α-Integrin expression and function modulates presentation of cell surface calreticulin.
Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the prophagocytic signal for macrophages. Cell adhesion is a physiologically relevant stimulus previously shown to increase calreticulin interaction with α-integrins via the juxtamembrane, cytosolic GFFKR motif. This study assessed whether integrin function can regulate surface calreticulin levels in ICD. We generated calreticulin-null T-lymphoblasts and confirmed the loss of surface calreticulin expression on cells treated with doxorubicin, an ICD inducer. Reconstituted expression with full-length calreticulin targeted to the endoplasmic reticulum (ER) successfully rescued doxorubicin-induced surface calreticulin. Reconstitution with a truncation mutant calreticulin targeted to the cytosol led to constitutively high surface calreticulin that was not further elevated by doxorubicin, suggesting calreticulin released from the stressed ER transits the cytosol before its translocation to the cell surface. When stimulated to engage integrin substrates, doxorubicin-treated wild-type T-lymphoblasts exhibited decreased surface calreticulin compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin was recapitulated for cells in suspension treated with a β1-integrin-activating antibody, 9EG7. Similarly, cells expressing a truncated α-integrin cytosolic tail, bearing only the juxtamembrane GFFKR calreticulin-binding motif, exhibited low surface calreticulin with doxorubicin treatment under non-adherent conditions. Using partial permeabilization techniques to distinguish between cytosolic and ER staining, we found that ICD inducers promoted the accumulation of cytosolic calreticulin with negligible change in total calreticulin, suggesting that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation. T-lymphoblasts co-treated with an ICD inducer and 9EG7 exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD inducer. This study reveals a previously uncharacterized function of integrins as negative regulators of ICD by suppressing presentation of cell surface calreticulin.
Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.|Department of Medicine, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.
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Liu
Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.
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Leclair
Department of Medicine, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.
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Monajemi
Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.
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Sly
Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.|Michael Cuccione Childhood Cancer Research Program, BC Children's Hospital, Vancouver, BC, Canada V5Z 4H4.
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GS
Reid
Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.|Michael Cuccione Childhood Cancer Research Program, BC Children's Hospital, Vancouver, BC, Canada V5Z 4H4.
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CJ
Lim
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D001483:Base Sequence; D037282:Calreticulin / Q000235:genetics* / Q000276:immunology; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D004317:Doxorubicin / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D039422:Integrin alpha3 / Q000235:genetics* / Q000276:immunology; D039441:Integrin alpha4 / Q000235:genetics* / Q000276:immunology; D039482:Integrin alpha5 / Q000235:genetics* / Q000276:immunology; D019012:Integrin beta1 / Q000235:genetics / Q000276:immunology; D019169:Jurkat Cells; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D061251:Primary Cell Culture; D021381:Protein Transport; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000473:pathology
D000903:Antibiotics, Antineoplastic; D000906:Antibodies; C576483:CALR protein, human; D037282:Calreticulin; D039422:Integrin alpha3; D039482:Integrin alpha5; D019012:Integrin beta1; D039441:Integrin alpha4; D004317:Doxorubicin