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Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages.

Navasa N, Martin-Ruiz I, Atondo E, Sutherland JD, Angel Pascual-Itoiz M, Carreras-González A, Izadi H, Tomás-Cortázar J, Ayaz F, Martin-Martin N, Torres IM, Barrio R, Carracedo A, Olivera ER, Rincón M, Anguita J - Sci Rep (2015)

Bottom Line: IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages.The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression.These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Animal Sciences. University of Massachusetts Amherst. Amherst, MA 01003.

ABSTRACT
MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

No MeSH data available.


Related in: MedlinePlus

IFNγ induces the repression of MCJ in macrophages.(A) RAW cells were stimulated with live B. burgdorferi (m.o.i = 25) or 100 ng/mL of LPS for 16 h and analyzed by immunoblotting for MCJ protein levels. Actin levels were determined to ensure equal loads. (B) BMMs were stimulated with live B. burgdorferi for 16 h and analyzed for MCJ mRNA levels by qRT-PCR. The data shown correspond to the mean ± SE of 3 points per group. (C) RAW cells (RAW), BMMs, Hepa 2–7 cells (Hepa) or CD8+ T cells (CD8) were stimulated with 100 ng/mL of IFNγ for 24–48 h, followed by the analysis of MCJ protein levels by immunoblotting. GAPDH levels were determined to ensure equal protein loads. (D) RAW cells were stimulated for 72 h with 100 ng/mL of IFNγ in 8-well chamber slides, washed and stained for intracellular MCJ. The slides were analyzed by ApoTome fluorescence microscopy. (E) RAW cells stimulated with IFNγ were analyzed for the levels of the mitochondrial protein, VDAC1, by immunoblotting. (F) RAW, BMMs and Hepa cells were stimulated with 100 ng/mL of IL-6 for 24 h, followed by their analysis for MCJ protein content by immunoblotting.
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f2: IFNγ induces the repression of MCJ in macrophages.(A) RAW cells were stimulated with live B. burgdorferi (m.o.i = 25) or 100 ng/mL of LPS for 16 h and analyzed by immunoblotting for MCJ protein levels. Actin levels were determined to ensure equal loads. (B) BMMs were stimulated with live B. burgdorferi for 16 h and analyzed for MCJ mRNA levels by qRT-PCR. The data shown correspond to the mean ± SE of 3 points per group. (C) RAW cells (RAW), BMMs, Hepa 2–7 cells (Hepa) or CD8+ T cells (CD8) were stimulated with 100 ng/mL of IFNγ for 24–48 h, followed by the analysis of MCJ protein levels by immunoblotting. GAPDH levels were determined to ensure equal protein loads. (D) RAW cells were stimulated for 72 h with 100 ng/mL of IFNγ in 8-well chamber slides, washed and stained for intracellular MCJ. The slides were analyzed by ApoTome fluorescence microscopy. (E) RAW cells stimulated with IFNγ were analyzed for the levels of the mitochondrial protein, VDAC1, by immunoblotting. (F) RAW, BMMs and Hepa cells were stimulated with 100 ng/mL of IL-6 for 24 h, followed by their analysis for MCJ protein content by immunoblotting.

Mentions: In order to determine whether the interaction of macrophages with bacterial products results in reduced levels of MCJ, we stimulated RAW cells and BMMs with live B. burgdorferi and assessed the levels of MCJ. Stimulation with the spirochete did not affect MCJ protein (Fig. 2A) or mRNA (Fig. 2B) levels. LPS stimulation also failed to alter the levels of MCJ in macrophages (Fig. 2A). These data indicate that the regulation of the expression of MCJ occurs independently of pattern-recognition receptor (PRR) stimulation, including TLR4, TLR1/2 and other PRRs stimulated by the interaction of live B. burgdorferi with macrophages1112131415. Since IFNγ is a major contributor to macrophage function during cardiac infection with B. burgdorferi1617, we stimulated macrophages with IFNγ. Treatment with IFNγ resulted in lower levels of MCJ protein in both RAW cells and BMMs (Fig. 2C,D). Because MCJ is localized in mitochondria, we examined the effect of IFNγ on mitochondrial mass; however, no difference was observed as determined by levels of the mitochondrial protein, VDAC1 (Fig. 2E). The effect of IFNγ was selective of macrophages, because it did not affect MCJ levels in the murine tumor cell line, Hepa 1–6 or primary CD8+ T cells (Fig. 2C). IL-6 has been shown to downregulate MCJ levels in breast cancer cell lines2. Similarly, we found that IL-6 induced the downregulation of MCJ in Hepa liver cancer cells (Fig. 2F). However, IL-6 failed to downregulate MCJ expression in RAW cells or BMMs (Fig. 2F). These results show that MCJ expression in macrophages is selectively silenced by IFNγ.


Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages.

Navasa N, Martin-Ruiz I, Atondo E, Sutherland JD, Angel Pascual-Itoiz M, Carreras-González A, Izadi H, Tomás-Cortázar J, Ayaz F, Martin-Martin N, Torres IM, Barrio R, Carracedo A, Olivera ER, Rincón M, Anguita J - Sci Rep (2015)

IFNγ induces the repression of MCJ in macrophages.(A) RAW cells were stimulated with live B. burgdorferi (m.o.i = 25) or 100 ng/mL of LPS for 16 h and analyzed by immunoblotting for MCJ protein levels. Actin levels were determined to ensure equal loads. (B) BMMs were stimulated with live B. burgdorferi for 16 h and analyzed for MCJ mRNA levels by qRT-PCR. The data shown correspond to the mean ± SE of 3 points per group. (C) RAW cells (RAW), BMMs, Hepa 2–7 cells (Hepa) or CD8+ T cells (CD8) were stimulated with 100 ng/mL of IFNγ for 24–48 h, followed by the analysis of MCJ protein levels by immunoblotting. GAPDH levels were determined to ensure equal protein loads. (D) RAW cells were stimulated for 72 h with 100 ng/mL of IFNγ in 8-well chamber slides, washed and stained for intracellular MCJ. The slides were analyzed by ApoTome fluorescence microscopy. (E) RAW cells stimulated with IFNγ were analyzed for the levels of the mitochondrial protein, VDAC1, by immunoblotting. (F) RAW, BMMs and Hepa cells were stimulated with 100 ng/mL of IL-6 for 24 h, followed by their analysis for MCJ protein content by immunoblotting.
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f2: IFNγ induces the repression of MCJ in macrophages.(A) RAW cells were stimulated with live B. burgdorferi (m.o.i = 25) or 100 ng/mL of LPS for 16 h and analyzed by immunoblotting for MCJ protein levels. Actin levels were determined to ensure equal loads. (B) BMMs were stimulated with live B. burgdorferi for 16 h and analyzed for MCJ mRNA levels by qRT-PCR. The data shown correspond to the mean ± SE of 3 points per group. (C) RAW cells (RAW), BMMs, Hepa 2–7 cells (Hepa) or CD8+ T cells (CD8) were stimulated with 100 ng/mL of IFNγ for 24–48 h, followed by the analysis of MCJ protein levels by immunoblotting. GAPDH levels were determined to ensure equal protein loads. (D) RAW cells were stimulated for 72 h with 100 ng/mL of IFNγ in 8-well chamber slides, washed and stained for intracellular MCJ. The slides were analyzed by ApoTome fluorescence microscopy. (E) RAW cells stimulated with IFNγ were analyzed for the levels of the mitochondrial protein, VDAC1, by immunoblotting. (F) RAW, BMMs and Hepa cells were stimulated with 100 ng/mL of IL-6 for 24 h, followed by their analysis for MCJ protein content by immunoblotting.
Mentions: In order to determine whether the interaction of macrophages with bacterial products results in reduced levels of MCJ, we stimulated RAW cells and BMMs with live B. burgdorferi and assessed the levels of MCJ. Stimulation with the spirochete did not affect MCJ protein (Fig. 2A) or mRNA (Fig. 2B) levels. LPS stimulation also failed to alter the levels of MCJ in macrophages (Fig. 2A). These data indicate that the regulation of the expression of MCJ occurs independently of pattern-recognition receptor (PRR) stimulation, including TLR4, TLR1/2 and other PRRs stimulated by the interaction of live B. burgdorferi with macrophages1112131415. Since IFNγ is a major contributor to macrophage function during cardiac infection with B. burgdorferi1617, we stimulated macrophages with IFNγ. Treatment with IFNγ resulted in lower levels of MCJ protein in both RAW cells and BMMs (Fig. 2C,D). Because MCJ is localized in mitochondria, we examined the effect of IFNγ on mitochondrial mass; however, no difference was observed as determined by levels of the mitochondrial protein, VDAC1 (Fig. 2E). The effect of IFNγ was selective of macrophages, because it did not affect MCJ levels in the murine tumor cell line, Hepa 1–6 or primary CD8+ T cells (Fig. 2C). IL-6 has been shown to downregulate MCJ levels in breast cancer cell lines2. Similarly, we found that IL-6 induced the downregulation of MCJ in Hepa liver cancer cells (Fig. 2F). However, IL-6 failed to downregulate MCJ expression in RAW cells or BMMs (Fig. 2F). These results show that MCJ expression in macrophages is selectively silenced by IFNγ.

Bottom Line: IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages.The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression.These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Animal Sciences. University of Massachusetts Amherst. Amherst, MA 01003.

ABSTRACT
MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

No MeSH data available.


Related in: MedlinePlus