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. 2012 Nov 21;104(22):1750-64.
doi: 10.1093/jnci/djs418. Epub 2012 Nov 12.

Regulation of Mitochondrial Oxidative Metabolism by Tumor Suppressor FLCN

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Free PMC article

Regulation of Mitochondrial Oxidative Metabolism by Tumor Suppressor FLCN

Hisashi Hasumi et al. J Natl Cancer Inst. .
Free PMC article

Abstract

Background: Birt-Hogg-Dubé (BHD) syndrome is a hereditary hamartoma syndrome that predisposes patients to develop hair follicle tumors, lung cysts, and kidney cancer. Genetic studies of BHD patients have uncovered the causative gene, FLCN, but its function is incompletely understood.

Methods: Mice with conditional alleles of FLCN and/or peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), a transcriptional coactivator that regulates mitochondrial biogenesis, were crossbred with mice harboring either muscle creatine kinase (CKM) -Cre or myogenin (MYOG) -Cre transgenes to knock out FLCN and/or PPARGC1A in muscle, or cadherin 16 (CDH16)- Cre transgenes to knock out FLCN and/or PPARGC1A in kidney. Real-time polymerase chain reaction, immunoblotting, electron microscopy, and metabolic profiling assay were performed to evaluate mitochondrial biogenesis and function in muscle. Immunoblotting, electron microscopy, and histological analysis were used to investigate expression and the pathological role of PPARGC1A in FLCN-deficient kidney. Real-time polymerase chain reaction, oxygen consumption measurement, and flow cytometry were carried out using a FLCN-null kidney cancer cell line. All statistical analyses were two-sided.

Results: Muscle-targeted FLCN knockout mice underwent a pronounced metabolic shift toward oxidative phosphorylation, including increased mitochondrial biogenesis (FLCN ( f/f ) vs FLCN ( f/f ) /CKM-Cre: % mitochondrial area mean = 7.8% vs 17.8%; difference = 10.0%; 95% confidence interval = 5.7% to 14.3%; P < .001), and the observed increase in mitochondrial biogenesis was PPARGC1A dependent. Reconstitution of FLCN-null kidney cancer cells with wild-type FLCN suppressed mitochondrial metabolism and PPARGC1A expression. Kidney-targeted PPARGC1A inactivation partially rescued the enlarged kidney phenotype and abrogated the hyperplastic cells observed in the FLCN-deficient kidney.

Conclusion: FLCN deficiency and subsequent increased PPARGC1A expression result in increased mitochondrial function and oxidative metabolism as the source of cellular energy, which may give FLCN-null kidney cells a growth advantage and drive hyperplastic transformation.

Figures

Figure 1.
Figure 1.
Mitochondrial biogenesis in FLCN-deficient muscle. A) Mice carrying FLCN alleles flanked by loxP sites (floxed, f) were crossed with CKM (muscle creatine kinase) Cre transgenic mice to generate muscle-specific FLCN knockout mice (FLCN f/f /CKM– Cre). Pictures show representative images of muscle with indicated genotypes. B) Mitochondrial gene mRNA levels in quadriceps muscle tissue from littermate-matched mice were quantified with the indicated genes by real-time polymerase chain reaction. * Cytochrome C. Mean±95% confidence interval (CI). n=6. Student’s t test (two-sided). C) Lysates of quadriceps muscle from littermate-matched mice were immunoblotted with the indicated antibodies. *Cytochrome C. β-actin served as a loading control. D) Electron microscopy images were obtained from gastrocnemius muscle tissues of littermate matched mice. ×1000 (scale bar: 2 µm) and ×15 000 (scale bar: 500nm) magnified images. Arrows indicate mitochondria. Right bars represent percentage of mitochondrial area. Mean ± 95% confidence interval. Student’s t test (two-sided).
Figure 2.
Figure 2.
Mitochondrial function in FLCN-deficient muscle tissues. A) Relative tricarboxylic acid cycle metabolite values for citrate and malate were obtained in metabolite profiling analyses on quadriceps muscle tissues of indicated genotypes. Mean ± 95% confidence interval. Quadriceps, n = 6. Student’s t test (two-sided). B) Coenzymes for the electron transport chain were measured in quadriceps muscle tissues of indicated genotypes. Nicotinamide adenine dinucleotide, reduced (NADH) was measured using a commercially available kit, and a relative value of flavin adenine dinucleotide was obtained by metabolite profiling analyses. Mean ± 95% confidence interval. Quadriceps, n = 6. Student’s t test (two-sided). C) Respiratory capacity of isolated mitochondria was analyzed in muscle tissues of indicated genotypes. State 3 respiration of complex I– and complex IV–dependent respiration was measured by Seahorse XF96 analyzer using mitochondria isolated from the entire hind limb muscle. Mean ± 95% confidence interval. n = 4. Student’s t test (two-sided). D) Relative ATP amounts were obtained from metabolite profiling analyses on quadriceps muscle tissues of indicated genotypes. Mean ± 95% confidence interval. Quadriceps, n = 6. Student’s t test (two-sided).
Figure 3.
Figure 3.
Mitochondrial oxidative function of FLCN-null UOK257 renal cancer cells. A) FLCN-null UOK257 renal cancer cells were reconstituted with wild-type FLCN expression. The mRNA of mitochondrial genes was quantified by real-time polymerase chain reaction in FLCN-null UOK257 cells, which express wild-type FLCN in a doxycycline-dependent manner. * Cytochrome C. Mean ± 95% confidence interval of triplicates. Analysis of variance test. B) Baseline respiration and respiratory capacity were analyzed by Seahorse XF96 analyzer on FLCN-inducible UOK257 cells cultured with or without doxycycline. Oxygen consumption ratio of baseline respiration was measured in completely supplemented media, and oxygen consumption ratio of maximum respiration was measured after the addition of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Mean ± 95% confidence interval of six wells. Student’s t test (two-sided). C) Representative flow cytometry analysis of JC-1–stained FLCN inducible UOK257 cells, cultured with or without doxycycline. The FL2+, FL1 fraction (upper left quadrant) represents cells with high membrane potential, whereas the FL2, FL1+ fraction (lower right quadrant) represents cells with low membrane potential. D) Representative flow cytometry analysis of tetramethylrhodamine methyl ester (TMRM)–stained FLCN-inducible UOK257 cells, cultured with or without doxycycline.
Figure 4.
Figure 4.
PPARGC1A expression under FLCN deficiency. A) The mRNA of transcription factor and coactivator was quantified by real-time polymerase chain reaction on quadriceps of FLCN f/f and FLCN f/f /CKM– Cre mice. Mean ± 95% confidence interval. Quadriceps, n = 4. Student’s t test (two-sided). B) Lysates from quadriceps muscle of indicated genotypes were immunoblotted with PPARGC1A antibody. Quadriceps, n = 3. β-actin served as a loading control. C) FLCN expression was knocked down in C2C12 myoblasts using FLCN small interfering RNA (siRNA), and mRNA of indicated genes was quantified with real-time polymerase chain reaction. * Cytochrome C. Mean ± 95% confidence interval of triplicates. Analysis of variance test. D) PPARGC1A mRNA was quantified with real-time polymerase chain reaction on FLCN-inducible UOK257 cells cultured with or without doxycycline. Mean ± 95% confidence interval of triplicates. Analysis of variance test.
Figure 5.
Figure 5.
PPARGC1A inactivation in FLCN-deficient muscle. A) FLCN f/f /CKM– Cre mice were crossbred with mice carrying PPARGC1A alleles flanked by loxP sites (floxed, f), and FLCN f/f /PPARGC1A f/f /CKM– Cre mice were generated. A photograph shows representative images of muscle from mice with FLCN f/f, FLCN f/f /CKM– Cre and FLCN f/f /PPARGC1A f/f /CKM– Cre genotypes. B) The mRNA of mitochondrial genes was quantified with real-time polymerase chain reaction on quadriceps samples obtained from FLCN f/f, FLCN f/f /CKM– Cre and FLCN f/f /PPARGC1A f/f /CKM– Cre mice. * Cytochrome C. Mean ± 95% confidence interval. Quadriceps, n = 4. Student’s t test (two-sided). C) Lysates from quadriceps muscle tissues were immunoblotted with the indicated antibodies. α-tubulin served as a loading control. Protein levels in left panel were quantified by Li-Cor Odyssey imager as shown in right panel. * Cytochrome C. Mean ± 95% confidence interval. n = 2. Student’s t test (two-sided). D) The mRNA of angiogenic factors (VEGFb and ANGPT2) and unfolded protein response gene (ERdj4) was quantified with real-time polymerase chain reaction in quadriceps muscle tissues of indicated genotypes. Mean ± 95% confidence interval. Quadriceps, n = 4. Student’s t test (two-sided). E) A different muscle-specific Cre transgenic strain, myogenin (MYOG)– Cre transgenic mice, was crossbred with mice carrying floxed alleles of FLCN and PPARGC1A. The mRNA of PPARGC1A, mitochondrial genes (ATP5g, COX5a, and CYCS), angiogenic factor (VEGFb) and unfolded protein response gene (CHOP) was quantified with real-time polymerase chain reaction on quadriceps samples obtained from FLCN f/f, FLCN f/f /MYOG– Cre and FLCN f/f /PPARGC1A f/f /MYOG– Cre mice. * Cytochrome C. Mean ± 95% confidence interval. Quadriceps, n = 4. Student’s t test (two-sided). F) Mice were treated with rapamycin 2mg/kg daily from postnatal day 7 for 2 months. The mRNA of the indicated genes was quantified with real-time polymerase chain reaction. * Cytochrome C. Mean ± 95% confidence interval. Quadriceps, n = 5 for FLCN f/f group and n = 6 for FLCN f/f /CKM– Cre group. Student’s t test (two-sided).
Figure 6.
Figure 6.
PPARGC1A expression in human Birt-Hogg-Dubé (BHD)–associated kidney tumor and FLCN-deficient mouse kidney tumor. A) PPARGC1A mRNA in human BHD-associated kidney cancer was quantified with real-time polymerase chain reaction. Bars represent mean ± 95% confidence interval. Normal kidney from sporadic cases (n = 5) vs normal kidney from BHD patients (n = 4) vs BHD-associated kidney cancer (n = 5). Student’s t test (two-sided). B) Lysates of FLCN-deficient mouse kidney tumors, solid tumors that developed in heterozygous FLCN knockout mice at 18–24 months of age, were immunoblotted with the indicated antibodies. β-actin served as a loading control. Normal mouse kidney (n = 4) vs FLCN-deficient mouse kidney tumor (n = 5). C) Human BHD-associated kidney cancer and adjacent normal kidney were stained with an antibody to COX4, a downstream target of PPARGC1A. Green = COX4; Blue = 4’,6-diamidino-2-phenylindole. Right panel shows hematoxylin and eosin (H&E) staining of the corresponding area, and inserts show higher magnification images. Dotted lines indicate the border between normal kidney and kidney cancer. Four samples of human BHD-associated kidney cancer were stained and similar results were obtained. (Scale bar: 100 µm in low magnified image, 10 µm in high magnified image.) D) Electron microscopy images were obtained from renal medulla of normal kidney from patients with non-BHD (sporadic) and BHD-associated kidney cancer. ×500 (scale bar: 10µm) and ×5000 (scale bar: 500nm) magnified images. Arrows indicate mitochondria.
Figure 7.
Figure 7.
Pathological significance of upregulated PPARGC1A in FLCN-deficient kidney. A) Lysates of kidney from mice at 5 days of age with indicated genotypes were immunoblotted with the PPAGC1A antibody. α-tubulin served as a loading control. CDH16– Cre = cadherin 16–Cre. Protein level in left panel was quantified by Li-Cor Odyssey imager as shown in right panel. Mean ± 95% confidence interval. n = 2. Student’s t test (two-sided). B) FLCN f/f /CDH16– Cre mice were crossbred with mice carrying PPARGC1A alleles flanked by loxP sites (floxed, f), and FLCN f/f /PPARGC1A f/f / CDH16– Cre mice were generated. Images show representative kidneys of each genotype at 14 days of age. Scale bar: 1mm. C) Ratios of kidney to body weight of indicated genotypes were measured at 21 days of age. Mean ± 95% confidence interval of eight mice per genotype. Student’s t test (two-sided). D) Representative hematoxylin- and eosin-stained tissues of enlarged kidneys from indicated genotypes. Arrows indicate hyperplastic cells in FLCN-deficient kidney (FLCN f/f / CDH16– Cre). Scale bars: 1mm, 40 µm, 10 µm. E) Representative electron microscopy images were obtained from kidney tissues. ×500 (scale bar: 10 µm) and ×5000 (scale bar: 500nm) magnified images. Arrows indicate mitochondria. F) Percentage of mitochondrial area per cell was quantified for the indicated genotypes. n = 24 cells for FLCN f/f, n = 20 cells for FLCN f/f /PPARGC1A f/+ / CDH16– Cre, n = 48 cells for FLCN f/f /PPARGC1A f/f /CDH16– Cre. Mean ± 95% confidence interval. Student’s t test (two-sided).

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