Adult stem cells maintain the potential to self-renew and differentiate, processes critical to tissue remodeling and repair. The decision to self-renew or differentiate is regulated by the cellular microenvironment and systemic factors.1, 2 In bone remodeling, mesenchymal stromal/stem cells (MSCs) differentiate into osteoblasts, chondrocytes, or adipocytes based on changes in the microenvironment of MSCs in the bone marrow.3–5 Recent clinical data suggests that a component of osteoporosis is inadequate anabolism due to an insufficient supply of osteoblasts.6, 7 The prevention of osteoporosis is dependent on an equal balance of catabolism and anabolism such that segments of bone that are removed by osteoclasts in the resorption phase of the remodeling cycle are replaced by equivalent amounts of new bone formed by osteoblasts recruited to the same site8 and is therefore dependent on an adequate supply of osteoblasts.
Parathyroid hormone (PTH) is classically regarded as the primary regulator of calcium homeostasis but has also been shown to enhance bone remodeling with both anabolic and catabolic effects, dependent on intermittent or continuous administration, respectively.9–11 PTH administered once daily is currently the only anabolic drug approved by the U.S. Food and Drug Administration (FDA) for treatment of osteoporosis.12 However, the precise mechanisms of how intermittent PTH results in bone formation remain elusive. PTH binds to a specific seven-transmembrane G-protein coupled receptor, PTH type 1 receptor (PTH1R). PTH1R then activates either G-protein alpha s subunit (Gαs) or G-protein alpha q subunit (Gαq) leading to the production of cyclic adenosine monophosphate (cAMP) for activation of protein kinase A, or stimulation of phospholipase for protein kinase C activation, respectively.13, 14 PTH has been shown in vitro to enhance differentiation of MSCs into the osteoblast lineage,15 but the traditional signaling pathway of PTH does not seem to provide a satisfactory explanation for these anabolic effects. Accumulated evidence indicates that PTH anabolic effects instead result from the orchestration of the effects of local factors such as bone morphogenetic proteins (BMPs), Wnts, or transforming growth factor beta (TGFβ) through recruitment of their receptors to PTH1R and/or endocytosis.16–18 For example, PTH can induce endocytosis of a PTH1R/TGFβ type II receptor (TβRII) complex,19 resulting in spatial and temporal integrated coupling of bone resorption and formation.
BMPs are expressed within the bone marrow stroma and are the only known morphogens that are able to induce osteoblast and chondroblast differentiation from MSCs.4, 5 Upon ligand binding, BMP receptor type II (BMPRII) recruits type I receptor to form a complex and mediates type I receptor phosphorylation. Subsequently these bind with Co-Smad (Smad4) in the cytoplasm and the R-Smad-Co-Smad complex translocates to the nucleus, where it acts as either an activator or repressor for transcription of target genes.20, 21 More than 20 members of BMPs have been shown to transduce their signaling through common BMP receptors (BMPRs),3–5 resulting in altered fates of MSCs.22 BMP2 has specifically been shown to be a potent inducer of osteoblast differentiation in MSCs.3 The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that regulate the binding of BMPs to their receptors. These various extracellular antagonists, such as noggin, sclerostin, Sog/Chordin (Sog/Chd), and DAN family members, are likely contributing to the diversified effects observed with BMP signaling pathways in relation to stem cell fate23, 24 because they bind to BMPs directly or indirectly through other extracellular proteins to prevent BMPs from gaining access to their receptors.25, 26
Signals that eradicate the antagonist extracellular network may increase the access of BMPs to their receptors, thereby promoting differentiation of MSCs to the osteoblast lineage. We hypothesized that one mechanism of PTH-enhanced differentiation of MSCs into the osteoblast lineage is through enhancement of BMP signaling by modifying the extracellular signaling network. Low-density lipoprotein receptor-related protein 6 (LRP6) specifically functions in the canonical wnt pathway,27 and there has been increasing evidence of cross-talk between Wnt and BMP signals at the promoter level, in the cytoplasm, and in the extracellular space.28 In the extracellular space, truncation of the LRP6 extracellular domain results in constitutive activation of the canonical wnt pathway, implying that the extracellular domain exerts an inhibitory effect on signaling through this receptor.29 LRP6 also shares common antagonists with BMPs, such as sclerostin,30, 31 and antagonists of either BMP/Wnt pathways, such as noggin and sclerostin, bind to each other with high affinity (dissociation constant Kd = 2.92 × 10−9 M).32 LRP6 has also been shown to interact with the PTH signaling pathway by directly forming a complex with PTH1R.33 Therefore, we hypothesized that LRP6 may be a key element in the communication between PTH- and BMP-induced differentiation of MSCs into the osteoblast lineage.
In the current study, we report that PTH increases phosphorylated Smad1 (pSmad1) in MSCs in vitro and in vivo. PTH-induced endocytosis of a PTH1R/LRP6 complex results in enhancement of pSmad1. Deletion of LRP6 alone is sufficient to account for the enhancement of pSmad signaling, resulting in increased access of BMP ligands to their receptors, ultimately increasing the proportion of MSCs differentiating into the osteoblast lineage. Thus, our study shows that one mechanism of the anabolic action of PTH is through enhancement of the differentiation of MSCs toward the osteoblast lineage by enhancing BMP-Smad signaling in an LRP6-dependent mechanism.
Subjects and Methods
Mice, cell cultures, and reagents
C57BL/6J (wild-type) mice were purchased from Charles River (Wilmington, MA, USA) and Tgfβ1+/+Rag2−/− mice with an immunodeficient background were obtained from the Mouse Models of Human Cancers Consortium Repository, National Cancer Institute (Frederick, MD, USA). Tgfβ1+/+Rag2−/− mice were maintained as wild-type mice and used for bone marrow cavity transplantation. Lrp6flox/flox mice used for Lrp6flox/flox MSC isolation were obtained from the Van Andel Research Institute (Grand Rapids, MI, USA). All animals were maintained in the Animal Facility of the Johns Hopkins University School of Medicine. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Johns Hopkins University, Baltimore, MD, USA. Green fluorescent protein (GFP)-labeled mouse adult MSCs were obtained from the Texas A&M Health Science Center College of Medicine Institute (College Station, TX, USA). Cells (Passage 2) were then maintained in Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS; Atlanta Biologicals, Lawrenceville, GA, USA), 10% horse serum (HS; Thermo Scientific, Logan, UT, USA), and 1% penicillin-streptomycin (PS; Mediatech, Herndon, VA). HEK293, UMR106, and C2C12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Mediatech) with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% PS. All reagents, including plasmids, antibodies, ligands, and adenoviruses, are listed in Supplemental Material.
Transfections, immunoblotting, coimmunoprecipitation, luciferase reporter assays, and knockdown experiments
We performed transfections of DNA plasmids with Lipofectamine (Invitrogen) using the protocol recommended by the manufacturer. For immunoblotting against phospho-antibodies, cells were harvested after starvation for 12 to 20 hours. For colocalization assays, HEK293–yellow fluorescent protein (YFP)-BMPRII and HEK293–cyan fluorescent protein (CFP)-PTH1R cells were generated by stable expression of YFP-BMPRII and CFP-PTH1R with retrovirus. Immunoprecipitation and immunoblotting analysis of cell lysates were performed as described.33 Luciferase activities were assayed with the Dual-Luciferase assay kit (Promega, Madison, WI, USA) following the manufacturer's instructions. Luciferase activity was measured and normalized to internal controls as Renilla luciferase units (RLU). β-arrestin1/2 and LRP6 knockdown experiments were performed as described in Supplemental Material.
Immunofluorescence colocalization assay
For BMPRII and PTH1R colocalization assays, we seeded HEK293 cells expressing YFP-BMPRII or CFP-PTH1R and treated them with tetramethylrhodamine-labeled PTH (PTHTMR; Genemed Synthesis Inc. San Antonio, TX, USA) at 37°C for indicated times and specific concentrations as reported in Results. For PTH1R internalization assays after β-arrestin 1/2 knockdown experiments, C2C12 cells expressing CFP-PTH1R were transfected with specific β-arrestin1/2 small interfering RNA (siRNA) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were treated with or without 100 nM PTH (Bachem Bioscience, Inc. King of Prussia, PA, USA) at 37°C for 30 minutes, after a 48-hour transfection interval. All cells were washed and fixed with 4% paraformaldehyde. Cells were mounted and observed with a Zeiss LSM 510 Meta Confocal Microscope.
LRP6 internalization assay
For LRP6 internalization assays, HEK293 cells were transfected with vesicular stomatitis virus G (VSV-G) tagged LRP6 and Mesoderm development (Mesd) plasmids. After transfection for 24 hours, cells were incubated with 100 nM PTHTMR at 37°C for 30 minutes. After the internalization of VSVG-LRP6, cells were washed three times with cold PBS to stop endocytosis and fixed with 4% paraformaldehyde. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-VSVG antibody (Abcam, Cambridge, UK) and observed with a Zeiss LSM 510 Meta Confocal Microscope. LRP6 cell-surface distribution was also monitored by biotinylation of cell-surface proteins as described.34 After biotin labeling, cell lysates were immunoprecipitated with anti-LRP6 antibody and the immunoprecipitates were analyzed by immunoblotting with anti-LRP6 antibody (total LRP6) or streptavidin-horseradish peroxidase (HRP) (cell surface LRP6). LRP6 internalization assays with flow cytometry were employed as described in Supplemental Material.
Ligand cell-surface binding assay
BMP2 (Pepro Tech, Inc., Rocky Hill, NJ, USA) was labeled with 125I (2000 Ci/mmol) using the chloramines-T method as described for cell-surface binding assays.35 For non-radioligand binding assays, BMP2 was labeled with Alexa 647 using the Alexa Fluor microscale labeling kit (Invitrogen). GFP-labeled Sca-1+CD45–CD11b–MSCs (2 × 105 cells/well) were plated in six-well plates. After cells reached confluence, they were washed with PBS and incubated with 250 ng/mL Alexa 647-BMP2 with or without 100 nM PTH at 37°C for 30 minutes, followed by another incubation at 4°C for 4 hours. Cells were rinsed with Hanks' balanced salt solution (HBAH buffer) (0.5 mg/mL BSA, 0.1% NaN3, 20 mM HEPES, pH 7.0) six times and then analyzed with flow cytometry.
Biotinylation of cell-surface BMPRII
HEK293 cells were transfected with various plasmids and then cell-surface proteins were labeled with 0.5 mg/mL Sulfo-NHS-LC-Biotin (Thermo Scientific) at 4°C for 1 hour. After incubation, nonbinding biotin was quenched with 0.1 M glycine, followed by three washes in cold PBS. Cells were harvested in modified radioimmunoprecipitation assay (RIPA) buffer. Flag-BMPRII was immunoprecipitated with anti-Flag antibody and protein G sepharose (Sigma-Aldrich, St. Louis, MO, USA). Total protein and biotinylated BMPRII were analyzed by immunoblotting.
Alkaline phosphatase activity, mineralization, and colony forming unit-fibroblast and colony forming unit-osteoblast assays
For alkaline phosphatase (ALP) activity and mineralization assays, GFP-labeled Sca-1+CD45–CD11b–cells were plated on six-well plates at a density of 2 × 105 cells/well and cultured in osteogenic medium containing 10−7 M dexamethasone (Sigma-Aldrich), 10 mM β-glycerol phosphate (Sigma-Aldrich), and 50 µg/mL L-ascorbic acid (Sigma-Aldrich). One hundred–nanomolar (100 nM) PTH, or 50 ng/mL BMP2 and/or 50 ng/mL noggin (R&D Systems, Inc., Minneapolis, MN, USA) were presented from day 1 to day 7 and thereafter with each change of osteogenic medium for the entire culture period. Histochemical staining for ALP activity in the cells was performed using Fast BCIP/NBT Tablets (Sigma-Aldrich) according to the manufacturer's protocol at day 7 and 14. Alizarin Red staining for calcium deposits was performed at day 21. Colony forming unit-fibroblast (CFU-F) and colony forming unit-osteoblast (CFU-Ob) assays were performed as described.36
Isolation and culture of Sca-1+CD45–CD11b–MSCs
We isolated bone marrow nucleated cells from 6-week-old Lrp6flox/flox mice euthanized by cervical dislocation. Cells were cultured with α minimum essential medium (α-MEM; Mediatech, Inc.) supplemented with penicillin (Sigma-Aldrich; 100 U/mL), streptomycin sulfate (Sigma-Aldrich; 100 mg/mL), and 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 humidified incubator. After 72 hours of adhesion, nonadherent cells were discarded and adherent cells were cultured an additional 7 days with a single media change. The adherent cells were harvested and incubated at 4°C for 20 minutes with phycoerythrin (PE)-conjugated anti-Sca1, Peridinin Chlorophyll Protein Complex (PerCP)-conjugated anti-CD45, and Allophycocyanin (APC)-conjugated anti-CD11b antibodies, and sorted by fluorescence-activated cell sorting (FACS) (FACSAria; Becton Dickinson, San Jose, CA, USA). The sorted Sca-1+CD45–CD11b–Lrp6flox/flox MSCs were enriched by further culture. A similar procedure was performed on GFP-labeled MSCs to sort GFP-labeled Sca-1+CD45–CD11b–MSCs.
PTH single-injection and bone marrow cavity transplantation
For the PTH single-injection model, forty 2-month-old C57BL/6J male mice were randomly divided into four groups and each group was given subcutaneous administration of PTH(1-34) (Bachem, Inc.; 40 µg/kg) or vehicle (equivalent volume of 1 mM acetic acid in PBS) once. Mice were euthanized at the indicated time points after injection (n = 10). For the bone marrow cavity transplantation model, 2-month-old C57BL/6J (wild-type) mice with an immunodeficient background (Rag2−/−, male) were used as recipients. A total of 5 × 105 GFP-labeled MSCs were injected into the bone marrow cavity of the femora as described.37 After treatment with vehicle or PTH at indicated times, whole bone marrow cells were harvested and incubated with the antibodies listed in Supplemental Material. Expression of markers were analyzed on a FACSCalibur flow cytometer (Becton Dickinson) and analyzed with Flowjo 7.6 software (Tree Star). All FACS data were analyzed with two channels, FL-1(GFP) for GFP-labeled MSCs and FL-2(PE) for all other receptors or markers. Cells were gated on FL-1 channel into a GFP-positive subset. The expression of all other receptors or markers on a GFP-positive subset were further analyzed with one-parameter histogram on FL-2 channel (PE).
Histochemistry, immunohistochemistry, and histomorphometric analysis
Standard protocols were performed. Details of procedures are described in Supplemental Material.
We repeated all experiments at least three times. The data are presented as mean ± SEM. Data were analyzed using an analysis of variance or Student's t test, and p < 0.05 was considered statistically significant in all calculations.
PTH promotes phosphorylation of Smad1 in MSCs in vivo
To confirm findings from prior studies38 in which intermittent PTH administration in ovariectomized mice induced rapid phosphorylation of BMP2 target Smad1/5/8 in the periosteum, C57BL/6J wild-type mice were injected with a single dose of PTH and sacrificed at 0 minutes, 30 minutes, 2 hours, or 8 hours after injection. A single PTH injection resulted in an increased percentage of osteoblast-like cells with phosphorylation of Smad 1/5/8 at all time points (hereafter described as phosphorylation of Smad1 or pSmad1). The highest percentage of osteoblast-like cells with pSmad levels occurred 30 minutes postinjection. The phosphorylation of Smad1 persisted for up to 8 hours postinjection as pSmad1 activity remained statistically significantly elevated compared to baseline activity 2 and 8 hours after a single PTH injection (Fig. 1A and B). To specifically examine whether PTH could induce phosphorylation of Smad1 in MSCs, GFP-labeled Sca-1+CD45–CD11b–MSCs (Supplemental Fig. S1) were transplanted into the femur cavities of immunodeficient Rag2−/− mice for 3 days. A single PTH injection or vehicle was administered and bone marrow was harvested 30 minutes postinjection. FACS analysis for GFP and pSmad1 demonstrated a six-fold increase in phosphorylation of Smad1 with a single PTH injection compared to vehicle, increasing pSmad1+ MSCs from 11.93% to 71.81% (Fig. 1C and D). These results suggest that one step in the mechanism of PTH-promoted differentiation of MSCs to osteoblasts is through PTH-enhancement of phosphorylation of Smad1.
PTH antagonizes inhibition effect of noggin on phosphorylation of Smad1
Western blot analysis showed that PTH enhanced BMP-stimulated phosphorylation of Smad1 in a dose- and time-dependent manner in UMR106 cells (Fig. 2A and B). PTH also enhanced BMP-stimulated phosphorylation of p38, another downstream target of the BMP signaling pathway (Fig. 2C). In the presence of noggin, a well known extracellular BMP antagonist, BMP-stimulated phosphorylation of Smad1 was nearly completely diminished, but recovery was seen with PTH treatment, equivalent to levels of BMP-stimulated phosphorylation of Smad1 not in the presence of noggin in C2C12 cells and MSCs (Fig. 2D and E, respectively). Using the PTH1R-null osteocytic cell line OC59, the effect of PTH on BMP-stimulated phosphorylation of Smad1 was also abolished (Fig. 2F). Collectively, these data indicate that PTH enhances BMP-stimulated phosphorylation of Smad1 through modulation of BMP signaling at the extracellular or receptor level in a PTH1R-dependent manner.
Endocytosis of LRP6 mediates PTH-enhanced phosphorylation of Smad1 in Sca-1+CD45–CD11b–MSCs
Extracellular BMP antagonists such as sclerostin and Dickkopf-related protein 1 (DKK1)39 bind to the large extracellular domain of LRP6 containing 1351 amino acids to form an inhibitory network in regulating multiple signaling pathways, such as the canonical Wnt signaling pathway.40 To test the hypothesis that LRP6 may also have an inhibitory effect on the BMP signaling pathway, phosphorylation of Smad1 in the presence or absence of BMP2 in UMR106 cells with knockdown of LRP6 using siRNA was assessed. Compared to scrambled siRNA, knockdown of LRP6 enhanced phosphorylation of Smad1 30 minutes after treatment with BMP2, whereas treatment with PTH in the absence of LRP6 no longer resulted in further increases in pSmad1 (Fig. 3A, Supplemental Fig. S2). Next, to test if this effect was also present in MSCs and affected by PTH signaling, Sca-1+CD45–CD11b–MSCs were isolated from bone marrow of Lrp6flox/flox mice in which the LRP6 gene was deleted by adenovirus-mediated expression of Cre, then stimulated in vitro with PTH. Similar to the results in UMR106 cells, deletion of LRP6 elevated the level of pSmad1 in both the vehicle or PTH-treated LRP6-deficient MSCs compared to their respective controls, but PTH treatment failed to further enhance pSmad1 levels without LRP6 (Fig. 3B). As PTH1R has been shown to downregulate the activity of other receptors such as TβRII via endocytosis,19 we next examined whether the observed LRP6-dependent PTH-enhanced BMP signaling was a result of endocytosis of LRP6. HEK293 cells were transfected with LRP6 plasmid and treated with vehicle or PTH. The level of cell-surface LRP6 was analyzed by colocalization, cell-surface biotinylation, and FACS analysis. PTH rapidly decreased the cell-surface level of LRP6 (Supplemental Fig. S3A and B), indicating PTH-induced endocytosis of LRP6. Moreover, immuno-colocalization experiments demonstrated that PTHTMR colocalized with LRP6 in the presence of PTH1R, and internalization of the colocalized complex of LRP6 occurred within 5 minutes of administration of PTHTMR. The internalization of LRP6 was not observed without expression of PTH1R, indicating that PTH-induced internalization of LRP6 is PTH1R-dependent (Fig. 3C). Consistent with these results, cell-surface biotinylation showed that PTH decreased endogenous cell-surface LRP6 significantly at 30 minutes in UMR106 cells (Fig. 3D), but not in PTH1R-deficient OC59 cells (Supplemental Fig. S4). The cell-surface expression of both PTH1R and LRP6 was also statistically significantly decreased 30 minutes after PTH injection in transplantation of MSCs by FACS analysis compared with vehicle injection (Fig. 3E–H).
β-arrestin is a key mediator of PTH1R endocytosis.41–43 Therefore, we examined whether blockade of PTH1R endocytosis by decreasing β-arrestin could antagonize the PTH effect on phosphorylation of Smad1. Knockdown of β-arrestin using siRNA decreased both PTH-induced PTH1R endocytosis (Fig. 4A) and PTH signaling downstream phosphorylated extracellular signal-regulated kinase (pERK) (Fig. 4B). In the presence of β-arrestin, PTH enhanced pSmad1 in BMP-treated cells (Fig. 4C, column 2 versus column 3, p < 0.05). However, in the absence of β-arrestin, phosphorylation of Smad1 was not enhanced by PTH treatment (Fig. 4C, column 5 versus column 6, p = not significant [NS]). Similarly, treatment with chlorpromazine (CP), an endocytosis inhibitor,44 also inhibited PTH-enhanced phosphorylation of Smad1 in C2C12 cells (Fig. 4D) and MSCs (Fig. 4E). These results suggest that BMP-stimulated phosphorylation of Smad1 is independent of β-arrestin; however, PTH-enhancement of pSmad is dependent on β-arrestin, confirming that PTH-triggered endocytosis of the PTH1R/LRP6 complex is one mechanism of PTH-enhanced phosphorylation of Smad1.
Endocytosis increases the access of BMPs to receptors
We then sought to examine how PTH-induced endocytosis of PTH1R/LRP6 was enhancing BMP2-stimulated phosphorylation of Smad1. Confocal imaging for YFP-BMPRII and PTHTMR in HEK293 cells showed that PTH did not induce endocytosis of BMPRII after PTH treatment (Fig. 5A). Similarly, GFP-labeled Sca-1+CD45–CD11b–MSCs that were transplanted into femur cavities of immunodeficient Rag2−/− mice did not demonstrate any affect on the cell-surface level of BMPRII after PTH treatment compared to vehicle, by FACS analysis (Fig. 5B and C). However, cell-surface protein biotinylation demonstrated that knockdown of LRP6 increased the exposure of endogenous cell-surface BMPRII significantly in C2C12 cells (Fig. 5D). 125I-labeled BMP2 (125I-BMP2) cell surface binding assay in the presence or absence of PTH demonstrated that PTH significantly enhanced cell-surface binding of 125I-BMP2 in a dose- and time-dependent manner (Fig. 5E and F, respectively). Similar results of increased binding of BMP2 to its receptor in the presence of PTH were seen in Sca-1+CD45–CD11b–MSCs (Fig. 5G). These results provide further evidence that PTH-induced endocytosis of PTH1R/LRP6 increases the binding of BMP2 to BMP receptors.
PTH-enhanced BMP signaling stimulates commitment of Sca-1+CD45–CD11b–MSCs to osteoblast differentiation
To examine the effects of PTH-enhanced phosphorylation of Smad1 on lineage commitment of GFP-labeled Sca-1+CD45–CD11b–MSCs, cells were seeded at clonal density to obtain discrete colonies (CFU-Fs) and treated with various combinations of BMP2, PTH, and noggin. None of the treatments exerted significant effects on the formation of CFU-Fs (Fig. 6A and B). However, inducing the CFUs with osteogenic medium and assessing for calcium deposition (CFU-Ob) demonstrated significantly increased CFU-Obs with BMP2 (Fig. 6C, column 3). PTH further increased the BMP2-induced CFU-Obs significantly (Fig. 6C, column 4). Interestingly, when noggin was added to either culture condition, this effect was almost abrogated, decreasing levels below baseline control and implying alternative signaling mechanisms beyond those explored in our study. However, in the presence of BMP2 and noggin, PTH-enhanced MSCs osteogenic differentiation in CFU-Obs increased to the control levels (Fig. 6A and C, column 6). These data are consistent with previous results (Fig. 2D and E). Similar results were observed in the measurements of the effects of PTH on ALP activity and mineralization of MSCs (Supplemental Fig. S5). To further confirm whether PTH-enhanced BMP signaling contributed to osteoblast differentiation of MSCs in vivo, GFP-labeled Sca-1+CD45–CD11b–MSCs were transplanted into the femur marrow cavity of Rag2−/− mice. Mice were injected with vehicle or PTH once daily for 1 week (Fig. 6D). The bone marrow was harvested for FACS analysis. Osterix+ GFP-labeled MSCs were increased from 25.52% to 44.81% in the PTH-injected mice relative to vehicle-injected group, further indicating that PTH induces osteoblast differentiation of MSCs in vivo (Fig. 6E and F). Furthermore, immunostaining of osterix in GFP-labeled MSCs indicated that PTH can promote osteoblast differentiation of MSCs on the bone surface (Supplemental Fig. S6). Altogether, these results demonstrate that one mechanism of PTH-promotion of osteoblast differentiation from MSCs is through enhancement of BMP2 signaling by endocytosis of a PTH1R/LRP6 complex, resulting in increased binding of BMP2 to its receptor.
The capacity of MSC self-renewal and multipotency is maintained in the bone marrow microenvironment.45, 46 The decision of adult stem cells to differentiate is regulated by signals from the microenvironment. BMPs play a critical role in control of differentiation of MSCs to specific lineages, including osteoblasts,47, 48 chondrocytes,49 and adipocytes.50 The activation of BMP signaling is tightly tempered by a family of soluble, extracellular-secreted BMP antagonists23 and is often integrated with other signaling pathways depending on cellular context.51–53 BMP antagonists including noggin and sclerostin act as central regulators of many complex cellular events through interaction with multiple molecular targets such as LRP6.30 It is known that these BMP antagonists are involved in activation of both the Wnt signaling cascade54, 55 and the BMP signaling pathways24 required for the maintenance of MSCs in the bone marrow. In this study, we observed inhibition of BMP signaling induced by noggin in C2C12 cells and MSCs. This inhibition was antagonized by PTH and resulted in enhancement of pSmad1 signaling via BMP2.
Our data also showed PTH-enhancement of pSmad signaling through an endocytic process. Endocytosis plays a critical role in initiating and spreading signals by determining which specific subpathway to activate or to terminate based on the internalization of plasma membranes, cell-surface receptors, and diverse soluble molecules. In some systems, the very outcome of cell signaling depends on how the various components of the signaling machinery are integrated and trafficked. For PTH signaling, β-arrestin2 mediates endocytosis of PTH1R via clatherin-coated pits, which not only mediates desensitization of G protein signaling but also effects the integration of different signals by inhibiting signal transduction.56 In this study, PTH-enhanced pSmad1 levels were attenuated by knockdown of β-arrestin. Simultaneously, these results were validated by decreased pERK with β-arrestin knockdown, which is downstream in the PTH signaling pathway. To further confirm the role of PTH1R endocytosis in the PTH-enhanced BMP signaling pathway, both C2C12 cell lines and MSCs were treated with the endocytosis inhibitor, chlorpromazine (CP), in combination with BMP and/or PTH and confirmed decreased pSmad1 levels.
LRP6 contains a large extracellular domain and binds to several Wnt signaling antagonists including sclerostin,30 DKK1,57 Wise,58 and R-spondin.59 Interestingly, sclerostin is an antagonist for both Wnt and BMP signaling pathways. Additionally, we previously found that LRP6 is a key component for PTH signaling via interaction with PTH1R on the cell membrane and acts by formation of a ternary complex, containing PTH, PTH1R, and LRP6, promoting rapid phosphorylation of LRP6, resulting in the recruitment of axin to LRP6 and inhibition of glycogen synthase kinase 3 (GSK-3) activity and stabilization of β-catenin.33 Finally, both DKK1 and sclerostin act as negative regulators for PTH-induced cAMP production.60 Thus, we hypothesized that LRP6 may act as a central organizer for the extracellular antagonist network for the negative regulation of both Wnt and BMP signaling pathways. Our results indicate that PTH disrupts the LRP6-organized antagonist network by a unique mechanism; ie, inducing endocytosis of the PTH1R-LRP6-antagonist complex. PTH-induced endocytosis of PTH1R/LRP6 complex was evident by FACS analysis of cell-surface LRP6, immunofluorescence colocalization of both proteins, and analysis of cell-surface biotinylated LRP6. Additionally, pSmad1 enhancement was seen with knockdown of LRP6 alone, but was abrogated in PTH1R-deficient cells.
The exact mechanism of how LRP6 endocytosis enhances BMP signaling, whether through direct reduction of BMP antagonists or downregulation of wnt cascades via endocytosis, remains to be elucidated. Importantly, we show that deletion of endogenous LRP6 in MSCs increased the exposure of BMPRII to its ligand at the cell surface and that PTH enhanced the binding affinity of BMP2 to its receptor in a dose- and time-dependent manner. Prior studies afford both as plausible hypotheses. Multiple interactions exist between BMP and Wnt antagonists.28–32 In embryonic stem cells, Wnt signaling inhibits BMP signaling.61 Kahlet and Westendorf62 have shown that the Wnt signaling pathway directly represses transcription of some genes including the osteocalcin gene mediated by Runx2, directly inhibiting osteoblast differentiation. Figure 7 outlines our working model with the knowledge known to date. In bone growth and development, BMP activity is tightly controlled by many antagonists spatially and temporally. With PTH treatment, a PTH1R/LRP6 complex is formed and its endocytosis results in increased binding affinity of BMP to its receptor, which promotes BMP-Smad signaling and MSC differentiation.
For this study, we limited testing our hypothesis to BMP2 and noggin. BMP2 is the most effective osteoinducer among all the BMPs tested in vitro and in vivo, and of the known antagonists, noggin has been shown to have a high specificity and affinity for BMP2 (Kd = 2 × 10−11 M).63 Additionally, low concentrations of noggin have been shown to inhibit BMP effects dramatically.64 Our data demonstrate one mechanism of PTH-enhancement of BMP-pSmad1 signaling leading to MSCs differentiation; however, there are likely other factors besides BMP2 and noggin involved. Specifically, the fifth column of Fig. 6A and C shows that treatment with both BMP2 and noggin suppresses CFU-Ob formation to a lower level than when the cells were not treated with BMP2, indicating that either there is endogenous BMP production by these cells or the differentiation is occurring through a non-BMP pathway. Many other authors have examined the effects of various other BMPs on MSC proliferation and differentiation, which may be contributing to this effect. Sammons and colleagues65 found that a combination of BMP6, PTH, and vitamin D3 could induce osteoblast differentiation in normal human adult bone marrow–derived MSCs. Pountos and colleagues66 also showed that treatment of elderly human osteoporotic trabecular bone–derived MSC differentiation to the osteoblast lineage could be enhanced with BMP-2, BMP-7, PTH, and platelet-derived growth factor (PDGF). Therefore, it is likely that other BMPs may also play a similar role and may be explored in future research.
PTH, a systemic hormone controlling calcium homeostasis, stimulates the activities of both osteoclasts and osteoblasts and alters the microenvironment in the bone marrow. PTH stimulates bone formation when injected daily and is the only anabolic drug for the treatment of osteoporosis patients.67, 68 Intensive studies on the mechanisms by which PTH exerts its bone anabolic actions have focused on its effects on osteoblasts.17 The current study instead focuses on maintaining the supply of osteoblasts, revealing that PTH stimulates the commitment of MSCs to the osteoblast lineage by enhancing BMP signaling. In addition to producing cAMP as its downstream signaling, PTH orchestrates the signaling of other local factors in bone marrow to regulate the activities of osteoblasts or osteoblast progenitors. PTH enhancement of BMP signaling is a part of the integration of the signaling networks of local factors for the spatial-temporal regulation of MSC differentiation.
All authors state that they have no conflicts of interest.
We thank Dr. Bart O Williams (Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute) for providing the Lrp6flox/flox mice. This work was supported by NIH grant DK057501 to XC, DK083350 to MW, and T32DK007751 to JC.
Authors' roles: BY, WL, and XC designed the experiments. BY, XLZ, CZY, and LLX carried out the experiments and analyzed the data. MW and JC gave detailed comments on the manuscript. BY, JC, and XC wrote the manuscript.