p53 inhibits the osteogenic differentiation but does not induce senescence in human dental follicle cells

Abstract

Replicative senescence causes a reduced osteogenic differentiation potential of senescent dental follicle cells (DFCs). The transcription factor p53 is often involved in the induction of cellular senescence, but little is known about its role in DFCs. This study examined for the first time the role of p53 compared to its pro-proliferative antagonist E2F-1 in terms of osteogenic differentiation potential and induction of senescence. Protein expression of E2F-1 decreased during cell aging, while p53 was expressed constitutively. Gene silencing of E2F1 (E2F-1) inhibited the proliferation rate of DFCs and increased the induction of cellular senescence. The induction of cellular senescence is regulated independently of the gene expression of TP53 (p53), since its gene expression depends on the expression of E2F1. Moreover, gene silencing of TP53 induced E2F1 gene expression and increased cell proliferation, but did not affect the rate of induction of cellular senescence. TP53 knockdown further induced the alkaline phosphatase and mineralization in DFCs. However, the simultaneous silencing of TP53 and E2F1 did not inhibit the inductive effect of TP53 knockdown on osteogenic differentiation, indicating that this effect is independent of E2F-1. In summary, our results suggest that p53 inhibits osteogenic differentiation and cell proliferation in senescent DFCs, but is not significantly involved in senescence induction.

Introduction

As part of the tooth germ the dental follicle is involved in tooth eruption and supplies progenitor cells for the development of the periodontal attachment apparatus. However, it is lost after tooth eruption, but this tissue can be isolated from impacted wisdom teeth after extraction (Wise et al., 1999; Diekwisch, 2001; Tummers and Thesleff, 2009). Stem and progenitor cells inside the dental follicle, which are decisive for the development of the periodontium, can be isolated and propagated in cell culture. Unfortunately, after more than 12 passages, DFCs begin to lose their potential for self-renewal and osteogenic differentiation (Morsczeck et al., 2016). Recently, we have shown that the occurrence of cellular senescence is related to the expression of p16 and Wnt5a (Morsczeck et al., 2018, 2019). However, none of these both factors is related to the diminished osteogenic differentiation potential of senescent DFCs.

Although it is unlikely that the senescence marker p16 prevents the osteogenic differentiation (Morsczeck et al., 2018), it can be assumed that factors involved in the induction of cellular senescence may also be implicated in the inhibition of osteogenic differentiation. A recent model suggests that p53 is among others a key factor for cellular senescence in cancer cells (Laine et al., 2013; Laine and Westermarck, 2014). In this model the transcription factor E2F-1 counteracts cellular senescence and prevents a pro-senescence therapy in p53-defective cancer cells. E2F-1, which also plays a crucial role in cell cycle progression, supports cell proliferation and supports cancer progression. In contrast, expression of p53 promotes the induction of cellular senescence (Laine et al., 2013). However, the roles of p53 in the control of aging is ambiguous and depends on a number of factors including protein acetylation (Vigneron and Vousden, 2010a). A recent study has shown that the presence of p53 is required for the inhibition of osteogenic differentiation in senescent mesenchymal stem cells after ionizing radiation. Interestingly, E2F-1 is expressed in the calvaria and participates in the differentiation of osteoblasts (Berman et al., 2008) and, in addition, overexpression of E2F-1 shifts the onset of endochondral bone formation (Scheijen et al., 2003). So, p53 and E2F-1 can be considered as contrary regulators of the osteogenic differentiation. While cellular senescence occurs in the extended cell culture, almost nothing is known about protein functions of p53 and E2F-1 and the induction of cellular senescence in DFCs and its putative roles for controlling the osteogenic differentiation.

We had assumed that p53 supports cellular senescence and inhibits osteogenic differentiation of senescent DFCs. This study investigated the roles of p53 and E2F-1 for the induction of senescence and their influence on osteogenic differentiation in senescent DFCs for the first time.