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The handle http://hdl.handle.net/1887/20733 holds various files of this Leiden University dissertation.
Author: Zhang, Xiaofei
Title: Molecular mechanisms of novel regulators in cytokine signal transduction Issue Date: 2013-04-10
Chapter 5
LRP8 Mediates Wnt/β-Catenin Signaling and Controls Osteoblast Differentiation
Juan Zhang,* Xiaofei Zhang,* Long Zhang, Fangfang Zhou, Maarten van Dinther, and Peter ten Dijke
Department of Molecular Cell Biology and Center for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands
*JZ and XZ contributed equally to this work
Address correspondence to: Peter ten Dijke, PhD, Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Center
Journal of Bone and Mineral Research 2012 Oct; 27(10):2065-74
Abstract
The Wnt/β-catenin signaling pathway plays a pivotal role in regulating osteoblast differentiation and bone formation. Here, we identify low-density lipoprotein (LDL) receptor-related protein 8 (LRP8) as a positive regulator of Wnt/β-catenin signaling. In a small interfering RNA (siRNA) screen, LRP8 was shown to be required for Wnt/β-catenin–
induced transcriptional reporter activity. We found that ectopic expression of LRP8 increased Wnt-induced transcriptional responses, and promoted Wnt-induced β-catenin accumulation. Moreover, knockdown of LRP8 resulted in a decrease in β-catenin levels and suppression of Wnt/β-catenin–induced Axin2 transcription. Functional studies in KS483 osteoprogenitor cells showed that LRP8 depletion resulted in impaired activation of endogenous Wnt-induced genes and decreased osteoblast differentiation and mineralization, whereas LRP8 ectopic expression had the opposite effect. These results identify LRP8 as a novel positive factor of canonical Wnt signaling pathway and show its involvement in Wnt-induced osteoblast differentiation.
Keywords: β-catenin; LRP8; osteoblast differentiation; signal transduction; Wnt
Introduction
Canonical Wnt signaling has been shown to play key roles in embryonic development and maintaining homeostasis in adults (Angers and Moon, 2009; Niehrs and Shen, 2010). Wnts control many different cellular functions, including stem cell self-renewal and differentiation (Beffert et al., 2006; Clevers, 2006). Wnts signals are transduced across the plasma membrane via seven transmembrane domain high-affinity receptors of the Frizzled (Fzd/Fz) family (Angers and Moon, 2009). Frizzled proteins belong to the G protein coupled receptors, which mediate both canonical Wnt/β-catenin signaling and β-catenin–
independent intracellular Wnt signaling (Angers and Moon, 2009). Low-density lipoprotein (LDL) receptor-related proteins 5 and 6 (LRP5/6), members of the low-density lipoprotein receptor (LDLR) family, are single-pass co-receptors that interact directly, albeit poorly, with Wnts, and form a Wnt-induced ternary complex with Fz that mediates β-catenin signaling (Angers and Moon, 2009; Cong et al., 2004; He et al., 2004; Tamai et al., 2000;
Tolwinski et al., 2003). LRP5/6 have an overall homology of 71% (Milat and Ng, 2009).
They are essential for Wnt/β-catenin signaling and are part of the LDL receptor family with a number of distinct conserved motifs, including complement-like cysteine-rich repeat motif (the LDL repeat), epidermal growth factor (EGF) receptor-like repeats, tyrosine- tryptophan-threonine- aspartate (YWTD) repeats that form a β-propeller structure critical for proper ligand binding, and proline-proline-prolineserine-serine-proline (PPPSP) motifs that are phosphorylated and important for downstream pathway activation (Niehrs and Shen, 2010; Rey and Ellies, 2010; Tamai et al., 2004; Zeng et al., 2005).
In the absence of Wnt ligands, β-catenin forms a multiprotein complex with casein kinase 1, glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coli (APC), and Axin, which drive β-catenin ubiquitination and degradation (Ikeda et al., 1998; Liu et al., 2002). In the presence of Wnt ligands, the Fz/LRP5/6 complex activates heterotrimeric G proteins and Dishevelled, followed by recruitment of the Axin-GSK3 complex and formation of signalosomes (Niehrs and Shen, 2010). LRP5/6 becomes phosphorylated at multiple sites by Casein kinase 1γ, upon which β-catenin is released from the degradation complex, and accumulates and then translocates to the nucleus. Nuclear β-catenin can form a complex with T-cell factor/lymphoid enhancer factor (TCF/LEF) family transcriptional factors and initiate transcriptional responses (Behrens et al., 1996; Novak and Dedhar, 1999).
Increasing evidence shows that Wnt/β-catenin signaling plays a critical role in osteoblast differentiation and bone formation (Milat and Ng, 2009). Conditional loss of β- catenin function during intramembranous and endochondral ossification induced osteochondro progenitor cells not to differentiate into osteoblasts but instead into chondrocytes (Day et al., 2005; Hill et al., 2005). Human genetic studies and characterization of transgenic mice showed that loss of function mutations in LRP5 resulted in a decrease in bone formation (Gong et al., 2001), and gain of function mutations in LRP5 caused high bone density in humans (Boyden et al., 2002; Little et al., 2002).
LRP6 heterozygous mice and missense mutations both showed decreased osteoblast
formation and bone formation (Holmen et al., 2004). Different Wnt proteins including Wnt3a, Wnt1, and Wnt2, were able to induce alkaline phosphatase (ALP) activity, an early marker of osteoblast differentiation, in several cell lines (Gong et al., 2001; Rawadi et al., 2003). Wnt induced the activation of Runt-related transcription factor 2 (RunX2), an osteogenic transcriptional factor, which is essential in osteoblast differentiation (Gaur et al., 2005; Kobayashi and Kronenberg, 2005; Komori, 2005).
LRP8 (also known as apolipoprotein E receptor 2) also belongs to the LDLR family (Kim et al., 1996). LRP8 consists of seven conserved LDL repeats and three EGF receptor–like domains, which are similar to LRP5/6 (Rey and Ellies, 2010). Unlike LRP5/6, which includes four YWTD β-propellers, LRP8 only contains one b-propeller (Rey and Ellies, 2010). Besides, LRP8 has one NPxY motif in the intracellular domain, which mediates the interaction with the phosphotyrosine-binding (PTB) domain–containing proteins (Cuitino et al., 2005; Hoe et al., 2006; Morimura and Ogawa, 2009). LRP8 was identified and characterized in the brain (Kim et al., 1996). Besides the brain, LRP8 is also expressed abundantly in the placenta, the ovaries, and the epididymis (Andersen et al., 2003; Kim et al., 1996; Novak et al., 1996; Stockinger et al., 2000). LRP8, as one of the cell-surface receptors for Reelin, functions as an important regulator of neuron migration in brain development (D'Arcangelo et al., 1999; Trommsdorff et al., 1999). Reelin signals through LRP8 or through very-low-density lipoprotein receptor (VLDLR), induces the interaction between LRP8 and adaptor protein Disabled-1 (Dab1) and tyrosine phosphorylation of Dab1, and further, results in the recruitment of phosphatidylinositol-3- kinase (PI3K) and GSK3b (Bock et al., 2003; D'Arcangelo et al., 1999; Schneider and Nimpf, 2003; Strasser et al., 2004). Interaction between LRP8 and Reelin also initiates internalization of Reelin in the clathrin-mediated endocytosis pathway (Duit et al., 2010).
LRP8 single-knockout mice were grossly normal, but showed reduced male fertility and suffered accelerated cell death during normal aging (Beffert et al., 2006; Trommsdorff et al., 1999); LRP8 and VLDLR double-knockout mice displayed disorganized neurons, and cortical lamination was affected in brain development (Trommsdorff et al., 1999). As a receptor for apolipoprotein E, the LRP8 gene polymorphism might associate with the developing Alzheimer’s disease (AD) (Ma et al., 2002).
Although LRP8 is in the LDLR family and shares conserved domains with LRP5/6, whether LRP8 is involved in Wnt signaling is unknown. In this work, we showed that LRP8 functioned as a positive regulator of the Wnt/β-catenin signaling pathway. Moreover, functional studies revealed that LRP8 promoted Wnt3a-induced osteoblast differentiation.
Materials and Methods
Cell culture
We cultured KS483 cells in a minimal essential medium (α-MEM; Gibco BRL, Carlsbad, CA, USA), and we cultured C2C12 cells, HEK293T cells, and L cells in DMEM (Gibco
BRL) supplemented with penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS; Gibco BRL) in an atmosphere containing 5% CO2 at 378C.
Plasmid constructs
We amplified a human LRP8 (BC051836) open reading frame by polymerase chain reaction (PCR) using high-fidelity polymerase Phusion (Thermo Fisher Scientific, Hudson, NH, USA) from an ImaGenes cDNA clone (clone ID: IRATp970B08105D) and inserted it into the EcoR V site of pLV-bc-CMV-puro lenti vector. Flag tagged full-length LRP8 (FL) or deletion construct (DC) was amplified by PCR from full-length LRP8 and was inserted into the Spe I site of a modified pCR3.1 vector with a flag tag at the C terminus (Maretto et al., 2003). Primers used for cloning are available upon request. Wnt-responsive luciferase reporter Bat-luc, Axin1-Myc, Axin2-Myc, and Wnt3a-Myc constructs have been described (Li et al., 2009; Maretto et al., 2003; van Bezooijen et al., 2007). Mouse LRP8 shRNAs (LRP8-1, TRCN0000177833; LRP8-2, TRCN0000176636) lentiviral transduction particles were purchased from Sigma Mission Library (St. Louis, MO, USA). All constructions were confirmed by DNA sequencing.
Transfections and luciferase reporter activity
We seeded C2C12 or KS483 cells into 96-well or 24-well plates and transiently transfected them with Wnt-responsive Luciferase reporter Bat-luc and small interfering RNA (siRNA) using DharmaFECT Duo reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. LacZ plasmid was cotransfected as internal reference.
Posttransfection, cells were starved with DMEM for 8 hours and then stimulated with control or Wnt3a conditioned medium (CM) for 16 hours. Cells were lysed and luciferase activities were measured according to the manufacturer’s instructions (Luciferase Reporter Assay System; Promega, San Luis Obispo, CA, USA). Experiments were performed in triplicate. Control siRNA (ON-TARGETplus control pool, non targeting pool, D-001810- 10), b-catenin (NM_007614) siRNA (ON-TARGETplus SMARTpool L-040628-00), LRP8 (NM_053073) siRNA (LRP8-1, J-046407-09 and LRP8-2, J-046407-10) were purchased from Dharmacon (Thermo Fisher Scientific).
RNA isolation and quantitative real-time PCR See Supplementary Materials and Methods.
Lentiviral transduction and stable cell line generation
We produced lentiviruses by transfecting indicated plasmids, together with helper plasmids pCMV-VSVG, pMDLg-RRE (gag/pol), and pRSV-REV, into HEK293T cells as described (Zhang et al., 2011). Cell supernatants were harvested 48 hours post transfection and thereafter used for cell infection. At 48 hours after infection, KS483 cells were cultured in medium containing 4 mg/mL puromycin (Sigma-Aldrich, St, Louis, MO, USA) for 1 week to generate stable cell lines expressing LRP8 or short hairpin RNAs (shRNAs) selectively targeting LRP8.
Western blot, immunofluorescence, and immunoprecipitation assay See Supplementary Materials and Methods.
ALP staining and mineralization assay See Supplementary Materials and Methods.
Statistical analysis
We used Student’s t test for statistical analysis, and p<0.05 was considered to be statistically significant.
Results
LRP8 mediates Wnt/β-catenin–induced responses
LRP8 mediates Wnt/β-catenin–induced responses To identify novel membrane-associated regulators of Wnt/β-catenin signaling, we performed an siRNA screen to find membrane- associated proteins that might regulate Wnt3a/β-catenin–induced transcriptional reporter activity. Each siRNA selectively targeting a transcript encoding a transmembrane or membrane-associated protein was transfected separately, and in total 113 genes were screened. For targeting each gene, a mix of four independent siRNAs was used. To achieve this, mouse C2C12 cells, which were transfected with the Wnt-responsive luciferase reporter Bat-luc and internal reference LacZ plus siRNA mix, were starved for 8 hours after 16-hour transfection, stimulated with Wnt3a conditioned medium (CM) for 16 hours, and then luciferase activities were measured. In this screen, β-catenin siRNA served as a positive control and it dramatically decreased Wnt3a-induced reporter activity (Figure S1A and S1B). Of note, siRNA against the canonical Wnt co-receptors LRP5 and LRP6 partially inhibited Wnt3a-induced reporter activity (Figure S1A and S1B). Similarly, siRNA-mediated depletion of LRP8 led to suppression of Wnt3a-induced reporter activity (Figure S1A and S1B).
Double-depletion of LRP8 with LRP5 or LRP6 further inhibited Wnt3a-induced reporter activity, compared with LRP5 or LRP6 single-knockdown (Figure S1B). Triple- depletion of LRP5, LRP6, and LRP8 inhibited Wnt3a-induced reporter activity as strongly as siRNA-mediated knockdown of β-catenin (Figure S1B). These results suggest that LRP8 is a mediator of Wnt/β-catenin signaling. We confirmed our findings obtained with siRNA mix by transfecting mouse KS483 osteoprogenitor cells with two independent LRP8 siRNAs. Consistent with the results obtained in our screen, LRP8 depletion in KS483 cells also decreased reporter activity driven by Wnt3a (Figure 1A). We also confirmed the initial siRNA-induced LRP8 depletion results with two additional, independent small hairpin RNAs (shRNAs) that silenced LRP8 by targeting different regions in the LRP8 gene.
Similar to the results with initial siRNA mix, silencing LRP8 expression strongly inhibited the Wnt3amediated induction of reporter activity (Figure 1C). Depletion of LRP8 by two independent siRNAs in C2C12 cells also inhibited Wnt3a-induced reporter activity (Figure
S2A). Taken together, these results suggest that LRP8 may serve as a positive regulator required for Wnt/β-catenin signaling.
Figure 1 LRP8 is required for Wnt3a-induced transcriptional reporter activation in KS483 cells. (A) KS483 cells were transfected overnight with indicated siRNAs together with Wnt reporter Bat-luc. Subsequently, the cells were starved for 8 hrs, and then stimulated with control conditioned medium or Wnt3a conditioned medium (Wnt3a CM). After 16 hrs, luciferase reporter activity was measured. Each experiment was performed in triplicate, and the data represent mean ±SD of three independent experiments after normalization with β-gal activity. (B) Total RNA was also prepared from experiment A and was subjected to quantitative real time-PCR analysis. The levels of LRP8 mRNA were determined and normalized to the amount of GAPDH mRNA. (C) KS483 cells stably expressing LRP8 shRNA were transfected with Wnt-responsive luciferase reporter Bat-luc, starved and then stimulated with indicated conditioned media (control CM and Wnt3A CM). After 16 hrs the reporter activity was analyzed as described above. (D) Total RNA was also prepared from experiment C and this was subjected to quantitative real time-PCR analysis. The relative mRNA expression of LRP8 was analyzed as described above.
All the experiments above were performed at least 3 times.
LRP8 positively regulates Wnt/β-catenin signaling
To gain further insight into the function of LRP8 in Wnt/β-catenin signaling, we examined the effect of expression of the Wnt/β-catenin direct target gene, Axin2, in KS483 cells by quantitative real-time PCR (QRT-PCR) analysis upon misexpression of LRP8. Consistent with the previous transcriptional reporter assay results, LRP8 knockdown in KS483 cells
significantly inhibited Wnt3a-induced Axin2 expression in both siRNA transient transfected cells and shRNAs stable infected cells (Figure 2A and 2B). Depletion of LRP8 in C2C12 cells also showed decreased Wnt3a-induced Axin2 expression (Figure S2B). In line with this, ectopic expression of LRP8 in both KS483 and C2C12 cells facilitated Wnt3a-mediated enhancement of Axin2 expression (Figure 2C and Figure S2C). Taken together, these findings suggest that LRP8 positively regulates canonical Wnt pathways and is a critical component for efficient Wnt/β-catenin signaling.
Figure 2 LRP8 positively regulates Wnt3a-induced Axin2 expression in KS483 cells. (A) KS483 cells transfected with indicated siRNAs were starved and then treated with control conditioned medium (control CM) or Wnt3a conditioned medium (Wnt3a CM) for 6 hrs and harvested for real time-PCR analysis. The relative mRNA expression of Axin2 (left panel) and LRP8 (right panel) were analyzed and normalized with the amount of GAPDH mRNA. (B-C) KS483 cell stably expression LRP8 shRNA (B) or LRP8(C) were starved and then treated with control CM or Wnt3a CM for 6 hrs, and then harvested for quantitative real-time PCR analysis. The relative mRNA expression of Axin2 (left panel) and LRP8 (right panel) were analyzed and normalized with the amount of GAPDH mRNA as described above. All the experiments above were performed at least 3 times.
Figure 3 LRP8 promotes β-catenin stabilization and nuclear accumulation in KS483 cells. (A) KS483 cells that stably expressed LRP8 shRNAs (left) or LRP8 (right) were seeded into a 6-well plate and then starved with DMEM for 8 hours, then treated with Wnt3a CM for indicated time points. After treatment, cells were lysed and analyzed by Western blotting. GAPDH was included as a loading control. The relative β-catenin expression level (normalized to GAPDH) was quantified, and these results are shown below the Western blot results. (B) KS483 cells that stably expressed LRP8 or empty vector were seeded in a 24-well plate, treated with control or Wnt3a CM for 2 hours, and then stained for β-catenin as shown in Supplemental Materials and Methods. The cells were stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize their nuclei. Arrows indicate nuclear staining of b- catenin in control CM treated KS483 cells. (C) HEK293T cells were transfected with vector, Flag-tagged LRP8, or Flag-tagged LRP8 with very few Wnt3a-Myc. At 48 hours after transfection, cell lysates were immunoprecipitated (IP) with anti-Flag antibody. Association of endogenous Axin was analyzed by immunoblotting (IB). Aliquots of the total cell lysates were loaded as input controls. (D) (top) The scheme depicts the respective domain contained in LRP8 and its deletion (ΔC); (bottom) HEK293T cells were transfected with Myc-tagged Axin1 (left) or Axin2 (right) and Flag-tagged full-length (FL), or the LRP8 deletion mutant ΔC. At 48 hours post transfection, cells were harvested for IP assays. Full-length LRP8 or its deletion mutant ΔC was IP with anti-Flag antibody. LRP8- associated Axin was detected with anti-Myc IB. The input total cell lysates is
shown as control. LBD=ligand binding domain; β-pro=β-propeller; TM=transmembrane; IB=immunoblotting;
IP=immunoprecipitated.
LRP8 mediates Wnt-induced β-catenin stabilization and nuclear accumulation
To investigate the mechanism by which LRP8 regulates canonical Wnt/β-catenin signaling, we examined the effect of LRP8 misexpression on Wnt3a-induced β-catenin expression overtime. As shown, both basal expression and Wnt3a-induced accumulation of β-catenin were reduced in LRP8 stably depleted cells and enhanced in LRP8 ectopic-expressed stable cell lines (Figure 3A). This effect occurred at the posttranscriptional level as the mRNA expression level of β-catenin remained the same in both LRP8 knockdown and overexpression stable cells (Figure S1C and S1D). As basal β-catenin levels are relatively high in KS483 cells compared with other cells such as C2C12 cells, we only observed moderate canonical Wnt activation in KS483 cells (data not shown).
To further show that LRP8 can mediate the Wnt-induced stabilization of β-catenin, we examined β-catenin subcellular localization. Confocal microscopy showed that, whereas β-catenin mainly remains membrane-associated in control cells, LRP8 infection alone promoted β-catenin nuclear signaling (Figure 3B). This observation was further amplified upon Wnt3a treatment (Figure 3B). The data above (Figure 3A and 3B) revealed that LRP8 can mediate Wnt3a-induced β-catenin stabilization and nuclear accumulation.
To address the mechanism of how LRP8 is involved in β-catenin stabilization and nuclear accumulation, we tested the interaction between LRP8 and components in β- catenin destruction complex. By performing co-immunoprecipitation assays, we found that LRP8 could associate with (endogenous) Axin in the presence of Wnt3a (Figure 3C and 3D). Previously, LRP5 was also found to interact with Axin via its intracellular domain (Mao et al., 2001). To map the region in LRP8 involved in Axin binding, LRP8−with and without its intracellular domain−was analyzed for its ability to co-precipitate with Axin1 and Axin2. As shown in Figure 3D, in contrast with full-length LRP8, LRP8 lacking its intracellular domain had no interaction with Axin1 and Axin2. Taken together, these data suggest that LRP8 mediates Wnt induced β-catenin stabilization and nuclear accumulation through interaction with Axin.
LRP8 promotes Wnt3a-induced osteoblast differentiation
Wnt/β-catenin signaling plays an important role in osteoblast differentiation (9). Wnt3a was shown to enhance ALP activity, an early marker of osteoblast differentiation, through β-catenin signaling (Rawadi et al., 2003). We therefore examined whether Wnt3a-induced ALP activity is affected by LRP8 misexpression. For analysis of ALP activity, KS483 cells were grown confluent and stimulated with control CM or Wnt3a CM for 4 days.
Histochemical staining revealed that Wnt3a-induced ALP activity was severely mitigated upon β-catenin knockdown (Figure 4A). We observed that the Wnt3a-induced ALP activity increase was significantly lower in cells transfected with LRP8 siRNA or expressing shRNAs than in those cells transfected with control siRNA or expressing shRNAs (Figure 4A). In addition, the basal ALP activity induced was inhibited by LRP8 knockdown (Figure 4A). Consistent with these findings, both basal and Wnt3a-induced ALP activity
were strongly potentiated by ectopic LRP8 expression (Figure 4B). Histochemical staining also showed that Wnt3a-induced ALP activity in C2C12 cells was potentiated by LRP8 overexpression (Figure S2D). To further consolidate the effect of LRP8 in Wnt3a induced osteoblast differentiation, we investigated LRP8’s effect in regulating Wnt3/β-catenin- induced expression of RunX2 and collagen type I alpha 1 (Col1a1), which are early-stage makers for osteoblast differentiation (Gaur et al., 2005; Komori, 2010). As expected, LRP8 depletion inhibited Wnt3a-induced activation of RunX2 and Col1a1 whereas LRP8 overexpression enhanced both basal and Wnt3a-induced activation of RunX2 and Col1a1 (Figure 4C and 4D). Therefore, these data show that LRP8 is a critical mediator of Wnt/β- catenin signaling and Wnt3a-induced early-stage osteoblast differentiation.
LRP8 promotes Wnt3a-induced mineralization of osteoprogenitor cells
As a (late) marker for osteogenic differentiation we analyzed calcium deposition and formation of mineralized matrix by Alizarin red S staining in KS483 osteoprogenitor cells.
Confluent KS483 cells were treated with control CM or Wnt3a CM for the first 4 days, and then refreshed with osteogenic medium for another 8 days (Figure 5A). Wnt3a-induced calcium deposition and mineralized matrix formation were found to be significantly decreased by LRP8 depletion (Figure 5B) and to be enhanced by LRP8 overexpression (Figure 5C). QRT-PCR analysis confirmed that LRP8 stable expression could facilitate Wnt3a to enhance the expression of bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN), which are late-stage markers of osteoblast differentiation (Figure 5D) (Bellows et al., 1999; Gorski, 1992). Taken together, these data suggest that LRP8 is a critical determinant for Wnt3a-induced mineralization and late-stage osteoblast differentiation.
Discussion
In this study, we performed an siRNA library screen and we identified LRP8 as a novel membrane-associated regulator of canonical Wnt/β-catenin signaling using a transcriptional reporter assay. We found that LRP8, which shares the same conserved domains with Wnt co-receptor LRP5/6, also functions as a bona fide Wnt co-receptor.
LRP8 was found to be a critical determinant for the expression of the Wnt3a-induced target gene Axin2 and for nuclear β-catenin accumulation (Figure 2 and Figure 3). The observed nuclear β-catenin distribution in LRP8-overexpression stable cell lines without exogenous treatment suggests that LRP8 may stimulate β-catenin nuclear accumulation by Wnt in serum or Wnt produced by cells (Figure 3B).
Figure 4 LRP8 promotes Wnt3a-induced early stage osteoblast differentiation in KS483 cells. LRP8 promotes Wnt3a-induced early stage osteoblast differentiation in KS483 cells. (A) (left) KS483 cells that were transfected with indicated siRNAs (top), or stably expressed LRP8 shRNAs (bottom) were treated with the indicated condition medium (CM) for 4 days. Subsequently, the cells were harvested for histochemical staining to determine ALP activity. (middle) Representative microscopic pictures of left panel. (right) The histochemically stained KS483 cells were dissolved in NaOH/EtOH solution and absorbance was measured at 540 nm. (B) (left) KS483 cells that stably expressed LRP8 were treated with indicated condition medium (CM) for 4 days, and then the cells were harvested for histochemical staining to determine ALP activity. (middle) Representative microscopic pictures of left panel. (right) The histochemical stained KS483 cells were dissolved in NaOH/EtOH solution and absorbance was measured under 540 nm. (C, D) KS483 cells that stably expressed LRP8 shRNAs
(C) or LRP8 (D) were seeded into a 12-well plate, and then starved for 8 hours, and then stimulated with indicated condition medium (CM) for 16 hours. Cells were harvested for quantitative real-time PCR (QRT-PCT) analysis.
The relative mRNA expression of RunX2 and Col1a1 were analyzed as described in Supplemental Materials and Methods. All the experiments were performed at least three times.
Figure 5 LRP8 promotes Wnt3a-induced mineralization in KS483 osteoprogenitor cells. (A) For mineralization assay, KS483 cells were seeded into 24-well plate or 12-well plate. When the cells reached confluence, indicated conditioned medium (Control CM or Wnt3a CM) were added for the first 4 days (with refreshment of medium after two days). Subsequently, the cells were cultured for another 8 days in osteogenic medium, which contains 0.2 mM ascorbic acid and 10 mM β-glycerolphosphate (with medium refreshment after 4 days). Finally, the cells were fixed with 3.7% formaldehyde, washed with PBS and stained with 2% alizarin red S solution for several minutes. (B-C) KS483 cells that stably expressed with indicated shRNAs (B) or LRP8 (C) were treated with control CM or Wnt3a CM followed by osteogenic culture medium as described in (A). (Left panel) Representative microscopic pictures of alizarin red S solution stained cells were taken. (Right panel) Relative quantification of alizarin red S (ARS) staining as described in “Materials and Methods”. Experiments
were performed in duplicated wells. All the experiments above were performed at least 3 times. (D) KS483 cells which stably expressed LRP8 were treated in the same way as described in (C), and then harvested for quantitative real-time PCR analysis. The relative mRNA expression of BSP, OPN and OCN were analyzed as described before.
All the experiments above were performed at least 3 times.
Importantly, LRP8 was found to play a pivotal role in Wnt3ainduced osteoblast differentiation, as measured by ALP activity and mineralization, and in induction of early and late osteoblast marker gene expression (Figure 4 and Figure 5). The mechanism of how Wnt/β-catenin functions in osteoblast differentiation and the bone formation process is still unclear. Several studies have suggested that Wnt is positive in the early stage but inhibitory in the late stage of osteoblast differentiation (Milat and Ng, 2009; van der Horst et al., 2005). Our results are in line with this biphasic role of Wnts. To show the positive effects of Wnt (and LRP8) on osteoblast differentiation, we only treated the confluent cells for 4 days with Wnt3a, followed by treatment with osteogenic medium for another 8 days in the absence of Wnt stimulation. If we continuously treated the cells with Wnt, we could not stimulate osteoblast differentiation (data not shown).
LRP8 knockout mice did not reveal a strong deficiency in bone formation. This can be caused by redundant function of LRP5, LRP6, and LRP8, and functional compensation by LRP5 and LRP6 in the absence of LRP8. Double-knockout of LRP8 and VLDL mice were smaller when compared with wild-type mice. The double knockout of LRP8 and LRP5/6 or LRP8 knock-in mice might provide more information about LRP8 functions in bone formation.
In our study, we used mouse myoblast C2C12 and KS483 osteoprogenitor cells to study LRP8 function. Based on QRT-PCR analysis, LRP5, LRP6, and LRP8 have moderate expression in both cell lines (data not shown). Single-depletion of LRP8 (or LRP5 and LRP6) in both cell lines decreases Wnt-induced signaling and osteoblast differentiation. Moreover, LRP5/6/8 triple-knockdown completely inhibited Wnt-induced signaling in C2C12 cells (Figure S1B). These results suggest that LRP5, LRP6, and LRP8 are all required for efficient signaling and are suboptimally expressed.
Wnt co-receptor LRP5/6 and Fz family members cooperate to mediate Wnt-induced β-catenin signaling (Bhanot et al., 1996; Tamai et al., 2000). A number of reports have shown that several Wnt proteins bind directly to β-propeller of LRP5/6 (Itasaki et al., 2003;
Kato et al., 2002; Tamai et al., 2000). LRP8 has one conserved β-propeller in the extracellular domain, but its sequence is quite divergent from the β-propellers that are present in LRP5/6. Also, LRP8 has a relatively long N-terminal (ligand binding) domain when compared with LRP5/6 (Rey and Ellies, 2010). Our data showed that LRP8 interacts directly with Wnt3a (Figure S3B). However, unlike LRP5/6, LRP8 interacts with Wnt3a through the N-terminal domain and not through the domain containing its single β- propeller motif (Figure S3B). Thus, the interaction of Wnt3a with LRP8, when compared with LRP5/6, is different. Moreover, we found that LRP8 directly interacts with Frizzled5 through both N-terminal and C-terminal regions (Figure S3C). Together, the results indicate that LRP8 interacts with Wnt3a and Frizzled and functions as a Wnt co-receptor.
Similar to what has been reported for LRP5/6 (Mao et al., 2001; Tolwinski et al., 2003), we found that LRP8 interacted with endogenous Axin when cells were challenged with Wnt3a (Figure 3C). We also found that the intracellular domain of LRP8 is essential for this interaction (Figure 3D). Therefore, the interaction between LRP8 and Axin might disrupt Axin, casein kinase 1, glycogen synthase kinase (GSK) 3β, and adenomatous polyposis coli (APC) destruction complex for β-catenin degradation, which results in the stabilization of β-catenin (Ikeda et al., 1998; Liu et al., 2002; Tamai et al., 2004). However, LRP8 may function distinctly from LRP5/6 on β-catenin because LRP8 contains no PPPSP motif. Further studies are needed to understand how β-catenin degradation is mediated by Wnt-induced LRP8 activation.
In conclusion, we identified and characterized LRP8 as a novel membrane- associated regulator of canonical Wnt/β-catenin signaling. Functional studies revealed that LRP8 promotes Wnt3a-induced osteoblast differentiation.
Disclosures
All authors state that they have no conflicts of interest.
Acknowledgments
We are grateful to Dr. C. Löwik for KS483 cells, Dr. Duanqing Pei for pCR3.1 vector, and Dr. Shengcai Lin for Axin1-Myc, Axin2-Myc plasmids, and Axin antibody. We thank David de Gorter for stimulating discussions, and Midory Thorikay and Martijn JWE Rabelink for excellent technical assistance. This work was supported by the Netherlands Organization for Scientific Research (NWO-MW), European Union FP7 grant TALOS, and Centre for Biomedical Genetics.
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Supplementary Information
Supplementary Materials and Methods
Plasmid constructs
Flag-tagged LRP8 deletion constructs (D1-D3) were amplified by PCR from full length LRP8, and then inserted into Spe I site of modified pCR3.1 vector with flag tag at the C terminus. Primers for LRP8 D1-D3 were as follows. D1 forward: 5′-
GGACTAGTACCATGGGCCTCCCCGAGCCGGGCCC-3′, D1 reverse: 5′-
GGACTAGTCAGTAGGTCCATCTCGTAGCCAG-3’. D2 forward: 5′-
GGACTAGTACCATGACCAAGAACTGCAAGG-3′. D2 reverse: 5′-
GGACTAGTATGGAAGATGACAATGTCATGTGG-3′. D3 forward: 5′-
GGACTAGTACCATGGAGCTGAAGCAGCCAAG-3′. D3 reverse: 5′-
GGACTAGTGGGTAGTCCATCATCTTCAAGGCTT-3′. Frizzled5-HA construct was gifted by Dr. Xi He (He et al., 1997). LRP1 (NM_008512) siRNA (ON-TARGETplus SMARTpool, L-040764-00), LRP2 (XM_921143) siRNA (ON-TARGETplus SMARTpool, L-045961-01), LRP3 (NM_001024707) siRNA (ON-TARGETplus SMARTpool, L-167228-00), LRP4 (NM_172668) siRNA (ON-TARGETplus SMARTpool, L-063003-01), LRP5 (NM_008513) siRNA (ON-TARGETplus SMARTpool, L-040650-01), LRP6 (NM_008514) siRNA (ON-TARGETplus SMARTpool, L-040651-01), LRP8 (NM_053073) siRNA (ON-TARGETplus SMARTpool, L-046407-01), LRP10 (NM_022993) siRNA (ON-TARGETplus SMARTpool, L-063305-01), LRP11 (NM_172784) siRNA (ON-TARGETplus SMARTpool, L-056420-01), and LRP12 (NM_172814) siRNA (ON-TARGETplus SMARTpool, L-055299-01) were purchased from Dharmcon (Thermo Fisher Scientific, Hudson, NH, USA). All constructions were confirmed by DNA sequencing.
RNA isolation and quantitative real-time PCR (QRT-PCR)
Total RNA was isolated using the NucleoSpin RNA II Kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions. Reverse-transcriptase (RT)-PCR was performed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, St.
Leon-Rot, Germany) according to the manufacturer’s instructions. Quantitative real-time PCR reactions were performed using the StepOne plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Results were normalized by measuring GAPDH expression. Primers used for QRT-PCR were:
mGAPDH forward, 5′-AACTTTGGCATTGTGGAAGG-3′
mGAPDH reverse, 5′-ACACATTGGGGGTAGGAACA-3′
LRP8 forward, 5′- GGCGGCTGTGAATACCTGTG-3′
LRP8 reverse, 5′- TGCTGGGAGTGGTTGCTGGT-3′
mAxin2 forward, 5′-GGTTCCGGCTATGTCTTTGC-3′
mAxin2 reverse, 5′-CAGTGCGTCGCTGGATAACTC-3′
mRunX2 forward, 5′-GAATGCTTCATTCGCCTCAC-3′
mRunX2 reverse, 5′-GTGACCTGCAGAGATTAACC-3′
mCol1a1 forward, 5′-GCCTTGGAGGAAACTTTGCTT-3' mCol1a1 reverse, 5′-GCACGGAAACTCCAGCTGAT-3′
mBSP forward, 5′-AGGGAACTGACCAGTGTTGG-3′
mBSP reverse, 5′-ACTCAACGGTGCTGCTTTTT-3′
mOPN forward, 5′-TGCACCCAGATCCTATAGCC-3′
mOPN reverse, 5′-CTCCATCGTCATCATCATCG-3′
mOCN forward, 5′-AGACTCCGGCGCTACCTT-3′
mOCN reverse, 5′-CTCGTCACAAGCAGGGTTAAG-3′
mβ-catenin forward, 5′-TCATGCGCTCCCCTCAGATG-3′
mβ-catenin reverse, 5′-AATCCACTGGTGACCCAAGC-3′
Western blot, immunofluorescence and immunoprecipitation assay
For Western blot, cells were seeded into 6-well plate and starved with DMEM for 8 hrs, and stimulated with indicated conditioned media for indicated time periods. Cells were lysed in radio-immunoprecipitation assay buffer (RIPA, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 0.1% Triton X-100), and proteins separated by 10% SDS-PAGE, and transferred onto Hybond™-C Extra membrane (Amersham Biosciences, UK). The membranes were then blotted with 5% nonfat milk and incubated with anti-GAPDH (1:5000, Millipore, USA), anti-β-catenin (1:10000, BD Transduction Laboratories, USA), anti-Flag (1:10000, Sigma-Aldich, St, Louis, MO, USA), anti-Myc (1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-Axin (gifted by Dr. Shengcai Lin) antibodies followed by horseradish peroxidase-conjugated anti-mouse (1:5000, GE Healthcare, UK) secondary antibodies. The membranes were then washed extensively and developed with enhanced chemiluminescence (ECL) (Thermo Scientific, USA).
For immunofluorescence, cells grown on coverslips were fixed with 3.7%
formaldehyde in phosphate-buffered saline (PBS), washed, blocked in 10% normal goat serum, then stained with anti-β-catenin antibody (BD Transduction Laboratories, USA), and then stained with Alexa Fluor 594 goat anti-mouse IgG (Invitrogen, USA). The images were acquired by a Leica confocal system. For immunoprecipitation (IP), the indicated plasmids were transfected into HEK293T cells by calcium phosphate or Lipofectamine (Invitrogen, Carlsbad, CA). 48 h post transfection cells were lysed in 400 μl of TNE buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NonidetP-40, 1 mM EDTA, protease inhibitor mixture (Sigma-Aldich, St, Louis, MO, USA)) on ice. Cell lysates were cleared by centrifugation at 13,000g for 10 min at 4 °C. Cleared cell lysates (60 μl) were saved as input for direct Western blotting analysis, and the remaining samples were transferred to a new tube containing 40 μl of anti-Flag-conjugated-agarose beads (Sigma-Aldich, St, Louis, MO, USA). The anti-Flag beads were then washed 4 times with TNE buffer and then eluted by boiling for 5 min in 2% SDS loading buffer (with 5% β-mercaptoethanol). After
centrifugation, supernatants were loaded onto SDS-PAGE and analysed by Western blotting.
Alkaline phosphatase staining and mineralization assay
For alkaline phosphatase (ALP) staining assay, C2C12 or KS483 cells were seeded into 24- well plate, and when the cells reached confluence, indicated conditioned medium were added for another 3-4 days. After cells were fixed by 3.7% formaldehyde, naphotol AS- MX phosphate (Sigma-Aldich, St, Louis, MO, USA) and fast blue RR salt (Sigma-Aldich, St, Louis, MO, USA) were added into the cells for histochemical examination of ALP activity. For quantification, the stained cells were dissolved in NaOH/EtOH solution and then the ALP activities were accessed at 545 nm absorbance.
For mineralization assay, KS483 cells were seeded into 24-well plate or 12-well plate, and when the cells reached confluence, indicated conditioned medium were added for the first 4 days, the medium was renewed every two days; and then osteogenic medium, which contains 0.2 mM ascorbic acid (Sigma-Aldich, St, Louis, MO, USA) and 10 mM β- glycerolphosphate (Sigma-Aldich, St, Louis, MO, USA), was added for another 8 days.
The medium was renewed after 4 days. After cells were fixed with 3.7% formaldehyde, cells were washed with PBS and stained with 2% alizarin red S solution (Sigma-Aldich, St, Louis, MO, USA) for several minutes and the washed with distilled water. To quantify the staining, KS483 cells with alizarin red S were extracted with 100 mM cetylpyridinium chloride (Sigma-Aldich, St, Louis, MO, USA) for at least 3 hrs at room temperature. The absorbance of the extracted Alizarin red S staining was measured at 570 nm.
Supplementary Acknowledgements
Authors are grateful to Dr. Xi He for Frizzled5-HA construct.
Supplementary References
He, X., Saint-Jeannet, J.P., Wang, Y., Nathans, J., Dawid, I., and Varmus, H. (1997). A member of the Frizzled protein family mediating axis induction by Wnt-5A. Science 275, 1652-1654.
Jeong, Y.H., Sekiya, M., Hirata, M., Ye, M., Yamagishi, A., Lee, S.M., Kang, M.J., Hosoda, A., Fukumura, T., Kim, D.H., et al. (2010). The low-density lipoprotein receptor-related protein 10 is a negative regulator of the canonical Wnt/β-catenin signaling pathway. Biochem Biophys Res Commun 392, 495-499.
Supplementary Figures
Figure S1 LRP8 is required for Wnt3a-induced transcriptional reporter activation in mouse C2C12 cells.
(A) Identification of LRP8 as a positive regulator of canonical Wnt canonical signaling pathway. Mouse C2C12 cells which were transfected with Wnt-responsive transcriptional luciferase reporter Bat-luc, internal reference LacZ expression plasmid, and the indicated siRNAs that selectively target a gene encoding a membrane associated protein (in this figure results targeting LRP1, 2, 3, 4, 5, 6, 8, 10, 11, 12 are depicted; in total 113 genes were screened, a mix of four independent siRNAs were used for each gene), were starved for 8 hrs, and then treated with control conditioned medium (CM) or Wnt3a CM for 16 hrs. Luciferase activities were measured and analyzed. In this screen, β-catenin siRNA served as a positive control and it dramatically decreased Wnt3a- induced reporter activity. LRP10 knockdown strongly potentiated Wnt3a-induced transcription reporter activity, which is consistent with recent report that ectopic expression of LRP10 potently inhibits Wnt/β-catenin signaling (Jeong et al., 2010). (B) Function of LRP8 in Wnt canonical signaling in C2C12 cells is redundant with LRP5 and 6. Mouse C2C12 cells were transfected with Wnt-responsive luciferase reporter Bat-luc, internal reference LacZ expression plasmid and with the indicated siRNAs selectively targeting LRP5/6/8. Subsequently, the cells were starved for 8 hrs, and then treated with control or Wnt3a CM for 16 hrs. Luciferase activities were measured and analyzed. (C-D) Depletion and overexpression LRP8 in KS483 cells doesn’t change mRNA expression level of β- catenin. KS483 cells which stably expressed LRP8 shRNAs (C) or LRP8 (D) were seeded into 12-well plate, and then staved for 8 hrs, and then stimulated with indicated condition medium (CM) with 16 hrs. Cells were harvested for quantitative Real-Time PCR (QRT-PCR) analysis. The relative mRNA expression of β-catenin was analyzed as described before. All the experiments above were performed at least 3 times.
Figure S2 LRP8 promotes Wnt3a-induced early stage osteoblast differentiation in mouse C2C12 cells. (A) LRP8 is required for Wnt3a-induced transcriptional reporter activation in C2C12 cells. C2C12 cells were transfected overnight with indicated siRNAs together with Wnt transcriptional reporter Bat-luc. Subsequently, the cells were starved for 8 hrs, and then stimulated with control or Wnt3a conditioned medium (CM). After 16 hrs, luciferase reporter activity was measured. Each experiment was performed in triplicate, and normalized with β-gal activity. (B-C) LRP8 positively regulates Wnt3a-induced Axin2 expression in C2C12 cells. C2C12 cells transfected with indicated siRNAs (B, Left panel), or C2C12 cells stably expression LRP8 shRNAs (B, Right panel) or LRP8 (C) were starved and then treated with control or Wnt3a CM for 6 hrs and harvested for QRT-PCR analysis. The relative mRNA expression of Axin2 and LRP8 were analyzed and normalized with the amount of GAPDH mRNA. All the experiments above were performed at least 3 times. (D) LRP8 promotes Wnt3a-induced early stage osteoblast differentiation in C2C12 cells. C2C12 cells stably expression LRP8 were treated with the indicated condition medium (CM) for 3 days. Subsequently, the cells were harvested for histochemical staining to determine ALP activity.
Figure S3 LRP8 binds to Wnt3a and Frizzled5. (A) The scheme depicts the respective domains that are present in LRP8 and its deletion expression constructs (D1-D3). LBD, ligand binding domain; β-pro, β-propeller; TM, transmembrane. (B) LRP8 interacts with Wnt3a through its N terminal region (that includes LBD). Flag-tagged full length or deletion mutants of LRP8 with Myc-tagged Wnt3a were co-expressed in HEK293T cells, and then immunoprecipitated (IP) with anti-Flag resin and associating proteins detected with the indicated antibodies by immunoblotting (IB). (C) LRP8 interacts with Frizzled5 through both N terminal and C terminal regions. Full length or deletion mutants of LRP8 with the Flag-tagged and HA-tagged Frizzled5 were co-expressed in HEK293T cells, immunoprecipitated (IP) with anti-Flag resin, and immunoprecipitates analysed by IB with the indicated antibodies.
