GAS5 protects against osteoporosis by targeting UPF1/SMAD7 axis in osteoblast differentiation
- Department of Orthopedics, The Eighth Affiliated Hospital, Sun Yat-sen University, China
- Department of Orthopedics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, China
- Department of Orthopedics, Zhujiang Hospital, Southern Medical University, China
- Center for Biotherapy,The Eighth Affiliated Hospital, Sun Yat-sen University, China
- Center for Biotherapy, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, China
Figures
Figure 1 with 2 supplements
GAS5 decreased in osteoporosis and positively correlated with the osteoblast differentiation.
(A) COL1A1 and BGLAP immunohistochemical staining of the femur head from hip dysplasia and osteoporosis patients. The red arrow shows typical staining. Scale bar, (upper, 500 µm; lower, 50 µm). (B) …
Figure 1—figure supplement 1
Gene marker expression of BMSCs osteoblast differentiation.
Relative mRNA expression of GAS5, BGLAP, ALPL, and RUNX2 along with the day of human BMSCs osteogenic differentiation.
Figure 1—figure supplement 2
The relation of GAS5 and the other makers of MSCs in the osteoblast differentiation.
(A) The correlation of GAS5 and ALP assay during osteogenic differentiation of MSCs (n = 15). (B, C, D) The correlation of GAS5 and ALPL, BGLAP, and RUNX2 during osteogenic differentiation of MSCs …
Figure 2 with 2 supplements
Decreasing GAS5 inhibited the osteoblast differentiation in vitro and in vivo.
(A) GAS5 siRNA knockdown efficiency tested by qRT-PCR. (B) ARS staining and quantification in the GAS5 knockdown or control group. Scale bar, 500 µm. (C) ALP staining and ALP tests in the GAS5 …
Figure 2—figure supplement 1
Western blot analysis and quantification of osteogenesis markers.
(A) Western blot analysis and quantification of osteogenesis markers (COL1A1, RUNX2, BGLAP) in GAS5 knockdown or overexpression conditions. (B) Quantification of Western blot of osteogenesis markers …
Figure 2—figure supplement 2
The quantification of BGLAP immunohistochemical staining.
The quantification of BGLAP immunohistochemical staining of in vivo bone formation tests in GAS5 knockdown or overexpression treatment.
Figure 3 with 2 supplements
Increasing GAS5 in BMSCs promotes osteoblast differentiation in vitro and in vivo.
(A) Relative GAS5 expression in the overexpression and control groups. (B) ARS staining and quantification in the GAS5 overexpression or control group. Scale bar, 50 µm. (C) ALP staining and ALP …
Figure 3—figure supplement 1
Western blot analysis and quantification of osteogenesis markers.
(A) Western blot analysis and quantification of osteogenesis markers (COL1A1, RUNX2, BGLAP) in GAS5 knockdown or overexpression conditions. (B) Quantification of western blot of osteogenesis markers …
Figure 3—figure supplement 2
The quantification of BGLAP immunohistochemical staining.
The quantification of BGLAP immunohistochemical staining of in vivo bone formation tests in GAS5 knockdown or overexpression treatment.
Figure 4 with 2 supplements
GAS5 functions by interacting with the UPF1 protein.
(A) Coomassie brilliant blue staining of RNA pull-down proteins in the GAS5 sense or antisense group. (B) The interaction between GAS5 and UPF1 was confirmed by Western blotting. (C, D) RIP assay …
Figure 4—figure supplement 1
QRT-PCR analysis of the GAS5 adjacent genes in GAS5 knockdown conditions.
ZBTB37 (zinc finger and BTB domain containing 37), RC3H1 (ring finger and CCCH-type domains 1), SERPINC1 (serpin family C member 1), ZBTB37 (zinc finger and BTB domain containing 37), TNN (tenascin …
Figure 4—figure supplement 2
RNA fold model of GAS5.
The model was predicted on the RNAfold web server website (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi). The rainbow color means the base-pair probabilities of the predicted folded …
Figure 5 with 2 supplements
GAS5 interacts with UPF1 to accelerate SMAD7 decay.
(A) Western blot analysis of the activation pathway (pSMAD1/5/8, SMAD1) in GAS5 knockdown or overexpression conditions. (B) SMAD7 was regulated by GAS5 knockdown or overexpression by western blot. (C…
Figure 5—figure supplement 1
Western blot analysis of the pathway.
(A, B) Western blot analysis and quantification of the pathway (catenin, ERK, SMAD2/3, SMURF1, SMURF2) in GAS5 knockdown or overexpression conditions. (C) Quantification for western blot analysis of …
Figure 5—figure supplement 2
Western blot analysis of the molecular in BMP pathway.
(A, B, C, D, E, F) Western blot analysis and quantification of BMP2, BMP4, BMP7, and SMAD6 in GAS5 knockdown or overexpression conditions.
Figure 6 with 3 supplements
Gas5 heterozygous mice exhibit an osteoporotic phenotype and impaired bone repair capacity.
(A) Representative micro-CT analysis of the three-dimensional reconstruction graph for Gas5+/- (n = 6) and WT mice (n = 6). (B) Representative H and E staining (upper) and Bglap immunohistochemical …
Figure 6—figure supplement 1
Relative gene expression in mouse.
(A, B) Relative Gas5 expression in the tails of Gas5+/+ (n = 6) and Gas5+/- (n = 6) mice analyzed by qRT-PCR and agarose gel electrophoresis. (C) Relative mRNA expression of Gas5, Upf1 and Smad7 in …
Figure 6—figure supplement 2
The quantification of Bglap immunohistochemical staining.
The quantification of Bglap immunohistochemical staining of Gas5+/-mice and WT mice.
Figure 6—figure supplement 3
Western blot analysis of the pathway.
Western blot analysis of the pathway (β-catenin, ERK) in the protein from the tibia of Gas5+/+ (n = 6) and Gas5+/- mice (n = 6).
Figure 7 with 1 supplement
Systemic transfection (tail vein injection) of Gas5-overexpressing adenoviruses alleviated bone loss in osteoporosis.
(A) Micro-CT analysis of the control mice (n = 6), osteoporosis mouse model (n = 6) or Gas5-overexpressing adenovirus-treated mice (n = 6). (B) H and E staining and Bglap immunohistochemical …
Figure 7—figure supplement 1
The quantification of Bglap immunohistochemical staining.
The quantification of Bglap immunohistochemical staining of the control mice (n = 6), osteoporosis mouse model (n = 6) or Gas5-overexpressing adenovirus-treated mice.
Author response image 1
(A)The SMAD7 mRNA abundance by RIP assay with anti-UPF1 antibody; (B) the SMAD6 mRNA abundance by RIP assay with anti-UPF1 antibody.
Author response image 2
Functional verification of 2 genes and 4 bands in the MMS.
A) SMAD1/5/8 pathway changes after siRNA knockdown of the two selected proteins. B) Quantification of western blot for two proteins. C) The bands which selected for the mass spectrum (red arrow). …
Additional files
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Supplementary file 1
Primers of the analyzed genes.
- https://cdn.elifesciences.org/articles/59079/elife-59079-supp1-v2.docx
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Supplementary file 2
The siRNA sequences of the analyzed genes.
- https://cdn.elifesciences.org/articles/59079/elife-59079-supp2-v2.docx
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Supplementary file 3
Characteristics of the study subjects.
- https://cdn.elifesciences.org/articles/59079/elife-59079-supp3-v2.docx
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Supplementary file 4
Characteristics of the 15 healthy donors from Center for Biotherapy, Sun Yat-sen Memorial Hospital.
- https://cdn.elifesciences.org/articles/59079/elife-59079-supp4-v2.docx
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Supplementary file 5
Data of mass spectrometry.
- https://cdn.elifesciences.org/articles/59079/elife-59079-supp5-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/59079/elife-59079-transrepform-v2.pdf
