Disruption of alpha-actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis.
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 alpha-actinin (ROD-GFP) that competitively displaces endogenous alpha-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 alpha-actinin-GFP expression protected, cells from TNF-alpha-induced apoptosis. Further investigation revealed that activation of TNF-alpha-induced survival signals, specifically Akt phosphorylation and NF-kappaB activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-alpha-induced apoptosis. Thus we conclude that alpha-actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF-alpha-induced survival signaling.
Triplett
Jason W
JW
Dept. of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Pavalko
Fredrick M
FM
D000185:Actinin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000199:Actins; D016023:Integrins; D050505:Mutant Proteins; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000185:Actinin; D019000:Phosphotyrosine; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases