Wnt/β-catenin Signaling Regulates Dental Pulp Stem Cell Properties Erica Scheller¹, Jia Chang², Cun-Yu Wang¹,²

¹ Biologic and Materials Sciences, University of Michigan School of Dentistry, ²Division of Oral Biology and Medicine, UCLA School of Dentistry.

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Objectives: Dental pulp stem cells (DPSCs) are a unique precursor population isolated from post-natal human dental pulp and have the ability to regenerate a reparative dentin-like complex. Canonical Wnt signaling plays a critical role in tooth development and stem cell self-renewal through β-catenin. While DPSCs have great potential for dentin regeneration and tooth repair, molecular regulation of DPSC differentiation is poorly understood. The objective of this study was to examine whether Wnt signaling regulates DPSC differentiation and stem cell properties.

Methods: DPSCs isolated from human adult third molar were stably transduced with canonical Wnt-1 or the active form of β-catenin using retrovirus-mediated infection. Following selection, confluent cell layers were induced to differentiate toward an odontoblast-like lineage with ascorbic acid, dexamethasone and β-glycerophosphate. Post-differentiation analysis included staining for calcium mineral and alkaline phosphatase (ALP) and analysis of osteogenic lineage markers osteonectin, osteopontin, bone sialoprotein, and collagen I.

Results: Northern blot analysis found that Wnt-1 strongly induced the expression of matricellular protein osteopontin (OPN) and modestly enhanced the expression of type I collagen in DPSCs. Unexpectedly, Wnt-1 inhibited alkaline phosphatase (ALP) activity and the formation of mineralized nodules by more than 80% when compared to control DPSCs. Moreover, over-expression of β-catenin was also sufficient to suppress their differentiation and mineralization.

Conclusion: Our results are the first demonstration that canonical Wnt/β-catenin signaling negatively regulates the odontoblast-like differentiation of DPSCs and may help to maintain their stem cell properties. Studies of crystal nucleation suggest that OPN is a potent inhibitor of mineral crystal growth at high concentrations. Since Wnt-1 strongly induced OPN expression, it is possible that the induction of OPN by Wnt-1 may play a role in the suppression of mineral formation in DPSCs. This work was supported by grants R01DE-016513 and T32DE-007057 from the National Institute of Dental and Craniofacial Research.

NG2, novel proapoptotic receptor, opposes integrin α4 to mediate anoikis
Nam E. Joo*, Taizo Watanabe*, Chunyuan Chen*, Martha Chekenya†, William B. Stallcup‡ and Yvonne L. Kapila*

*Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109; †Norlux Neuro-Oncology, Department of Biomedicine, University of Bergen, Jonas Lies Vei 91, N-5009 Bergen, Norway; and ‡The Burnham Institute for Medical Research, Cancer Research Center, La Jolla, California 92037

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 Background: Disruption of cell–matrix interactions can lead to anoikis—apoptosis due to loss of matrix contacts. Altered fibronectin (FN) matrix or FN fragments as are found in periodontal disease induce anoikis of primary human fibroblasts by a novel signaling pathway. This is characterized by reduced focal adhesion kinase (FAK) phosphorylation and transcriptional downregulation of p53. However, the receptors involved are unknown. pFAK is regulated by nerve/glial antigen 2 (NG2) receptor signaling through PKCα, a point at which signals from integrins and proteoglycans may converge.

Objective: We tested the hypothesis that NG2 acts in concert with integrin α4β1 to regulate anoikis and that PKCα and FAK signaling are control points where these two receptor-mediated signaling pathways converge.

Methods: We used immunoblotting, immunofluorescence and flow cytometry to determine protein expression levels and a flow cytometry based apoptotic assay. Fibroblasts were transfected with NG2, integrin α4, and PKCα  cDNAs or siRNA to examine the regulation of pFAK mediated by NG2 and integrin α4.

Results: Altered FN induced anoikis by upregulating NG2 and downregulating integrin α4. Suppressing NG2 expression or overexpressing α4 rescued cells from anoikis. NG2 overexpression alone induced apoptosis and, by reducing pFAK, increased anoikis induced by an altered matrix. NG2 overexpression or an altered matrix also suppressed PKCα  expression, but overexpressing integrin α4 enhanced pFAK independently of PKCα. Cotransfection with NG2 cDNA and integrin α4 siRNA did not lower PKCα and pFAK levels more than transfection with either alone. PKCα was upstream of pFAK, as silencing PKCα decreased pFAK. PKCα  overexpression reversed this behavior and rescued cells from anoikis.

Conclusion: NG2 is a novel proapoptotic receptor, and NG2 and integrin α4 oppositely regulate anoikis. NG2 and integrin α4 regulate pFAK by PKCα-dependent and -independent pathways, respectively. These mechanisms may underlie periodontal disease pathogenesis (Study supported by NIH grant R01 DE013725 to YLK).