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Shh signaling, negatively regulated by BMP signaling, inhibits the osteo/dentinogenic differentiation potentials of mesenchymal stem cells from apical papilla

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Abstract

Mesenchymal stem cells (MSCs) derived from dental tissues show promise for use in tooth-related tissue regeneration, but the molecular mechanisms underlying their directed differentiation remain unclear, limiting their usefulness. Sonic Hedgehog (Shh) signaling is a major signaling pathway that regulates cell differentiation and osteogenesis. We found that when Shh signaling was activated by human recombinant SHH-N protein or by overexpression of active mutant M2-Smoothened (SMO) in stem cells from apical papilla (SCAPs), GLI1, a key downstream transcription factor and a marker of Shh signaling, was upregulated. Subsequently, in vitro osteo/dentinogenic differentiation and in vivo osteogenesis were inhibited in SCAPs. Moreover, the expression of GLI1 and SMO were downregulated by BMP signaling while osteo/dentinogenic differentiation in SCAPs was upregulated. These results provide insights into the role of Shh signaling in the directed differentiation of MSCs derived from dental tissues and suggest possible target genes for optimizing the use of stem cells of dental origin for tissue regeneration applications.

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Acknowledgments

The authors thank Dr. Cun-Yu Wang of the University of California, Los Angeles, School of Dentistry, for the kind gift of the human full-length M2-SMO-pCDNA3.1 plasmid., the National Natural Science Foundation of China (81070798 to Z. P. Fan, 81170931 to J. Du, 81070843 to Z. C. Shan), the Funding Project to Science Facility in Institutions of Higher Learning Under the Jurisdiction of Beijing Municipality (PXM2011_014226_07_000066 to Z. P. Fan), the Program for New Century Excellent Talents in University (NCET-12-0611 to Z. P. Fan).

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Authors and Affiliations

  1. Laboratory of Molecular Signaling and Stem Cells Therapy, Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Tiantan Xili No.4, Dongcheng District, Beijing, 100050, China

    Qingsong Jiang, Juan Du, Yushi Ma, Ping Ma, Juan Du & Zhipeng Fan

  2. Department of Prosthodontics, Capital Medical University School of Stomatology, Tiantan Xili No.4, Dongcheng District, Beijing, 100050, China

    Qingsong Jiang

  3. Department of Stomatology, Qingdao Municipal Medical Group, Donghai Zhonglu No.5, Shinan District, Qingdao, 266071, Shandong, China

    Xiaonan Yin

  4. Department of Oral and Maxillofacial Surgery, Capital Medical University School of Stomatology, Tiantan Xili No.4, Dongcheng District, Beijing, 100050, China

    Zhaochen Shan

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  1. Qingsong Jiang
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  2. Juan Du
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Corresponding authors

Correspondence to Juan Du or Zhipeng Fan.

Additional information

The first two authors have contributed equally to this work.

The seventh author, Juan Du, is the corresponding author.

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Supplementary Fig. 1

Human recombinant SHH-N protein inhibits ALP activity and mineralization in stem cells from periodontal ligaments (PDLSCs). a Human recombinant SHH-N protein inhibits ALP activity in PDLSCs. b, c SHH-N protein inhibits mineralization in PDLSCs. The student’s t test was performed to determine statistical significance. All error bars represent s.d. (n = 3). **P < 0.01. (TIFF 1095 kb)

Supplementary Fig. 2

The expressions of BSP were not affected after depletion of GLI1. a The knock-down of GLI1 in SCAPs. SCAPs were infected with lentiviruses expressing GLI1 shRNA (GLI1sh) or Scramsh. After selection with 2 μg/ml puromycin for 7 days, GLI1 expression was determined by real-time RT-PCR. GAPDH was used as an internal control. b BSP mRNA expression was not significantly changed after depletion of GLI1. The student’s t test was performed to determine statistical significance. All error bars represent s.d. (n = 3). **P < 0.01. (TIFF 118 kb)

Supplementary Fig. 3

The expressions of GLI1 and SMO were not affected after depletion of SMAD4. a The knock-down of SMAD4 in SCAPs. SCAPs were infected with lentiviruses expressing SMAD4 shRNA (SMAD4sh) or Scramsh. After selection with 2 μg/ml puromycin for 7 days, SMAD4 expression was determined by Western Blot. An α-tubulin antibody was used as an internal control. b DLX5 mRNA expression was downregulated after depletion of SMAD4, GAPDH was used as an internal control. c GLI1 mRNA expression was not significantly changed after depletion of SMAD4. d SMO mRNA expression was not significantly changed after depletion of SMAD4. The student’s t test was performed to determine statistical significance. All error bars represent s.d. (n = 3). **P < 0.01. (TIFF 1749 kb)

Supplementary Fig. 4

The expression of SMO and GLI1 is downregulated and the expression of BMP2 is upregulated during osteo/dentinogenic differentiation of PDLSCs. a-d, PDLSCs were induced to differentiate by culture in osteogenic medium, and gene expression was determined at the indicated time points using real-time RT-PCR. GAPDH was used as an internal control. a SMO mRNA expression was downregulated at 10 and 14 days after osteogenic induction. b GLI1 mRNA expression was downregulated at 10 and 14 days after osteogenic induction. c BMP2 mRNA expression was upregulated at 7, 10, and 14 days after osteogenic induction. d BMP4 mRNA expression was not significantly changed after osteogenic induction. The one-way ANOVA was performed to determine statistical significance. All error bars represent s.d. (n = 3). *P < 0.05. **P < 0.01. (TIFF 184 kb)

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Jiang, Q., Du, J., Yin, X. et al. Shh signaling, negatively regulated by BMP signaling, inhibits the osteo/dentinogenic differentiation potentials of mesenchymal stem cells from apical papilla. Mol Cell Biochem 383, 85–93 (2013). https://doi.org/10.1007/s11010-013-1757-9

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