Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05

    • P and p are cyclin dependent kinase inhibitors that play

    2018-11-08

    P21 and p16 are cyclin-dependent kinase inhibitors that play important roles in cell-cycle regulation and tumor suppression (Poole et al., 2004; Shapiro et al., 1998), and both are highly expressed upon senescence in MSCs. Knockdown of p21 in late-passage MSCs increases stem cell properties, including the proliferation rate, differential potentials, and expression of stemness markers, and also enhances the capacity for in vivo bone repair (Yew et al., 2011). Knockdown of p16 has also been shown to reverse the senescent state and promote the proliferation ability (Shibata et al., 2007). RB encodes the retinoblastoma (RB) protein, which controls cell-cycle progression from G1 into S phase by binding to E2F and inhibiting its activity (Li et al., 1994). It is also involved in the control of stem cell properties (Galderisi et al., 2006). The role of RB in the cellular senescence of MSCs in vitro has been studied in expanded human MSCs (Alessio et al., 2013). Moreover, the fate choice of MSCs in vivo has been studied using an oncogenic transformation mouse model that induces liposarcoma through RB deletion (Calo et al., 2010). However, the mechanism that RB mediates to inhibit replicative senescence and promote the differentiation potential of human MSCs has not been studied.
    Results
    Discussion MSCs have a wide range of clinical uses, including tissue engineering, cell therapy, immunomodulatory therapy, and anticancer cell therapy (Gómez-Barrena et al., 2011; Krampera et al., 2007). Since all of the above clinical applications require large-scale production of MSCs, a number of culture expansion techniques have been developed. However, with these techniques, MSCs have been shown to enter the senescent state and simultaneously lose their proliferation and differentiation abilities after a few passages of expansion in culture (Shibata et al., 2007; Yew et al., 2011). We demonstrated that RB maintains MSC properties by upregulating DNMT1, and thereby represses the expression of senescence markers such as p21 and p16 by increasing the DNA methylation levels of their promoters (Tsai et al., 2012). The current findings might be used to modify the properties of expanded MSCs for use in future clinical therapies. Traditionally, RB has been considered to negatively regulate cell-cycle progression by binding to E2F1 and arresting Z-VEID-FMK in G1 phase. RB binds to E2F1 and thereby inhibits E2F1 transcriptional activity (Galderisi et al., 2006; Weinberg, 1995). While being phosphorylated, RB would liberate E2F1 and stop inhibiting the expression of E2F1 target genes, thereby letting a cell enter into S phase (Galderisi et al., 2006). Our data show that RB knockdown increased the proliferation of MSCs at the beginning, suggesting that RB inhibited cell-cycle progression. However, after a few passages of expansion, the growth curve of MSCs with RB knockdown dropped abruptly and then the proliferation rate of the MSCs with RB knockdown became slower than that of MSCs expressing control shRNAs. This dramatic change in growth might be explained by the role of RB in maintaining stem cell properties. Bone marrow stem cells, like other primitive stem cells, possess the property of mitotic quiescence, which means that a cell can temporarily exit from the cell cycle and divide slowly or rarely (Galderisi et al., 2006; Morrison et al., 1997). In this work, early-passage MSCs transduced without or with control shRNAs (representing the cells containing RB) were able to stay in the quiescent state and exhibited a growth curve that dropped comparatively slowly. In contrast, late-passage MSCs or MSCs with RB knockdown could leave the quiescent state, quickly enter cell-cycle progression, and then become exhausted in the senescent state after a few passages of subculture. Together, these data suggest that RB might maintain MSCs in the quiescent state and prevent them from undergoing premature senescence. It was previously reported that the DNMT1 promoter could be repressed by the RB tumor suppressor in the mouse embryonal carcinoma cell line P-19 (Bigey et al., 2000). However, the same group of authors also demonstrated that RB and c-JUN could form an AP-1 complex by binding to the noncanonical AP-1 site and activating the promoter of DNMT1 (Slack et al., 2001). RB might have the opposite roles in regulating DNMT1 promoter activity. Our data showed that DNMT1 was expressed in parallel with RB expression in MSCs, and accordingly decreased and increased with RB knockdown and overexpression, respectively. Similarly to previous findings (Bigey et al., 2000; Slack et al., 2001), we demonstrated that RB binds with c-JUN, rather than E2F1, to increase the expression of DNMT1. As expected, knockdown c-JUN in early-passage MSCs blocked RB-dependent expression of DNMT1. Interestingly, we also demonstrated that RB-dependent upregulation of MSC properties was mediated through upregulation of DNMT1. Together, these data suggest that RB plays a positive role in regulating MSC properties through a pathway that involves E2F1, c-JUN, DNMT1, p21, and p16, and maintains early-passage MSCs in a relatively mitotic quiescent state, thereby protecting the cells against rapid exhaustion and premature senescence (Figure 5E).