Interaction between S100A8/A9 and annexin A6 is involved in the calcium-induced cell surface exposition of S100A8/A9.
The calcium binding S100A8/A9 complex (MRP8/14; calgranulin) is considered as an important proinflammatory mediator in acute and chronic inflammation and has recently gained attention as a molecular marker up-regulated in various human cancers. Here, we report that S100A8/A9 is expressed in breast cancer cell lines and is up-regulated by interleukin-1beta and tumor necrosis factor-alpha in SKBR3 and MCF-7 cells. We identified the phospholipid-binding protein annexin A6 as a potential S100A8/A9 binding protein by affinity chromatography. This finding was verified by Southwestern overlay experiments and by coimmunoprecipitation with the S100A8/A9-specific monoclonal antibody 27E10. Immunocytochemical experiments demonstrated that S100A8/A9 and annexin A6 colocalize in SKBR3 breast cancer cells predominantly in membranous structures. Upon calcium influx both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells. Subcellular fractionation studies suggested that after A23187 stimulation membrane association of S100A8/A9 is not enhanced. However, both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells upon calcium influx. Experiments with artificial liposomes indicated that S100A8/A9 is able to associate with membranes independently of both annexin A6 and independently of calcium. Finally, cell surface expression of S100A8/A9 could not be observed in A23187-treated A431 and HaCaT cells. Both cell lines are known to be devoid of annexin A6. Repression of annexin A6 expression by small interfering RNA in SKBR3 cells abolishes the cell surface exposition of S100A8/A9 upon calcium influx, suggesting that annexin A6 contributes to the calcium-dependent cell surface exposition of the membrane associated-S100A8/A9 complex.
Bode
Günther
G
Institute for Immunology, Roentgenstrasse 21, University of Muenster, D-48149 Muenster, Germany.
Lüken
Aloys
A
Kerkhoff
Claus
C
Roth
Johannes
J
Ludwig
Stephan
S
Nacken
Wolfgang
W
D017317:Annexin A6 / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D040501:Calgranulin A / Q000235:genetics / Q000378:metabolism*; D040502:Calgranulin B / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D011485:Protein Binding
D017317:Annexin A6; D040501:Calgranulin A; D040502:Calgranulin B; D002118:Calcium