Abstract
The retinoblastoma protein (pRb) is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.
Highlights
The retinoblastoma tumor suppressor protein is a cell cycle repressor inactivated in most human cancers [1,2,3,4,5]
While the cell cycle regulatory pathway centered on pRb is inactivated in most human cancers [1], pRb itself is inactivated with high frequency in a subset of human tumors, including retinoblastomas, osteosarcomas, and small cell lung carcinomas [4]. pRb can be indirectly inactivated in other tumor types as a consequence of alterations targeting genes coding for any of its several upstream regulators such as CDK4, cyclin D and p16ink4a [6]
Our studies confirm and extend these reports by showing that abrogating pRb expression in murine osteoblasts leads to aberrant expression of surface cadherins, disruption of adherens junctions, and loss of contact-dependent growth arrest
Summary
The retinoblastoma tumor suppressor protein (pRb) is a cell cycle repressor inactivated in most human cancers [1,2,3,4,5]. While the cell cycle regulatory pathway centered on pRb is inactivated in most human cancers [1], pRb itself is inactivated with high frequency in a subset of human tumors, including retinoblastomas, osteosarcomas, and small cell lung carcinomas [4]. Studies conducted in retinoblastomas, osteosarcomas, and small cell lung carcinomas point to an additional role for pRb as a regulator of cell adhesion. These tumor types show high frequencies of pRb inactivation and are composed of cells that lack stable adherens junctions, which are cadherin- and catenin-containing membrane complexes required for cell adhesion. A strong correlation has been found in retinoblastomas and osteosarcomas between abnormal adherens junctions and invasive capacity [8,9], underscoring the notion that disruption of adherens junctions-mediated cell adhesion is intimately related to metastasis
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