Oxidative-induced calcium mobilization is dependent on annexin VI release from lipid rafts.
BACKGROUND
Oxidative stress results in macrophage reprogramming through the formation of focal adhesion-like complexes on lipid rafts. Although the cellular mechanisms responsible for this reprogramming remain unknown, oxidative stress is known to result in a transient increase in intracellular calcium. This transient flux is thought to occur through the membrane dissociation of the calcium-bound protein annexin VI. The purpose of this study is to clarify the source of the calcium, and determine if it is responsible for the formation of focal adhesion-like complexes during oxidative stress through the activation of calcium/calmodulin dependent protein kinase II (CaMK II).


METHODS
THP-1 cells were stimulated with hydrogen peroxide. Selected cells were pretreated with methyl beta-cyclodextrin (MbetaCD), a cholesterol-depleting agent; 1,2-bis aminophenoxy ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator; or autocamtide 2-related inhibitory peptide, a CaMK II inhibitor. Intracellular calcium flux was determined by a Fluo-3 technique. Lipid raft and cellular protein were extracted and analyzed for active CaMK II, annexin VI, and components of focal adhesion-like complexes.


RESULTS
Hydrogen peroxide exposure led to mobilization of annexin VI from lipid rafts to the cytosol, which was followed by an increase in cytosolic calcium, phosphorylation of CaMK II, and formation of focal adhesion-like complexes. Cholesterol depletion from lipid rafts attenuated all of these effects. 1,2-bis Aminophenoxy ethane-N,N,N',N'-tetraacetic acid and autocamtide 2-related inhibitory peptide pretreatment attenuated CaMK II phopshorylation and formation of focal ahdesionlike complexes.


CONCLUSIONS
Macrophage reprogramming during oxidative stress occurs through the cytosolic mobilization of annexin VI from lipid rafts. As a result, bound calcium dissociates, resulting in the activation of CaMK II and the formation of focal adhesion-like complexes.
Cuschieri
Joseph
J
Department of Surgery, University of Washington, Seattle, USA. jcuschie@u.washington.edu
Bulger
Eileen
E
Garcia
Iris
I
Maier
Ronald V
RV
D017317:Annexin A6 / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*
D017317:Annexin A6; D016877:Oxidants; D006861:Hydrogen Peroxide; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium