Renal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 Signaling

Summary The Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors.


Inventory of Supplemental Information
Table S1 is related to Figure 4, provided separately as an Excel file Table S2 is

Cell fractionation
To prepare cytoplasmic and nuclear extracts for immunoblotting, cell pellets were resuspended in hypotonic buffer (20 mM HEPES, pH 8.0, 10 mM KCl, 1 mM MgCl2, 0.1% Triton X-100 and 20% glycerol) and incubated on ice for 10 min. Supernatants collected after centrifugation at 1,500 g for 5 min were used as cytoplasmic fractions. Pellets were washed twice in hypotonic buffer, centrifuged at 5,000 g for 5 min and lysed on ice for 1 h in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40) followed by centrifugation at 21,000 g for 10 min. All buffers used were supplemented with 'Complete Protease Inhibitor Cocktail' (Roche).

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Proteins were separated by SDS-PAGE and visualized by subsequent Coomassie staining.
Gel bands of appropriate size were excised and destained overnight in 50% methanol, 5% acetic acid in water. Proteins were reduced with 10 mM dithiothreitol and then alkylated using 30 mM 4-vinylpyridine for 30 min at 20 o C. Proteins were digested with 1 μg elastase, trypsin or chymotrypsin in 50 mM ammonium bicarbonate overnight at 37⁰C and resulting peptides were extracted from the gel with 50% acetonitrile, 5% acetic acid in water.
Resulting peptides were analysed on an Acquity nano UPLC system (Waters) supplemented with a 25 cm C18 column, 1.7 µm particle size (Waters) online coupled to an LTQ Orbitrap Velos (Thermo Scientific). Peptides were eluted by applying a 60 min linear gradient from 5% buffer A (0.1% formic acid in water) to 40% buffer B (0.1% formic acid in acetonitrile) at a flowrate of 250 nl/min (approx. 6000 psi). Full MS scans were performed at a resolution of 30.000. Collision-induced dissociation was performed on the twenty most abundant ions per full MS scan using an isolation width of 1.0 Da. Fragment ions were acquired at a resolution of 7.500. All fragmented precursor ions were actively excluded from repeated MS/MS analysis for 15 s. Raw data was converted to Mascot generic files using msconvert (Kessner et al., 2008) and database searches were performed with MASCOT (Perkins et al., 1999) and CPFP 1.3.0 (Trudgian et al., 2010).

Pathology and Immunohistochemistry
Tissues were fixed in 10% neutral buffered formalin, dehydrated and processed for paraffin wax embedding and sectioning (5μm). Haematoxylin & eosin (H&E) sections were generated for all samples and analysed for routine pathology by two independent pathologists (MEB and GWS). Cyst frequency was determined for each genotype at specified ages in five low power (x10) fields with a minimum of n=5 per group. Immunohistochemistry (IHC) was carried out using the EnVision kit (Dako) as per manufacturer's protocol as described in (Bardella et al., 2011).

Metabolite analysis (CE-TOFMS)
Frozen samples were homogenized by a cell disrupter (MS-100R; TOMY, Tokyo, Japan) at 2°C, after adding 500 µL of methanol that contained internal standards [20 µM each of methionine sulfone and 2-(N-morpholino)-ethanesulfonic acid (MES)]. The homogenate was then mixed with 200 µl of Milli-Q water and 500 µl of chloroform and centrifuged at 9100 g for 4 hr at 4°C. Subsequently, the aqueous solution was centrifugally filtered through a 5-kDa cut-off filter (Millipore) to remove proteins. The filtrate was centrifugally concentrated and dissolved in 50 µL Milli-Q water containing reference compounds (200 µM each of 3aminopyrrolidine and trimesate). Furthermore, prior to CE-TOFMS analysis, the sample solution for cation and anion was diluted (five times) and (two times) with Milli-Q water, respectively. The concentration of each metabolite was calculated as previously described (Soga et al., 2009).

Immunofluorescence and confocal analysis
Immunofluorescence (IF) and confocal analysis was carried out as previously described (O'Flaherty et al., 2010).

Quantitative reverse transcription PCR
Quantitative reverse transcription PCR analysis was carried out as previously described. (O'Flaherty et al., 2010). Normalization was to β-actin mRNA and relative gene expression was calculated using the ΔΔCT method (Livak and Schmittgen, 2001).

Short interfering RNA (siRNA) knockdown
Cells were transfected with ON-TARGET plus siRNA smartpools (Dharmacon) at a final concentration of 25 nM following the manufacturer's protocol. A non-targeting SMARTpool was included as a control in each assay. Knockdown of protein was determined using immunoblotting and Q-PCR.