Modulation of Canonical Wnt Signaling by the Extracellular Matrix Component Biglycan

Modulation of canonical Wnt signaling by the extracellular matrix component biglycan

Agnes D. Berendsena, Larry W. Fishera, Tina M. Kiltsa, Rick T. Owensb, Pamela G. Robeya, J. Silvio Gutkindc, and Marian F. Younga,1

aCraniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892; bLifeCell Corporation, Branchburg, NJ 08876; and cOral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892

Edited by Darwin J. Prockop, Texas A&M Health Science Center, Temple, TX, and approved September 9, 2011 (received for review July 1, 2011) Although extracellular control of canonical Wnt signaling is crucial is stabilized within the cytosol and then translocated to the nu- for tissue homeostasis, the role of the extracellular microenviron- cleus, where it accumulates and activates lymphoid enhancer- ment in modulating this signaling pathway is largely unknown. In binding factor/T cell-specific factor (TCF)-mediated gene tran- the present study, we show that a member of the small leucine- scription. The extracellular microenvironment that regulates rich proteoglycan family, biglycan, enhances canonical Wnt sig- this pathway by modulating the activity of Wnt proteins and/or naling by mediating Wnt function via its core protein. Immuno- their antagonists remains largely unknown. In this regard, precipitation analysis revealed that biglycan interacts with both several different proteoglycans and/or their glycosaminoglycan the canonical Wnt ligand Wnt3a and the Wnt coreceptor low- chain components that are abundantly present at the cell sur- density lipoprotein receptor-related protein 6 (LRP6), possibly face have been reported to stimulate the Wnt/β-catenin path- via the formation of a trimeric complex. Biglycan-deficient cells way. These include free heparan sulfate (4), heparin chains (5–7), treated with exogenous Wnt3a had less Wnt3a retained in cell and heparan sulfate proteoglycans, such as glypican-3 (8) and layers compared with WT cells. Furthermore, the Wnt-induced syndecan-1 (9). levels of LRP6 phosphorylation and expression of several Wnt The canonical Wnt signaling pathway appears to be particu- target genes were blunted in biglycan-deficient cells. Both larly important for bone biology. Mutations in either LRP5 or recombinant biglycan proteoglycan and biglycan core protein LRP6 or their downstream signaling targets have been associated increased Wnt-induced β-catenin/T cell-specific factor–mediated with several bone-related diseases (10). Wnt signaling has been transcriptional activity, and this activity was completely inhibited proposed to control bone mass through diverse mechanisms, by Dickkopf 1. Interestingly, recombinant biglycan was able to including renewal of stem cells (11), stimulation of preosteoblast rescue impaired Wnt signaling caused by a previously described replication (12), and enhancement of osteoblast activity (13). missense mutation in the extracellular domain of human LRP6 Wnt signaling in mature osteoblasts has been shown to control (R611C). Furthermore, biglycan’s modulation of canonical Wnt sig- bone resorption by regulating the expression and secretion of naling affected the functional activities of osteoprogenitor cells, the osteoclastogenesis inhibitor osteoprotegerin (14). Despite including the RUNX2-mediated transcriptional activity and cal- the significant progress made over the past decade, our under- cium deposition. Use of a transplant system and a fracture heal- standing of the role of the extracellular microenvironment in ing model revealed that expression of Wnt-induced secreted modulating Wnt/β-catenin signaling in bone remains limited. In protein 1 was decreased in bone formed by biglycan-deficient this regard, we are particularly interested in one of the members cells, further suggesting reduced Wnt signaling in vivo. We pro- of the small leucine-rich proteoglycan family, biglycan (15), be- pose that biglycan may serve as a reservoir for Wnt in the peri- cause of its pronounced expression in bone, where it is concen- cellular space and modulate Wnt availability for activation of the trated in the pericellular space (16). Previous studies in our canonical Wnt pathway. laboratory have shown that mice deficient in biglycan develop age-related osteopenia resulting from a bone formation defect mineralization | regeneration | skeleton involving a reduced number of osteoblasts at the bone surface (17). In addition, expression of BGN (located on the X chro- anonical Wnt signaling regulates diverse biological processes mosome) may be related to stature in humans, given that Cduring development and tissue homeostasis (1). Further- patients with Turner syndrome (XO) have short stature and low more, defective Wnt signaling plays major roles in diseases such levels of BGN, whereas patients with supernumerary sex chro- as cancer (2) and osteoporosis (3). The extracellular control of mosomes have increased limb length and high levels of BGN Wnt signaling is dependent on both the extracellular storage of (18). In vitro studies revealed that biglycan’s modulation of Wnt proteins and the secretion of Wnt antagonists, such as se- growth factor activities, including both TGF-β (19) and bone creted frizzled-related proteins, Wnt inhibitory factor 1, sclero- morphogenetic protein 2/4 (20) signaling in osteoprogenitor stin, and Dickkopf (Dkk) family members (1). In addition to cells, likely contributes to the mechanism through which biglycan binding signaling molecules, matrix components also can be ac- affects bone formation. Although biglycan and canonical Wnt tively involved in modulating the activation of Wnt/β-catenin signaling are both associated with skeletal tissues, whether this signaling. Activation of the canonical pathway starts by binding proteoglycan plays a direct role in modulating this signaling of Wnt to the frizzled receptor and its coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) or its close relative LRP5. This binding results in LRP6 phosphorylation and Author contributions: A.D.B., L.W.F., J.S.G., and M.F.Y. designed research; A.D.B. and T.M.K. performed research; L.W.F., R.T.O., P.G.R., and J.S.G. contributed new reagents/ activation and recruitment of the Axin complex, which is com- analytic tools; A.D.B., L.W.F., P.G.R., J.S.G., and M.F.Y. analyzed data; and A.D.B. and posed of the scaffolding protein Axin, the tumor-suppressor M.F.Y. wrote the paper. protein adenomatous polyposis coli, casein kinase 1, and glyco- The authors declare no conflict of interest. gen synthase kinase 3, to the receptors. This complex is re- This article is a PNAS Direct Submission. sponsible for the constitutive proteasome-mediated degradation 1To whom correspondence should be addressed. E-mail: [email protected]. β of cytoplasmic -catenin protein in the absence of a Wnt signal. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. However, on activation of the Wnt/β-catenin pathway, β-catenin 1073/pnas.1110629108/-/DCSupplemental.

17022–17027 | PNAS | October 11, 2011 | vol. 108 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1110629108 Downloaded by guest on September 26, 2021 pathway is not known. In the present study, we used bone as the interaction between biglycan and Wnt3a. To confirm a direct a model system to identify a novel player, biglycan, in the ex- interaction between biglycan core proteins and Wnt3a, we tracellular microenvironment that modulates canonical Wnt immunoprecipitated mixtures of recombinant human biglycan signaling. (nonglycanated) with recombinant human Wnt3a using antibodies specific to biglycan, followed by immunoblotting with Results biglycan- and Wnt3a-specific antibodies. We found that biglycan Biglycan Interacts with Wnt3a and LRP6 via Its Core Protein. Given core proteins directly interacted with Wnt3a (Fig. 1B). that biglycan was previously reported to bind growth factors, Because Wnt3a was previously reported to bind and activate such as TGF-β,wefirst assessed whether biglycan could directly canonical Wnt signaling through the LRP6 receptor (22), we interact with Wnt3a, a prototypical, well-characterized activator hypothesized that biglycan also could interact with this receptor of the β-catenin-dependent canonical pathway (21). Immuno- to locally enhance Wnt function. Immunoprecipitation of cell precipitation of mixtures of recombinant human biglycan (with lysates of HEK-293T cells overexpressing V5-tagged human and without glycanation) with recombinant human Wnt3a using biglycan and V5-tagged human LRP6 using antibodies specific antibodies specific to the C terminus of Wnt3a pulled down for biglycan or LRP6, followed by immunoblotting of pull-down Wnt3a only in the absence of biglycan (Fig. 1A, Left). This result lysates with V5-specific antibodies, revealed that biglycan indeed suggests that biglycan blocks the ability of the antibodies against was bound to LRP6 (Fig. 1 C and D). Furthermore, we found Wnt3a to pull down Wnt3a, thus indirectly suggesting that that immunoprecipitation of cell lysates using antibodies specific biglycan interacts with Wnt3a at the C terminus. This suggestion for LRP6 resulted in the pull-down of LRP6, biglycan, and Wnt3 fi is further supported by the nding that antibodies to the N-ter- when cells overexpressed HA-tagged human/mouse Wnt3 in minal region of Wnt3a pulled down Wnt3a from the mixture addition to V5-tagged human biglycan and V5-tagged human of recombinant biglycan (nonglycanated) and Wnt3a (Fig. 1A, LRP6 (Fig. 1E). The possible formation of a trimeric complex of Right). Because biglycan was not found in the pull-down of these proteins suggests a role for biglycan in the presentation of Wnt3a, we assumed that the antibodies against Wnt3a affected Wnt to the LRP6 receptor, thereby modulating the canonical Wnt pathway.

Biglycan-Deficient Calvarial Cells Retain Less Wnt3a in Cell Layers and Have Reduced Wnt-Induced Phosphorylation of LRP6 and β-Catenin/ TCF-Mediated Transcriptional Activity. Immunoblot analysis revealed that cells obtained from calvaria of newborn WT mice had increased levels of biglycan synthesis with time in culture (Fig. 2A). Treatment of lysate proteins with chondroitinase ABC resulted in increased detection of biglycan core proteins, indicating synthesis of both glycanated and nonglycanated biglycan core proteins by the calvarial cells (Fig. 2A). Because biglycan directly interacts with Wnt3a, we next determined whether levels of Wnt3a retained in the calvarial cell layers and their surrounding matrix were decreased by the absence of biglycan. Immunoblotting revealed that Wnt3a was decreased by ∼60% in the cell layers of biglycan-deficient cells (Fig. 2B), suggesting that the proteoglycan may serve to retain Wnt3a at the cell surface/ pericellular environment. We then determined whether reduced levels of Wnt3a in the calvarial cell layer in the absence of biglycan affected the phosphorylation of LRP6 at S1490 (pLRP6S1490), an event that is required for full induction of β-catenin activity (23). Immunoblotting showed that the Wnt3a- induced pLRP6S1490 appeared less in biglycan-deficient cells compared with WT cells (Fig. 2B), suggesting reduced activation of the canonical Wnt pathway in the absence of biglycan. We next assessed whether biglycan could modulate the canonical Wnt pathway at the transcriptional level. Because the crux of the canonical mechanism is known to be the nuclear CELL BIOLOGY accumulation of β-catenin (24), we analyzed whether Wnt3a could induce a translocation of β-catenin from the cytoplasm to the nucleus in WT and biglycan-deficient calvarial cells. Immu- nofluorescence analysis showed that Wnt3a induced a nuclear Fig. 1. Biglycan interacts with Wnt3a and LRP6 via its core protein. (A and β fi B) Immunoblotting with biglycan or Wnt3a antibodies of immunoprecipi- localization of -catenin in both WT and biglycan-de cient cell tates (IPs) collected using biglycan and Wnt3a antibodies after mixing of types (Fig. S1), indicating activation of the canonical Wnt recombinant human biglycan (with and without glycanation) with pathway. However, there was no clear difference between the recombinant human Wnt3a protein. (A) IP using antibodies to the C termi- cell types in the number of cells demonstrating Wnt-induced nus of Wnt3a (Left) or with antibodies to the N-terminal region of Wnt3a translocation of β-catenin. In the nucleus, β-catenin complexes (Right). (B) IP using antibodies to Bgn. (C and D) IPs of cell lysates of HEK- with TCF/lymphoid enhancer-binding factor transcription fac- 293T cells overexpressing V5-tagged human biglycan and V5-tagged human tors, thereby regulating the expression of genes controlling LRP6 using antibodies for biglycan (C) or LRP6 (D), followed by immuno- proliferation and cell cycle progression (24). Real-time RT-PCR blotting of pull-down lysates with V5-specific antibodies. (E) IPs of cell lysates of HEK-293T cells overexpressing V5-tagged human biglycan, V5- revealed that the Wnt3a-induced expression of several Wnt tagged human LRP6, and HA-tagged human/mouse Wnt3 using antibodies target genes encoding Axin2, Cyclin D1, WISP1, and MMP14 for LRP6, followed by immunoblotting of pull-down lysates with V5- and HA- was blunted in the absence of biglycan, with a 25–60% reduction specific antibodies. after 4 d of Wnt3a treatment (Fig. 2C).

Berendsen et al. PNAS | October 11, 2011 | vol. 108 | no. 41 | 17023 Downloaded by guest on September 26, 2021 Considering our finding that biglycan affected the Wnt-associated signaling cascade but had no effect on its own, we spec- ulated that the mechanism by which biglycan enhances Wnt signaling involves direct mediation of Wnt function. To test this hypothesis, we assessed whether the Wnt signaling antagonist Dkk1, which is known to bind LRP6 with high affinity and pre- vent formation of the Frizzled-Wnt-LRP6 complex in response to Wnts (27), could inhibit biglycan’s effect on Wnt signaling. Recombinant Dkk1 dose-dependently inhibited Wnt3a-induced β-catenin reporter activity, including biglycan-enhanced activity (Fig. 3C), indicating that biglycan directly mediates Wnt function. To test whether biglycan modulates extracellular Wnt function, we next examined intracellular activation of the Wnt pathway in β-catenin reporter cells in the presence or absence of biglycan core proteins by use of lithium chloride (LiCl), an inhibitor of glycogen synthase kinase-3β, whose function is to target β-catenin for destruction (28). Inhibition of glycogen synthase kinase-3β prevents the constitutive proteasome-mediated degradation of cytoplasmic β-catenin, resulting in the accumulation and nuclear translocation of β-catenin, where it induces β-catenin/TCF-mediated transcriptional activity. Recombi- nant biglycan core proteins had no effect on the dose-dependent LiCl-induced β-catenin reporter activity (Fig. 3D), confirming that biglycan mediates Wnt signaling in the extracellular environment and does not affect intracellular events.

Biglycan Core Protein Rescues Impaired Wnt Signaling Caused by a Missense Mutation in the Extracellular Domain of Human LRP6 (R611C). A missense mutation in the extracellular domain of LRP6 was previously linked to a family with early coronary dis- fi Fig. 2. Biglycan-de cient calvarial cells retain less Wnt3a in cell layers and ease and osteoporosis in humans (29). This missense mutation, demonstrate both reduced Wnt-induced phosphorylation of LRP6 and which substitutes cysteine for arginine (R611C) at a highly reduced β-catenin/TCF-mediated transcriptional activity. (A) Immunoblot analysis for biglycan core protein after treatment of an equal volume of ly- conserved residue of an epidermal growth factor-like domain sate proteins obtained from WT calvarial cell layers with chondroitinase ABC. (Fig. 4A), was shown to impair Wnt signaling in vitro. Because (B) Immunoblot analysis for Wnt3a and phosphorylation of LRP6 (pLRP6S1490) recombinant biglycan was able to enhance Wnt signaling medi- on protein extracts obtained from WT and biglycan-deficient calvarial cells ated by LRP6, we hypothesized that biglycan could rescue de- after treatment with Wnt3a. (C) Relative expression levels of Wnt target fective Wnt signaling caused by the LRP6-R611C mutation. genes measured by real-time RT-PCR in WT and biglycan-deficient calvarial Overexpression of LRP6-R611C in β-catenin reporter cells cells after treatment with Wnt3a. Values represent mean relative expression ± resulted in an ∼30–65% reduction in β-catenin reporter activity SD (n = 3), shown as Wnt3a-induced fold change in gene expression. Gene (depending on the Wnt3a concentration) compared with cells expression was normalized to untreated controls at each time point (in- overexpressing LRP6-WT (Fig. 4 B and C). Recombinant bi- dicated by the dotted line). *P < 0.05; **P < 0.01; ***P < 0.001. glycan core protein increased the Wnt3a-induced reporter activity in a dose-dependent manner (Fig. 4B), but just to a limited Both Biglycan Proteoglycan and Biglycan Core Protein Enhance Wnt- extent in cells expressing only endogenous LRP6 (indicated by Induced β-Catenin/TCF-Mediated Transcriptional Activity via LRP6, LRP6-MT). Immunoblotting (using antibodies to V5-fusion and This Activity Is Inhibited by Dkk1. To further elucidate the peptide) showed comparable expression levels of LRP6-R611C β mechanism by which biglycan affects canonical Wnt signaling, we and LRP6-WT in the -catenin reporter cells. Furthermore, immunoprecipitation using antibodies to biglycan (Fig. 4D) and analyzed the effect of recombinant biglycan (25) on Wnt sig- LRP6 (Fig. 4E), followed by detection with V5-specific anti- naling in a β-catenin reporter cell line (HEK-293T cells stably ’ fl bodies, revealed that biglycan s interaction with LRP6 was not transfected with the SuperTOP ash reporter plasmid, in which affected by the R611C missense mutation in the LRP6 receptor. fi fl several TCF4-binding repeats drive re y luciferase expression) Given the finding that biglycan enhanced Wnt-mediated sig- (26). Recombinant biglycan alone had no effect on the reporter naling in cells, we assessed whether biglycan would affect Wnt- activity, but in the presence of increasing amounts of Wnt3a, induced osteogenic commitment of osteoprogenitor cells. Re- both recombinant biglycan proteoglycan and biglycan core pro- combinant biglycan core protein stimulated the Wnt-induced teins increased the Wnt-induced β-catenin reporter activity in the reporter activity driven by RUNX2-responsive promoter se- cells overexpressing LRP6-WT (Fig. 3A). Biglycan core protein quences in murine preosteoblast MC3T3-E1 cells overexpressing was more effective than biglycan proteoglycan in enhancing Wnt LRP6-WT (Fig. 4F). signaling, indicating that the functional sites likely reside in the fi core protein, and that the glycosaminoglycan chains possibly Biglycan-De cient Cells Have Reduced Wnt-Induced Mineralization and Show Less Wnt-Induced Secreted Protein 1 Expression in Bone interfere with biglycan’s effect on Wnt signaling. Recombinant Formed in Transplants and During Fracture Healing. We further biglycan proteoglycan and biglycan core proteins also increased addressed the possibility that biglycan’s modulation of canonical β the Wnt-induced -catenin reporter activity in cells over- Wnt signaling could be involved in controlling functional activities expressing LRP6 mutant (LRP6-MT; in which all serine/threo- of osteoprogenitor cells. To directly assess the Wnt-induced ef- nine residues in all PPPS/TP motifs were replaced with alanine) fects on osteogenic differentiation of bone marrow stromal cells (Fig. 3B), which most likely is mediated by endogenously ex- (BMSCs), we analyzed the levels of calcium deposition by WT and pressed LRP5/6 in these cells. biglycan-deficient cells in cultures. Alizarin red staining revealed

17024 | www.pnas.org/cgi/doi/10.1073/pnas.1110629108 Berendsen et al. Downloaded by guest on September 26, 2021 Fig. 3. Both biglycan proteoglycan and biglycan core protein enhance Wnt-induced β-catenin/TCF-mediated transcriptional activity via LRP6, and this activity is inhibited by Dkk1. (A) Effect of biglycan proteoglycan (PG) and biglycan core protein on Wnt3a-induced reporter activity in β-catenin reporter cells overexpressing LRP6-WT or inactive LRP6-MT. Values represent mean relative luminescence ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, control (no biglycan) vs. biglycan treatment. (B) Effect of biglycan PG and biglycan core protein on Wnt3a- induced reporter activity in β-catenin reporter cells overexpressing LRP6-MT. Values represent mean relative luminescence ± SD (n = 3). *P < 0.05; **P < 0.01, control (no biglycan) vs. biglycan treatment. (C) Effect of Dkk1 on Wnt3a- induced reporter activity in β-catenin reporter cells in the presence of biglycan core protein. Values represent mean relative luminescence ± SD (n = 3). *P < 0.05, +Wnt3a/−biglycan core vs. +Wnt3a/ +biglycan core at 0 and 10 ng/mL Dkk1. (D) Effect of LiCl-induced β-catenin reporter activity in β-catenin reporter cells in the presence of biglycan core protein. NaCl was used for control purposes. Values represent mean relative luminescence ± SD (n =3).

that biglycan-deficient BMSCs had less Wnt-induced calcium becular structures in the absence of biglycan. Interestingly, the deposition after 21 d of culture and demonstrated no significant biglycan-deficient transplants had marrow spaces that contained increase in response to exogenously added Wnt3a (Fig. 5A). To less fat but increased numbers of hematopoietic cells (Fig. 5C), assess whether biglycan also modulates canonical Wnt signaling in similar to what is found in biglycan-deficient mice (17). Immu- osteoprogenitor cells in an in vivo context, we seeded WT and nostaining for Wnt-induced secreted protein 1 (WISP1), a pro- biglycan-deficient BMSCs in gelatin sponges, transplanted them tein known to be up-regulated by Wnt (30), revealed lower s.c. into immunocompromised mice, and harvested them after WISP1 expression levels in the bone of transplants in the ab- 6 wk. Micro-CT analysis revealed significantly lower trabecular- sence of biglycan (Fig. 5D), suggesting reduced Wnt signaling in like bone formation and bone mineral density in the biglycan- biglycan-deficient osteoprogenitor cells. To further address deficient transplants (Fig. 5B). H&E-stained sections through the biglycan’s effect on canonical Wnt signaling in vivo, we analyzed center of the transplants confirmed the reduced levels of tra- WISP1 expression levels during bone formation in a fracture CELL BIOLOGY

Fig. 4. Biglycan core protein rescues impaired β-catenin/TCF-mediated transcriptional activity caused by the R611C missense mutation in LRP6 and stimulates Wnt-induced RUNX2 transcriptional activity. (A) Location of the R611C mutation in LRP6. (B) Effect of biglycan core protein on Wnt3a-induced β-catenin reporter activity in β-catenin reporter cells overexpressing LRP6-WT or LRP6-R611C. Values represent the mean relative luminescence ± SD (n = 3). *P < 0.05; **P < 0.01, LRP6-R611C (no biglycan) vs. LRP6-R611C (biglycan-treated) and LRP6-WT (no biglycan). (C) Wnt3a-induced β-catenin reporter activity in cells expressing only endogenous LRP6 (indicated by LRP6-MT) compared with cells overexpressing LRP6-WT or LRP6-R611C. Values represent mean relative luminescence ± SD (n = 3). *P < 0.05; **P < 0.01. (D and E) Immunodetection of the V5 tag (fused with biglycan, LRP6-WT, and LRP6-R611C) of IPs collected using biglycan (D) or LRP6 (E) antibodies after harvesting of β-catenin reporter cells overexpressing biglycan, LRP6-WT, LRP6-R611C, or a combination of these. (F) Effect of biglycan core protein on Wnt3a-induced p6OSE2-luc reporter activity in MC3T3-E1 cells. Values represent mean relative luminescence ± SD (n =6). **P < 0.01.

Berendsen et al. PNAS | October 11, 2011 | vol. 108 | no. 41 | 17025 Downloaded by guest on September 26, 2021 These data indicate that biglycan’s modulation of canonical Wnt signaling might be involved in the osteogenic differentiation of osteoprogenitor cells, which may ultimately affect the bone formation process. Discussion The present study describes a mechanism by which biglycan in the extracellular microenvironment contributes to the control of the canonical Wnt signaling pathway. We have identified that this small leucine-rich proteoglycan is a modulator of Wnt signaling through a direct interaction of its core protein with Wnt ligand and its coreceptor, LRP6. In the absence of biglycan, less Wnt was retained in cell layers, apparently resulting in reduced activation of the Wnt/β-catenin pathway. Recombinant biglycan’s enhanced Wnt-induced signaling through LRP6 was mediated by its core protein. The enhanced Wnt-induced signaling could be completely inhibited by the extracellular Wnt inhibitor Dkk1, confirming biglycan’s extracellular role in mediating Wnt function. Although we used bone as a model system in this study, our findings may have important implications for two reasons, given biglycan’s wide tissue distribution (15, 16, 32). First, biglycan is likely to play a physiological role in the control of Wnt/β-catenin signaling. Second, biglycan has potential as a valuable biological agent in the treatment of Wnt-related diseases. Although the precise mechanism through which biglycan enhances Wnt/β-catenin signaling is not yet clear, we expect that biglycan’s core protein plays a crucial role in this process by mediating Wnt function through the formation of a possible trimeric complex with both Wnt and LRP6. This supposition is supported by our findings that biglycan alone had no effect on Wnt/β-catenin signaling, and that the extracellular Wnt inhibitor Dkk1 could completely inhibit biglycan’s effect on Wnt signaling. Possible mechanisms through which biglycan affects the canonical Wnt pathway through LRP6 include modulation of Wnt’s availability to bind to the coreceptor complex of LRP6 and Frizzled proteins and/or participation in the formation of this receptor complex. Further studies are needed to elucidate this mechanism in more detail. In light of biglycan’s ability to mediate Wnt3a function, it is tempting to speculate that biglycan also may bind and mediate the function of other canonical (such as Wnt1) or noncanonical Wnt proteins and/or Wnt antagonists. Fig. 5. Biglycan-deficient cells have reduced Wnt-induced mineralization This possibility is intriguing, given that other Wnt-binding pro- in cultures and show less WISP1 expression in trabecular structures formed teoglycans, such as glypicans, are able to bind several Wnt in transplants and during fracture healing. (A) Wnt-induced calcium de- ligands (5, 33, 34). Biglycan also may modulate Wnt signaling not position assessed by alizarin red staining of WT and biglycan-deficient only through LRP6, but also via other Wnt coreceptors, such as BMSCs. Values represent mean relative alizarin red staining ± SD (n =3). LRP5, Derailed/RYK, ROR, Frizzled, and FRL1/crypto; how- < < *P 0.05; **P 0.01. (B)Micro-CTimagesofWT(Upper) and biglycan- ever, attributing such potential roles for biglycan remains spec- deficient (Lower) transplants and quantitative assessment of the bone mineral density of trabecular structures. (C) H&E staining of sections of WT ulative at this point. (Upper) and biglycan-deficient (Lower) transplants. (D) Immunostaining for The role of the extracellular microenvironment in regulating WISP1 on sections through the center of WT (Upper) and biglycan-deficient Wnt signaling is likely to be highly specific to maintain tissue (Lower) transplants. Arrows indicate positive staining in the trabecular-like homeostasis. In this respect, closely related matrix proteins may bone formed in the transplants. (E) H&E staining of sections of WT (Upper) have differential effects on this signaling pathway. These differ- and biglycan-deficient (Lower) femurs at 14 d postfracture. White arrows ential effects are illustrated by the previously reported finding indicate the fracture area. (F) Immunostaining for WISP1 on sections that decorin, a closely related family member of biglycan (15), through WT (Upper) and biglycan-deficient (Lower)femursat14dpost- also affects β-catenin signaling (35). But decorin functions by fracture showing WISP1 expression surrounding the woven bone formed interacting with the Met receptor, which, in contrast to biglycan, in the callus area. (G) Relative WISP1 gene expression levels measured by β fi leads to decreased -catenin/TCF-mediated transcriptional ac- real-time RT-PCR in WT and biglycan-de cient callus areas of femurs at β 7 d postfracture. Values represent mean relative expression ± SD (n =4). tivity through suppression of intracellular levels of -catenin in *P < 0.05. tumor cells. Although increased decorin expression levels in the absence of biglycan in osteoprogenitor cells has been reported (20), total β-catenin levels were not affected in these cells, healing model, given that WISP1 was previously shown to making a substantial role for decorin in β-catenin signaling in be expressed during skeletal development and fracture repair these cells unlikely. (31). WISP1 expression was reduced in woven bone in the callus Recombinant human biglycan (rhBGN) could be a potentially area of femurs obtained from biglycan-deficient mice at 14 interesting therapeutic agent in treating Wnt-related diseases by d postfracture (Fig. 5 E and F). Moreover, WISP1 gene ex- locally enhancing canonical Wnt signaling. Systemically delivered pression levels were significantly decreased in callus areas of rhBGN was reported to reduce muscle pathology in an mdx femurs of biglycan-deficient mice at 7 d postfracture (Fig. 5G). mouse model of Duchenne muscular dystrophy (36). RhBGN

17026 | www.pnas.org/cgi/doi/10.1073/pnas.1110629108 Berendsen et al. Downloaded by guest on September 26, 2021 was shown to be well tolerated in animals dosed for as long as In conclusion, we have identified biglycan as component in the 3 mo, indicating its potential value for therapy. Although the extracellular microenvironment that modulates canonical Wnt present study only highlights a role for rhBGN in enhancing signaling. Our findings shed new light on the thus-far limited Wnt/β-catenin signaling in cells in culture, we propose that knowledge of the tight extracellular control of signaling pathways rhBGN may exert similar effects on Wnt activities in cells in vivo. in tissue homeostasis. Furthermore, understanding the mecha- This information may lead to new treatment modalities for Wnt- nisms through which matrix proteins modulate canonical Wnt related diseases. In this respect, it is of crucial importance to signaling may lead to new therapeutic approaches for the tar- assess the contribution of defective Wnt signaling to the char- geting and cure of Wnt-related diseases. acteristic features of the disease. For example, a previously characterized family harboring a missense mutation in LRP6 Materials and Methods substituting cysteine for arginine at a highly conserved residue of The LRP6-R611C mutant construct was generated by site-directed muta- an epidermal growth factor-like domain (R611C), which has genesis of the WT LRP6 sequence using the QuikChange II XL Site-Directed been linked to impaired Wnt signaling in vitro (29). Additional Mutagenesis Kit (Agilent) in accordance with the manufacturer’s instruc- studies by that group revealed that the mechanisms by which the tions. The following oligonucleotides were used to generate the mutant mutation imparts its effects are likely to be Wnt-independent, at construct: forward, 5′-ctcagggcctttgctgtgcttgc-3′; reverse, 5′-gcaagcacagca- least in part. The mechanisms of hypercholesterolemia in LRP6- aaggccctgag-3′. fi R611C mutation carriers likely involve altered receptor af nity Details regarding animals, cell culture, plasmids, DNA transfections and for LDL and its subcellular localization, along with reduced reporter assays, immunoprecipitation, immunoblotting and immunofluo- cellular LDL clearance (37). Furthermore, atherosclerosis in rescence analysis, quantitative RT-PCR, primer sets (Table S1), in vitro calcium these mutation carriers may be due to enhanced vascular smooth accumulation, in vivo osteogenesis assay, the fracture healing model, and muscle cell proliferation in response to PDGF (38). Thus, this statistical analysis are provided in SI Materials and Methods. may indicate the involvement of signaling pathways other than Wnt in the contribution of this mutation to osteoporosis, as was ACKNOWLEDGMENTS. We thank Li Li, Azusa Maeda, Emily Pinnow, and uncovered in hypercholesterolemia and atherosclerosis defects. Brian Sworder, Craniofacial and Skeletal Diseases Branch, National Institute for Dental and Craniofacial Research, for technical assistance. This research However, we cannot rule out the possibility that a Wnt-mediated was supported in part by the Division of Intramural Research, National mechanism that affects the proliferation or apoptosis of osteo- Institute for Dental and Craniofacial Research, of the Intramural Research progenitor cells may be involved in the effects of this mutation. Program of the National Institutes of Health.

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