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Journal of Dental Research
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Wnt/β-catenin Inhibits Dental Pulp Stem Cell Differentiation

E.L. Scheller1, J. Chang2 and C.Y. Wang1,2,*

1 Laboratory of Molecular Signaling, Department of Biologic and Materials Sciences, School of Dentistry, The University of Michigan, Ann Arbor, MI 48109, USA; and
2 Laboratory of Molecular Signaling, Division of Oral Biology and Medicine, UCLA School of Dentistry, Box 951668, 33-030A CHS, 10833 Le Conte Ave., Los Angeles, CA 90095-1668, USA


Figure 1
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  		Figure 1. Wnt signaling induced β-catenin in DPSCs. (A) DPSCs stably expressing hemagglutinin (HA)-tagged Wnt-1 were established. DPSCs were stably transduced with retroviruses expressing Wnt-1 or control vector. Wnt-1 expression was determined by Western blot analysis. (B) Wnt-1 stabilized β-catenin in DPSCs. The cytosolic and nuclear extracts from both DPSC/V and DPSC/Wnt-1 cells were probed with anti-β-catenin monoclonal antibodies. For loading control, the cytosolic extracts (CE) were probed with anti-{alpha}-tubulin monoclonal antibodies, and nuclear extracts (NE) were probed with anti-TFIIB polyclonal antibodies. (C) Wnt-1 did not affect the proliferation of DPSCs. Both DPSC/Wnt-1 and DPSC/V cells were grown with or without serum. Cell numbers were counted in triplicate at the indicated times. CE, cytosolic extract; NE, nuclear extract; HA, hemagglutinin; TFIIB, transcription factor IIB.

 

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  		Figure 2. Wnt-1 strongly induced OPN expression and maintained extracellular matrix proteins. (A) Wnt-1 slightly enhanced type I alpha collagen expression by DPSCs during differentiation. (B) Wnt-1 strongly induced OPN expression in DPSCs. (C) Expression of ON was similar in both DPSC/V and DPSC/Wnt-1 cells. The total RNA from DPSC/Wnt-1 and DPSC/V cells was probed with 32P-labeled cDNA probes for type I alpha collagen, OPN, and ON. (D) Wnt-1 partially inhibited BSP expression in DPSCs, as determined by qPCR analysis. The results represent average values from triplicate assays, and the experiments were repeated twice. OPN, osteopontin; ON, osteonectin; BSP, bone sialoprotein.

 

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  		Figure 3. Wnt-1 inhibits mineral formation by DPSCs. (A) Wnt-1 inhibits ALP activity in DPSCs after 5 days of culture in differentiating medium. The experiments were performed twice, and the results represent average values from triplicate assays. (B) Secreted Wnt-1 inhibited ALP activity. Mixed cultures were established with various ratios of DPSC/V to DPSC/Wnt-1 cells as indicated and grown in differentiating medium for 12 days. Results were quantified by densitometry to compare staining intensities. Sample staining was repeated in duplicate. DPSC/V results were set to 100% staining and DPSC/Wnt-1 results to 0% staining for standardization of the mixed cultures. (C) Wnt-1 inhibited mineral formation of DPSCs after 14 days of culture in differentiation medium. Both DPSC/V and DPSC/Wnt-1 cells were induced to differentiate with dexamethasone and β-GP. Two wks after induction, cells were stained with Alizarin red. The experiments were performed twice, and the results represent average values from triplicate assays. ALP, alkaline phosphatase; β-GP, β-glycerophosphate.

 

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  		Figure 4. β-catenin inhibits mineral formation by DPSCs. (A) DPSCs stably expressing HA-tagged β-catenin were established. The expression of β-catenin was determined by Western blot analysis. (B) β-catenin inhibited ALP activity. The experiments were repeated twice, and the results represent average values from triplicate assays. (C) β-catenin inhibited mineral formation of DPSCs, as determined by Alizarin red staining. Cells from panels B and C were cultured in differentiation medium for 10 days. The experiments were repeated twice, and the results represent average values from triplicate assays. ALP, alkaline phosphatase; HA, hemagglutinin.

 

Journal of Dental Research, Vol. 87, No. 2, 126-130 (2008)
DOI: 10.1177/154405910808700206
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