Sulfhydryl modification by 4,4?-dithiodipyridine induces calcium mobilization in human osteoblast-like cells

Abstract

The effect of oxidants on Ca 2+ movement in osteoblasts is unclear. In this study, we show that 4,4¢-dithiodipyridine (4,4¢-DTDP), a reactive disulphide that mobilizes Ca 2+ in muscle, induces an increase in cytoplasmic free-Ca 2+ concentrations ([Ca 2+ ] i ) in MG63 human osteosarcoma cells loaded with the Ca 2+ -sensitive dye fura-2. 4,4¢-DTDP acted in a concentrationdependent manner with an EC 50 of 10 lM. Removing extracellular Ca 2+ reduced the Ca 2+ signal by 35%. In Ca 2+ -free medium, the 4,4¢-DTDP-induced [Ca 2+ ] i increase was not changed by depleting store Ca 2+ with 50 lM brefeldin A (a Golgi apparatus permeabilizer), by 2 lM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), by 1 lM thapsigargin (an inhibitor of the endoplasmic reticulum Ca 2+ pump) or by 5 lM ryanodine. Ca 2+ signals induced by 4,4¢-DTDP in Ca 2+ -containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 4,4¢-DTDP-induced Ca 2+ release was inhibited by a thiolselective reducing reagent, dithiothreitol (0.05-2.5 mM), in a concentration-dependent manner. Collectively, this study shows that 4,4¢-DTDP induced [Ca 2+ ] i increases in human osteosarcoma cells via releasing store Ca 2+ from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The store Ca 2+ release induced by 4,4¢-DTDP appears to be associated with thiol oxidation. Furthermore, overnight incubation with 4,4¢-DTDP inhibited cell activity in a concentrationdependent manner.

Enhancement of bone formation with a synthetic matrix containing bone morphogenetic protein-2 by the addition of calcium citrate

“…Calcium ions have been known to be intracellular messengers responsible for controlling various cellular processes, including mitosis, cell death, movement, differentiation and bone regeneration [6, 9, 16, 23, 29]. Maeno [10] observed that 2–4 mM of Ca 2+ was suitable for proliferation and survival of osteoblasts, whereas slightly higher concentrations (6–8 mM) favoured osteoblast differentiation and matrix mineralization in both 2‐ and 3‐dimensional cultures.…”

Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells

“…The inositol-1,4,5-trisphosphate-sensitive Ca 2+ store is an important Ca 2+ store that releases Ca 2+ into the cytosol when cells are stimulated by endogenous substances such as histamine (Lee et al, 2001a). But exogenous reagents can release Ca 2+ from inositol 1,4,5-trisphosphateinsensitive stores (Kuo et al, 2003;Jan et al, 2003).…”

“…MG63 osteosarcoma cells have characteristics similar to human osteoblasts and have been used as a model for research on human osteoblasts (Rezzonico et al, 2002). Many endogenous and exogenous reagents can stimulate MG63 cells by evoking a [Ca 2+ ] i increase, such as 2,2′-dithiodipyridine (Kuo et al, 2003), carvedilol (Liu and Jan, 2004), and desipramine . The inositol-1,4,5-trisphosphate-sensitive Ca 2+ store is an important Ca 2+ store that releases Ca 2+ into the cytosol when cells are stimulated by endogenous substances such as histamine (Lee et al, 2001a).…”

Effects of MK‐886, a leukotriene synthesis inhibitor, on [Ca2+]i and apoptosis in MG63 human osteosarcoma cells

“…In osteoblastic cells, Babich et al [1997] [Chang et al, 2005], 4,4 0 -dithiodipyridine [Kuo et al, 2003], CP55,940 [Lu et al, 2002b], and tamoxifen [Lu et al, 2002a]. Additionally, it appears that phospholipase C-dependent pathways did not play a role in MK-886-induced Ca MK-886 is cytotoxic in several cell types including Jurkat T lymphocytes [Deshpande and Kehrer., 2006], HT-29 cells [Kleban et al, 2006], HL-60 cells [Stika et al, 2006], gastric cancer cells [Fan et al, 2004], and rat arterial myocytes [Smirnov et al, 1998].…”

“…MG63 cells have characteristics similar to human osteoblasts and are used as a model for research on human osteoblasts [Rezzonico et al, 2002]. Many endogenous and exogenous reagents can stimulate MG63 cells by evoking a [Ca 21 ] i increase, such as 2,2 0 -dithiodipyridine [Kuo et al, 2003], carvedilol [Liu and Jan, 2004], and desipramine [Lee et al, 2001]. Exogenous ligands can also release Ca 21 from inositol 1,4,5-trisphosphate-insensitive stores [Kuo et al, 2003;Jan et al, 2003].…”