Reduction in vinculin levels with Rb1 loss is responsible for altered differentiation in preosteoblasts.

We have shown previously that loss of Rb1 in osteoblasts results in changes to gap junctional intercellular communication and a decrease in membrane proteins such as connexin 43 and cadherin 11. These cells also start expressing adipocyte specific transcription factors and marker genes while continuing to express osteoblast specific differentiated features. The mechanistic reason for this shift in differentiation behavior is not known. Recent studies have suggested a role for vinculin, a protein component of focal adhesions, in the transduction of signals to YAP-TAZ transcription factors to suppress adipocyte differentiation and allow osteoblast differentiation. We found loss of Rb1 expression in osteoblasts dramatically reduced vinculin quantity, with less distribution within focal adhesions when tested by immunofluorescence. Vinculin levels steadily increased during in vitro osteoblast differentiation and were about 6-8-fold higher in differentiated osteoblasts. Rb1 deficient cells showed an increase albeit at reduced levels (2-4-fold) during differentiation. A corresponding increase in YAP-TAZ activity and expression were seen in control osteoblasts and a stunted but similar response was present in Rb1 deficient osteoblasts. The fact that Rb1 deficient osteoblasts respond by increasing vinculin levels suggests that they can maintain the expected functional changes at a reduced level. To demonstrate that the concentration of vinculin was the determining factor, we either over expressed or knocked down vinculin which resulted in appropriately altering YAP/TAZ activity. Increased vinculin expression enhanced osteogenic while inhibiting adipocytic gene expression. During osteoblast differentiation, TAZ can be clearly visualized in the nucleus in osteoblasts' whereas with Rb1 deficiency the distribution was diffuse within the cells. The reduction in vinculin expression, combined with a feeble response in YAP-TAZ signaling activity, may explain the lack of complete inhibition of adipocyte specific gene expression and a generation of a mixed phenotype seen with loss of Rb1 function. As the adipocyte master transcription factor PPAR-gamma levels are increased by Rb1 loss, we knockdown PPAR-gamma which resulted in an increase to vinculin expression, showing that the PPAR-gamma induced activation of adipocyte differentiation pathway is responsible for reducing vinculin expression in RbKD cells.