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Def6 regulates endogenous type-I interferon responses in osteoblasts and suppresses osteogenesis

  1. Zhonghao Deng
  2. Courtney Ng
  3. Kazuki Inoue
  4. Ziyu Chen
  5. Yuhan Xia
  6. Xiaoyu Hu
  7. Matthew Greenblatt
  8. Alessandra Pernis
  9. Baohong Zhao  Is a corresponding author
  1. Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, United States
  2. Department of Medicine, Weill Cornell Medical College, United States
  3. Institute for Immunology and School of Medicine, Tsinghua University, China
  4. Pathology and Laboratory Medicine, Weill Cornell Medical College, United States
  5. Research Division, Hospital for Special Surgery, United States
  6. Autoimmunity and Inflammation Program, Hospital for Special Surgery, United States
  7. Graduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, United States
  8. Graduate Program in Cell and Development Biology, Weill Cornell Graduate School of Medical Sciences, United States
Research Article
Cite this article as: eLife 2020;9:e59659 doi: 10.7554/eLife.59659
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 5 supplements
Def6 absence accelerates osteoblast differentiation.

(A, B) Expression of Def6 during osteoblast differentiation using WT and Def6-/- calvarial osteoblast cells at the indicated time points by quantitative real-time PCR (qPCR) analysis of mRNA expression (A) or immunoblot analysis of protein levels (B). (C) ALP staining and (D) Alizarin red staining (upper panel) and its quantification (lower panel) of WT and Def6-/- calvarial osteoblast differentiation at day 15 in osteogenic medium. (E) qPCR analysis of mRNA expression of Alpl, Bsp and Bglap during WT and Def6-/- calvarial osteoblast differentiation process. Data are mean ± SEM. **p<0.01. n.s., not statistically significant.

Figure 1—figure supplement 1
Def6 expression in osteoblasts in vivo and in osteoblast cell lines.

(A) Immunofluorescence staining of Def6 (green) on the slices of femurs from 8-week-old WT and Def6-/- mice. Def6-/- mice were used as a negative control for Def6 staining. The slices were counterstained with DAPI (blue). White arrows point to osteoblasts on the bone surface. BM, bone marrow. Scale bar: 50 µm. (B–C) Immunoblot analysis of Def6 expression in ST2 cells (B) and MC3T3-E1 cells (C) during osteoblast differentiation. p38 or GAPDH were used as a loading control.

Figure 1—figure supplement 2
RNAseq–based expression heatmap of the marker genes of osteoclasts and macrophages (left panel) and volcano plot (right panel) of RNA-seq analysis of differentially expressed genes in WT and Def6-/- calvarial osteoblasts.
Figure 1—figure supplement 3
CD45 selection does not affect Def6 role in osteoblast differentiation.

CD45 negative calvarial osteoblasts were selected using CD45 magnetic beads according to the manufacturer's instruction, and used to test osteoblast differentiation and gene expression. (A) ALP staining at day 9 and (B) Alizarin red staining at day 21 (left panel) and its quantification (right panel) of CD45 negative WT and Def6-/- calvarial osteoblast differentiation in osteogenic medium. (C, D) qPCR analysis of mRNA expression of osteoblast marker genes Alpl, Bsp and Bglap (C) and ISGs Mx1, Ifit1, Ifit2 and Eif2ak2 (D) during CD45 negative WT and Def6-/- calvarial osteoblast differentiation process. Data are mean ± SD. *p<0.05. **p<0.01.

Figure 1—figure supplement 3—source data 1

Source data for Figure 1—figure supplement 3.

https://cdn.elifesciences.org/articles/59659/elife-59659-fig1-figsupp3-data1-v1.xlsx
Figure 1—figure supplement 4
Bone marrow derived macrophages (BMMs) from WT and Def6-/- mice were co-cultured with CD45 negative WT calvarial osteoblasts using transwell inserts, in which BMMs were cultured in the upper compartment of the well and osteoblasts were on the bottom compartment of the well.

(A) ALP staining at day 7 and (B) qPCR analysis of osteoblast marker gene expression at the indicated times. n.s., not statistically significant.

Figure 1—figure supplement 4—source data 1

Source data for Figure 1—figure supplement 4.

https://cdn.elifesciences.org/articles/59659/elife-59659-fig1-figsupp4-data1-v1.xlsx
Figure 1—figure supplement 5
Immunoblot analysis of Def6 expression after TNFα (40 ng/ml) treatment for 24 hr on the WT and Def6-/- calvarial osteoblasts.

p38 was used as a loading control.

Figure 2 with 1 supplement
Def6 deficiency enhances osteogenesis in vivo.

(A) Images of calcein double labeling of the tibiae of WT and Def6-/- littermate mice. (B) Bone morphometric analysis of mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS) after calcein double labeling of the tibiae of WT and Def6-/- littermate male mice. n = 6. (C) Images of Masson-Goldner staining of tibiae of WT and Def6-/- littermate mice. The bones show green, osteoid matrix appears dark orange on the surface of the bone beneath the osteoblasts, osteoblasts (Ob) are stained orange lining on the bone surface, and bone marrow cells appear red in the photograph. (D) Bone morphometric analysis of osteoblast surface per bone surface (Ob.S/BS) and osteoblast number per bone perimeter (N.Ob/B.Pm) of the tibiae of WT and Def6-/- littermate male mice. n = 8. Scale bars = 50 µm. Data are mean ± SEM. *p<0.05, **p<0.01. n.s., not statistically significant.

Figure 2—figure supplement 1
Def6 exhibits osteoporosis in lumbar bones.

(A) Images of Von Kossa staining of lumbar from WT and Def6-/- mice. (B) Bone morphometric analysis of bone volume/tissue vol (BV/TV) of lumbar from WT and Def6-/- male mice. n = 6. Data are mean ± SEM. **p<0.01.

Figure 2—figure supplement 1—source data 1

Source data for Figure 2—figure supplement 1.

https://cdn.elifesciences.org/articles/59659/elife-59659-fig2-figsupp1-data1-v1.xlsx
Def6 deficiency decreases the expression of interferon stimulated genes (ISGs), which are induced during osteoblast differentiation.

(A) Volcano plot of RNA-seq analysis of differentially expressed genes using the mRNAs isolated from the WT and Def6-/- calvarial osteoblast cells stimulated with osteogenic medium for 12 days. Blue dots show genes more highly expressed in Def6-/- osteoblasts than WT osteoblasts with significant (p<0.05) and greater than 1.2-fold changes. Red dots show genes more highly expressed in WT osteoblasts than Def6-/- osteoblasts with significant (p<0.05) and greater than 1.2-fold changes. (B) Pathway analysis of significantly differentially expressed genes. Upper panel shows enriched pathways in WT and lower panel shows enriched pathways in Def6-/- osteoblasts. (C) RNAseq–based expression heatmaps of the interferon stimulated genes (left panel) and osteoblast genes (right panel) regulated by Def6 deficiency based on the differentially expressed genes from (A). Row z-scores of CPMs of genes were shown in the heatmap. #1, replicate 1. #2, replicate 2. (D) Gene set enrichment analysis (GSEA) of differentially expressed genes from WT and Def6-/- calvarial osteoblast cells stimulated with osteogenic medium for 12 days ranked by NES scores. Type-I IFN response genes and osteoblast genes are mostly enriched in WT and Def6-/- osteoblast cells, respectively (p<0.0001 and FDR < 0.0001). The enrichment plots are shown. NES, normalized enrichment score. (E) qPCR analysis of type-I IFN response gene expression during osteoblast cell differentiation. Data are mean ± SEM. **p<0.01. n.s., not statistically significant.

Def6 suppresses osteogenesis via endogenous IFNβ-mediated autocrine feedback inhibition.

qPCR analysis of type-I IFN response gene expression (A) and osteoblast marker gene expression (B) during osteoblast cell differentiation in the absence or presence of IFNβ blocking antibody (10 U/ml). Control IgG, 10 U/ml. (C) Alizarin red staining (left panel) and its quantification (right panel) of WT and Def6-/- calvarial osteoblast differentiation at day 16 in the osteogenic medium in the absence or presence of IFNβ blocking antibody (10 U/ml). Control IgG, 10 U/ml. Data are mean ± SEM. *p<0.05. n.s., not statistically significant.

Figure 5 with 1 supplement
Def6 regulates cellular response of osteoblast cells to IFNβ.

(A–D) Immunoblot analysis of type-I IFN receptor expression during osteoblast differentiation (A), the induction of p-STAT1 and 3 (B, C) and expression of STAT1 (D) in WT and Def6-/- osteoblast cells stimulated with mouse recombinant IFN-β (10 U/ml) at the indicated time points. p38 was used as a loading control. The relative density of each band to its corresponding loading control p38 band was calculated by Image J software, and then was normalized to the WT controls at time 0 (the 1st lanes). The relative density for p-STAT3 to total STAT3 was also calculated. (E) qPCR analysis of type-I IFN response gene expression after indicated time points of treatment with IFN-β (10 U/ml) in WT and Def6-/- osteoblast cells. (F) Osteoblast differentiation is induced by the osteogenic medium in the absence or presence of mouse recombinant IFN-β in the WT and Def6-/- osteoblast cells. Alizarin red staining at day 16 (left panel) and its quantification (right panel) were performed. Data are mean ± SEM. *p<0.05, **p<0.01. n.s., not statistically significant. (G) A model showing that the Def6-IFN axis regulates both osteoclast-mediated bone resorption (Binder et al., 2017) and osteoblast-mediated bone formation (current study) in bone homeostasis. Def6 deletion enhances both bone resorption (Binder et al., 2017) and formation (current study) via attenuated type-I IFN-mediated feedback inhibition of the differentiation of both cell types, leading to a high turn-over osteoporotic phenotype in Def6-/- mice.

Figure 5—figure supplement 1
qPCR analysis of mRNA expression of Ifnar1 and Ifnar2 during osteoblast differentiation.
Figure 5—figure supplement 1—source data 1

Source data for Figure 5—figure supplement 1.

https://cdn.elifesciences.org/articles/59659/elife-59659-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 2 supplements
Def6 deficiency inhibits endogenous expression of IFN-β and ISG genes via downregulation of PKR in osteoblasts.

(A–B) ELISA analysis of IFN-β level in mouse serum (A) and in CD45 negative calvarial osteoblast cell cultures (B) from WT and Def6-/- mice (n = 5). (C) ALP staining at day 7 and (D) Alizarin red staining at day 21 (left panel) and its quantification (right panel) of WT and Ifnar1-/- (IFNαβR KO) calvarial osteoblast differentiation in osteogenic medium. (E) qPCR analysis of mRNA relative expression of osteoblast marker genes and ISGs during WT and Ifnar1-/- (IFNαβR KO) calvarial osteoblast differentiation process. (F) Immunoblot analysis of PKR expression in WT and Def6-/- calvarial osteoblasts. p38 was used as a loading control. (G) Immunoblot analysis of PKR expression after knockdown of PKR by PKR-LNA induced gene silencing in ST2 cells. p38 was used as a loading control. (H–J) qPCR analysis of mRNA expression of Ifnb and ISGs (H), ALP staining, scale bar: 500 µm (I) and qPCR analysis of mRNA expression of osteoblastic genes (J) in ST2 cells with or without PKR knockdown. (K) Immunoblot analysis of PKR expression after overexpression of PKR by retroviral transduction for 24 hr. p38 was used as a loading control. (L) qPCR analysis of mRNA expression of Ifnb, ISGs and osteoblast markers. Data are mean ± SD. *p<0.05. **p<0.01. n.s., not statistically significant.

Figure 6—figure supplement 1
qPCR analysis of mRNA expression of Ifnb during osteoblast differentiation.
Figure 6—figure supplement 1—source data 1

Source data for Figure 6—figure supplement 1.

https://cdn.elifesciences.org/articles/59659/elife-59659-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
ELISA analysis of IFNα level in the serum of 8 week old WT and Def6-/- mice.

n = 5.

Figure 6—figure supplement 2—source data 1

Source data for Figure 6—figure supplement 2.

https://cdn.elifesciences.org/articles/59659/elife-59659-fig6-figsupp2-data1-v1.xlsx

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(M. musculus)
Def6-/-PMID:16470246RRID:MGI:3628445
Genetic reagent
(M. musculus)
Ifnar-/-PMID:8009221RRID:MGI:3765898
Cell line (Homo sapiens)Platinum-E (Plat-E)Cell BiolabsCat# RV-101
RRID:CVCL_B488
Has been authenticated by STR
profiling and tested
negative for mycoplasma
Cell line (M. musculus)ST2DSMZCat# ACC333
RRID:CVCL_2205
Has been authenticated by STR
profiling and tested
negative for mycoplasma
Cell line (M. musculus)MC3T3-E1ATCCCat# CRL-2594
RRID:CVCL_5437
Has been authenticated by STR
profiling and tested
negative for mycoplasma
Antibodyanti-IBP/Def6
(rabbit polyclonal)
PMID:12923183WB (1:1000)
IF (1:100)
Antibodyanti-IFN-α/βRα
(mouse monoclonal)
Santa Cruz BiotechnologyCat# sc-7391
RRID:AB_2122749
WB (1:1000)
Antibodyanti-mouse IFN Beta,
neutralizing
(rabbit polyclonal)
PBL Assay ScienceCat# 32400–1
RRID:AB_387872
10 U/ml
Antibodyanti-Stat1
(rabbit polyclonal)
Santa Cruz BiotechnologyCat# sc-346
RRID:AB_632435
WB (1:1000)
Antibodyanti-Stat3
(rabbit monoclonal)
Cell Signaling TechnologyCat# 12640
RRID:AB_2629499
WB (1:1000)
Antibodyanti- Phospho-Stat1
(Ser727) (rabbit polyclonal)
Cell Signaling TechnologyCat# 9177
RRID:AB_2197983
WB (1:1000)
Antibodyanti- Phospho-Stat1
(Tyr701) (rabbit polyclonal)
Cell Signaling TechnologyCat# 9171
RRID:AB_331591
WB (1:1000)
Antibodyanti-Phospho-Stat3 (Tyr705)
(rabbit polyclonal)
Cell Signaling TechnologyCat# 9131
RRID:AB_331586
WB (1:1000)
Antibodyanti-PKR
(mouse monoclonal)
Santa Cruz BiotechnologyCat# sc-6282
RRID:AB_628150
WB (1:1000)
Antibodyanti-p38α
(rabbit polyclonal)
Santa Cruz BiotechnologyCat# sc-535
RRID:AB_632138
WB (1:3000)
Antibodyanti-Gapdh
(rabbit polyclonal)
Santa Cruz BiotechnologyCat# sc-25778
RRID:AB_10167668
WB (1:3000)
Recombinant DNA
reagent
pMXs-IRES-GFP
(plasmid)
Cell BiolabsCat# RTV-013
Recombinant DNA
reagent
pMXs-mPKR-FLAG-IG
(plasmid)
AddgeneCat# 101792
RRID:Addgene_101792
Sequence-based
reagent
siEif2ak2 LNAQiagenCat#30060040 nM for knockdown
Peptide, recombinant
protein
Recombinant mouse IFNβPBL Assay ScienceCat#12400–110 U/ml
Peptide, recombinant
protein
Recombinant Murine TNFαPeprotechCat# 315-01A40 ng/ml
Peptide, recombinant
protein
Recombinant Murine M-CSFPeprotechCat# 315–0220 ng/ml
Commercial assay or kitVeriKine-HS Mouse
IFN Beta Serum ELISA Kit
PBL Assay ScienceCat#42410–1
Commercial assay or kitMouse IFN Alpha All
Subtype ELISA Kit
PBL Assay ScienceCat#42115–1
Chemical compound,
drug
FuGENE 6
Transfection Reagent
PromegaCat# E2691
Chemical compound,
drug
TransIT-TKO
Transfection Reagent
MirusCat#MIR2150
Software, algorithmHISAT2PMID:31375807RRID:SCR_015530http://daehwankimlab.github.io/hisat2/
Software, algorithmHTseqPMID:25260700RRID:SCR_005514https://htseq.readthedocs.io/en/master/#
Software, algorithmedgeRPMID:19910308
PMID:22287627
RRID:SCR_012802https://bioconductor.org/packages/release/bioc/html/edgeR.html
Software, algorithmpheatmaphttps://cran.r-project.org/web/packages/
pheatmap/index.html
RRID:SCR_016418https://cran.r-project.org/web/packages/pheatmap/index.html
Software, algorithmEnrichrPMID:23586463
PMID:27141961
RRID:SCR_001575https://maayanlab.cloud/Enrichr/
Software, algorithmImageJ
(v1.50i)
PMID:22930834RRID:SCR_003070https://imagej.nih.gov/ij/index.html
Software, algorithmGraphpad Prism 8GraphPad SoftwareRRID:SCR_002798
Software, algorithmOsteomeasureOsteoMetrics, Inc
OtherProLong Gold Antifade
Mountant with DAPI
Thermo Fisher scientificCat#P36941

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