Mechanism of cyclosporin-induced inhibition of intracellular collagen degradation.
The immunosuppressant cyclosporin A (CsA) markedly inhibits collagen degradation by an intracellular phagocytic pathway in fibroblasts, an effect that can lead to massive gingival overgrowth. We used a collagen bead model of collagen phagocytosis to determine whether CsA inhibits internalization by blocking efflux of calcium from endoplasmic reticulum (ER) and mitochondrial calcium stores. CsA caused dose-dependent inhibition of phagocytosis of collagen-coated (but not bovine serum albumin-coated) beads. Chelation of intracellular Ca(2+) with BAPTA/AM or inhibition of Ca(2+)-ATPase of ER stores with thapsigargin reduced collagen bead phagocytosis. Measurement of intracellular calcium by ratio fluorometry showed increases in response to collagen-coated beads. Preincubation with CsA or thapsigargin caused a >3-fold decrease in intracellular calcium elevations in response to stimulation with collagen beads. Direct measurements of Ca(2+) in mitochondrial and ER stores showed that CsA only slightly inhibited collagen bead-induced discharge of calcium from mitochondria, but almost completely blocked discharge from ER stores. We reduced the numbers of mitochondria with chronic ethidium bromide treatment to test for the importance of ER/mitochondrial interactions. In these cells, CsA delayed collagen bead-induced calcium discharge from mitochondria. Collectively, these data indicate that CsA inhibits collagen phagocytosis by blocking calcium release from ER stores and may perturb functional interactions between the ER and mitochondria that regulate calcium stores.
Canadian Institutes of Health Research Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
P D
PD
Arora
L
L
Silvestri
B
B
Ganss
J
J
Sodek
C A
CA
McCulloch
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003094:Collagen / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D015398:Signal Transduction; D013696:Temperature; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D012710:Serum Albumin, Bovine; D004533:Egtazic Acid; D019284:Thapsigargin; D016572:Cyclosporine; D003094:Collagen; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium