Impact of Retinoblastoma Protein Loss on Osteoblast Migration and Differentiation.
retinoblastoma protein pathway is inactivated in a wide range of human cancers. RB1
itself, however, is frequently inactivated only in a subset of tumors including
retinoblastoma and osteosarcoma (OS). The fact that RB1 loss favors tumor formation in
certain tissues suggests that pRB might have tissue-specific functions in addition to
its central role in cell cycle regulation. ... read moreIndeed pRB is crucial for both commitment to
the osteoblast lineage and late stages of osteoblast differentiation. RB1 knockout (KO)
calvarial osteoblasts contain an increased pool of osteoprogenitors that could
differentiate into adipocytes as well as osteoblasts. In addition, pRB interacts with
RUNX2 and enhances RUNX2-dependent transcription of osteoblast- specific genes. One
characteristic of OS as well as other tumors in which RB1 is frequently inactivated is
the lack of N-cadherin mediated cell-cell adhesions as a result of alterations in the
expression and localization of N-cadherin. In contrast to OS, N-cadherin is highly
expressed in osteoblasts and important for osteoblast differentiation and function.
Perturbation of these interactions impairs in vitro mineralization of osteoblasts and
osteoblast-specific gene expression. Therefore loss of N-cadherin-mediated cell-cell
adhesions could contribute to the undifferentiated and invasive nature of osteosarcoma.
The frequent inactivation of RB1 and parallel loss of N-cadherin expression in OS
prompted me to ask whether these observations are directly related to each other. In
this study I observed reduced N-cadherin expression in germline RB1 KO calvarial
osteoblasts. In addition, we sought to understand if low N-cadherin expression affected
migration and contributed to the adipogenic potential of RB1 KO osteoblasts. Whereas
primary RB1 KO osteoblasts were not necessarily more migratory, RB1 KO cell lines
derived from primary osteoblasts had increased migration potential compared to RB1 WT
counterparts. These altered migratory properties of RB1 KO osteoblasts showed a
dependence on IGF-I expression and receptor signaling, which are potentially intrinsic
to the progenitor cells immortalized upon loss of pRB (expression). We demonstrate that
adipogenic potential in RB1 KO cell lines is correlated with N-cadherin expression. Cell
lines that undergo adipogenic differentiation downregulate N-cadherin expression,
whereas those that don't maintain high levels of N-cadherin. We also show that
individual RB1 KO cell lines consist of cells with varying degrees of N-cadherin
expression, and adipogenic potential is enriched in the N-cadherin low population.
Finally we show that acute loss of pRB doesn't affect N-cadherin expression or migration
nor confer adipogenic potential to RB WT calvarial cells. Our findings suggest that
these traits are properties of a particular cell type rather than a direct consequence
of pRB loss. We propose that during osteoblast differentiation there is a transient cell
state with low N-cadherin expression and this is maintained by IGF-I in an autocrine or
paracrine fashion. Low N-cadherin expression could allow this cell to adopt either
osteogenic or adipogenic fate and prime this cell for migration which could become
apparent with additional alterations. Loss of pRB at this stage will prevent proper
differentiation and deregulate cell cycle control resulting in the persistence and
expansion of this cell type. Additional mutations could transform these cells and lead
to osteosarcoma formation.
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Genetics.
Advisor: Philip Hinds.
Committee: Richard Etten, Charlotte Kuperwasser, and Grace Gill.
Keyword: Genetics.read less