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Mimitin - a novel cytokine-regulated mitochondrial protein.

Wegrzyn P, Yarwood SJ, Fiegler N, Bzowska M, Koj A, Mizgalska D, Malicki S, Pajak M, Kasza A, Kachamakova-Trojanowska N, Bereta J, Jura J, Jura J - BMC Cell Biol. (2009)

Bottom Line: This was accompanied by a slight decrease in proliferation of HepG2 cells.Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin.We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. p.wegrzyn@uj.edu.pl

ABSTRACT

Background: The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin - a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners.

Results: By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18-24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway.

Conclusion: Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of apoptotic caspases 3/7, suggesting that this protein may indirectly participate in apoptosis.

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Signalling pathway engaged in IL-1-dependent activation of mimitin gene. A. Representative Northern blots: HepG2 cells stably transfected with retroviral vector pCFG5-IEG2 encoding nondegradable mutant of IκBα, and cells with an empty vector (mock-control) were stimulated with IL-1 (12 h) and the level of mimitin transcript was measured. Results obtained for both types of cells were normalized to 18S rRNA level. B. The graph represents data calculated from three independent Northern blot experiments. Error bars indicate the values of standard deviation (**p < 0.02). C. HepG2 cells were pretreated with: 10 μM U0126, 10 μM SP600125, or 10 μM ZM336372 for 30 min and then stimulated with 15 ng/ml IL-1 for 12 h. The level of mRNA coding for mimitin was estimated in comparison to control (unstimulated cells) by real-time PCR. Error bars indicate standard deviation from three independent experiments (*p < 0.05, **p < 0.02). D. Western blot analysis confirming activity of inhibitors of MAPK kinases. HepG2 cells were pretreated for 30 min with inhibitors (as in Fig. 2C), stimulated with 15 ng/ml IL-1 for 10 min and then cell lysates were analyzed by Western blotting with anti-P-Erk, anti-P-JNK and anti-P-p38 antibodies. The blot was later stripped and reprobed with an α-tubulin antibody (bottom panel) to ensure equal loading.
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Figure 2: Signalling pathway engaged in IL-1-dependent activation of mimitin gene. A. Representative Northern blots: HepG2 cells stably transfected with retroviral vector pCFG5-IEG2 encoding nondegradable mutant of IκBα, and cells with an empty vector (mock-control) were stimulated with IL-1 (12 h) and the level of mimitin transcript was measured. Results obtained for both types of cells were normalized to 18S rRNA level. B. The graph represents data calculated from three independent Northern blot experiments. Error bars indicate the values of standard deviation (**p < 0.02). C. HepG2 cells were pretreated with: 10 μM U0126, 10 μM SP600125, or 10 μM ZM336372 for 30 min and then stimulated with 15 ng/ml IL-1 for 12 h. The level of mRNA coding for mimitin was estimated in comparison to control (unstimulated cells) by real-time PCR. Error bars indicate standard deviation from three independent experiments (*p < 0.05, **p < 0.02). D. Western blot analysis confirming activity of inhibitors of MAPK kinases. HepG2 cells were pretreated for 30 min with inhibitors (as in Fig. 2C), stimulated with 15 ng/ml IL-1 for 10 min and then cell lysates were analyzed by Western blotting with anti-P-Erk, anti-P-JNK and anti-P-p38 antibodies. The blot was later stripped and reprobed with an α-tubulin antibody (bottom panel) to ensure equal loading.

Mentions: To investigate the role of NFκB signalling pathway in activation of mimitin gene by IL-1 we used a cell line stably transfected with retroviral vector pCFG5-IEG2 encoding a non-degradable mutant of the inhibitor of NFκB (mIκBα). Cells stably transfected with the empty vector served as a control. The control and mIκBα-expressing cells were stimulated with IL-1 for 12 h. To monitor the level of mimitin transcript Northern blot analysis was carried out. As shown in Fig. 2A and 2B, the level of mimitin transcript in HepG2 cells expressing mIκBα and stimulated with IL-1 is the same as it is in case of IL-1 stimulated control. Thus, the NFκB signalling pathway appears not to be involved in the expression of IL-1-dependent activation of mimitin gene. It is known, however, that another signalling pathway important in the expression of IL-1-regulated genes involves MAP kinases. To check if this pathway is engaged in mimitin regulation three potent, selective, pharmacological inhibitors of MAPKs cascade were used: U0126 for ERK, SP600125 for JNK, ZM336372 for p38 The specificity of each inhibitor was verified by Western blotting with antisera against phosphorylated forms of individual kinases (Fig. 2D), and a lack of cytotoxic effects was confirmed by the MTT assay (data not shown). As shown in Fig. 2C all these inhibitors abolished the IL-1-induced elevation of mimitin mRNA level, confirming the involvement of MAP kinases in the stimulation of mimitin gene expression by IL-1.


Mimitin - a novel cytokine-regulated mitochondrial protein.

Wegrzyn P, Yarwood SJ, Fiegler N, Bzowska M, Koj A, Mizgalska D, Malicki S, Pajak M, Kasza A, Kachamakova-Trojanowska N, Bereta J, Jura J, Jura J - BMC Cell Biol. (2009)

Signalling pathway engaged in IL-1-dependent activation of mimitin gene. A. Representative Northern blots: HepG2 cells stably transfected with retroviral vector pCFG5-IEG2 encoding nondegradable mutant of IκBα, and cells with an empty vector (mock-control) were stimulated with IL-1 (12 h) and the level of mimitin transcript was measured. Results obtained for both types of cells were normalized to 18S rRNA level. B. The graph represents data calculated from three independent Northern blot experiments. Error bars indicate the values of standard deviation (**p < 0.02). C. HepG2 cells were pretreated with: 10 μM U0126, 10 μM SP600125, or 10 μM ZM336372 for 30 min and then stimulated with 15 ng/ml IL-1 for 12 h. The level of mRNA coding for mimitin was estimated in comparison to control (unstimulated cells) by real-time PCR. Error bars indicate standard deviation from three independent experiments (*p < 0.05, **p < 0.02). D. Western blot analysis confirming activity of inhibitors of MAPK kinases. HepG2 cells were pretreated for 30 min with inhibitors (as in Fig. 2C), stimulated with 15 ng/ml IL-1 for 10 min and then cell lysates were analyzed by Western blotting with anti-P-Erk, anti-P-JNK and anti-P-p38 antibodies. The blot was later stripped and reprobed with an α-tubulin antibody (bottom panel) to ensure equal loading.
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Related In: Results  -  Collection

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Figure 2: Signalling pathway engaged in IL-1-dependent activation of mimitin gene. A. Representative Northern blots: HepG2 cells stably transfected with retroviral vector pCFG5-IEG2 encoding nondegradable mutant of IκBα, and cells with an empty vector (mock-control) were stimulated with IL-1 (12 h) and the level of mimitin transcript was measured. Results obtained for both types of cells were normalized to 18S rRNA level. B. The graph represents data calculated from three independent Northern blot experiments. Error bars indicate the values of standard deviation (**p < 0.02). C. HepG2 cells were pretreated with: 10 μM U0126, 10 μM SP600125, or 10 μM ZM336372 for 30 min and then stimulated with 15 ng/ml IL-1 for 12 h. The level of mRNA coding for mimitin was estimated in comparison to control (unstimulated cells) by real-time PCR. Error bars indicate standard deviation from three independent experiments (*p < 0.05, **p < 0.02). D. Western blot analysis confirming activity of inhibitors of MAPK kinases. HepG2 cells were pretreated for 30 min with inhibitors (as in Fig. 2C), stimulated with 15 ng/ml IL-1 for 10 min and then cell lysates were analyzed by Western blotting with anti-P-Erk, anti-P-JNK and anti-P-p38 antibodies. The blot was later stripped and reprobed with an α-tubulin antibody (bottom panel) to ensure equal loading.
Mentions: To investigate the role of NFκB signalling pathway in activation of mimitin gene by IL-1 we used a cell line stably transfected with retroviral vector pCFG5-IEG2 encoding a non-degradable mutant of the inhibitor of NFκB (mIκBα). Cells stably transfected with the empty vector served as a control. The control and mIκBα-expressing cells were stimulated with IL-1 for 12 h. To monitor the level of mimitin transcript Northern blot analysis was carried out. As shown in Fig. 2A and 2B, the level of mimitin transcript in HepG2 cells expressing mIκBα and stimulated with IL-1 is the same as it is in case of IL-1 stimulated control. Thus, the NFκB signalling pathway appears not to be involved in the expression of IL-1-dependent activation of mimitin gene. It is known, however, that another signalling pathway important in the expression of IL-1-regulated genes involves MAP kinases. To check if this pathway is engaged in mimitin regulation three potent, selective, pharmacological inhibitors of MAPKs cascade were used: U0126 for ERK, SP600125 for JNK, ZM336372 for p38 The specificity of each inhibitor was verified by Western blotting with antisera against phosphorylated forms of individual kinases (Fig. 2D), and a lack of cytotoxic effects was confirmed by the MTT assay (data not shown). As shown in Fig. 2C all these inhibitors abolished the IL-1-induced elevation of mimitin mRNA level, confirming the involvement of MAP kinases in the stimulation of mimitin gene expression by IL-1.

Bottom Line: This was accompanied by a slight decrease in proliferation of HepG2 cells.Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin.We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. p.wegrzyn@uj.edu.pl

ABSTRACT

Background: The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin - a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners.

Results: By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18-24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway.

Conclusion: Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of apoptotic caspases 3/7, suggesting that this protein may indirectly participate in apoptosis.

Show MeSH
Related in: MedlinePlus