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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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Mimitin gene expression in HepG2 cells stimulated with IL-1, IL-6 or with both cytokines. Cells were stimulated with the cytokines and the mimitin transcript level was evaluated by Northern blotting (A). The data are presented relatively to the mimitin transcript level in unstimulated control cells harvested after 4, 12, and 24 h. A lower panel shows representative results from Northern blot hybridization and ethidium bromide-stained 28S rRNA. Changes in the mimitin protein level estimated by ELISA were calculated relatively to unstimulated control cells (B). Vertical bars indicate the values of standard error of three independent experiments (*p < 0.05, **p < 0.02, ***p < 0.01).
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Figure 1: Mimitin gene expression in HepG2 cells stimulated with IL-1, IL-6 or with both cytokines. Cells were stimulated with the cytokines and the mimitin transcript level was evaluated by Northern blotting (A). The data are presented relatively to the mimitin transcript level in unstimulated control cells harvested after 4, 12, and 24 h. A lower panel shows representative results from Northern blot hybridization and ethidium bromide-stained 28S rRNA. Changes in the mimitin protein level estimated by ELISA were calculated relatively to unstimulated control cells (B). Vertical bars indicate the values of standard error of three independent experiments (*p < 0.05, **p < 0.02, ***p < 0.01).

Mentions: The mimitin transcript was initially detected by us [3] by differential display analysis in HepG2 cells stimulated with IL-1. To study the importance of proinflammatory cytokines in mimitin gene expression HepG2 cells were stimulated with IL-1, IL-6 or a mixture of both cytokines. Changes in gene expression were evaluated at the transcript and protein levels (Fig. 1A and 1B). In Northern blot analysis the densitometric values of bands corresponding to mimitin transcript were measured for control (unstimulated) cells and cells stimulated with IL-1 or IL-6. In case of cytokine mixture, cells were prestimulated with IL-1 and then stimulated with IL-6 to simulate the physiological cascade of events. In all cases exposure to cytokines led to an increase in mimitin mRNA abundance. The highest transcript level observed after 12 h of stimulation with IL-1 or IL-6 exceeded control levels by 1.9 and 1.5 times, respectively (Fig. 1A). In the case of the two cytokines combined we observed 1.6-fold up-regulation of mimitin mRNA after 27/24 h of cytokine exposure (Fig. 1A).


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)

Mimitin gene expression in HepG2 cells stimulated with IL-1, IL-6 or with both cytokines. Cells were stimulated with the cytokines and the mimitin transcript level was evaluated by Northern blotting (A). The data are presented relatively to the mimitin transcript level in unstimulated control cells harvested after 4, 12, and 24 h. A lower panel shows representative results from Northern blot hybridization and ethidium bromide-stained 28S rRNA. Changes in the mimitin protein level estimated by ELISA were calculated relatively to unstimulated control cells (B). Vertical bars indicate the values of standard error of three independent experiments (*p < 0.05, **p < 0.02, ***p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667391&req=5

Figure 1: Mimitin gene expression in HepG2 cells stimulated with IL-1, IL-6 or with both cytokines. Cells were stimulated with the cytokines and the mimitin transcript level was evaluated by Northern blotting (A). The data are presented relatively to the mimitin transcript level in unstimulated control cells harvested after 4, 12, and 24 h. A lower panel shows representative results from Northern blot hybridization and ethidium bromide-stained 28S rRNA. Changes in the mimitin protein level estimated by ELISA were calculated relatively to unstimulated control cells (B). Vertical bars indicate the values of standard error of three independent experiments (*p < 0.05, **p < 0.02, ***p < 0.01).
Mentions: The mimitin transcript was initially detected by us [3] by differential display analysis in HepG2 cells stimulated with IL-1. To study the importance of proinflammatory cytokines in mimitin gene expression HepG2 cells were stimulated with IL-1, IL-6 or a mixture of both cytokines. Changes in gene expression were evaluated at the transcript and protein levels (Fig. 1A and 1B). In Northern blot analysis the densitometric values of bands corresponding to mimitin transcript were measured for control (unstimulated) cells and cells stimulated with IL-1 or IL-6. In case of cytokine mixture, cells were prestimulated with IL-1 and then stimulated with IL-6 to simulate the physiological cascade of events. In all cases exposure to cytokines led to an increase in mimitin mRNA abundance. The highest transcript level observed after 12 h of stimulation with IL-1 or IL-6 exceeded control levels by 1.9 and 1.5 times, respectively (Fig. 1A). In the case of the two cytokines combined we observed 1.6-fold up-regulation of mimitin mRNA after 27/24 h of cytokine exposure (Fig. 1A).

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