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A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

Adam J, Yang M, Bauerschmidt C, Kitagawa M, O'Flaherty L, Maheswaran P, Özkan G, Sahgal N, Baban D, Kato K, Saito K, Iino K, Igarashi K, Stratford M, Pugh C, Tennant DA, Ludwig C, Davies B, Ratcliffe PJ, El-Bahrawy M, Ashrafian H, Soga T, Pollard PJ - Cell Rep (2013)

Bottom Line: On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism.Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls.Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Metabolism Group, Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, UK.

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Generation and Analyses of FH (FL) (FH) and FH(-MLS) (FHcyt)-Expressing Transgenic Mice, Related to Figure 2(A) Restriction digest profile of CB92-CAGGS-FH(FL) (FH) and CB92-CAGGS-FH(-MLS) (FHcyt), showing the predicted fragments. (MLS = mitochondrial leader sequence).(B and C) show the plasmid maps of the two exchange vectors, including the sequenced regions and the locations of the restriction sites used for the restriction analysis.(D) The exchanged Rosa26 loci (Chen et al., 2011) are shown with the positions of the genotyping primers.(E) PCR genotyping for exchange at the 3′ end using primer combination AttL-PolyF1 and Rosa3HR-R (Clones ending in 1, 2 or 3 contain the full length construct and clones ended in 4, 5 or 6 contain the MLS deleted construct).(F) PCR genotyping for exchange at the 5′ end using primer combination ExPGK3 and ExNeo2 (top panel) and PCR genotyping for the presence of the transgenic construct allowing the two variant (full length and MLS deleted) to be distinguished on the basis of size (bottom panel).(G) Verification by immunoblot of the presence of FH-V5 in tissues of transgenic mice. Such verification was confirmed in tissues from three mice for both FH(FL) (FH) and FH(-MLS) (FHcyt).(H) Confirmation by immunofluorescence of the localization of FH-V5 and FHcyt-V5 in renal tubular cells. Scale bar = 5 μm.
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figs1: Generation and Analyses of FH (FL) (FH) and FH(-MLS) (FHcyt)-Expressing Transgenic Mice, Related to Figure 2(A) Restriction digest profile of CB92-CAGGS-FH(FL) (FH) and CB92-CAGGS-FH(-MLS) (FHcyt), showing the predicted fragments. (MLS = mitochondrial leader sequence).(B and C) show the plasmid maps of the two exchange vectors, including the sequenced regions and the locations of the restriction sites used for the restriction analysis.(D) The exchanged Rosa26 loci (Chen et al., 2011) are shown with the positions of the genotyping primers.(E) PCR genotyping for exchange at the 3′ end using primer combination AttL-PolyF1 and Rosa3HR-R (Clones ending in 1, 2 or 3 contain the full length construct and clones ended in 4, 5 or 6 contain the MLS deleted construct).(F) PCR genotyping for exchange at the 5′ end using primer combination ExPGK3 and ExNeo2 (top panel) and PCR genotyping for the presence of the transgenic construct allowing the two variant (full length and MLS deleted) to be distinguished on the basis of size (bottom panel).(G) Verification by immunoblot of the presence of FH-V5 in tissues of transgenic mice. Such verification was confirmed in tissues from three mice for both FH(FL) (FH) and FH(-MLS) (FHcyt).(H) Confirmation by immunofluorescence of the localization of FH-V5 and FHcyt-V5 in renal tubular cells. Scale bar = 5 μm.

Mentions: Given that the part of the urea cycle affected by fumarate accumulation functions in the cytosol (Shambaugh, 1977), we hypothesized that cytosolic FH may be important in the pathogenesis of HLRCC. Previously, we demonstrated that expression of cytosolic FH in FH1KO MEFs reduced fumarate levels significantly with concomitant loss of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NRF2) and hypoxia-inducible factor (HIF) expression, but did not restore defects in oxidative metabolism (Adam et al., 2011; O’Flaherty et al., 2010). To investigate the in vivo role of cytosolic FH, we constructed two transgenic mouse lines stably expressing either FH or FHcyt (excluded from the mitochondria) with a C-terminal V5 affinity tag and under the control of the CAG promoter (Niwa et al., 1991). Equivalent expression between both lines was ensured by targeting the FH transgenes to the Rosa26 locus (Zambrowicz et al., 1997) using integrase-mediated cassette exchange (Chen et al., 2011) (Figures 2A and S1). Targeting fidelity was assessed using PCR (Figure S1), and FH protein localization was confirmed in embryonic stem (ES) cells by immunofluorescence (Figures 2B and 2C). Transgenic expression of FH-V5 was analyzed by immunoblotting and immunofluorescence (Figure S1). Similar to HLRCC patients with renal cancer, mice with kidney-specific FH1 deletion develop hyperplastic renal cysts (Pollard et al., 2007). We intercrossed FH1KO mice with both transgenic lines (FH1KO+FH and FH1KO+FHcyt). Macroscopic analyses of kidneys from 30-week-old mice (Figure 2D) indicated that expression of either transgene was sufficient to ameliorate the increased renal mass in FH1KO mice, and microscopic analysis at three time points (13, 20, and 30 weeks) confirmed that transgenic expression of cytosolic FH was sufficient to suppress cyst development (Figures 2E–2H).


A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

Adam J, Yang M, Bauerschmidt C, Kitagawa M, O'Flaherty L, Maheswaran P, Özkan G, Sahgal N, Baban D, Kato K, Saito K, Iino K, Igarashi K, Stratford M, Pugh C, Tennant DA, Ludwig C, Davies B, Ratcliffe PJ, El-Bahrawy M, Ashrafian H, Soga T, Pollard PJ - Cell Rep (2013)

Generation and Analyses of FH (FL) (FH) and FH(-MLS) (FHcyt)-Expressing Transgenic Mice, Related to Figure 2(A) Restriction digest profile of CB92-CAGGS-FH(FL) (FH) and CB92-CAGGS-FH(-MLS) (FHcyt), showing the predicted fragments. (MLS = mitochondrial leader sequence).(B and C) show the plasmid maps of the two exchange vectors, including the sequenced regions and the locations of the restriction sites used for the restriction analysis.(D) The exchanged Rosa26 loci (Chen et al., 2011) are shown with the positions of the genotyping primers.(E) PCR genotyping for exchange at the 3′ end using primer combination AttL-PolyF1 and Rosa3HR-R (Clones ending in 1, 2 or 3 contain the full length construct and clones ended in 4, 5 or 6 contain the MLS deleted construct).(F) PCR genotyping for exchange at the 5′ end using primer combination ExPGK3 and ExNeo2 (top panel) and PCR genotyping for the presence of the transgenic construct allowing the two variant (full length and MLS deleted) to be distinguished on the basis of size (bottom panel).(G) Verification by immunoblot of the presence of FH-V5 in tissues of transgenic mice. Such verification was confirmed in tissues from three mice for both FH(FL) (FH) and FH(-MLS) (FHcyt).(H) Confirmation by immunofluorescence of the localization of FH-V5 and FHcyt-V5 in renal tubular cells. Scale bar = 5 μm.
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figs1: Generation and Analyses of FH (FL) (FH) and FH(-MLS) (FHcyt)-Expressing Transgenic Mice, Related to Figure 2(A) Restriction digest profile of CB92-CAGGS-FH(FL) (FH) and CB92-CAGGS-FH(-MLS) (FHcyt), showing the predicted fragments. (MLS = mitochondrial leader sequence).(B and C) show the plasmid maps of the two exchange vectors, including the sequenced regions and the locations of the restriction sites used for the restriction analysis.(D) The exchanged Rosa26 loci (Chen et al., 2011) are shown with the positions of the genotyping primers.(E) PCR genotyping for exchange at the 3′ end using primer combination AttL-PolyF1 and Rosa3HR-R (Clones ending in 1, 2 or 3 contain the full length construct and clones ended in 4, 5 or 6 contain the MLS deleted construct).(F) PCR genotyping for exchange at the 5′ end using primer combination ExPGK3 and ExNeo2 (top panel) and PCR genotyping for the presence of the transgenic construct allowing the two variant (full length and MLS deleted) to be distinguished on the basis of size (bottom panel).(G) Verification by immunoblot of the presence of FH-V5 in tissues of transgenic mice. Such verification was confirmed in tissues from three mice for both FH(FL) (FH) and FH(-MLS) (FHcyt).(H) Confirmation by immunofluorescence of the localization of FH-V5 and FHcyt-V5 in renal tubular cells. Scale bar = 5 μm.
Mentions: Given that the part of the urea cycle affected by fumarate accumulation functions in the cytosol (Shambaugh, 1977), we hypothesized that cytosolic FH may be important in the pathogenesis of HLRCC. Previously, we demonstrated that expression of cytosolic FH in FH1KO MEFs reduced fumarate levels significantly with concomitant loss of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NRF2) and hypoxia-inducible factor (HIF) expression, but did not restore defects in oxidative metabolism (Adam et al., 2011; O’Flaherty et al., 2010). To investigate the in vivo role of cytosolic FH, we constructed two transgenic mouse lines stably expressing either FH or FHcyt (excluded from the mitochondria) with a C-terminal V5 affinity tag and under the control of the CAG promoter (Niwa et al., 1991). Equivalent expression between both lines was ensured by targeting the FH transgenes to the Rosa26 locus (Zambrowicz et al., 1997) using integrase-mediated cassette exchange (Chen et al., 2011) (Figures 2A and S1). Targeting fidelity was assessed using PCR (Figure S1), and FH protein localization was confirmed in embryonic stem (ES) cells by immunofluorescence (Figures 2B and 2C). Transgenic expression of FH-V5 was analyzed by immunoblotting and immunofluorescence (Figure S1). Similar to HLRCC patients with renal cancer, mice with kidney-specific FH1 deletion develop hyperplastic renal cysts (Pollard et al., 2007). We intercrossed FH1KO mice with both transgenic lines (FH1KO+FH and FH1KO+FHcyt). Macroscopic analyses of kidneys from 30-week-old mice (Figure 2D) indicated that expression of either transgene was sufficient to ameliorate the increased renal mass in FH1KO mice, and microscopic analysis at three time points (13, 20, and 30 weeks) confirmed that transgenic expression of cytosolic FH was sufficient to suppress cyst development (Figures 2E–2H).

Bottom Line: On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism.Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls.Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Metabolism Group, Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, UK.

Show MeSH
Related in: MedlinePlus