J Korean Med Sci.  2012 May;27(5):465-470. 10.3346/jkms.2012.27.5.465.

Depletion of Mitochondrial DNA Stabilizes C1qTNF-Related Protein 6 mRNA in Muscle Cells

• Kim MJ
• Lee W
• Park EJ
• Park SY

• KMID: 1372785
• DOI: http://doi.org/10.3346/jkms.2012.27.5.465

Abstract

Mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of several metabolic diseases. Recently, we demonstrated that C1qTNF-related protein-6 (CTRP6) is involved in fatty acid metabolism in muscle cells. In this study, we showed that expression of CTRP6 was up-regulated in mtDNA-depleted C2C12 cells, which displayed a marked decrease in cellular mtDNA and ATP content. Replacement of mtDNA normalized the expression level of CTRP6 similar to that in normal C2C12 cells, indicating that CTRP6 expression was up-regulated by mtDNA depletion. However, CTRP6 promoter activity remained unchanged in mtDNA-depleted cells. We also found that mtDNA depletion inhibited decay of CTRP6 mRNA. Taken together, mtDNA depletion induces an increase in CTRP6 expression by increasing mRNA stability.

Keyword

DNA, Mitochondrial; CTRP6; C1qTNF-6; mRNA Stability; C2C12 Cells

MeSH Terms

Adiponectin/*genetics/metabolism
Animals
Cell Line
DNA, Mitochondrial/*metabolism
Mice
Promoter Regions, Genetic
RNA Stability
RNA, Messenger/*metabolism
Up-Regulation

Figure

• 

  Fig. 1 Establishment and characterization of mtDNA-depleted C2C12 cells. (A) Genomic DNA content. Genomic DNA was isolated from control, mtDNA-depleted, and reverted C2C12 cells, and mtDNA-encoded genes, including cytochrome c oxidase subunit I (COX-I) and cytochrome c oxidase subunit III (COX-III), were amplified by PCR. COX-IV was used as a nuclear DNA-encoded control. A representative result of at least three independent experiments is shown. (B) Contents of mRNAs transcribed from mtDNA and nuclear DNA. Total RNA was extracted from control, mtDNA-depleted, and reverted C2C12 cells, and levels of COX-I, COX-III, and COX-IV transcripts were analyzed by quantitative real-time PCR. The mRNA levels were expressed as a percentage of that observed in control C2C12 cells, which were set as 100%. Results are expressed as the means ± SD of three independent experiments. t-test: *P < 0.01 vs control C2C12 cells. (C) Cellular ATP content. Total cellular ATP levels were analyzed using a somatic cell ATP assay kit (Sigma). ATP content was expressed as a percentage of that observed in control C2C12 cells. Results are expressed as the means ± SD of three independent experiments. t-test: *P < 0.01 vs control C2C12 cells.

• 

  Fig. 2 Expression of CTRP6 mRNA and protein in control, mtDNA-depleted, and reverted C2C12 cells. (A) Total RNA was isolated from control, mtDNA-depleted, and reverted C2C12 cells, and the level of CTRP6 mRNA was analyzed by quantitative real-time PCR. The relative values are expressed as a fold increase of that observed in control C2C12 cells. Results are expressed as the means ± SD of three independent experiments. t-test: *P < 0.01 vs control C2C12 cells. (B) Cell lysates were prepared from control, mtDNA-depleted, and reverted C2C12 cells. Equal amounts (20 µg proteins) of cell lysates were separated via 10% SDS-PAGE and subjected to immunoblotting with anti-CTRP6 or anti-actin antibody. A representative result of three independent experiments is shown.

• 

  Fig. 3 Analysis of the mouse CTRP6 promoter activity in control, mtDNA-depleted, and reverted C2C12 cells. Expression of a luciferase reporter gene driven by the mouse CTRP6 promoter was measured after transfection of control, mtDNA-depleted, and reverted C2C12 cells with pCTRP6-luc and pSV40-luc. Luciferase activity was normalized to Renilla luciferase activity and shown as a fold increase of that observed in control C2C12 cells, which was set as 1. Results are expressed as the means ± SD of three independent experiments.

• 

  Fig. 4 Stability of CTRP6 mRNA in control, mtDNA-depleted, and reverted C2C12 cells. (A) Control, mtDNA-depleted, and reverted C2C12 cells were treated with 12.5 µg/mL actinomycin D for the indicated times, followed by extraction and analysis of CTRP6 mRNA via RT-PCR. β-actin was used as an internal control. A representative result of three independent experiments is shown. (B) The levels of CTRP6 mRNA at the indicated time were analyzed by quantitative real-time PCR. The relative levels of CTRP6 mRNA are expressed as a percentage of that observed at time 0, which was set as 100%. Results are expressed as the means ± SD of three independent experiments. t-test: *P < 0.01 vs control C2C12 cells.

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