PGC-1alpha/beta upregulation is associated with improved oxidative phosphorylation in cells harboring nonsense mtDNA mutations

Abstract

We have studied the functional effects of nonsense mitochondrial DNA (mtDNA) mutations in the COXI and ND5 genes in a colorectal tumor cell line. Surprisingly, these cells had an efficient oxidative phosphorylation (OXPHOS); however, when mitochondria from these cells were transferred to an osteosarcoma nuclear background (osteosarcoma cybrids), the rate of respiration markedly declined suggesting that the phenotypic expression of the mtDNA mutations was prevented by the colorectal tumor nuclear background. We found that there was a significant increase in the steady-state levels of PGC-1alpha and PGC-1beta transcriptional coactivators in these cells and a parallel increase in the steady-state levels of several mitochondrial proteins. Accordingly, adenoviral-mediated overexpression of PGC-1alpha and PGC-1beta in the osteosarcoma cybrids stimulated mitochondrial respiration suggesting that an upregulation of PGC-1alpha/beta coactivators can partially rescue an OXPHOS defect. In conclusion, upregulation of PGC-1alpha and PGC-1beta in the colorectal tumor cells can be part of an adaptation mechanism to help overcome the severe consequences of mtDNA mutations on OXPHOS.

Figure 1. mtDNA mutation load in colorectal and osteosarcoma cell lines

To assess the detection limits of the ‘last cycle hot’ PCR assay, plasmids containing mtDNA fragments harboring either the wild-type or mutated sequence were constructed for both the COXI and ND5 regions. Mutated and wild-type plasmids were mixed at known ratios and 10 ng of the sample was subjected to the ‘last cycle hot PCR’ followed by digestion with the appropriate restriction endonuclease (see Materials and Methods). (A) Analyses for the COXI G6264A mutation in COXI plasmid mixtures, the colorectal V425 and the osteosarcoma cybrid (RVA1). (B) A similar analysis for the 12418insA mutation in ND5 plasmid mixtures and the same cell lines. (C) Standard curve depicting the correlation between percentage input wild-type COXI- and ND5-containing plasmids and the percentage wild-type signal detected in the ‘last cycle hot PCR’/RFLP results. (D) Amplification (left) and ‘last cycle hot PCR’ analysis followed by restriction endonuclease digestion (right) of COXI mRNA amplified by RT-PCR.

Figure 2. V425 colorectal tumor cell line suppressess a defective respiratory phenotype associated with nonsense mtDNA mutations

(A) The activities of enzyme complexes I + III, II + III and IV in colorectal and osteosarcoma cell lines. The change in all enzyme complex activities was found to be significant (P < 0.05). (B) The O₂ consumption measurements in colorectal and osteosarcoma cell lines. (C) After permeabilization with digitonin and addition of ADP, the oxidation of glutamate plus malate (site I substrates), succinate (site II substrate) and ascorbate plus NNN′N′-tetramethyl-p-phenylenediamine (TMPD) (site IV substrates) were determined. Specific inhibitors for complex I (rotenone), complex III (antimycin A) and complex IV (KCN) were added at the indicated times. (C) shows that the ascorbate/TMPD-driven respiration in V425 was more efficient than expected considering the potential severity of a homoplasmic stop codon mutation in COXI. (D) When the V425 mtDNA was transferred to an osteosarcoma nuclear background (RVA1 cybrid), the rate of respiration markedly declined. The osteosarcoma control (W20) is shown for comparison. Error bars are SD of the mean for n ≥ 3 determinations. Comparisons with the control cell line were performed by the Student’s t-test. Experiments shown in (C) and (D) were performed a single time.

Figure 3. Mitochondrial protein expression in colorectal and osteosarcoma cell lines

(A) Mitochondrial protein synthesis was investigated in different cell lines as described in methods. Band assignment was performed according to Chomyn (60). An abnormal band of ~13 kDa (*) was observed in V425, RVA1 and RVC1 suggesting that it is the truncated product associated with the COXI stop codon mutation. (B) Steady-state levels of complex I subunits (ND1 and ND39), complex II SDH(Fp) subunit, core I subunit of complex III, cytochrome c, complex IV subunits (COX I, COX II and COX IV), complex V subunits (ATPase α and ATPase β), mitochondrial porin (VDAC1) and prohibitin analyzed by western blots. Mitochondrial preparations were used for this analysis. (C) Steady-state levels of Bcl-2 and Bcl-_xL in colorectal tumor and osteosarcoma cell lines. Total cell homogenates were used for this analysis. (D) Immunolocalization of cytochrome c. V425 and V429 cells were incubated with Mitotracker (CMX-ROS), fixed and cytochrome c detected by indirect immunofluorescence. All exposures were identical between V425 and V429. The complete color images (cytochrome c and Mitotracker) can be found in Supplementary Material, Figure S1.

Figure 4. KCN titration of endogenous respiration and COX activity

(A) The endogenous respiration was determined at different KCN concentrations. Respiration was rapidly abolished in V425 with the addition of KCN. (B) Threshold plot (percentage COX inhibition versus percentage O₂ consumption rate) showing a lack of respiratory control threshold by COX in V425.

Figure 5. Functional consequences of V425 mtDNA mutations on ATP synthesis, ΔΨm and cytosolic Ca²⁺ buffering

(A) Endogenous respiratory rates of different cell lines in the presence or absence of oligomycin and CCCP. (B) Rate of oligomycin-sensitive ATP synthesis in different cell lines. Mitochondrial ATP synthesis was measured in permeabilized cells incubated with pyruvate/malate (see Materials and Methods). (C) Relative ΔΨm in V425 and V429 assessed as JC1 aggregate/monomer ratios. (D) Mitochondrial calcium uptake in V425 and V429 cells. Fura-2 was used to monitor the cytosolic calcium levels in the absence or presence of 100 µm ATP. Error bars are SD of the mean for n ≥ 3 determinations. Comparisons with the control cell line were performed by the Student’s t-test.

Figure 6. Transcriptional coactivators PGC-1α and PGC-1β are markedly upregulated in V425

(A) Steady-state levels of transcription factors expected to affect mitochondrial function. We could not detect an increase in NRF-1 or NRF2α proteins in V425. The steady-state levels of mitochondrial transcription factor, mtTFA were relatively reduced in V425. (B) Results of TaqMan® quantitative RT-PCR. The data is expressed as a relative fold-change from the control (W20 RNA), relative to mRNA concentration (top) or β-actin levels (bottom). The levels of PGC-1α and PGC-1β transcripts were found to be highly upregulated, whereas the levels of NRF1 transcripts were reduced in V425 when compared with V429. Error bars are SD of the mean for n ≥ 3 determinations. Comparisons with the control cell line were performed by the Student’s t-test.

Figure 7. Overexpression of PGC-1α and PGC-1β stimulates mitochondrial respiration in RVA1 cybrid

(A) Endogenous and ascorbate/TMPD-driven respiration in osteosarcoma cybrid (RVA1) overexpressing GFP, PGC-1α, PGC-1β and PGC-1α + PGC-1β. A significant increase in respiration was observed in RVA1 overexpressing PGC-1α or PGC-1β when compared with the uninfected control or GFP overexpressing cells (P < 0.05). Overexpression of PGC-1α + PGC-1β stimulated respiration in a synergistic manner. (B) Endogenous and ascorbate/TMPD-driven respiration in control osteosarcoma (W20) cells. Error bars are SD of the mean for n ≥ 3 determinations. Comparisons with the control cell line were performed by the Student’s t-test. (C) Steady-state levels of mitochondrial proteins in W20 and RVA1 cells overexpressing PGC-1α, PGC-1β and PGC-1α + PGC-1β. The steady-state levels of SDH(Fp), COX IV, COX Vb and ATPase α subunits were increased in RVA1 overexpressing PGC-1α/β when compared with the GFP overexpressing cells. Some of these proteins were also increased in W20 overexpressing PGC-1α + PGC-1β. (D) The panel shows the quantitation of western blot results shown in panel C. The signal intensity for mitochondrial proteins was normalized by the respective tubulin control. The fold-change in the steady-state levels of mitochondrial proteins in PGC-1α/β overexpressing cells is expressed relative to the respective GFP overexpressing control. (E) Spectrophotometric determination of COX activity in W20 (left) and RVA1 (right) cells overexpressing GFP, PGC-1α or PGC-1β. A significant increase in COX activity was observed in cells overexpressing PGC-1α when compared with the GFP overexpressing cells (* = P < 0.05 compared with the GFP control). (F) Spectrophotometric determination of citrate synthase activity. A significant increase in citrate synthase activity was observed in W20 (left) and RVA1 (right) cells overexpressing PGC-1α or PGC-1β when compared with the GFP overexpressing cells (* = P < 0.05 for all samples compared with the respective GFP controls). Error bars are SD of the mean for n ≥ 3 determinations. Comparisons with the control cell line were performed by the Student’s t-test.

Figure 8. Schematic representation of nuclear-mitochondrial communication response to OXPHOS defect in V425 colorectal cancer cells

V425 colorectal cancer cells harbor a severe defect in COX activity and a partial defect in mitochondrial respiration, membrane potential (ΔΨm) and cytoslic calcium [Ca²⁺]_c buffering ability. Partial defect in ΔΨm and [Ca²⁺]_c buffering ability can stimulate the Ca²⁺ signaling cascade which in turn might activate genes involved in tumor invasion and metastasis. The defect in the activity of complex IV (and possibly also complex I) signals the nucleus to activate the expression of PGC-1 family of transcriptional coactivators that regulate OXPHOS gene expression. Increased expression of PGC-1α and PGC-1β coactivators in turn stimulates respiration by increasing mitochondrial biogenesis, which in V425 appears to involve direct coactivation of a broad but specific set of OXPHOS genes (such as SDH, COX IV, Cyt c, VDAC1 and Bcl-_xL). This paradigm provides a link between deleterious mtDNA mutations, partial OXPHOS defects and cancer progression.