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Mitofusin 2-deficiency suppresses cell proliferation through disturbance of autophagy.

Ding Y, Gao H, Zhao L, Wang X, Zheng M - PLoS ONE (2015)

Bottom Line: In the present study, we found that knockdown of Mfn2 by shRNA led to impaired autophagic degradation, inhibited mitochondrial oxygen consumption rate and cell glycolysis, reduced ATP production, and suppressed cell proliferation.Inhibition of autophagic degradation mimicked Mfn2-deficiency mediated cell proliferation suppression, while enhancement of autophagosome maturation restored the suppressed cell proliferation by Mfn2-deficiency.Thus, our findings revealed the role of Mfn2 in regulating cell proliferation and mitochondrial metabolism, and shed new light on understanding the mechanisms of Mfn2 deficiency related diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Health Science Center, Peking University, Beijing, China.

ABSTRACT
Mitofusin2 (Mfn2), a mitochondrial outer membrane protein serving primarily as a mitochondrial fusion protein, has multiple functions in regulating cell biological processes. Defects of Mfn2 were found in diabetes, obesity, and neurodegenerative diseases. In the present study, we found that knockdown of Mfn2 by shRNA led to impaired autophagic degradation, inhibited mitochondrial oxygen consumption rate and cell glycolysis, reduced ATP production, and suppressed cell proliferation. Inhibition of autophagic degradation mimicked Mfn2-deficiency mediated cell proliferation suppression, while enhancement of autophagosome maturation restored the suppressed cell proliferation by Mfn2-deficiency. Thus, our findings revealed the role of Mfn2 in regulating cell proliferation and mitochondrial metabolism, and shed new light on understanding the mechanisms of Mfn2 deficiency related diseases.

No MeSH data available.


Related in: MedlinePlus

Mfn2 knockdown inhibits cell proliferation.(A) Western blotting showing Mfn2 and Mfn1 protein levels in HeLa cells infected with adenovirus containing scrambled RNA or two sets of Mfn2 shRNAs. β-actin was used as protein loading control. (B) Confocal imagings of HeLa cells stained with mitoTrackor showing mitochondrial morphology. Scale bar: 10 μm. (C) Cell counting kit-8 (CCK8) assay of HeLa cells without infection or infected with adenovirus containing scrambled RNA or Mfn2 shRNA. (D) CCK8 assay of T/G HA-VSMC cells infected with adenovirus containing scrambled RNA or Mfn2 shRNA at time point as indicated after infection. (E) 3-(4,5–dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of HeLa cells infected with scrambled RNA, Mfn2 shRNA, or Mfn2 shRNA co-infected with Mfn2 cDNA. (F) Cell apoptosis assay by flow cytometry in HeLa cells infected with scrambled RNA or Mfn2 shRNA. (G) Fluorescence activated cell sorting analysis by flow cytometry to measure cell cycle distribution in HeLa cells infected with scrambled RNA, Mfn2 shRNA, or Mfn2 shRNA co-infected with Mfn2 cDNA, and (H) the histogram plot. n = 3–6 independent experiments for each group. *, p<0.05 versus scramble control.
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pone.0121328.g001: Mfn2 knockdown inhibits cell proliferation.(A) Western blotting showing Mfn2 and Mfn1 protein levels in HeLa cells infected with adenovirus containing scrambled RNA or two sets of Mfn2 shRNAs. β-actin was used as protein loading control. (B) Confocal imagings of HeLa cells stained with mitoTrackor showing mitochondrial morphology. Scale bar: 10 μm. (C) Cell counting kit-8 (CCK8) assay of HeLa cells without infection or infected with adenovirus containing scrambled RNA or Mfn2 shRNA. (D) CCK8 assay of T/G HA-VSMC cells infected with adenovirus containing scrambled RNA or Mfn2 shRNA at time point as indicated after infection. (E) 3-(4,5–dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of HeLa cells infected with scrambled RNA, Mfn2 shRNA, or Mfn2 shRNA co-infected with Mfn2 cDNA. (F) Cell apoptosis assay by flow cytometry in HeLa cells infected with scrambled RNA or Mfn2 shRNA. (G) Fluorescence activated cell sorting analysis by flow cytometry to measure cell cycle distribution in HeLa cells infected with scrambled RNA, Mfn2 shRNA, or Mfn2 shRNA co-infected with Mfn2 cDNA, and (H) the histogram plot. n = 3–6 independent experiments for each group. *, p<0.05 versus scramble control.

Mentions: To determine the effect of Mfn2 loss-of-function on cell proliferation, we infected HeLa cells with adenovirus containing short hairpin RNA of Mfn2 (Mfn2 shRNA) to knockdown Mfn2, or with adenovirus containing scramble RNA as control. Cells infected with Mfn2 shRNA for 48 hours significantly decreased Mfn2 protein level to about 36% of that in the scramble control cells, as indicated by western blot analysis (Fig. 1A), whereas the protein level of its homologue mitofusin 1(Mfn1), another outer mitochondrial membrane fusion protein, was not altered by Mfn2 shRNA. Down-regulation of Mfn2 led to mitochondrial fragmentation as compared with the thread-shaped mitochondria in scramble control cells (Fig. 1B & S1 Fig.), in consistent with previous reports that deficiency of Mfn2 causes mitochondrial fragmentation due to decreased mitochondrial fusion[27,30]. Surprisingly, in contrast to our expectation, cells infected with Mfn2 shRNA showed inhibited cell proliferation rate at as early as 1 day after Mfn2 shRNA infection, to 86.7±2.16% of cells without adenovirus infection or 85.5±2.09% of cells infected with scramble RNA, and to 74.7±7.43%, 70.8±3.09%, and 65.8±4.69% of scramble RNA control cells at day 2, day 3, and day 4 after Mfn2 shRNA infection, respectively, as detected by CCK8, a cell counting kit-8 assay (Fig. 1C). Unlike the inhibitory function of Mfn2 knockdown on cell proliferation, knockdown of Mfn1 by siRNA showed no effect on cell proliferation in spite of the fragmented mitochondria (S2 Fig.). Similar growth suppressive effect by Mfn2 knockdown was detected in a human smooth muscle cell line T/G HA-VSMC (Fig. 1D). We also employed another cell proliferative assay 3-(4,5–dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to confirm the role of Mfn2 in cell proliferation. Mfn2 knockdown resulted in decreased MTT absorbance to 78±3.17%, 73.1±5.78%, 67.6±1.2%, and 63.8±9.56% of that in scramble RNA cells at day 1, day 2, day 3, and day 4 after infection, respectively, and this blunted cell growth by Mfn2 knockdown was totally restored by co-expression of Mfn2 cDNA with Mfn2 shRNA (Fig. 1E), to a protein level comparable of that in scramble control cells (data not shown). Direct counting cell numbers showed similar suppressive results in Mfn2 shRNA transfected cells (S3 Fig.). Our data here shown that Mfn2-deficiency inhibits HeLa cell and smooth muscle proliferation.


Mitofusin 2-deficiency suppresses cell proliferation through disturbance of autophagy.

Ding Y, Gao H, Zhao L, Wang X, Zheng M - PLoS ONE (2015)

Mfn2 knockdown inhibits cell proliferation.(A) Western blotting showing Mfn2 and Mfn1 protein levels in HeLa cells infected with adenovirus containing scrambled RNA or two sets of Mfn2 shRNAs. β-actin was used as protein loading control. (B) Confocal imagings of HeLa cells stained with mitoTrackor showing mitochondrial morphology. Scale bar: 10 μm. (C) Cell counting kit-8 (CCK8) assay of HeLa cells without infection or infected with adenovirus containing scrambled RNA or Mfn2 shRNA. (D) CCK8 assay of T/G HA-VSMC cells infected with adenovirus containing scrambled RNA or Mfn2 shRNA at time point as indicated after infection. (E) 3-(4,5–dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of HeLa cells infected with scrambled RNA, Mfn2 shRNA, or Mfn2 shRNA co-infected with Mfn2 cDNA. (F) Cell apoptosis assay by flow cytometry in HeLa cells infected with scrambled RNA or Mfn2 shRNA. (G) Fluorescence activated cell sorting analysis by flow cytometry to measure cell cycle distribution in HeLa cells infected with scrambled RNA, Mfn2 shRNA, or Mfn2 shRNA co-infected with Mfn2 cDNA, and (H) the histogram plot. n = 3–6 independent experiments for each group. *, p<0.05 versus scramble control.
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pone.0121328.g001: Mfn2 knockdown inhibits cell proliferation.(A) Western blotting showing Mfn2 and Mfn1 protein levels in HeLa cells infected with adenovirus containing scrambled RNA or two sets of Mfn2 shRNAs. β-actin was used as protein loading control. (B) Confocal imagings of HeLa cells stained with mitoTrackor showing mitochondrial morphology. Scale bar: 10 μm. (C) Cell counting kit-8 (CCK8) assay of HeLa cells without infection or infected with adenovirus containing scrambled RNA or Mfn2 shRNA. (D) CCK8 assay of T/G HA-VSMC cells infected with adenovirus containing scrambled RNA or Mfn2 shRNA at time point as indicated after infection. (E) 3-(4,5–dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of HeLa cells infected with scrambled RNA, Mfn2 shRNA, or Mfn2 shRNA co-infected with Mfn2 cDNA. (F) Cell apoptosis assay by flow cytometry in HeLa cells infected with scrambled RNA or Mfn2 shRNA. (G) Fluorescence activated cell sorting analysis by flow cytometry to measure cell cycle distribution in HeLa cells infected with scrambled RNA, Mfn2 shRNA, or Mfn2 shRNA co-infected with Mfn2 cDNA, and (H) the histogram plot. n = 3–6 independent experiments for each group. *, p<0.05 versus scramble control.
Mentions: To determine the effect of Mfn2 loss-of-function on cell proliferation, we infected HeLa cells with adenovirus containing short hairpin RNA of Mfn2 (Mfn2 shRNA) to knockdown Mfn2, or with adenovirus containing scramble RNA as control. Cells infected with Mfn2 shRNA for 48 hours significantly decreased Mfn2 protein level to about 36% of that in the scramble control cells, as indicated by western blot analysis (Fig. 1A), whereas the protein level of its homologue mitofusin 1(Mfn1), another outer mitochondrial membrane fusion protein, was not altered by Mfn2 shRNA. Down-regulation of Mfn2 led to mitochondrial fragmentation as compared with the thread-shaped mitochondria in scramble control cells (Fig. 1B & S1 Fig.), in consistent with previous reports that deficiency of Mfn2 causes mitochondrial fragmentation due to decreased mitochondrial fusion[27,30]. Surprisingly, in contrast to our expectation, cells infected with Mfn2 shRNA showed inhibited cell proliferation rate at as early as 1 day after Mfn2 shRNA infection, to 86.7±2.16% of cells without adenovirus infection or 85.5±2.09% of cells infected with scramble RNA, and to 74.7±7.43%, 70.8±3.09%, and 65.8±4.69% of scramble RNA control cells at day 2, day 3, and day 4 after Mfn2 shRNA infection, respectively, as detected by CCK8, a cell counting kit-8 assay (Fig. 1C). Unlike the inhibitory function of Mfn2 knockdown on cell proliferation, knockdown of Mfn1 by siRNA showed no effect on cell proliferation in spite of the fragmented mitochondria (S2 Fig.). Similar growth suppressive effect by Mfn2 knockdown was detected in a human smooth muscle cell line T/G HA-VSMC (Fig. 1D). We also employed another cell proliferative assay 3-(4,5–dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to confirm the role of Mfn2 in cell proliferation. Mfn2 knockdown resulted in decreased MTT absorbance to 78±3.17%, 73.1±5.78%, 67.6±1.2%, and 63.8±9.56% of that in scramble RNA cells at day 1, day 2, day 3, and day 4 after infection, respectively, and this blunted cell growth by Mfn2 knockdown was totally restored by co-expression of Mfn2 cDNA with Mfn2 shRNA (Fig. 1E), to a protein level comparable of that in scramble control cells (data not shown). Direct counting cell numbers showed similar suppressive results in Mfn2 shRNA transfected cells (S3 Fig.). Our data here shown that Mfn2-deficiency inhibits HeLa cell and smooth muscle proliferation.

Bottom Line: In the present study, we found that knockdown of Mfn2 by shRNA led to impaired autophagic degradation, inhibited mitochondrial oxygen consumption rate and cell glycolysis, reduced ATP production, and suppressed cell proliferation.Inhibition of autophagic degradation mimicked Mfn2-deficiency mediated cell proliferation suppression, while enhancement of autophagosome maturation restored the suppressed cell proliferation by Mfn2-deficiency.Thus, our findings revealed the role of Mfn2 in regulating cell proliferation and mitochondrial metabolism, and shed new light on understanding the mechanisms of Mfn2 deficiency related diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Health Science Center, Peking University, Beijing, China.

ABSTRACT
Mitofusin2 (Mfn2), a mitochondrial outer membrane protein serving primarily as a mitochondrial fusion protein, has multiple functions in regulating cell biological processes. Defects of Mfn2 were found in diabetes, obesity, and neurodegenerative diseases. In the present study, we found that knockdown of Mfn2 by shRNA led to impaired autophagic degradation, inhibited mitochondrial oxygen consumption rate and cell glycolysis, reduced ATP production, and suppressed cell proliferation. Inhibition of autophagic degradation mimicked Mfn2-deficiency mediated cell proliferation suppression, while enhancement of autophagosome maturation restored the suppressed cell proliferation by Mfn2-deficiency. Thus, our findings revealed the role of Mfn2 in regulating cell proliferation and mitochondrial metabolism, and shed new light on understanding the mechanisms of Mfn2 deficiency related diseases.

No MeSH data available.


Related in: MedlinePlus