Labile zinc and zinc transporter ZnT4 in mast cell granules: role in regulation of caspase activation and NF-kappaB translocation.
The granules of mast cells and other inflammatory cells are known to be rich in zinc (Zn), a potent caspase inhibitor. The functions of granular Zn, its mechanism of uptake, and its relationship to caspase activation in apoptosis are unclear. The granules of a variety of mast cell types fluoresced intensely with the Zn-specific fluorophore Zinquin, and fluorescence was quenched by functional depletion of Zn using a membrane-permeable Zn chelator N, N, N', N'-tetrakis (2-pyridyl-methyl)ethylenediamine (TPEN). Zn levels were also depleted by various mast cell activators, including IgE/anti-IgE, and Zn was rapidly replenished during subsequent culture, suggesting an active uptake mechanism. In support of the latter, mast cells contained high levels of the vesicular Zn transporter ZnT(4), especially in the more apical granules. Immunofluorescence and immunogold labeling studies revealed significant pools of procaspase-3 and -4 in mast cell granules and their release during degranulation. Functional depletion of Zn by chelation with TPEN, but not by degranulation, resulted in greatly increased susceptibility of mast cells to toxin-induced caspase activation, as detected using a fluorogenic substrate assay. Release of caspases during degranulation was accompanied by a decreased susceptibility to toxins. Zn depletion by chelation, but not by degranulation, also resulted in nuclear translocation of the antiapoptotic, proinflammatory transcription factor NF-kappaB. These findings implicate a role for ZnT(4) in mast cell Zn homeostasis and suggest that granule pools of Zn may be distinct from those regulating activation of procaspase-3 and NF-kappaB.
Ho
Lien H
LH
Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011.
Ruffin
Richard E
RE
Murgia
Chiara
C
Li
Lixin
L
Krilis
Steven A
SA
Zalewski
Peter D
PD
D021581:Active Transport, Cell Nucleus; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D053471:Caspases, Initiator; D027682:Cation Transport Proteins; D015550:Cell Degranulation; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000737:chemistry*; D004789:Enzyme Activation; D006706:Homeostasis; D006801:Humans; D008407:Mast Cells / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism; D014118:Toxins, Biological / Q000494:pharmacology; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D016328:NF-kappa B; C491597:SLC30A4 protein, human; D014118:Toxins, Biological; C505407:CASP3 protein, human; C093516:CASP4 protein, human; D053148:Caspase 3; D020169:Caspases; D053471:Caspases, Initiator; D015032:Zinc