Impaired bone development and increased mesenchymal progenitor cells in calvaria of RB1 ^−/− mice

Abstract

We have previously shown that the retinoblastoma protein (pRb) can activate expression of Runx2-dependent, bone-specific genes in cultured cells. We now show that pRb also plays a role early in osteogenesis, and that in primary RB1 ؊/؊ calvarial cells there is an increased osteoprogenitor pool. To understand pRb's function in vivo, we generated a conditional RB1-KO mouse in which pRb expression is efficiently extinguished in osteoblasts. These animals display an apparent developmental defect in bones, most strikingly in the calvaria. Cultured RB1 ؊/؊ calvarial osteoblasts fail to cease proliferation upon reaching confluence or following differentiation. Re-plating assays of primary RB1 ؊/؊ calvarial cells after differentiation showed a clear adipogenic ability with increased multipotency. RB1 ؊/؊ osteoblasts display a severe reduction in levels of mRNAs expressed late in differentiation. In this study, we present strong evidence that pRb has multiple regulatory roles in osteogenesis. Furthermore, in the absence of RB1 ؊/؊ there is a larger pool of multipotent cells compared with the WT counterpart. This increased pool of osteoprogenitor cells may be susceptible to additional transforming events leading to osteosarcoma, and is therefore key to understanding RB1 as a target in malignancy.differentiation ͉ retinoblastoma protein ͉ osteoprogenitors

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

“…For example, downregulation of p53 has been associated with increased selfrenewal, and upregulation of stem cell markers, such as Nanog, ABCB1 and CD44 (Jerry et al, 2008). Other studies have shown that lower levels of p53 results in increased mammary stem/progenitors (Tao et al, 2011) and lack of pRb has been shown to promote expansion of multi-potent stem cells (Gutierrez et al, 2008). In addition, the ST antigen has been shown to activate Notch, Hedgehog and Wnt pathways (Ali-Seyed et al, 2006) all of which are associated with stem cell self-renewal as well as breast carcinogenesis (Liu et al, 2005).…”

The retinoblastoma tumor suppressor and stem cell biology

“…Rb inactivation in lineage-committed immature osteoblasts in mice is not sufficient to initiate tumors but potentiates the effects of loss of p53 (Walkley et al 2008). However, loss of Rb in multipotent mesenchymal progenitors, which can give rise to cells in the bone lineage, leads to increased numbers of these progenitors (Gutierrez et al 2008), suggesting that stem cells before the preosteoblast stage may also be sensitive to loss of RB and may also serve as a cell of origin for osteosarcoma. Indeed, in mice, deletion of Rb and p53 in mesenchymal stem/progenitor cells expressing the Sca1 marker is sufficient to initiate metastatic tumors closely resembling human osteosarcoma (Berman et al 2008).…”

The Retinoblastoma Protein in Osteogenesis and Osteosarcoma Formation

“…Conditional deletion of pRb in osteoprogenitor cells in mice resulted in an increased pool of mesenchymal progenitor cells in calvaria of pRb-deficient mice. These pRb-deficient progenitors showed clear adipogenic ability with increased multipotency (Calo et al, 2010;Gutierrez et al, 2008), suggesting that pRb loss resulted in an inability to irreversibly enter the osteogenic differentiation pathway. Interestingly, the ossification defects observed in pRb deficient mice can be suppressed by deletion of E2F1 , suggesting that the impaired osteogenesis observed upon pRb loss could be a consequence of overproliferating osteoprogenitors that are unable to undergo irreversible cell cycle withdrawal in order to commit to a specific cell lineage.…”

“…This may explain the commonly observed absence of Osteocalcin expression in osteosarcoma tumors, as well as their poorly differentiated state. The increased pool of poorly differentiated and rapidly dividing osteoprogenitors may also be susceptible to additional transforming events that could cooperate with pRb loss to further advance the genesis of osteosarcomas (Gutierrez et al, 2008). In addition, as described above, pRb expression has been shown to be important for the expression of OB-cadherin, which is the cadherin type that is unique to the fully mature and differentiated osteoblast, as well as for the establishment of the cell-to-cell adhesion that is so important for osteoblast differentiation and function (Sosa-García et al, 2010).…”