BMP-9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/β-catenin signalling
Correction(s) for this article
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BMP9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/β-catenin signalling
- Volume 27Issue 8Journal of Cellular and Molecular Medicine
- pages: 1155-1156
- First Published online: April 12, 2023
Ni Tang
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorWen-Xin Song
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorJinyong Luo
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorXiaoji Luo
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorJin Chen
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorKatie A. Sharff
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorYang Bi
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorBai-Cheng He
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorJia-Yi Huang
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorGao-Hui Zhu
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorYu-Xi Su
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorWei Jiang
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorMin Tang
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Search for more papers by this authorYun He
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorYi Wang
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorLiang Chen
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorGuo-Wei Zuo
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorJikun Shen
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorXiaochuan Pan
Department of Radiology, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorRussell R. Reid
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorHue H. Luu
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorRex C. Haydon
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorCorresponding Author
Tong-Chuan He
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Correspondence to: T.-C. HE, M.D., Ph.D., Molecular Oncology Laboratory, The University of Chicago Medical Center, 5841 South Maryland Avenue, MC 3079, Chicago, IL 60637, USATel.: (773) 702-7169Fax: (773) 834-4598E-mail: [email protected]Search for more papers by this authorNi Tang
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorWen-Xin Song
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorJinyong Luo
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorXiaoji Luo
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorJin Chen
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorKatie A. Sharff
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorYang Bi
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorBai-Cheng He
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorJia-Yi Huang
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorGao-Hui Zhu
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorYu-Xi Su
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorWei Jiang
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorMin Tang
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Search for more papers by this authorYun He
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorYi Wang
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorLiang Chen
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorGuo-Wei Zuo
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorJikun Shen
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorXiaochuan Pan
Department of Radiology, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorRussell R. Reid
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorHue H. Luu
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorRex C. Haydon
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Search for more papers by this authorCorresponding Author
Tong-Chuan He
The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
Correspondence to: T.-C. HE, M.D., Ph.D., Molecular Oncology Laboratory, The University of Chicago Medical Center, 5841 South Maryland Avenue, MC 3079, Chicago, IL 60637, USATel.: (773) 702-7169Fax: (773) 834-4598E-mail: [email protected]Search for more papers by this authorAbstract
Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-β/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/β-catenin signalling plays an important role in BMP-9-induced osteogenic differentiation of MSCs. Wnt3A and BMP-9 enhanced each other’s ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. β-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers. Furthermore, β-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP-9 induced recruitment of both Runx2 and β-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between β-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs.
Supporting Information
Fig. S1 (A) BMP-9-induced alkaline phosphatase (ALP) activity is not affected by overexpression of sLRP-5 or sLRP-6 in mouse embryonic fibroblasts (MEFs). Subconfluent primary MEF cells were co-infected with AdBMP-9 and AdR-sLRP-5, AdR-sLRP-6, or AdGFP alone. At 10 days after infection, cells were subjected to histochemical staining of ALP activity. (B) BMP-9-induced ALP activity was inhibited as a result of silencing β-catenin expression. C3H101/2 cells were co-infected with AdBMP-9 and three different titres of AdR-simBC (or AdGFP or AdR-simBC alone). ALP activity was assessed histochemically at 7 days after infection.
Fig. S2 (A) Both FrzB and simBC inhibit BMP-9-induced expression of the late osteogenic markers osteocalcin and osteopontin. Mouse embryonic fibroblasts (MEFs) were co-infected with AdBMP-9 and AdR-simBC, AdFrzB, or AdGFP for 10 days. Total RNA was isolated for RT-PCR and qPCR analysis using primers specific for mouse osteocalcin and osteopontin. Experiments were done in triplicate. (B) Silencing of -catenin and FrzB overexpression inhibit BMP-9-induced mineralization. C3H10T1/2 and MEF cells were co-infected with AdBMP-9 and AdFrzB, AdR-simBC, or AdGFP. At 21 days after infection, cells were fixed and subjected to Alizarin Red S staining. Representative macrographic images are shown. (C) and (D) FrzB overexpression inhibits BMP-9-induced ectopic bone formation. MEF cells were co-infected with AdBMP-9 and varying titres of AdFrzB, or AdGFP for 15 hrs and subjected to subcutaneous injection into athymic mice. At 5 weeks after implantation, animals were killed and bony masses were retrieved. Representative gross images are shown (C). The retrieved masses were further subjected to microCT scanning. Representative reconstructed 3-dimmensional images are shown (D).
Fig. S3 FrzB inhibits BMP-9-induced ectopic bone formation and mineralization. Mouse embryonic fibroblast (MEF) cells were coinfected with AdBMP-9 and AdFrzB (both low and high titres were used) or AdRFP for 15 hrs. The infected cells were collected and subjected to subcutaneous injection into athymic mice. At 5 weeks after implantation, animals were killed, and bony masses were retrieved, decalcified and subjected to haematoxylin and eosin staining (parts a to c), Masson?s Trichrome staining (parts d to f) and Alcian Blue staining (parts g to i). Representative images are shown. For the Trichrome stain, decalcified ossified matrix stained dark red, whereas osteoid or cartilage matrix stained blue. For the Alcian Blue stain, cartilage stained blue. OC, osteoid or cartilage-like matrix; OM, ossified or mineralized matrix. Magnification, 150×.
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