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Impact of glutathione peroxidase-1 deficiency on macrophage foam cell formation and proliferation: implications for atherogenesis.

Cheng F, Torzewski M, Degreif A, Rossmann H, Canisius A, Lackner KJ - PLoS ONE (2013)

Bottom Line: Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process.GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE(-/-) mice.The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1(-/-)ApoE(-/-) mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany.

ABSTRACT
Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE(-/-) mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1) to analyze which cells express GPx-1 within atherosclerotic lesions and (2) to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE(-/-) mice after 12 weeks on a Western type diet revealed that both macrophages and - even though to a less extent - smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1(-/-)ApoE(-/-) mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. The present study demonstrates that GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK (ERK1/2) pathway encouraging further studies on new therapeutic strategies against atherosclerosis.

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Effects of MCSF on the phosphorylation of MAPKs.After pre-incubation for 3 days with 10 ng/ml MCSF, peritoneal macrophages were incubated for 5 and 15 min with 10 ng/ml MCSF. Cellular protein was extracted and protein samples (0.4 mg/ml) were analyzed by Western blot with specific antibodies: anti-phosphorylated MEK1/2 or anti-MEK1/2 (A, right), anti-phosphorylated ERK1/2 or anti-ERK1/2 (B, right), anti-phosphorylated p90RSK or anti-RSK1/2/3 (C, right), anti-phosphorylated p38 MAPK or anti-p38 MAPK (D, right) and anti-phosphorylated SAPK/JNK or anti-SAPK/JNK (E, right) antibodies (representative experiments). ß-Actin or Actin were used as control. Quantitative results were calculated by band densitometry with the intensity of phosphorylated MEK1/2, ERK1/2, p90RSK, p38 MAPK and SAPK/JNK normalized to the total MEK1/2, ERK1/2, RSK1/2/3, p38 MAPK and SAPK/JNK (A–E, left panels). Data represent mean ± SD of 3 separate experiments. *, **indicate statistically significant differences (*p<0.05, **p<0.01) compared with cells without MCSF treatment.
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pone-0072063-g004: Effects of MCSF on the phosphorylation of MAPKs.After pre-incubation for 3 days with 10 ng/ml MCSF, peritoneal macrophages were incubated for 5 and 15 min with 10 ng/ml MCSF. Cellular protein was extracted and protein samples (0.4 mg/ml) were analyzed by Western blot with specific antibodies: anti-phosphorylated MEK1/2 or anti-MEK1/2 (A, right), anti-phosphorylated ERK1/2 or anti-ERK1/2 (B, right), anti-phosphorylated p90RSK or anti-RSK1/2/3 (C, right), anti-phosphorylated p38 MAPK or anti-p38 MAPK (D, right) and anti-phosphorylated SAPK/JNK or anti-SAPK/JNK (E, right) antibodies (representative experiments). ß-Actin or Actin were used as control. Quantitative results were calculated by band densitometry with the intensity of phosphorylated MEK1/2, ERK1/2, p90RSK, p38 MAPK and SAPK/JNK normalized to the total MEK1/2, ERK1/2, RSK1/2/3, p38 MAPK and SAPK/JNK (A–E, left panels). Data represent mean ± SD of 3 separate experiments. *, **indicate statistically significant differences (*p<0.05, **p<0.01) compared with cells without MCSF treatment.

Mentions: To explain the effect of MCSF on MAPKs activation in macrophages from GPx-1 deficient mice, we determined the level of ERK1/2, p38 MAPK and SAPK/JNK phosphorylation in peritoneal macrophages using an antibody raised against both phosphorylation sites required for activation of ERK1/2 (Figure 4 B, right), p38 MAPK (Figure 4 D, right) and SAPK/JNK (Figure 4 E, right). MCSF induced early ERK1/2 phosphorylation significantly more in macrophages of GPx-1−/−ApoE−/− mice than in ApoE−/− control mice after 5 min (p<0,05). After 15 min this difference was no longer significant (Figure 4 B, left). MCSF did not increase p38 MAPK (Figure 4 D, left) and SAPK/JNK (Figure 4 E, left) phosphorylation significantly. Since ERK1/2 are activated by MEK1/2 and p90RSK is an important downstream substrate of ERK1/2, we tested further whether MEK1/2 and p90RSK are also activated by MCSF. Using an antibody reacting with phosphorylated MEK1/2 and p90RSK, MEK1/2 (Figure 4 A) and p90RSK (Figure 4 C), phosphorylation was found to be enhanced significantly in macrophages of GPx-1−/−ApoE−/− mice compared with ApoE−/− control mice at 5 min (p<0,05), and then MEK1/2 declined to basal level after 15 min.


Impact of glutathione peroxidase-1 deficiency on macrophage foam cell formation and proliferation: implications for atherogenesis.

Cheng F, Torzewski M, Degreif A, Rossmann H, Canisius A, Lackner KJ - PLoS ONE (2013)

Effects of MCSF on the phosphorylation of MAPKs.After pre-incubation for 3 days with 10 ng/ml MCSF, peritoneal macrophages were incubated for 5 and 15 min with 10 ng/ml MCSF. Cellular protein was extracted and protein samples (0.4 mg/ml) were analyzed by Western blot with specific antibodies: anti-phosphorylated MEK1/2 or anti-MEK1/2 (A, right), anti-phosphorylated ERK1/2 or anti-ERK1/2 (B, right), anti-phosphorylated p90RSK or anti-RSK1/2/3 (C, right), anti-phosphorylated p38 MAPK or anti-p38 MAPK (D, right) and anti-phosphorylated SAPK/JNK or anti-SAPK/JNK (E, right) antibodies (representative experiments). ß-Actin or Actin were used as control. Quantitative results were calculated by band densitometry with the intensity of phosphorylated MEK1/2, ERK1/2, p90RSK, p38 MAPK and SAPK/JNK normalized to the total MEK1/2, ERK1/2, RSK1/2/3, p38 MAPK and SAPK/JNK (A–E, left panels). Data represent mean ± SD of 3 separate experiments. *, **indicate statistically significant differences (*p<0.05, **p<0.01) compared with cells without MCSF treatment.
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Related In: Results  -  Collection

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pone-0072063-g004: Effects of MCSF on the phosphorylation of MAPKs.After pre-incubation for 3 days with 10 ng/ml MCSF, peritoneal macrophages were incubated for 5 and 15 min with 10 ng/ml MCSF. Cellular protein was extracted and protein samples (0.4 mg/ml) were analyzed by Western blot with specific antibodies: anti-phosphorylated MEK1/2 or anti-MEK1/2 (A, right), anti-phosphorylated ERK1/2 or anti-ERK1/2 (B, right), anti-phosphorylated p90RSK or anti-RSK1/2/3 (C, right), anti-phosphorylated p38 MAPK or anti-p38 MAPK (D, right) and anti-phosphorylated SAPK/JNK or anti-SAPK/JNK (E, right) antibodies (representative experiments). ß-Actin or Actin were used as control. Quantitative results were calculated by band densitometry with the intensity of phosphorylated MEK1/2, ERK1/2, p90RSK, p38 MAPK and SAPK/JNK normalized to the total MEK1/2, ERK1/2, RSK1/2/3, p38 MAPK and SAPK/JNK (A–E, left panels). Data represent mean ± SD of 3 separate experiments. *, **indicate statistically significant differences (*p<0.05, **p<0.01) compared with cells without MCSF treatment.
Mentions: To explain the effect of MCSF on MAPKs activation in macrophages from GPx-1 deficient mice, we determined the level of ERK1/2, p38 MAPK and SAPK/JNK phosphorylation in peritoneal macrophages using an antibody raised against both phosphorylation sites required for activation of ERK1/2 (Figure 4 B, right), p38 MAPK (Figure 4 D, right) and SAPK/JNK (Figure 4 E, right). MCSF induced early ERK1/2 phosphorylation significantly more in macrophages of GPx-1−/−ApoE−/− mice than in ApoE−/− control mice after 5 min (p<0,05). After 15 min this difference was no longer significant (Figure 4 B, left). MCSF did not increase p38 MAPK (Figure 4 D, left) and SAPK/JNK (Figure 4 E, left) phosphorylation significantly. Since ERK1/2 are activated by MEK1/2 and p90RSK is an important downstream substrate of ERK1/2, we tested further whether MEK1/2 and p90RSK are also activated by MCSF. Using an antibody reacting with phosphorylated MEK1/2 and p90RSK, MEK1/2 (Figure 4 A) and p90RSK (Figure 4 C), phosphorylation was found to be enhanced significantly in macrophages of GPx-1−/−ApoE−/− mice compared with ApoE−/− control mice at 5 min (p<0,05), and then MEK1/2 declined to basal level after 15 min.

Bottom Line: Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process.GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE(-/-) mice.The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1(-/-)ApoE(-/-) mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany.

ABSTRACT
Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE(-/-) mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1) to analyze which cells express GPx-1 within atherosclerotic lesions and (2) to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE(-/-) mice after 12 weeks on a Western type diet revealed that both macrophages and - even though to a less extent - smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1(-/-)ApoE(-/-) mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. The present study demonstrates that GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK (ERK1/2) pathway encouraging further studies on new therapeutic strategies against atherosclerosis.

Show MeSH
Related in: MedlinePlus