Loss of Function of the Retinoblastoma Gene Affects Gap Junctional Intercellular Communication and Cell Fate in Osteoblasts

Simple Summary Certain tumor suppressor genes, such as p53 and Retinoblastoma gene (RB1), that normally function to maintain order and prevent the occurrence of cancer in normal tissue are usually found to be mutated at a high rate in bone cancers (osteosarcomas). In this manuscript we have investigated some of Rb1's functions by creating a deficiency in the expression of the Rb1 in bone cells to determine how this affects their ability to maintain bone-specific gene expression and function. We found that loss of Rb1 function in cells predisposed them to express features of both adipocytes (fat cells) and osteoblasts (bone cells). Normally, when these osteoblasts are induced to grow and function specifically as bone tissue, they significantly increase their communication with their neighboring cells. When the Rb1 gene function is reduced, there is a loss of this communication. The membrane protein essential to mediate this communication, connexin 43 (Cx43), is also reduced. One of the reasons why loss of Rb1 function might cause a change in the behavior of osteoblasts, relates to the increased expression of a master regulator of adipocyte formation-a protein called PPAR-gamma. We found the expression of PPAR-gamma to be increased by about 7-9-fold in Rb1-deficient cells. As this master regulator of adipocyte differentiation could potentially induce the changes seen, we reduced PPAR-gamma levels in control osteoblasts and Rb1-deficient cells to determine if the adipocyte features could be reversed. We found that normal osteoblasts and Rb1-deficient osteoblasts were able to revert to a predominantly osteoblast-specific expression when PPAR-gamma was reduced. However, the changes to communication and Cx43 expression were not reversed in Rb1-deficient cells. This suggests that either normal Rb1 function and/or other events in the process of osteoblast differentiation regulate appropriate cellular communication, and not PPAR-gamma in normal bone cells.Abstract Loss of function of the Retinoblastoma gene (RB1) due to mutations is commonly seen in human osteosarcomas. One of the Rb1 gene functions is to facilitate cell fate from mesenchymal stem cells to osteoblasts and prevent adipocyte differentiations. In this study, we demonstrate that a stable reduction of Rb1 expression (RbKD) in murine osteoblasts causes them to express higher levels of PPAR-gamma and other adipocyte-specific transcription factors while retaining high expression of osteoblast-specific transcription factors, Runx2/Cbfa1 and SP7/Osterix. Inhibition of gap junctional intercellular communication (GJIC) in osteoblasts is another mechanism that causes osteoblasts to transdifferentiate to adipocytes. We found that preosteoblasts exposed to osteoblast differentiating media (DP media) increased GJIC. RbKD cells showed reduced GJIC along with a reduction in expression of Cx43, the protein that mediates GJIC. Other membrane associated adhesion protein Cadherin 11 (Cad11) was also decreased. Since PPAR-gamma is increased with Rb1 loss, we wondered if the reduction of this transcription factor would reverse the changes observed. Reduction of PPAR-gamma in control osteoblasts slightly increased bone-specific expression and reduced adipocytic expression as expected along with an increase in Cad11 and Cx43 expression. GJIC remained high and was unaffected by a reduction in PPAR-gamma in control cells. Knockdown of PPAR-gamma in RbKD cells reduced adipocyte gene expression, while osteoblast-specific expression showed improvement. Cx43, Cad11 and GJIC remained unaffected by PPAR-gamma reduction. Our observations suggest that increased PPAR-gamma that happens with Rb1 loss only affects osteoblast-adipocyte-specific gene expression but does not completely reverse Cx43 gene expression or GJIC. Therefore, these effects may represent independent events triggered by Rb1loss and/or the differentiation process.