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. 2009 Mar 31:10:23.
doi: 10.1186/1471-2121-10-23.

Mimitin - a novel cytokine-regulated mitochondrial protein

Paulina Wegrzyn  1 , Stephen J Yarwood, Nathalie Fiegler, Monika Bzowska, Aleksander Koj, Danuta Mizgalska, Stanisław Malicki, Magdalena Pajak, Aneta Kasza, Neli Kachamakova-Trojanowska, Joanna Bereta, Jacek Jura, Jolanta Jura
Affiliations

Mimitin - a novel cytokine-regulated mitochondrial protein

Paulina Wegrzyn et al. BMC Cell Biol. .

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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Figures

Figure 1
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).
Figure 2
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.
Figure 3
Figure 3
Abundance of mimitin transcripts in human tissues. 32P-labelled molecular probe for mimitin or beta actin transcript (control) was used for hyridization with commercial membrane loaded with 2 μg of mRNA isolated from 12 different human tissues: 1. brain, 2. heart, 3. skeletal muscles, 4. colon, 5. thymus, 6. spleen, 7. kidney, 8. liver, 9. small intestine, 10. placenta, 11. lung, 12. peripheral blood leukocytes. For mimitin gene, a single trasncript of 640 nt is detected; for beta actin a single 2.0 kb band is present in all lanes. A 1.8 kb actin isoform is visible in the heart and skeletal muscle lanes.
Figure 4
Figure 4
Subcellular localization of mimitin. Subcellular fractionation of HepG2 cells was done with Qproteome Kit (A) and the obtained cytosolic (lane 1), membrane (lane 2), nuclear (lane 3) and cytoskeletal proteins (lane 4) were checked for the presence of appropriate markers with monospecific antibodies. Western blotting showed the presence of mimitin exclusively in the membrane fraction. In additional experiments cytosolic (lane 5), mitochondrial (lane 6), microsomal (lane 7) and crude nuclear fraction containing unlysed cells (lane 8), were isolated (see Materials and Methods). This result confirmed the presence of mimitin solely in mitochondria.
Figure 5
Figure 5
Coimmunoprecipitation of mimitin with MAP1S. In HepG2 cells MAP1S with c-Myc tag and mimitin genes were overexpressed. The coimmunoprecipitation analysis was performed with ProFoundTM c-Myc Tag IP/Co-IP Kit. Proteins present in cells extracts and eluates were detected in Western blot with antibodies specific for Mimitin and specific for Myc tag in case of MAP1S.
Figure 6
Figure 6
Effect of mimitin gene silencing on caspase 3/7 activities. HepG2 cells were transfected with mimitin-siRNA, GAPDH-siRNA, negative control siRNA, or transfection reagent (siPORT NeoFX) (blank). Apoptosis was induced 72 h post transfection by treatment of cells with TNF/CHX for 5 h. Caspase 3/7 activity was measured using Caspase-GloTM3/7 assay kit. A. Changes in caspase activity in cells with TNF/CHX-induced apoptosis were compared to cells treated with TNF/CHX and transfected with a negative control siRNA (assumed as equal 1). In each case the value of caspase 3/7 activity after TNF/CHX treatment was divided by the value of basal activity of non-stimulated cells. Values represent the mean of three independent experiments with error bars showing standard error (*p < 0.05). B. Representative Western blot analysis with 20 μg protein from caspase activity assay using anti-GAPDH and anti-Mimitin antibodies. Samples marked (+) were stimulated with TNF/CHX while samples marked (-) were not stimulated.
Figure 7
Figure 7
Overexpression of mimitin decreases caspase 3/7 activities. HepG2 cells were transfected either with pcDNA3 empty vector, or pcDNA3mim and then not-treated (control) or treated with TNF/CHX. Caspase activity was determined using Caspase-GloTM3/7 reagent. A. Graph presents changes in the relative changes in caspase 3/7 activity. In each case the value of caspase 3/7 activity after TNF/CHX treatment was divided by the value of basal activity of nonstimulated cells. Caspase activity in cells overexpressing mimitin was compared to cells transfected with an empty vector pcDNA3 (assumed as equal 1). The values represent the mean of 3 independent experiments ± SD with *p < 0.05. B. Western blot confirming overexpression of mimitin in cells transfected with pcDNA3mim. The blot was later stripped and reprobed with an α-tubulin antibody (lower panel) to ensure equal loading. RT-PCR results show transcript level for neomycin resistance gene (exogenous gene in pcDNA3) as a control of transfection efficiency and transcript level for elongation factor 2 (endogenous gene) as a control of RNA amount in tested samples.
Figure 8
Figure 8
Effect of mimitin gene silencing on proliferation of HepG2 cells. Cells were transfected with silencing RNA specific for mimitin (50 nM), or for GAPDH (100 nM, positive control), or unspecific siRNA (negative control, 50 nM) or were treated with transfection reagent siPORT NeoFX alone (blank). At 72 h post transfection cells were incubated with BrdU (5 μM) for 3 h and incorporation was determined with Cell Proliferation ELISA. A. Cell proliferation is shown relative to the non-sepcific siRNA (control). Values were calculated from absorbance readings at 450 nm and represent the mean of three independent experiments with standard deviation (*p < 0.05, **p < 0.02 compared to control). B. Representative Western blot analysis with 20 μg protein from simultaneously seeded 12-well plates using anti-GAPDH or anti-mimitin antibodies.
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