnt-stimulated signaling. Fz8CRDinhibited Wnt3a-induced phosphorylation of LRP6 (Fig.4D, lane 8), but did not inhibit the effect of PTH (Fig. 4D,lane 4). In contrast, LRP6N blocked PTH-stimulatedLRP6 phosphorylation (Fig. 4E). The results indicate thatPTH-induced formation of PTH1R–LRP6 complexthrough their extracellular domains is essential for phos-phorylation of LRP6.

Because the levels of �-catenin were increased in os-teoblasts of rats with injection of a single dose of PTH(Fig. 1E,F), we tested whether the amounts of phosphor-ylated LRP6 were enhanced in osteoblasts from the sametissue. Immunostaining with an antibody specific for thephosphorylated PPPSP demonstrated that PTH-stimu-lated phosphorylation of LRP6 in preosteoblasts or os-teoblasts at the surface of trabecular bone (Fig. 4F, sec-ond and third rows), whereas the amount of total LRP6protein remained unchanged (Fig. 4F, first row). The tem-poral pattern of phosphorylation of the PPPSP motifswas similar to that of �-catenin (cf Figs. 1E, 4F [secondand third rows] and Figs. 1F, 4G). Thus, PTH increasesthe abundance of �-catenin in osteoblasts in vivothrough phosphorylation of LRP6.

PKA is required in PTH-, but not in Wnt-activatedLRP6–�-catenin signaling

As the activation of �-catenin signaling by PTH in os-teoblasts seems to be independent of Wnt, we attemptedto investigate the mechanism responsible for the PTHeffects. PTH activates cAMP-dependent PKA, which issufficient for initiation of signals mediating PTH actionin osteoblasts. We assessed whether PKA participates inPTH-activated LRP6–�-catenin signaling. Binding ofintact PTH (1–84) or PTH (1–34) to PTH1R activatesPKA. However, the native C-terminal fragments of PTHbind PTH1R but do not activate PKA (Kronenberg et al.1998; Gensure et al. 2005; Murray et al. 2005). The C-terminal fragments PTH (7–84) and PTH (39–84) weremuch less effective than PTH (1–84) in activating�-catenin signaling (Fig. 5A), altering the stability of �-catenin (Fig. 5B), and inducing axin–LRP6 binding (Fig.5C). These results suggest that cAMP–PKA activation isinvolved in activation of LRP6. The minimum effectsinduced by PTH (7–84) and PTH (39–84) (Fig. 5A–C) may

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be mediated by other signaling components than PKA asthe unrelated peptide of the similar length did not exertsuch effect (data not shown). PKI-(14–22), a specific in-hibitor of PKA-directed phosphorylation, inhibited PTH-induced LRP6 phosphorylation (Fig. 5D). Moreover, thePKA inhibitors, PKI-(14–22) and H89 reduced the bindingof axin to LRP6 (Fig. 5E), �-catenin stabilization (Fig. 5F,

lane 3), and �-catenin-dependent transcription activity(Fig. 5G), further indicating that PKA activity is essentialfor PTH-activated LRP6–�-catenin signaling. However,H89 did not affect Wnt3a-stimulated LRP6 phosphoryla-tion (Fig. 5H), �-catenin stabilization (Fig. 5F, lane 5), and�-catenin-dependent transcription activity (Fig. 5I). Theresults provide further evidence to support the concept

Figure 3. Extracellular domain of LRP6 interacts with PTH1R. (A) Interaction of the N-terminal domain of LRP6 with PTH1R. HEK293 cells were transfected with indicated plasmids and treated with or without 10−8 M PTH (1–84). IP was done to identify theinteraction. (B,C) Surface binding of LRP6N in the presence of PTH treatment. HEK 293 cells were transfected with HA-PTH1R andtreated 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) Representativeimages are shown. (C) IgG CM or LRP6N CM surface binding rates; i.e., the ratios of the number of cells showing in green to thenumber of cells showing in red were calculated. (D) Soluble LRP6N disrupts PTH1R binding to endogenous LRP6. UMR-106 cells weretreated 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 PBSand cell lysates were collected. The endogenous PTH1R-associated LRP6 was determined by Western blotting of the anti-PTH1Rimmunoprecipitates. PI, preimmune serum control. (E) Inhibition of PTH-induced TCF4/LEF activation by soluble LRP6N. UMR-106cells 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 withcontrol), 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 LiCltreatment. Luciferase activity was measured and normalized to internal controls as Renilla luciferase units (RLU). (*) P < 0.001 (incomparison with control), n = 3. (G) DKK1 reduced Wnt3a- or PTH-induced �-catenin stabilization in HEK293 cells as determined byWestern blotting analysis. (H) Inhibition of PTH-induced TCF4/LEF activation by DKK1 and Sclerostin. UMR-106 cells were trans-fected 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 withWnt3a or PTH treatment), n = 3.






that both PTH and Wnt activate LRP6–�-catenin signal-ing in osteoblasts, but do so through distinct pathways.

Discussion

The data reported here demonstrate that PTH activates�-catenin signaling in osteoblasts in vitro and in vivo bydirect recruitment of LRP6 to PTH/PTH1R complex.PTH ligand induces direct interaction of the extracellu-lar domain of LRP6 with PTH1R at the cell surface. As aresult, the PPPSP motifs of LRP6 are phosphorylated andaxin is recruited to the phosphorylated PPPSP motifs,leading to stabilization of �-catenin (Fig. 5J). The extra-cellular domain of LRP6, which inhibits LRP6–PTH1Rcomplex formation, blocks LRP6 phosphorylation and

�-catenin activation. In addition, DKK1 and sclerostin,antagonists of LRP6 by direct binding to the extracellulardomain of LRP6, inhibit PTH-induced axin–LRP6 bind-ing (Supplemental Fig. 3). Thus, PTH-induced recruit-ment of LRP6 to PTH1R is essential in activation of�-catenin signaling. This PTH-activated LRP6–�-cateninpathway is likely a direct effect rather than via a Wntligand-dependent process because the phosphorylation ofLRP6 by PTH is rapid. In addition, Fz8CRD, the com-petitive inhibitor of the Wnt receptor Fz, is not able toblock the action of PTH.

LRP6 is a well-recognized coreceptor for Wnt, and itsfunction in osteoblasts has been considered primarily interms of its effect on Wnt signaling (Baron et al. 2006;Balemans and Van 2007; Glass and Karsenty 2007). It

Figure 4. PTH-induced phosphorylation of LRP6 andaxin recruitment. (A) UMR-106 cells were serum de-prived and treated with 10−8 M PTH (1–84). Phosphor-ylated endogenous LRP6 was identified by Westernblotting analysis of the anti-LRP6 immunoprecipitateswith Ab1490. (B) PTH induced the recruitment of axin1to 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 de-tection of axin1 and LRP6 levels by Western blottinganalysis. (C) LRP6–axin binding in HEK 293 cells. Cellswere transfected with PTH1R, HA-tagged axin, andVSVG-tagged LRP6 and treated with 10−8 M PTH(1–84). The axin-associated LRP6 was determined byWestern blotting of the anti-VSVG immunoprecipi-tates. (D) Fz8CRD does not inhibit PTH-induced LRP6phosphorylation. HEK293 (for lanes 1–4) or MEF (forlanes 5–8) cells were transfected with VSVG-taggedLRP6, treated with control CM or Fz8CRD CM for 2 h,and then with 10−8 M PTH (1–84) or Wnt3a. The phos-phorylated LRP6 was detected by Western blottinganalysis of the anti-VSVG immunoprecipitates withAb1490. (E) UMR-106 cells were serum deprived,treated with control CM or LRP6N CM for 1 h, andthen with 10−8 M PTH (1–84) for another 15 min. Phos-phorylated endogenous LRP6 was identified by Westernblotting analysis of the anti-LRP6 immunoprecipitateswith Ab1490. (F,G) Immunohistochemical analysis ofphosphorylated LRP6 levels in femur sections from5-mo-old male rats at the indicated time points afterPTH (1–34) injection (40 µg/kg). Representative of sec-tions immunohistochemically stained with antibody tototal LRP6 (top row) and phosphorylated LRP6 (Ab1490,middle and bottom rows), and counterstained withhematoxylin viewed at lower power (middle row) andhigher power (bottom row). The metaphyseal area ofdistal femurs was examined. (F) Double asterisks marklocations in the low-power images that are shown inthe high-power fields below. The phosphorylated LRP6-positive osteoblasts were counted in a blinded fashionusing OsteoMeasure Software (OsteoMetrics, Inc.) fromthree random high-power fields per specimens at me-taphysis subjacent to diaphyseal hematopoietic bonemarrow, and a total of six specimens in each group wereused. (G) The quantification of phosphorylated LRP6-positive osteoblasts is presented as percentage of totalosteoblasts. (*) P < 0.005; (**) P < 0.001 (in comparisonwith control), n = 6.

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would be of considerable interest to further determinethe roles of LRP6 and �-catenin in PTH-mediated effects

on the osteoblasts function. We found that knockdownof LRP6 blocked PTH-stimulated phosphorylation of

Figure 5. PKA mediates PTH-, but notWnt3a-activated LRP6–�-catenin signal-ing. (A) Failure of PTH C-terminal ligandsto stimulate TCF4/LEF activity. UMR-106cells were transfected with TCF4-Lucplasmid, treated with 10−8 M PTH ligandsand subject to a luciferase assay. (*)P < 0.05 (in comparison with control),n = 3; (**) P < 0.005 (in comparison withcontrol), n = 3. (B) Failure of PTH C-termi-nal ligands to stabilize �-catenin. UMR-106 cells were treated with PTH ligandsfor 1 h. Cytosolic fractions were preparedfor detection of �-catenin protein by West-ern blot analysis. (C) Effect of PTH C-ter-minal ligands on LRP6–axin binding. Cellswere transfected with indicated plasmidsand treated with PTH ligands. The LRP6-associated axin was determined by West-ern blotting of the anti-VSVG immunopre-cipitates. (WCL) Whole-cell lysates. (D)Inhibition of PTH-induced LRP6 phos-phorylation by PKA inhibitor. Cells weretransfected with VSVG-LRP6 and PTH1R,metabolically labeled with [32P] phos-phate, and treated with PKI-(14–22) for 1 hbefore adding PTH (1–34) for another 15min. VSVG-LRP6 was immunoprecipi-tated from cell lysates. Proteins were re-solved by SDS-PAGE and visualized by au-toradiography. (E) Inhibition of the bindingof axin with LRP6 by PKA inhibitors.Cells were transfected with indicated plas-mids and pretreated with H89 or PKI-(14–22) for 1 h before adding PTH (1–84) foranother 30 min. The LRP6-associated axinwas determined as in C. (F) Inhibition ofPTH-induced, but not Wnt3a-induced �-catenin stabilization by PKA inhibitor.UMR-106 cells were treated with 10−8 MPTH (1–34) or 50 ng/mL recombinantmouse Wnt3a (rmWnt3a) together withvehicle (control) or H89. Cytosolic frac-tions were prepared for detection of�-catenin protein by Western blot analy-sis. (G) Inhibition of PTH-activated TCF4/LEF activity by PKA inhibitors. UMR-106cells were transfected with TCF4-Lucplasmid, treated with 10−8 M PTH to-gether with vehicle (control), H89, orPKI-(14–22), and subject to a luciferaseassay. (*) P < 0.005 (in comparison withPTH treatment group), I = 3. (H) Failure ofPKA inhibitor to inhibit Wnt3a-induced

LRP6 phosphorylation. MEFs were transfected with VSVG-LRP6 and pretreated with increasing doses of H89 for 1 h before addingcontrol CM or Wnt3a CM for another 30 min. The phosphorylated LRP6 was detected by Western blotting analysis of the anti-VSVGimmunoprecipitates with Ab1490. (I) Failure of PKA inhibitors to inhibit Wnt3a-stimulated TCF4/LEF activity. UMR-106 cells weretransfected 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–�-cateninpathway activation in response to PTH. Upon PTH ligand binding to its receptor PTH1R, LRP6 are recruited and form complexes withPTH/PTH1R, thus positioning LRP6 in close proximity with PTH1R. In parallel, PKA is activated downstream from PTH1R andmediate the phosphorylation of LRP6, which leads to the recruitment of axin and stabilization of �-catenin.






CREB at Ser133 and ERK (Supplemental Fig. 4), theknown direct PKA or PKC targets (Tyson et al. 1999;Gesty-Palmer et al. 2006), and importantly, inhibitedPTH-stimulated mRNA expression of osteocalcin andRANKL, osteoblast differentiation markers (Fig. 2D,E).Moreover, we analyzed mouse models of long-term ad-ministration of PTH, which has paradoxical effects inthat PTH stimulates bone formation when injecteddaily, but causes severe bone loss if continually infused(Supplemental Fig. 5A,B). Intermittent, but not continu-ous, administration of PTH causes phosphorylation ofLRP6 and stabilization of �-catenin in osteoblasts(Supplemental Fig. 5C). In addition to stabilization of�-catenin by PTH and Wnts, TGF� also stimulates pro-liferation of osteoprogenitors through stabilization of �-catenin (Jian et al. 2006). Thus, it appears that �-cateninis one of the common mediators of osteoblastic boneformation induced by different extracellular signals.

PTH is also known to inhibit osteocyte expressionof sclerostin (Bellido et al. 2005; Keller and Kneissel2005; Leupin et al. 2007), which is an inhibitor of Wnt-activated �-catenin signaling via directly binding toLRP6 and a negative regulator of bone formation (Win-kler et al. 2003; van Bezooijen et al. 2004; Li et al. 2008).Sclerostin also inhibits PTH-induced axin–LRP6 bind-ing, implying that suppression of sclerostin by PTHwould increase the availability of LRP6 to facilitate PTHsignaling in a positive feedback fashion. The activationof PKA by PTH may occur in parallel to mediate thephosphorylation of LRP6 and axin recruitment for acti-vation of �-catenin (Fig. 5J). Sequence analysis revealsfour PKA consensus sites in the cytoplasmic domain ofLRP6. Our results demonstrate that LRP6 can be directlyphosphorylated by PKA catalytic subunit (SupplementalFig. 6), and PKA inhibitor H89 inhibited PTH-inducedphosphorylation of LRP6 as well as its binding to axin(Fig. 5D,E). These data suggest that the functional bind-ing of LRP6 to axin is PKA phosphorylation-dependent.Therefore, both recruitment of LRP6 to PTH1R andphosphorylation of LRP6 by PKA are involved in PTH-induced stabilization of �-catenin. The fact that PTH-stimulated bone formation can still occur in LRP5-defi-cient mice establishes that LRP5 alone is not essentialfor the stimulatory effects of PTH on bone formation(Sawakami et al. 2006; Iwaniec et al. 2007). In our studies,PTH directly phosphorylates the intracellular domain ofLRP6, but not LRP5 (Supplemental Fig. 6). Forskolin, aPKA agonist, induced binding of axin to LRP6, but not toLRP5 (Supplemental Fig. 7), further indicating that LRP6may act differently from LRP5 in PTH actions in osteo-blasts. The distinct roles of these coreceptors in PTHfunction and whether their mutations cause bone defectsby altering PTH signaling remain to be investigated.

Materials and methods

cDNA constructs

PTH1R tagged with HA was subcloned into pCDNA3.1. cDNAsfrom human LRP5 (J. Mao et al. 2001) and LRP6 (Tamai et al.

2000) tagged with HA and VSVG were subcloned into pCMVand pCS2+, respectively. LRP6N + T (LRP6 N-terminal plus thetransmembrane domain) and LRP6T + C (LRP6 transmembranedomain plus C-terminal) tagged with VSVG were subcloned intopCS2 + . LRP6N + 1479m, LRP6N + 1490m, LRP6N + 1493m, andLRP6N + 1496m were generated by mutagenesis of either theserine (at aa1490 or aa1496) or threonin (at aa1479 or aa1493) toalanine. LRP6N-IgG was generated by fusing the LRP6 extracel-lular domain with IgG (Tamai et al. 2000). si-GFP (Wan et al.2005) and si-LRP6 plasmids were generated using a BS/U6 vec-tor. Briefly, a 22-nucleotide (nt) oligo (oligo 1) corresponding tonucleotides 2981–3002 of the human LRP6 coding region wasfirst inserted into the BS/U6 vector digested with ApaI (blunted)and HindIII. The inverted motif that contains the 6-nt spacerand five Ts (oligo 2) was then subcloned into the HindIII andEcoRI sites of the intermediate plasmid to generate BS/U6/LRP5/6.

Primary osteoblast isolation and culture

Osteoblasts were isolated by digestion of calvaria of newbornmice as decribed (Wang et al. 2007). Briefly, calvaria were incu-bated with 10 mL of digestion solution containing 1.8 mg/mL ofcollagenase type I (Worthington Biochemical Corp.) for 15 minat 37°C under constant agitation. The supernatant was thenharvested, replaced with fresh collagenase, and the digestionrepeated an additional four times. Digestion solutions contain-ing the osteoblasts were pooled together. After centrifugation,osteoblasts were obtained and cultured in �-MEM containing10% FBS, and 1% penicillin/streptomycin at 37°C in a humidi-fied incubator supplied with 5% CO2.

Cell culture, conditioned media, transfection, and luciferasereporter assays

HEK293, UMR-106, and mouse embryonic fibroblast (MEF)cells were maintained in DMEM with 10% FCS. Mouse Wnt3aconditioned medium (Wnt3a CM) was produced from mouse Lcells stably transfected with mouse Wnt3a (American Type Cul-ture Collection) and control conditioned medium (Control CM)was from nontransfected L cells. IgG, LRP6N-IgG, DKK1,Sclerostin, VSVG-LRP6N and Myc-Fz8CRD conditioned mediawere produced from HEK 293 cells transfected with the indi-vidual plasmids. Transfections were carried out using lipofect-amine reagent (Invitrogen). Luciferase assays were carried out ineither UMR-106 or HEK 293 cells as described previously (Wanet al. 2005), with 0.3 µg of TCF-Luc reporter plasmid plus 50 ngof Renilla luciferase plasmid (internal control) per well in the12-well plate. Experiments were repeated at least three timeswith triplicate for each experiment.

Cell fractionation, co-IP, and Western blot analysis

Cells were harvested in cavitation buffer (5 mM HEPES at pH7.4, 3 mM MgCl2, 1 mM EGTA, 250 mM sucrose) containingprotease and phosphatase inhibitors and homogenized by nitro-gen cavitation (200 p.s.i., for 5 min) in a cell disruption bomb(Parr Instrument Co.). The cell homogenate was centrifugedtwice at 700g for 10 min to pellet the nuclei. The supernatantwas further centrifuged at 100,000g (Beckman SW50.1 rotor) for1 h to separate the membrane and cytosol fractions, and theresulting membrane pellet was washed three times with cavi-tation buffer before use in the assays (Zhang et al. 1999). IP andWestern blot analysis of cell lysates were performed as de-scribed previously (Wan et al. 2005).

Wan et al.





Cell suface binding by immunofluorescence colocalizationassay

Cells were transfected with HA-PTH1R and treated with IgGconditioned medium or LRP6N-IgG conditioned medium for 1h followed by PTH (1–84) treatment for 15 min. Cells were thenwashed with PBS, fixed with 4% paraformaldehyde, permeabi-lized with 0.1% Triton X-100, and incubated with primary an-tibody followed by incubation with chromophore-conjugatedsecondary antibody. Digital pictures were taken using an Olym-pus, IX TRINOC camera fitted to an Olympus, IX70 InvertedResearch Microscope (Olympus) with objective lenses of Hoff-man Modulation Contrast, HMC 10 LWD PL FL, 0.3NA �/1,(OPTICS, Inc.) at room temperature, and processed using Mag-naFire SP imaging software (Optronics). A Zeiss TCs SP2 sys-tem was used for confocal imaging. The ratios of the number ofcells showing in green to the number of cells showing in redwere calculated. For each treatment, 100 cells on each of threedifferent slides were analyzed.

Metabolic 32P labeling and in vivo phosphorylation assays

Cells were transfected with expression plasmids and werewashed twice with phosphate-free DMEM containing 2% dia-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 thenwashed with ice-cold PBS and lysed with radioimmunoprecipi-tation assay buffer. VSVG-LRP6 was immunoprecipitated withanti-VSVG, and the resultant precipitates were separated by8.5% SDS-PAGE. Gels were dried and exposed to Biomax Mr orMS film (Eastman Kodak Co.). After autoradiographic analysis,dried gels were rehydrated with transfer buffer, and transferredonto PVDF membranes. For equal loading confirmation, thetransfected VSVG-LRP6 was visualized by the ECLPlus Westernblotting detection system (Amersham Biosciences).

Quantitative real-time PCR

Cells were homogenized using Trizol reagent (Invitrogen), andtotal RNA was extracted according to the manufacturer’s pro-tocol. cDNA was produced and quantitative real-time PCR wereperformed in an iCycler real-time PCR machine using iQ SYBRGreen supermix (Bio-Rad). Primers are as follows: GAPDH (for-ward, 5�-GGGTGTGAACCACGAGAAAT-3�; reverse, 5�-CCTTCCACAATGCCAAAGTT-3�), Osteocalcin (forward, 5�-CTTGGTGCACACCTAGCAGA-3�; reverse, 5�-CTCCCTCATGTGTTGTCCCT-3�), and RANKL (forward, 5�- CCAAGATCTCTAACATGACG-3�; reverse, 5�-CACCATCAGCTGAAGATAGT-3�). The quantity of RANKL and Osteocalcin mRNA ineach sample was normalized using the CT (threshold cycle)value obtained for the GAPDH mRNA amplifications.

FRET procedure

PTH1R and BMPRII cDNAs were cloned into ECFP-N1, andLRP6 and mLRP4T100 cDNAs were cloned into EYFP-N1(Clontech) expression vectors. These vectors were modified bysite-directed mutagenesis that prevents the self-dimerization(Bhatia et al. 2005). CFP and YFP were fused at the C termini ofthe receptors. Because CFP-PTH1R or CFP-BMPRII (the fluo-rescent FRET donors) is quenched when in the proximity ofYFP-LRP6 or YFP-mLRP4T100 (the acceptors), FRET efficiencycan be measured by comparing donor fluorescence pre- and post-photobleaching of the acceptor. An increase in donor fluores-cence after acceptor photobleaching indicates that donor and

acceptor fluorophores were within FRET range. HEK293 cellson coverslips in 35-mm dishes were cotransfected with 0.1 µg ofeach plasmid. Cells were observed using Leica TCS SP II AOBSlaser-scanning confocal microscope. An excitation wavelengthof 405 nm and an emission range of 416–492 nm, and an exci-tation wavelength of 514 nm and an emission range of 525–600nm were used to acquire images of CFP and YFP, respectively.YFP was photobleached by using full power of the 514 nm linefor 1–2 min. An image of CFP and YFP fluorescence after pho-tobleaching was obtained by using the respective filter sets.Images were representatives of three experiments. The FRETefficiencies were calculated according to

FRET Eff% =Donorpost − Donorpre

Donorpost

Xenopus embryo manipulation

RNAs for microinjection were synthesized using SP6 mMessagemMachine in vitro transcription kit (Ambion). RNAs were in-jected into the marginal zone region of two ventral blastomeresof four-cell stage embryos, and the phenotype of the embryoswas observed at the tadpole stages. The doses of RNAs usedwere 200 pg LRP6, 2 pg PTH, and 50 pg PTH1R.

Animals

The experimental protocol was reviewed and approved by theInstitutional Animal Care and Use Committee (IACUC) ofUniversity of Alabama at Birmingham. For the experimentsin which rats or mice were administered PTH as single-doseinjection, 5-mo-old male Sprague Dawley rats (Charles RiverLaboratories) or 2-mo-old male C57BL/J6 mice (The JacksonLaboratory) (six per group) were administered a single dose ofeither vehicle (1 mM acetic acid in sterile PBS) or PTH (1–34)(Bachem, Inc.) at 40 µg/kg in a volume of 100 µl. In the mousemodel, mouse recombinant Wnt3a (R&D Systems) was injectedat 25 µg/kg in a volume of 100 µL. All treatments were throughbolus intravenous injection via the tail vein. Rats/mice weresacrificed at 0.5, 2, 8, and 24 h after injection.

Immunohistochemical analysis of the bone tissue

Formalin-fixed femur or tibia tissue sections of 5 µm thicknessfrom rats or mice were processed with antigen retrieval andhydrogen peroxide treatment prior to incubation with primarymonoclonal antibody specific for �-catenin (BD Biosciences),goat polyclonal antibody sclerostin (R&D Systems), or rabbitpolyclonal phosphorylated LRP6 (Ab1490) for 1 h at room tem-perature or overnight at 4°C. Negative controls were obtainedby replacing the primary antibodies with irrelevant control iso-type IgG. Antibody detection was accomplished using the bio-tin-streptavidin horseradish peroxidase (for �-catenin andsclerostin) or alkaline phosphatase (for Ab1490) (EnVision Sys-tem, Dako). �-Catenin and sclerostin staining was based on per-oxidase (HRP) using DAB as chromogen. Phospho-LRP6 stain-ing was based on alkaline phosphotase (AP) using PermanentRed as chromogen. The sections were then counterstained withhematoxylin. Isotype-matched negative control antibodies(R&D Systems) were used under the same conditions. Osteo-blasts/preosteoblasts were observed at the bone surface withlarge, spherical, and basal mononucleus. Only those specimensin which >10% of the cells were stained were considered aspositive. In the rat model, numbers of total osteoblasts andnumbers of �-catenin- or p-LRP6-positive osteoblasts werecounted in three random high-power fields at metaphysis sub-






jacent to the epiphyseal growth plates or the diaphyseal hema-topoietic bone marrow per specimen, and a total of six speci-mens in each group were used. In the mouse model, numbers oftotal osteoblasts and numbers of �-catenin-positive osteoblastswere counted in three random high-power fields in a 2-mmsquare, 1 mm distal to the lowest point of the growth plate inthe secondary spongiosa. Numbers of total osteocytes and num-bers of sclerostin-positive osteocytes were counted in three ran-dom high-power fields per trabecular bone section or corticalbone section, and a total of six specimens in each group wereused.

Statistical analysis

Data were analyzed using Student’s t-test and are expressed asthe mean ± SEM.

Acknowledgments

Our special thanks go to Fiona Hunter, Thomas Clemens, andTim Nagy for critical reading of the manuscript. We thankDianqing Wu for pCMV-HA2-LRP5 and HA-axin plasmids,Thomas J. Gardella for pcDNA1-PTHR1 and pCDM8-PTH plas-mids, Jen-chih Hsieh for pRK5-IgG plamid, Guojun Bu formLRP4T100 cDNA, and Anna Bafico for specific MAb for LRP6.This work was supported by a grant from National Institutes ofHealth to Xu Cao (DK057501). The bone sections were per-formed at UAB Center for Metabolic Bone Diseases.

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