Abstract
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.
Highlights
Cyclosporin A (CsA)1 is a cyclic endecapeptide immunosuppressant that is widely used to prevent organ rejection after transplantation
Since cyclosporin A (CsA) binding to cyclophilin perturbs the function of the permeability transition pore (PTP) (6) and can independently inhibit calcium release from endoplasmic reticulum (ER) stores (5, 21), we considered that CsA may inhibit collagen phagocytosis by deregulating calcium homeostasis in mitochondrial and/or ER stores
To overcome the problems of phenotypic variability associated with primary isolates of gingival fibroblasts (35), we first rationalized the use of Rat2 fibroblasts, a stable and readily propagated cell line previously used for studies of collagen phagocytosis and that exhibits many of the characteristic features of periodontal fibroblasts (24)
Summary
CsA, cyclosporin A; PTP, permeability transition pore; ER, endoplasmic reticulum; CCCP, carbonyl cyanide m-chlorophenylhydrazone; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈtetraacetic acid acetoxymethyl ester; HGFs, human gingival fibroblasts; BSA, bovine serum albumin. In “professional” phagocytes such as neutrophils, efficient phagocytosis is dependent on calcium release from intracellular stores (16), which, in turn, is reliant on inositol 1,4,5trisphosphate receptor function to regulate calcium levels within endoplasmic reticulum (ER) stores (17). These receptors are translocated to periphagosomal sites during particle internalization (18), suggesting that phagocytosis may be affected by calcium levels in the intracellular stores. Since CsA binding to cyclophilin perturbs the function of the PTP (6) and can independently inhibit calcium release from ER stores (5, 21), we considered that CsA may inhibit collagen phagocytosis by deregulating calcium homeostasis in mitochondrial and/or ER stores. The data show that CsA inhibits the binding step of collagen phagocytosis through a calcium-regulated pathway involving ER and mitochondrial stores
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