Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass
- University of Texas Southwestern Medical Center, United States
- Tongji University, China
- Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
Figures
Figure 1
Osteolectin contains conserved integrin binding motifs and binds with high affinity to integrin α11β1.
(A, B) The human (A) and mouse (B) Osteolectin proteins contain RGD and LDT sequences. (C) Alignment of Osteolectin amino acid sequences shows that the RGD and LDT domains are evolutionarily …
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Figure 1—source data 1
Data for Figure 1.
- https://doi.org/10.7554/eLife.42274.004
Figure 2
Osteolectin activates Wnt pathway signaling in skeletal stem/progenitor cells.
(A) Western blot of cell culture supernatant from parental or Osteolectin deficient MC3T3-E1 cells and human bone marrow stromal cells (hBMSC#1 and hBMSC#2 cells) (this blot is representative of …
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Figure 2—source data 1
Data for Figure 2.
- https://doi.org/10.7554/eLife.42274.006
Figure 3
Osteogenic differentiation in response to Osteolectin requires β-catenin.
(A and B) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with PBS or 30 ng/ml recombinant mouse Osteolectin, as well as DMSO or 200 nM of …
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Figure 3—source data 1
Data for Data for Figure 3.
- https://doi.org/10.7554/eLife.42274.008
Figure 4
Integrin α11 is required for osteogenic differentiation and Wnt pathway activation in response to Osteolectin but exogenous collagen does not activate Wnt pathway signaling.
(A) Osteogenic differentiation in culture of parental or Itga11 deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells with PBS or recombinant mouse Osteolectin (n = 3 independent experiments). …
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Figure 4—source data 1
Data for Figure 4.
- https://doi.org/10.7554/eLife.42274.010
Figure 5 with 1 supplement
Conditional Itga11 deletion from LepR+cells accelerates the loss of trabecular bone during aging.
(A) Lepr-Cre; Itga11fl/fl mice were grossly normal and indistinguishable from littermate controls. (B – D) Body length (B), body mass (C) and femur length (D) did not significantly differ between Lep…
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Figure 5—source data 1
Data for Data for Figure 5.
- https://doi.org/10.7554/eLife.42274.012
Figure 5—figure supplement 1
Generation of an Itga11 floxed mice.
(A) DNA surrounding Itga11 exon3 was used as homologous arms for recombineering. The recombineering plasmid was modified by inserting loxP sites flanking exon3. Deletion of exon three leads to a …
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Figure 5—figure supplement 1—source data 1
Data for Figure 5—figure supplement 1.
- https://doi.org/10.7554/eLife.42274.014
Figure 6
Conditional Itga11 deletion from LepR+cells reduces cortical bone formation in adult mice.
(A) Representative microCT images of cortical bone in the mid-femur diaphysis of male Lepr-Cre; Itga11fl/fl mice and littermate controls at 2, 6, and 12 months of age. (B–F) microCT analysis of the …
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Figure 6—source data 1
Data for Figure 6.
- https://doi.org/10.7554/eLife.42274.016
Figure 7 with 1 supplement
Integrin α11 is required by bone marrow stromal cells to undergo osteogenesis in response to Osteolectin.
(A) CFU-F frequency and (B) cells per CFU-F colony formed by bone marrow cells from Lepr-Cre; Itga11fl/fl mice and littermate controls at 2 and 6 months of age (n = 6–8 mice per genotype per time …
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Figure 7—source data 1
Data for Figure 7.
- https://doi.org/10.7554/eLife.42274.018
Figure 7—figure supplement 1
Osteolectin promotes Wnt target gene transcription in mouse bone marrow stromal cells in an integrin α11-dependent manner.
(A) Bone marrow stromal cells from Lepr-Cre; Itga11fl/fl mice and sex-matched littermates at 2 months of age were cultured in osteogenic differentiation medium supplemented with PBS (control) or 30 …
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Figure 7—figure supplement 1—source data 1
Data for Figure 7—figure supplement 1.
- https://doi.org/10.7554/eLife.42274.020
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent(M.musculus) | Osteolectin-/- | PMID: 27976999 | ||
| Genetic reagent (M.musculus) | Lepr-Cre | PMID: 11283374 | JAX Stock (008320) | |
| Antibody | rabbit polyclonal anti-phospho-PI3 Kinase | Cell Signaling | 4228S | (1:1000) |
| Antibody | rabbit polyclonal anti-phospho-Akt (Ser473) | Cell Signaling | 4060S | (1:1000) |
| Antibody | rabbit polyclonal anti-phospho-GSK-3α/β | Cell Signaling | 9331S | (1:1000) |
| Antibody | rabbit polyclonal anti-β-Catenin | Cell Signaling | 8480S | (1:1000) |
| Antibody | rabbit polyclonal anti-anti-β-Actin | Cell Signaling | 8457S | (1:10000) |
| Antibody | rabbit polyclonal anti-GSK-3b | Cell Signaling | 9315S | (1:1000) |
| Antibody | goat polyclonal anti-rabbit IgG, HRP-linked antibody | Cell Signaling | 7074S | (1:5000) |
| Antibody | horse polyclonal anti-mouse IgG, HRP-linked antibody | Cell Signaling | 7076S | (1:5000) |
| Antibody | donkey polyclonal anti-sheep IgG, HRP-linked antibody | R and D Systems | HAF016 | (1:5000) |
| Antibody | rabbit polyclonal anti-phospho-FAK (Y397) | Cell Signaling | 3283S | (1:1000) |
| Antibody | rabbit polyclonal anti-Histone H3 | Cell Signaling | 4499S | (1:5000) |
| Antibody | goat polyclonal anti-mouse Osteolectin | R and D Systems | AF3729 | (1:1000) |
| Antibody | sheep polyclonal anti-human Osteolectin | R and D Systems | AF1904 | (1:1000) |
| Antibody | rabbit polyclonal anti-Adiponectin | Abcam | ab181699 | (1:1000) |
| Antibody | rabbit polyclonal anti-Integrin α11 | Abcam | ab198826 | (1:1000) |
| Antibody | rabbit polyclonal anti-FAK | Cell Signaling | 3285P | (1:1000) |
| Antibody | mouse monoclonal anti-6x-His Tag antibody | Thermo Fisher Scientific | MA1-135 | (1:1000) |
| Antibody | rat polyclonal anti-CD45-APC | Tonbo | 20–0451 | (1:200) |
| Antibody | rat polyclonal anti-Ter119-APC | Tonbo | 20–5921 | (1:200) |
| Antibody | rat polyclonal anti-CD31-APC | Biolegend | 102410 | (1:200) |
| Antibody | goat polyclonal anti-Mouse Leptin R, Biotin | R and D Systems | AF497 | (1:200) |
| Antibody | mouse monoclonal anti-Streptavidin PE | Biolegend | 410504 | (1:500) |
| Peptide, recombinant protein | Bovine serum albumin | Sigma-Aldrich | A3156 | |
| Peptide, recombinant protein | recombinant human pro-Collagen I α1 | R and D Systems | 6220 CL | |
| Peptide, recombinant protein | recombinant human osteolectin | PMID: 27976999 | ||
| Peptide, recombinant protein | recombinant mouse osteolectin | PMID: 27976999 | ||
| Peptide, recombinant protein | recombiant Integrin α11β1 protein | R and D Systems | 6357-AB | |
| Peptide, recombinant protein | recombiant Integrin α10β1 protein | R and D Systems | 5895-AB | |
| Peptide, recombinant protein | recombiant Integrin αVβ3 protein | R and D Systems | 3050-AV | |
| Peptide, recombinant protein | recombiant Integrin αVβ1 Protein | R and D Systems | 6579-AV | |
| Peptide, recombinant protein | recombiant Integrin α4β1 protein | R and D Systems | 5668-A4 | |
| Peptide, recombinant protein | recombiant Integrin α9β1 protein | R and D Systems | 5438-A9 | |
| Peptide, recombinant protein | recombiant Integrin αIIbβ3 Protein | R and D Systems | 7148-A2 | |
| Peptide, recombinant protein | recombiant Integrin αMβ2 Protein | R and D Systems | 4047-AM | |
| Chemical compound,drug | DAPI | Life Technologies | D1306 | |
| Chemical compound, drug | TRIzol LS Reagent | Invitrogen | 10296028 | |
| Chemical cmpound,drug | Collagenase, Type 1 | Worthington | LS004196 | |
| Chemical compound, drug | Dispase II | Roche Diagnostic | D4693 | |
| Chemical compound, drug | DNase I | Sigma-Aldrich | 10 104 159 001 | |
| Chemical compound, drug | IWR-1-endo | Sigma-Aldrich | 681669 | |
| Chemical compound, drug | AZD2858 | Selleck | S7253 | |
| Chemical compound, drug | Y-27632 Rock inhibitor | Selleck | S1049 | |
| Chemical compound, drug | 4% paraformaldehyde in PBS | Thermo Fisher Scientific | J19943-K2 | |
| Cell lines (M.musculus) | MC3T3-E1, subclone 4 | ATCC | CRL-2593 | |
| Cell lines (H.sapiens) | hBMSC#1 | ATCC | PCS-500–012 | |
| Cell lines (H.sapiens) | hBMSC#2 | Lonza | PT-2501 | |
| Commercial assay or kit | Cell Culture Contamination Detection Kit | Molecular Probes | C-7028 | |
| Commercial assay or kit | TMB stop solution | KPL | 95059–198 | |
| Commercial assay or kit | SureBlue TMB Microwell Peroxidase Substrate | KPL | 95059–282 | |
| Commercial assay or kit | HBSS, without Calcium and Magnesium | Corning | 21022CV | |
| Commercial assay or kit | HBSS, with Calcium and Magnesium | Corning | 21023CV | |
| Commercial assay or kit | CleanCap Cas9 mRNA | TriLink | L-7206 | |
| Commercial assay or kit | MEGAshortscript T7 Transcription Kit | Ambion | AM1354 | |
| Commercial assay or kit | MEGAclear Transcription Clean-Up Kit | Ambion | AM1908 | |
| Commercial assay or kit | EDTA-free Protease Inhibitor Cocktail | Sigma-Aldrich | 11 836 170 001 | |
| Commercial assay or kit | ECL Western Blotting Substrate | Pierce | 32106 | |
| Commercial assay or kit | SuperScript III Reverse Transcriptase | Invitrogen | 18080044 | |
| Commercial assay or kit | StemPro Osteogenesis Differentiation Kit | Gibco | A1007201 | |
| Commercial assay or kit | StemPro Adipogenesis Differentiation Kit | Gibco | A1007001 | |
| Commercial assay or kit | StemPro Chondrogenesis Differentiation Kit | Gibco | A1007101 | |
| Commercial assay or kit | MicroVue DPD ELISA Kit | Quidel | 8007 | |
| Commercial assay or kit | MicroVue Creatinine Assay Kit | Quidel | 8009 | |
| Commercial assay or kit | Rat/Mouse PINP ELISA kit | Immunodiagnostic Systems | AC-33F1 | |
| Commercial assay or kit | Nuclear/Cytosol Fractionation Kit | Biovision | K266 | |
| Other | Fetal bovine serum | Sigma-Aldrich | F2442 | |
| Other | Penicillin Streptomycin 100x solution | HyClone | SV3001 | |
| Other | DMEM, low glucose | Gibco | 11885084 | |
| Other | DMEM, high glucose | Gibco | D5671 |
Author response table 1
RNA-seq analysis of LepR+ cells from non-irradiated bone marrow as well as hematopoietic cells and unfractionated cells from irradiated bone marrow (which are high in adipocytes).
Itga11 is poorly expressed in adipocyte-rich bone marrow cells after irradiation (all data represent average FPKM values from multiple replicates).
| Cell type | Adipoq | Fabp4 | Plin1 | Itga11 | Osteolectin |
| LepR+ cells from bone marrow | 835 | 23 | 1.2 | 34 | 37 |
| Hematopoietic cells from irradiated bone marrow | 1.8 | 13 | 0.3 | 0.1 | 0.2 |
| Whole bone marrow cells after irradiation (high in adipocytes) | 92 | 1016 | 68 | 2.2 | 2.8 |
Additional files
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Supplementary file 1
Markers used for the flow cytometric isolation of bone marrow cell populations.
Stem and progenitor cell populations were isolated from mouse bone marrow by flow cytometry using the listed markers. Lineage (Lin) markers used to isolate Lineage negative cell populations were CD2, CD3, CD5, CD8, Ter119, Gr-1, and B220.
- https://doi.org/10.7554/eLife.42274.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.42274.022
