A Role for the Retinoblastoma Protein As a Regulator of Mouse Osteoblast Cell Adhesion: Implications for Osteogenesis and Osteosarcoma Formation

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

Oncomine was used for analysis of E2F1 and Pak1 expression in four common solid tumor types.

<p>Oncomine was used for analysis of E2F1 and Pak1 expression in four common solid tumor types.</p

The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

<div><p>We previously characterized the retinoblastoma tumor suppressor protein (Rb) as a regulator of adherens junction assembly and cell-to-cell adhesion in osteoblasts. This is a novel function since Rb is predominantly known as a cell cycle repressor. Herein, we characterized the molecular mechanisms by which Rb performs this function, hypothesizing that Rb controls the activity of known regulators of adherens junction assembly. We found that Rb represses the expression of the p21-activated protein kinase (Pak1), an effector of the small Rho GTPase Rac1. Rac1 is a well-known regulator of adherens junction assembly whose increased activity in cancer is linked to perturbations of intercellular adhesion. Using nuclear run-on and luciferase reporter transcription assays, we found that Pak1 repression by Rb is transcriptional, without affecting Pak1 mRNA and protein stability. Pak1 promoter bioinformatics showed multiple E2F1 binding sites within 155 base pairs of the transcriptional start site, and a Pak1-promoter region containing these E2F sites is susceptible to transcriptional inhibition by Rb. Chromatin immunoprecipitations showed that an Rb-E2F complex binds to the region of the Pak1 promoter containing the E2F1 binding sites, suggesting that Pak1 is an E2F target and that the repressive effect of Rb on Pak1 involves blocking the trans-activating capacity of E2F. A bioinformatics analysis showed elevated Pak1 expression in several solid tumors relative to adjacent normal tissue, with both Pak1 and E2F increased relative to normal tissue in breast cancer, supporting a cancer etiology for Pak1 up-regulation. Therefore, we propose that by repressing Pak1 expression, Rb prevents Rac1 hyperactivity usually associated with cancer and related to cytoskeletal derangements that disrupt cell adhesion, consequently enhancing cancer cell migratory capacity. This de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated with cancer progression and metastasis.</p></div

Pak1 silencing partially restores adherens junctions.

<p><b>(A)</b> Immunoblot analysis showing reduced Pak1 expression in Rb-/- MC3T3 cells infected with an adenovirus vector carrying an RNAi against Pak1, relative to a scrambled vector control. <b>(B)</b> Immunofluorescence labeling showing re-establishment of beta-catenin presence in the intercellular spaces after infection of Rb-/- MC3T3 cells with adeno-Pak1 RNAi (<i>top panel</i>, <i>arrow</i>), relative to Rb-/- MC3T3 cells infected with a scrambled control-carrying adenovirus (<i>middle panel</i>). Untreated Rb+/+ MC3T3 cells showing intercellular beta-catenin labeling are shown for comparison (<i>bottom panel</i>, <i>arrow</i>).</p

Conserved regions in the Pak1 promoter contain E2F binding sites.

<p><b>(A)</b> Alignment of a region of 1,000 bp (from -700 to +300 bp) of the mouse and human Pak1 promoters generated by the Data Base of Transcriptional Start Sites (DBTSS), showing 7 conserved regions labeled 0–6. The longest conserved stretch is region 0, which spans a 406-bp region from -186 to +220 with 66% identity. The table below the promoter diagram shows the start and stop positions for each conserved region in mouse (m) and human (h). <b>(B)</b> Schematic of the human and mouse Pak1 promoters showing 5 E2F1 binding sites (labeled as 1–3 in the human promoter and 4 and 5 in the mouse promoter), as identified by Genomatrix analysis. These E2F1 binding sites are in the conserved region (labeled as 0 in <b>A)</b>. The table shows the positions, strand, and sequences of each E2F1 binding site, with the core nucleotides in each binding site indicated capitalized in bold. <b>(C)</b> A Pak1 mouse promoter-Firefly luciferase construct containing the 2 E2F binding sites was transfected into Rb+/+ and Rb-/- MC3T3 cells, and promoter activation was measured by its luciferase activity and normalized against a co-transfected Renilla luciferase construct. The Pak1 promoter region containing the E2F binding sites was amplified using the forward (F) and reverse (R) primers illustrated in <b>(B)</b> and in bold in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142406#pone.0142406.s001" target="_blank">S1 Fig</a>. Transcriptional activity in Rb-/- cells was stronger than in Rb+/+ cells by a factor of 2.1 after normalization with Renilla luciferase, a value that is close to the 2.7-fold transcriptional induction that we observed in our nuclear run-on assays. *P < 0.05.</p

Oncomine was used for analysis of E2F1 and Pak1 expression in four common solid tumor types.

<p>Oncomine was used for analysis of E2F1 and Pak1 expression in four common solid tumor types.</p

Model linking Rb function to cell adhesion via control of Pak1 expression.

<p><b>(A)</b> Rb-expressing osteoblasts show significantly diminished Pak1 expression relative to their Rb-deficient counterparts since Rb binds to E2F1 in the Pak1 promoter and blocks its activity. This can be either by direct interference with E2F1’s trans-activating capacity or by recruitment of histone deacetylases to E2F1-sensitive promoters (<i>1</i>). <b>(B)</b> In the absence of Rb, the unrestricted E2F1 action induces transcription of the Pak1 gene (<i>1</i>) with consequent translation of the Pak1 protein (<i>2</i>). Once translated, Pak1 binds and is activated by a Rho GTPase such as Rac1 or Cdc42 to form an active complex (3), which then phosphorylates the merlin tumor suppressor in serine 518 (<i>4</i>), a phosphorylation that impairs merlin function. Because merlin acts mainly by promoting the stabilization of adherens junctions at the cell membrane, loss of merlin function by the Pak1-dependent phosphorylation will bring about a disruption of adherens junctions and therefore of intercellular adhesion.</p