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

The retinoblastoma protein or its regulators are altered in most human cancers. Although commonly thought of as solely a repressor of E2F-dependent transcription and cell cycle progression, pRb has gained notoriety in recent years as a key actor in cellular differentiation programs. In the June issue of Molecular Cell, Benevolenskaya et al. report that a long-known but poorly understood pRb interactor, RBP2, acts as an inhibitor of differentiation contributing to pRb's role as a coordinator of differentiation and cell cycle exit. Loss of pRb may unleash RBP2, maintaining cells in a poorly differentiated progenitor state that is prerequisite to tumor formation.