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15d-PGJ2 induces apoptosis of mouse oligodendrocyte precursor cells.

Xiang Z, Lin T, Reeves SA - J Neuroinflammation (2007)

Bottom Line: The cytotoxicity of 15d-PGJ2 was associated with the production of ROS and was inversely related to intracellular glutathione (GSH) levels.However, the cytotoxicity of 15d-PGJ2 was not decreased by the free radical scavengers ascorbic acid or alpha-tocopherol.Taken together, these results demonstrated that 15d-PGJ2 is toxic to early stage OP cells, suggesting that 15d-PGJ2 may represent a deleterious factor in the natural remyelination process in MS.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNS Signaling Laboratory, MassGeneral Institute for Neurodegenerative Disease (MIND), Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA. zxiang1@partners.org <zxiang1@partners.org>

ABSTRACT

Background: Prostaglandin (PG) production is associated with inflammation, a major feature in multiple sclerosis (MS) that is characterized by the loss of myelinating oligodendrocytes in the CNS. While PGs have been shown to have relevance in MS, it has not been determined whether PGs have a direct effect on cells within the oligodendrocyte lineage.

Methods: Undifferentiated or differentiated mouse oligodendrocyte precursor (mOP) cells were treated with PGE2, PGF2alpha, PGD2 or 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2). Cell growth and survival following treatment were examined using cytotoxicity assays and apoptosis criteria. The membrane receptors for PGD2 and the nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma, as well as reactive oxygen species (ROS) in the death mechanism were examined.

Results: PGE2 and PGF2alpha had minimal effects on the growth and survival of mOP cells. In contrast, PGD2 and 15d-PGJ2 induced apoptosis of undifferentiated mOP cells at relatively low micromolar concentrations. 15d-PGJ2 was less toxic to differentiated mOP cells. Apoptosis was independent of membrane receptors for PGD2 and the nuclear receptor PPARgamma. The cytotoxicity of 15d-PGJ2 was associated with the production of ROS and was inversely related to intracellular glutathione (GSH) levels. However, the cytotoxicity of 15d-PGJ2 was not decreased by the free radical scavengers ascorbic acid or alpha-tocopherol.

Conclusion: Taken together, these results demonstrated that 15d-PGJ2 is toxic to early stage OP cells, suggesting that 15d-PGJ2 may represent a deleterious factor in the natural remyelination process in MS.

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15d-PGJ2 cytotoxicity involves free radical production and is influenced by intracellular glutathione levels. (A) Time course of 15d-PGJ2-induced ROS production. mOP cells were preloaded with the fluorescent ROS probe DCFDA for 30 min, and then treated with 15d-PGJ2 (10 μM) for 1 h. ROS production was expressed as DCFDA fluorescence intensity (pixel value). (B) mOP cells were pre-treated or not with NAC (1 mM), Ascorbic acid (1 mM) or α-tocopherol (1 mM) for 1 h prior to treatment of 15d-PGJ2 (5 μM) for 24 h, toxicity was examined by counting the apoptotic cells with condensed nuclei. (C) mOP cells were treated or not with BSO (100 μM) for 4 h and the total level of intracellular GSH was measured. (D) mOP cells were treated with BSO (100 μM) for 1 h and then co-treated with 15d-PGJ2 (1 μM) for 24 h. Cells treated with BSO or 15d-PGJ2 alone or untreated were included as controls. Toxicity was examined by counting the apoptotic cells with condensed nuclei. Asterisks indicate significant difference (One-way ANOVA with Newman-Keuls or Dunnet posttest, or two way ANOVA with Bonferroni posttest, *(p < 0.05), **(p < 0.01) ***(p < 0.001); two-tailed t-test used in C).
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Figure 4: 15d-PGJ2 cytotoxicity involves free radical production and is influenced by intracellular glutathione levels. (A) Time course of 15d-PGJ2-induced ROS production. mOP cells were preloaded with the fluorescent ROS probe DCFDA for 30 min, and then treated with 15d-PGJ2 (10 μM) for 1 h. ROS production was expressed as DCFDA fluorescence intensity (pixel value). (B) mOP cells were pre-treated or not with NAC (1 mM), Ascorbic acid (1 mM) or α-tocopherol (1 mM) for 1 h prior to treatment of 15d-PGJ2 (5 μM) for 24 h, toxicity was examined by counting the apoptotic cells with condensed nuclei. (C) mOP cells were treated or not with BSO (100 μM) for 4 h and the total level of intracellular GSH was measured. (D) mOP cells were treated with BSO (100 μM) for 1 h and then co-treated with 15d-PGJ2 (1 μM) for 24 h. Cells treated with BSO or 15d-PGJ2 alone or untreated were included as controls. Toxicity was examined by counting the apoptotic cells with condensed nuclei. Asterisks indicate significant difference (One-way ANOVA with Newman-Keuls or Dunnet posttest, or two way ANOVA with Bonferroni posttest, *(p < 0.05), **(p < 0.01) ***(p < 0.001); two-tailed t-test used in C).

Mentions: Previous reports have suggested that 15d-PGJ2 may induce intracellular oxidative stress [38,39]. To examine whether there is increased ROS production in 15d-PGJ2-treated mOP cells we preloaded cells with the fluorescent ROS probe DCFDA prior to 15d-PGJ2-treatment. ROS production was significantly increased in mOP cells as early as 45 min after treatment with 15d-PGJ2 (10 μM) (Fig. 4A). Glutathione (GSH) is an important antioxidant that protects cells from oxidative damage by ROS. We therefore tested whether manipulations of the intracellular level of GSH could affect apoptotic cell death induced by 15d-PGJ2. Pretreatment of mOP cells with NAC, which is a precursor molecule for GSH synthesis and a reducing agent for oxidized GSH [40], provided ~60% protection against 15d-PGJ2 induced death, while pre-incubation with the antioxidants ascorbic acid or α-tocopherol did not provide protection (Fig. 4B). In contrast, application of buthionine sulfoximine (BSO), an inhibitor for γ-glutamylcysteinase synthatase [41] which depletes intracellular GSH (Fig. 4C), was toxic to mOP cells by itself and sensitized mOP cells to a lower concentration of 15d-PGJ2 (Fig. 4D). These results suggest that the toxicity of 15d-PGJ2 to mOP cells is related to intracellular GSH levels.


15d-PGJ2 induces apoptosis of mouse oligodendrocyte precursor cells.

Xiang Z, Lin T, Reeves SA - J Neuroinflammation (2007)

15d-PGJ2 cytotoxicity involves free radical production and is influenced by intracellular glutathione levels. (A) Time course of 15d-PGJ2-induced ROS production. mOP cells were preloaded with the fluorescent ROS probe DCFDA for 30 min, and then treated with 15d-PGJ2 (10 μM) for 1 h. ROS production was expressed as DCFDA fluorescence intensity (pixel value). (B) mOP cells were pre-treated or not with NAC (1 mM), Ascorbic acid (1 mM) or α-tocopherol (1 mM) for 1 h prior to treatment of 15d-PGJ2 (5 μM) for 24 h, toxicity was examined by counting the apoptotic cells with condensed nuclei. (C) mOP cells were treated or not with BSO (100 μM) for 4 h and the total level of intracellular GSH was measured. (D) mOP cells were treated with BSO (100 μM) for 1 h and then co-treated with 15d-PGJ2 (1 μM) for 24 h. Cells treated with BSO or 15d-PGJ2 alone or untreated were included as controls. Toxicity was examined by counting the apoptotic cells with condensed nuclei. Asterisks indicate significant difference (One-way ANOVA with Newman-Keuls or Dunnet posttest, or two way ANOVA with Bonferroni posttest, *(p < 0.05), **(p < 0.01) ***(p < 0.001); two-tailed t-test used in C).
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Related In: Results  -  Collection

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Figure 4: 15d-PGJ2 cytotoxicity involves free radical production and is influenced by intracellular glutathione levels. (A) Time course of 15d-PGJ2-induced ROS production. mOP cells were preloaded with the fluorescent ROS probe DCFDA for 30 min, and then treated with 15d-PGJ2 (10 μM) for 1 h. ROS production was expressed as DCFDA fluorescence intensity (pixel value). (B) mOP cells were pre-treated or not with NAC (1 mM), Ascorbic acid (1 mM) or α-tocopherol (1 mM) for 1 h prior to treatment of 15d-PGJ2 (5 μM) for 24 h, toxicity was examined by counting the apoptotic cells with condensed nuclei. (C) mOP cells were treated or not with BSO (100 μM) for 4 h and the total level of intracellular GSH was measured. (D) mOP cells were treated with BSO (100 μM) for 1 h and then co-treated with 15d-PGJ2 (1 μM) for 24 h. Cells treated with BSO or 15d-PGJ2 alone or untreated were included as controls. Toxicity was examined by counting the apoptotic cells with condensed nuclei. Asterisks indicate significant difference (One-way ANOVA with Newman-Keuls or Dunnet posttest, or two way ANOVA with Bonferroni posttest, *(p < 0.05), **(p < 0.01) ***(p < 0.001); two-tailed t-test used in C).
Mentions: Previous reports have suggested that 15d-PGJ2 may induce intracellular oxidative stress [38,39]. To examine whether there is increased ROS production in 15d-PGJ2-treated mOP cells we preloaded cells with the fluorescent ROS probe DCFDA prior to 15d-PGJ2-treatment. ROS production was significantly increased in mOP cells as early as 45 min after treatment with 15d-PGJ2 (10 μM) (Fig. 4A). Glutathione (GSH) is an important antioxidant that protects cells from oxidative damage by ROS. We therefore tested whether manipulations of the intracellular level of GSH could affect apoptotic cell death induced by 15d-PGJ2. Pretreatment of mOP cells with NAC, which is a precursor molecule for GSH synthesis and a reducing agent for oxidized GSH [40], provided ~60% protection against 15d-PGJ2 induced death, while pre-incubation with the antioxidants ascorbic acid or α-tocopherol did not provide protection (Fig. 4B). In contrast, application of buthionine sulfoximine (BSO), an inhibitor for γ-glutamylcysteinase synthatase [41] which depletes intracellular GSH (Fig. 4C), was toxic to mOP cells by itself and sensitized mOP cells to a lower concentration of 15d-PGJ2 (Fig. 4D). These results suggest that the toxicity of 15d-PGJ2 to mOP cells is related to intracellular GSH levels.

Bottom Line: The cytotoxicity of 15d-PGJ2 was associated with the production of ROS and was inversely related to intracellular glutathione (GSH) levels.However, the cytotoxicity of 15d-PGJ2 was not decreased by the free radical scavengers ascorbic acid or alpha-tocopherol.Taken together, these results demonstrated that 15d-PGJ2 is toxic to early stage OP cells, suggesting that 15d-PGJ2 may represent a deleterious factor in the natural remyelination process in MS.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNS Signaling Laboratory, MassGeneral Institute for Neurodegenerative Disease (MIND), Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA. zxiang1@partners.org <zxiang1@partners.org>

ABSTRACT

Background: Prostaglandin (PG) production is associated with inflammation, a major feature in multiple sclerosis (MS) that is characterized by the loss of myelinating oligodendrocytes in the CNS. While PGs have been shown to have relevance in MS, it has not been determined whether PGs have a direct effect on cells within the oligodendrocyte lineage.

Methods: Undifferentiated or differentiated mouse oligodendrocyte precursor (mOP) cells were treated with PGE2, PGF2alpha, PGD2 or 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2). Cell growth and survival following treatment were examined using cytotoxicity assays and apoptosis criteria. The membrane receptors for PGD2 and the nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma, as well as reactive oxygen species (ROS) in the death mechanism were examined.

Results: PGE2 and PGF2alpha had minimal effects on the growth and survival of mOP cells. In contrast, PGD2 and 15d-PGJ2 induced apoptosis of undifferentiated mOP cells at relatively low micromolar concentrations. 15d-PGJ2 was less toxic to differentiated mOP cells. Apoptosis was independent of membrane receptors for PGD2 and the nuclear receptor PPARgamma. The cytotoxicity of 15d-PGJ2 was associated with the production of ROS and was inversely related to intracellular glutathione (GSH) levels. However, the cytotoxicity of 15d-PGJ2 was not decreased by the free radical scavengers ascorbic acid or alpha-tocopherol.

Conclusion: Taken together, these results demonstrated that 15d-PGJ2 is toxic to early stage OP cells, suggesting that 15d-PGJ2 may represent a deleterious factor in the natural remyelination process in MS.

Show MeSH
Related in: MedlinePlus