Disruption of -actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis

Triplett, Jason W., and Fredrick M. Pavalko. Disruption of -actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis. Am J Physiol Cell Physiol 291: C909–C921, 2006. First published June 28, 2006; doi:10.1152/ajpcell.00113.2006.—Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of -actinin (ROD-GFP) that competitively displaces endogenous -actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas -actinin-GFP expression protected, cells from TNF-induced apoptosis. Further investigation revealed that activation of TNF-induced survival signals, specifically Akt phosphorylation and NFB activation, was inhibited in ROD-GFPexpressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-induced apoptosis. Thus we conclude that -actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF-induced survival signaling.