The Mechanism of GREM1's Effect on Osteogenic/Odontogenic Differentiation of Stem Cells from Apical Papilla

ZHU Xing-yan, DIAO shu, YANG Dong-mei, FAN Zhi-peng

Abstract

To study the effect of bone morphogenetic protein (BMP) antagonist Gremlin 1 (GREM1) on the function of stem cells from apical papilla (SCAPs) and explore its mechanism.   Methods   After isolation and culturing of stem cells from apical papilla in vitro, immunofluorescent staining was done to examine the subcellular localization of GREM1 in SCAPs. Transfection with lentiviral GREM1 shRNA was done to knock-down the GREM1. The SCAPs were subjected to osteogenic induction in both the GREM1 knockdown group and the control group, and the knockdown effect of GREM1 was examined using real time-PCR and Western blot. Two groups of cells were collected and the alkaline phosphatase (ALP) activity was measured 7 d after osteogenic induction. Alizarin red staining was done 3 weeks after osteogenic/odontogenic induction and real time-PCR was done after 0, 1, 2, 3 weeks of osteogenic induction to examine the expression of osteogenic/odontogenic marker genes, including osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and and the critical transcription factor osterix (OSX), Runt-related transcription factor 2 (RUNX2), and distal-less homebox 2 (DLX2). Two groups of cells were collected, and CCK-8 and CFSE assay were used to evaluate changes in cell proliferation. In addition, real time-PCR was used to examine the expression of senescence-related genes p53 and wide-type activated factor 1 (Waf1), a regulatory factor of the cell cycle, stemness associated gene krupple-like factor 4 (KLF4), and SRY related HMG box-2 (SOX2), and the expression of bone morphogenetic protein (BMP) 2, 4, 5, 6, 7, 9 after GREM1 knockdown.   Results   Immunofluorescence staining showed that the expression of GREM1 in the nucleus was higher than that in the cytoplasm. Real time-PCR and Western blot affirmed that GREM1 was knocked down steadily. The ALP activity of the GREM1 knockdown group was higher than that of the control group (P<0.05), and the alizarin red staining was lighter than that of the control group. The expression of OCN and DMP1 increased in the first, second and third week, OPN was increased in the second week, BSP increased in the third week, DSPP increased in the first week, and the difference was statistically significant (P<0.05). The key osteogenic transcription factors RUNX2, OSX, and DLX2 all increased at different stages, and the difference was statistically significant (P<0.05). CCK-8 and CFSE assay showed that the proliferation ability of the GREM1 knockdown group decreased (P<0.05). In the GREM1 knockdown group, the expression of BMP2, 6, and 7 increased, the expression of SOX2 and KLF4 increased, while the expression of p53 and Waf1 decreased (P<0.05).   Conclusions   The knockdown of GREM1 enhanced the osteogenic/odontogenic differentiation and stemness of SCAPs and inhibited the proliferation and senescence of SCAPs. Effects of GREM1 on the function of SCAPs maybe achieved through regulating the gene expression of BMP2, BMP6, and BMP7 at the mRNA level.

 

Keywords: Stem cells from the apical papilla, GREM1, Bone morphogenetic protein, Osteogenic/odontogenic differentiation

 

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