Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae

Abstract

During tooth root development, stem cells from apical papillae (SCAPs) are indispensable, and their abilities of proliferation, migration and odontoblast differentiation are linked to root formation. Leucine-rich repeat-containing GPCR 4 (LGR4) modulates the biological processes of proliferation and differentiation in multiple stem cells. In this study, we showed that LGR4 is expressed in all odontoblast cell lineage cells and Hertwig's epithelial root sheath (HERS) during the mouse root formation in vivo. In vitro we determined that LGR4 is involved in the Wnt/β-catenin signaling pathway regulating proliferation and odonto/osteogenic differentiation of SCAPs. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that LGR4 is expressed during odontogenic differentiation of SCAPs. CCK8 assays and in vitro scratch tests, together with cell cycle flow cytometric analysis, demonstrated that downregulation of LGR4 inhibited SCAPs proliferation, delayed migration and arrested cell cycle progression at the S and G2/M phases. ALP staining revealed that blockade of LGR4 decreased ALP activity. QRT-PCR and Western blot analysis demonstrated that LGR4 silencing reduced the expression of odonto/osteogenic markers (RUNX2, OSX, OPN, OCN and DSPP). Further Western blot and immunofluorescence studies clarified that inhibition of LGR4 disrupted β-catenin stabilization. Taken together, downregulation of LGR4 gene expression inhibited SCAPs proliferation, migration and odonto/osteogenic differentiation by blocking the Wnt/β-catenin signaling pathway. These results indicate that LGR4 might play a vital role in SCAPs proliferation and odontoblastic differentiation.

http://ift.tt/2hJIWx5