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

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-ɣ 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-ɣ is increased with Rb1 loss, we wondered if the reduction of this transcription factor would reverse the changes observed. Reduction of PPAR-ɣ 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-ɣ in control cells. Knockdown of PPAR-ɣ in RbKD cells reduced adipocyte gene expression, while osteoblast-specific expression showed improvement. Cx43, Cad11 and GJIC remained unaffected by PPAR-ɣ reduction. Our observations suggest that increased PPAR-ɣ 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.

Keywords

• Retinoblastoma gene
• osteoblast differentiation
• gap junctional intercellular communication
• connexin 43