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Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates: POSSIBLE LINKS BETWEEN CELL METABOLISM AND STABILIZATION OF HIF *

Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates:... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 7, pp. 4524 –4532, February 16, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates POSSIBLE LINKS BETWEEN CELL METABOLISM AND STABILIZATION OF HIF Received for publication, November 8, 2006 Published, JBC Papers in Press, December 19, 2006, DOI 10.1074/jbc.M610415200 ‡§¶ ‡§¶  ¶ ‡§¶ Peppi Koivunen , Maija Hirsila¨ , Anne M. Remes , Ilmo E. Hassinen , Kari I. Kivirikko , ‡§¶1 and Johanna Myllyharju ‡ § ¶ From the Collagen Research Unit, Biocenter Oulu, Departments of Medical Biochemistry and Molecular Biology and Neurology, University of Oulu, FIN-90014 Oulu, Finland The stability and transcriptional activity of the hypoxia-induc- get genes have important roles in many physiological and path- ible factors (HIFs) are regulated by two oxygen-dependent events ological events such as angiogenesis, vascular remodeling, that are catalyzed by three HIF prolyl 4-hydroxylases (HIF-P4Hs) erythropoiesis, glucose utilization, iron transport, cell prolifer- and one HIF asparaginyl hydroxylase (FIH). We have studied pos- ation, survival and apoptosis, and tumor progression (1–3). sible links between metabolic pathways and HIF hydroxylases by HIFs are  heterodimers, the human  subunit having three analyzing the abilities of citric acid cycle intermediates to inhibit isoforms, HIF-1 to HIF-3 (1–3). HIF-1 and HIF-2 are syn- purified human HIF-P4Hs and FIH. Fumarate and succinate were thesized constitutively, and hydroxylation of at least one of two identifiedas in vitroinhibitorsofallthreeHIF-P4Hs,fumaratehav- critical proline residues mediates their interaction with the von ing K values of 50–80 M and succinate 350–460 M, whereas Hippel-Lindau (VHL) E3 ubiquitin-ligase complex, which tar- neither inhibited FIH. Oxaloacetate was an additional inhibitor of gets them for rapid proteasomal degradation under normoxic all three HIF-P4Hs with K values of 400–1000 M and citrate of conditions (4–6). This hydroxylation is catalyzed in humans by HIF-P4H-3, citrate being the most effective inhibitor of FIH with a three cytoplasmic and nuclear HIF prolyl 4-hydroxylases (HIF- K of 110 M. Culturing of cells with fumarate diethyl or dimethyl P4Hs, also known as PHDs 1–3, HPHs 3-1, and EGLNs 2, 1, and ester, or a high concentration of monoethyl ester, stabilized 3, respectively) (7–9), which are distinct from the well charac- HIF-1 and increased production of vascular endothelial growth terized endoplasmic reticulum lumenal collagen P4Hs factor and erythropoietin. Similar, although much smaller, (C-P4Hs) (10, 11). All P4Hs are 2-oxoglutarate dioxygenases changes were found in cultured fibroblasts from a patient with and require Fe , 2-oxoglutarate, O , and ascorbate (1, 10). fumarate hydratase (FH) deficiency and upon silencing FH using Even small decreases in the O concentration (12, 13) will small interfering RNA. No such effects were seen upon culturing of inhibit the activities of the HIF-P4Hs so that HIF-1 and cells with succinate diethyl or dimethyl ester. As FIH was not inhib- HIF-2 escape degradation and dimerize with HIF-, this ited by fumarate, our data indicate that the transcriptional activity dimer then recognizing the HIF-responsive elements in numer- of HIF is quite high even when binding of the coactivator p300 is ous hypoxia-regulated target genes such as those for vascular prevented. Our data also support recent suggestions that the endothelial growth factor (VEGF), erythropoietin (Epo), glu- increased fumarate and succinate levels present in the FH and suc- cose transporters, and glycolytic enzymes (1–5). cinate dehydrogenase-deficient tumors, respectively, can inhibit Transcriptional activation in an oxygen-dependent manner the HIF-P4Hs with consequent stabilization of HIF-s and effects is another key event that regulates HIF activity. Hydroxylation on tumor pathology. of a critical asparagine residue in the C-terminal transactivation domain of HIF- blocks its interaction with the transcriptional coactivator p300 (14). The asparaginyl hydroxylase catalyzing The hypoxia-inducible transcription factors (HIFs) play a this modification is identical to a protein known as factor inhib- central role in the regulation of oxygen homeostasis. Their tar- iting HIF (FIH) (15, 16). It is also a 2-oxoglutarate dioxygenase and requires the same cosubstrates as the P4Hs (15) but its * This work was supported by Grants 200471 and 202469 from the Health catalytic and inhibitory properties are distinct from those of the Science Council and 44843 from the Finnish Centre of Excellence Pro- HIF-P4Hs (17). gramme 2000 –2005 of the Academy of Finland, the S. Juselius Foundation, and FibroGen Inc. (South San Francisco, CA). The costs of publication of this Certain citric acid cycle intermediates and related com- article were defrayed in part by the payment of page charges. This article pounds have recently been reported to inhibit activities of HIF- must therefore be hereby marked “advertisement” in accordance with 18 P4Hs, whereas no data are available on their effects on FIH. U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: P. O. Box 5000, University Succinate inhibited nonspecified total HIF-P4H activity in of Oulu, FIN-90014 Oulu, Finland. Tel.: 358-8-537-5740; Fax: 358-8-537- crude cell extracts in vitro with an apparent IC of 0.5 mM (18) 5811; E-mail: [email protected]. and purified HIF-P4H-2 in an in vitro VHL capture assay per- The abbreviations used are: HIF, hypoxia-inducible transcription factor; VHL, von Hippel-Lindau; HIF-P4H, HIF prolyl 4-hydroxylase; C-P4H, collagen formed under nonsaturating substrate concentrations and in prolyl 4-hydroxylase; VEGF, vascular endothelial growth factor; Epo, eryth- the presence of one succinate concentration with an apparent ropoietin; FIH, HIF asparaginyl hydroxylase; SDH, succinate dehydrogen- K of 9 M (19), whereas fumarate had in a similar assay an ase; FH, fumarate hydratase; siRNA, small interfering RNA; HEK293, human embryonic kidney 293. apparent K of 3 M (19). In agreement with these data, cultur- This is an Open Access article under the CC BY license. 4524 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 •FEBRUARY 16, 2007 Inhibition of HIF Prolyl 4-Hydroxylases ing of cancer cells with 20 mM succinate dimethyl ester for 48 h ues obtained are often somewhat different from those obtained (18) or with 2.5 mM succinate diethyl ester or 0.5 mM fumarate in assays involving capture of hydroxylated HIF- or a fragment monoethyl ester together with 3-nitropropionic acid (19), a of this by pVHL. (Pro-Pro-Gly) was used as the substrate for a dual inhibitor of succinate dehydrogenase (SDH) and fumarate purified recombinant human type I C-P4H (23). The K values hydratase (FH), led to stabilization of HIF-1, and a similar were determined by adding the 2-oxoglutarate analogues at effect was seen upon silencing of SDH or FH by the correspond- four constant concentrations while varying the concentration ing siRNA (18, 19). Oxaloacetate and pyruvate have likewise of 2-oxoglutarate or the peptide substrate. been reported to stabilize HIF-1 in cultured cancer cell lines Stabilization of HIF-1 by Diethyl Fumarate and Dimethyl and inactivate HIF-P4Hs in a manner reversible by ascorbate Fumarate in Cultured Cells—Human embryonic kidney (20, 21). (HEK293), hepatocellular carcinoma (Hep3B), and neuroblas- We have studied possible links between metabolic pathways toma (Kelly) cells, and fibroblasts (CRL-2086) were obtained and HIF hydroxylases by systematically analyzing the abilities from the American Type Culture Collection and cultured in of citric acid cycle intermediates and other aliphatic 2-oxoglut- Dulbecco’s (Biochrom) or Eagle’s (Sigma) minimal essential arate analogues to inhibit purified human HIF-P4Hs and FIH medium or in RPMI 1640 (Sigma) medium with 10% fetal under initial velocity conditions and in the presence of saturat- bovine serum (BioClear) and 50 g/ml ascorbate. They were 5 4 2 ing peptide substrate concentrations in vitro. Fumarate and then subcultured at a density of 1  10 or 1.6  10 cells/cm succinate were identified as inhibitors of all three HIF-P4Hs, in the case of fibroblasts, grown overnight, and incubated for fumarate having K values of 50–80 M and succinate 350–460 8–20 h with increasing concentrations of diethyl fumarate, di- M, whereas neither inhibited FIH to any significant extent methyl fumarate, monoethyl fumarate, diethyl succinate, or di- even at a 10 mM concentration. Oxaloacetate was an additional methyl succinate (Sigma). In control experiments the cells were inhibitor of all three HIF-P4Hs with K values of 400–1000 M treated with 300–500 M cobalt or subjected to hypoxia by and citrate was an inhibitor of HIF-P4H-3 with a K of 180 M, culturing in an airtight incubator (Billups-Rothenberg) under whereas pyruvate and many other 2-oxoglutarate analogues 1% O balanced with N . The viability of cells was determined 2 2 showed no inhibition of any of the HIF-P4Hs even at a 10 mM using trypan blue dye exclusion. Skin fibroblasts from a patient concentration, variations in ascorbate levels having no effect on with a homozygous loss of function mutation in the FH gene inhibition by oxaloacetate or pyruvate. FIH was inhibited most (24) and fibroblasts from a healthy control were cultured in effectively by citrate and oxaloacetate with K values of 110 and Dulbecco’s medium with 10% fetal bovine serum. Medium 400 M, respectively. Our further experiments using culturing samples were collected for analysis of the production of VEGF of cells with fumarate diethyl ester or dimethyl ester, which pass and Epo (see below) and total cell extracts were obtained by through cell membranes, led to stabilization of HIF-1 and to lysing the cells in 150 mM NaCl, 0.1% SDS, and 20 mM Tris-HCl, increases in VEGF production in all the cell types studied and in pH 6.8. Nuclear extracts were prepared by the NE-PER nuclear Epo production in human hepatocellular carcinoma Hep3B extraction reagent (Pierce). The extracts were analyzed by 8% cells. Similar, although much smaller, changes were found in SDS-PAGE under reducing conditions followed by Western cultured skin fibroblasts from a patient with autosomal reces- blotting with a monoclonal antibody against human HIF-1 TM sive FH deficiency and in cells transfected with FH siRNA. No (BD Biosciences) and ECL immunodetection. Antibodies to such effects were seen upon culturing of cells with succinate  tubulin (total extracts, Sigma) and  actin (nuclear extracts, diethyl ester or dimethyl ester. Novus Biologicals) were used as loading controls. RNA Interference—Four siRNAs of the FH coding sequence TM MATERIALS AND METHODS (set of 4 siGENOME duplexes, Dharmacon) were trans- Expression, Purification, and Activity Assays of Recombinant fected at 75 nM concentration into HEK293 cells using Lipo- TM HIF-P4Hs and FIH—FLAG His-tagged HIF-P4Hs 1–3 and FIH fectamine 2000 (Invitrogen) according to the manufactur- TM (17, 22) were expressed using recombinant baculoviruses in H5 er’s instructions. The Silencer GAPDH and Silencer insect cells cultured in suspension or on plates in Sf900IISFM negative control siRNAs (Ambion) were used as controls. serum-free medium (Invitrogen). The cells, seeded at a density Medium samples were collected 48 h after transfection, and of 1 10 /ml, were infected at a multiplicity of 5 and harvested total RNA and protein were isolated from the cells using the TM 72 h after infection, washed with a solution of 0.15 M NaCl and PARIS kit (Ambion). The ability of FH siRNAs to down- 0.02 M phosphate, pH 7.4, and homogenized in a 0.1 M NaCl, 0.1 regulate FH mRNA was studied by semiquantitative reverse TM M glycine, 10 M dithiothreitol, 0.1% Triton X-100, and 0.01 M transcriptase-PCR using the SMART PCR cDNA synthesis Tris buffer, pH 7.8. The soluble fractions were subjected to kit (BD Biosciences). PCR was carried out under the following purification with an anti-FLAG M2 affinity gel (Sigma) (17, 22). conditions: 1 min at 94 °C followed by 22 cycles of 1 min at HIF-P4H and FIH activities were assayed by methods based on 94 °C, 1 min at 60 °C, and 1 min at 72 °C. The primers used for measurement of the hydroxylation-coupled stoichiometric amplification were: 5-GGAGGTGTGACAGAACGCATGC- 14 14 release of CO from 2-oxo-[1- C]glutarate with synthetic CACC-3 and 5-GCTGCCTTGTCATACCCTATATGAGG-3. HIF-1 peptides as substrates and the K and IC values of the Stabilization of HIF-1 was analyzed by Western blotting of the m 50 purified enzymes were determined as described previously (12, protein samples as described above. 17, 22). This method makes it possible to measure HIF-P4H Analysis of the Production of VEGF and Epo—The VEGF and activities under true initial velocity conditions and using satu- Epo concentrations in the medium samples were measured rating peptide substrate concentrations, and therefore the val- using the Quantikine human VEGF and Epo immunoassays FEBRUARY 16, 2007• VOLUME 282 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4525 Inhibition of HIF Prolyl 4-Hydroxylases TABLE 1 K values of HIF-P4Hs, FIH, and C-P4H-I for 2-oxoglutarate and IC and K values for fumarate, succinate, oxaloacetate, and citrate m 50 i a,b a,b a,b c Compound Constant HIF-P4H-1 HIF-P4H-2 HIF-P4H-3 C-P4H-I FIH d e 2-Oxoglutarate K 2  0.4 1  0.2 12 420 25 Fumarate IC 120  10 80  30 60  30 ND 10000 K 80  10 60  20 50  15 190  30 10000 Succinate IC 830  540 510  310 570  170 ND 10000 K 350  20 460  70 430  200 400 10000 Oxaloacetate IC 1000  420 3800  1400 1200  350 ND 1400  370 K 400  130 1000  50 590  120 100 400  40 Citrate IC 6300  1300 4800  1600 550  200 ND 850  190 K ND 1800  320 180  80 450 110  30 Values are mean  S.D. from three to ten independent experiments. The IC values of HIF-P4Hs 1, 2, and 3 for pyruvate, malate, isocitrate, 2-oxoadipinate, 2-oxovalerate, 2-oxobutyrate, adipinate, glutarate, malonate, 3-oxoglutarate, levulinate, and lactate were at least 10 mM, except those of HIF-P4H-3 for glutarate and HIF-P4H-2 for 3-oxoglutarate, which were 6 and 5 mM, respectively. Similar values were obtained from at least two independent experiments. The IC value of FIH for isocitrate was about 5 mM, those for all the other compounds being at least 10 mM. Similar values were obtained from at least two independent experiments. Ref. 43. Ref. 17. ND, not determined. Ref. 26. Ref. 27. (R&D Systems) according to the manufacturer’s instructions. Inhibition of the purified recombinant HIF-P4Hs and FIH Statistical analyses were performed with SPSS 14.0 for was studied using six citric acid cycle intermediates and 10 Windows. other aliphatic 2-oxoglutarate analogues (Table 1). The IC Determination of Cellular Fumarate Concentrations—Equal values of the HIF-P4Hs and FIH were first determined in the numbers of HEK293 cells were incubated in the absence and presence of 10 and 50–100 M 2-oxoglutarate, respectively. presence of 40 M diethyl fumarate for 20 h, the culture Fumarate was found to be the most efficient inhibitor of the medium was carefully removed, and the cell pellets were imme- three HIF-P4Hs, with IC values of 120, 80, and 60 M for diately cooled in liquid N . The frozen pellets were lysed in 8% 2 HIF-P4Hs 1, 2, and 3, respectively, whereas succinate had IC cold perchloric acid and centrifuged. The supernatant was col- values of 830, 510, and 570 M (Table 1). The IC values of lected and the remaining pellet was washed and extracted with HIF-P4Hs 1, 2, and 3 for oxaloacetate were 1, 3.8, and 1.2 mM, 6% perchloric acid and centrifuged. The supernatants were respectively, and those for citrate 6.3, 4.8, and 0.55 mM, whereas pooled and neutralized by K CO and used to determine the 2 3 the IC values for pyruvate, malate, isocitrate, 2-oxoadipinate, amount of fumarate with fumarase, malate dehydrogenase, and 2-oxovalerate, 2-oxobutyrate, adipinate, glutarate, malonate, aspartate aminotransferase (Sigma) with a Fluoromax-2 spec- 3-oxoglutarate, levulinate, and lactate were at least 10 mM for all trofluorometer (HORIBA Jobin Yvon, Inc., Edison, NJ) as three isoenzymes, except that glutarate had an IC of about 6 described previously (25) with the exception that glycyl glycine, mM for HIF-P4H-3 and 3-oxoglutarate had an IC of about 5 pH 9, supplemented with glutamic acid was used as a buffer. mM for HIF-P4H-2 (Table 1). The maleate present in the commercial fumarase preparation FIH was inhibited most effectively by citrate, with an IC of was removed by gel filtration in a PD-10 column (GE Health- 850 M (Table 1). The IC values of FIH for oxaloacetate and care). The excitation wavelength was 340 nm and emission was isocitrate were about 1.4 and 5 mM, respectively, whereas those recorded at 460 nm. Samples of skin fibroblasts from a patient of all the other compounds tested, including fumarate, succi- with a homozygous FH mutation and from a healthy control nate, and pyruvate were at least 10 mM (Table 1). were prepared and analyzed as above. Inhibition of the HIF-P4Hs by fumarate, succinate, oxaloac- etate, and citrate, the compounds with the lowest IC values RESULTS among those tested, and of FIH by citrate and oxaloacetate, its Inhibition of HIF-P4Hs and FIH by Citric Acid Cycle Intermedi- most effective inhibitors, was analyzed in more detail. The ates and Certain Other Aliphatic 2-Oxoglutarate Analogues— mode of inhibition and K values were determined by adding FLAG His-tagged human HIF-P4Hs 1–3 and FIH were fumarate, succinate, oxaloacetate, or citrate at four constant expressed in insect cells, which were harvested 72 h after infec- concentrations while varying the concentration of 2-oxoglut- tion, homogenized in a buffer containing Triton X-100, and arate or the peptide substrate. The inhibition was found to be centrifuged. The recombinant enzymes were purified from the competitive with respect to 2-oxoglutarate (as shown for fuma- soluble fraction by anti-FLAG affinity chromatography and rate with HIF-P4Hs 2 and 3, succinate with HIF-P4H-1, and their activities were assayed by methods based on measurement citrate with FIH in Fig. 1, A–D) and noncompetitive with of the hydroxylation-coupled stoichiometric release of CO respect to the peptide substrate (data not shown). The K values from 2-oxo-[1- C]glutarate (12, 17, 22). The K values of the of HIF-P4Hs 1, 2, and 3 for fumarate were 80, 60, and 50 M, purified HIF-P4Hs for 2-oxoglutarate were found to be 1–2 M for isoenzymes 1 and 2, and 12 M for isoenzyme 3 (Table 1), respectively, and those for succinate 350, 460, and 430 M, these values being distinctly lower than those previously whereas the values for oxaloacetate ranged from 400 to 1000 reported for the HIF-P4Hs in crude insect cell extracts (12). M and the K values of HIF-P4H-2 and HIF-P4H-3 for citrate 4526 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 •FEBRUARY 16, 2007 Inhibition of HIF Prolyl 4-Hydroxylases FIGURE 1. Competitive inhibition of HIF-P4H-2 (A) and HIF-P4H-3 (B) by fumarate, HIF-P4H-1 by succinate (C), and FIH by citrate (D) with respect to 2-oxoglutarate. Inhibition of purified HIF hydroxylases was studied by adding fumarate, succinate, or citrate at 4 constant concentrations (indicated with the symbols given in the figures) while varying the concentration of 2-oxoglutarate. K values were calculated from the plots of the slopes of the lines at the inhibitor concentrations studied (insets), the intercept on the x axis being equal to K . Essentially identical K values were measured in at least three independent i i experiments (see Table 1). were 1800 and 180 M, respectively (Table 1). The K values of cinate diethyl or dimethyl ester, all of which are converted to FIH for citrate and oxaloacetate were 110 and 400 M, respec- fumarate or succinate by the action of cellular esterases (28, 29). tively, whereas those for fumarate and succinate were more These compounds gave no inhibition of purified HIF-P4Hs in than 10 mM (Table 1). vitro even at 10 mM concentrations (details not shown). In con- Succinate, oxaloacetate, and citrate have also been reported trol experiments, cells were treated with cobalt or cultured (26, 27) to inhibit the C-P4Hs, their K values for the C-P4H under hypoxia. Total cell extracts and nuclear extracts were isoenzyme I being 400, 100, and 450 M, respectively (Table 1). prepared and analyzed by 8% SDS-PAGE under reducing con- Our current data indicate that fumarate is an additional ditions followed by Western blotting with a HIF-1 antibody C-P4H-I inhibitor, with a K of 190 M (Table 1). and an  tubulin antibody as a loading control. Stabilization of As oxaloacetate and pyruvate have been reported to inhibit HIF-1 was seen in total extracts from the HEK293, Hep3B, the HIF-P4Hs in an in vitro VHL capture assay in the presence and Kelly cells and fibroblasts with 20–100, 10–100, 40–120, of nonsaturating ascorbate levels (30–100 M) (20, 21), we also and 100–150 M diethyl fumarate, respectively (Fig. 2, A–D), studied inhibition of the HIF-P4Hs by these two compounds in higher concentrations leading to cell detachment (as shown by the presence of varying ascorbate concentrations. However, the decreased staining for  tubulin). Similar results were obtained IC values obtained under these conditions were identical to with dimethyl fumarate (as shown for HEK293 cells in Fig. 2E), those determined in the presence of 2 mM ascorbate (details not whereas no stabilization of HIF-1 was seen with succinate shown). diethyl or dimethyl ester in a concentration range of 50 M to 5 Stabilization of HIF-1 by Fumarate Diethyl and Dimethyl mM (as shown for succinate dimethyl ester in HEK293 cells in Esters in Cultured Cells—To study whether fumarate and suc- Fig. 2F). Stabilization of HIF-1 by diethyl fumarate was also cinate stabilize HIF-1, we cultured human embryonic kidney seen in nuclear extracts (as shown for the HEK293, Hep3B, and (HEK293), hepatocellular carcinoma (Hep3B), and neuroblas- Kelly cells in Fig. 3, A–C). Fumarate monoethyl ester also sta- toma (Kelly) cells and fibroblasts (CRL-2086) for 20 h in the bilized HIF-1 (as shown for nuclear extracts from HEK293 presence of increasing concentrations of the membrane-per- cells in Fig. 3D), but the concentration required for a detectable meable fumarate diethyl, dimethyl or monoethyl ester, or suc- stabilization was 5 mM, being thus about 2 orders of magnitude FEBRUARY 16, 2007• VOLUME 282 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4527 Inhibition of HIF Prolyl 4-Hydroxylases FIGURE 3. Stabilization of HIF-1 by fumarate diethyl and monoethyl esters in cultured cells. HEK293 (A), Hep3B (B), and Kelly cells (C) were incu- bated for 20 h with increasing concentrations (M, indicated below the lanes) of fumarate diethyl ester. HEK293 cells were also incubated with increasing concentrations of fumarate monoethyl ester (D). Stabilization of HIF-1 in cells cultured under hypoxic conditions (lanes H,1%O ) is also shown. Nuclear extracts from cells were analyzed by 8% SDS-PAGE under reducing conditions followed by Western blotting with HIF-1 and  actin antibodies and ECL immunodetection. The exposure times of lanes H are 12, 12, and 6 times shorter than those of other lanes in A, C, and D, respectively. Lanes 5000 and H in D were run in a parallel gel at the same time as lanes 0 –1000. Similar results were obtained from at least three independent experiments. FIGURE 2. Stabilization of HIF-1 by fumarate diethyl and dimethyl esters and succinate dimethyl ester in cultured cells. HEK293 (A), Hep3B (B) and Kelly cells (C), and fibroblasts (D) were incubated for 20 h with increasing concentrations (M, indicated below the lanes) of membrane-permeable FIGURE 4. Stabilization of HIF-1 in cultured fibroblasts from a patient fumarate diethyl ester. HEK293 cells were also incubated with increasing con- with autosomal recessive FH deficiency. Skin fibroblasts from a patient centrations of fumarate dimethyl ester (E) and succinate dimethyl ester (F). with a homozygous Q376P mutation in the FH gene (FH ) and control fibro- Stabilization of HIF-1 in cells cultured under hypoxic conditions (lanes H,1% blasts (FH ) were cultured under normoxia (N) and hypoxia (H,1%O ). Total O ) is also shown. Total cell extracts were analyzed by 8% SDS-PAGE under cell extracts were analyzed by 8% SDS-PAGE under reducing conditions fol- reducing conditions followed by Western blotting with HIF-1 and  tubulin lowed by Western blotting with HIF-1 and  tubulin antibodies and ECL antibodies and ECL immunodetection. The exposure times of lanes H are 10, 4, immunodetection. The exposure times of lanes H detected by the HIF-1 12, and 20 times shorter than those of other lanes in A–D, respectively. Similar antibody are 10 times shorter than those of lanes N. Similar results were results were obtained from at least three independent experiments. obtained from at least three independent experiments. higher than the concentration of fumarate diethyl ester required In further experiments endogenous FH was silenced by for a similar extent of stabilization (compare with Fig. 3A). transfecting HEK293 cells with FH-specific siRNAs. FH mRNA Stabilization of HIF-1 in Cultured Fibroblasts from a level, analyzed by reverse transcriptase-PCR, was found to be Patient with Autosomal Recessive FH Deficiency and in HEK293 lower in the FH siRNA-transfected cells than control-trans- Cells Transfected with FH siRNA—To study whether lack of fected ones (Fig. 5A), and a very weak but reproducible stabili- endogenous FH leads to stabilization of HIF-1, skin fibroblasts zation of the HIF-1 was seen in the FH siRNA-transfected from a patient with a homozygous Q376P mutation in the FH cells (Fig. 5B). gene (24) were cultured and analyzed as above. The level of FH Increased VEGF Production by Diethyl Fumarate, Dimethyl activity in the mitochondrial and cytosolic fractions of cultured Fumarate, and FH Deficiency in Cultured Cells—To study fibroblasts from this patient is only 0.5% of the control mean whether the stabilized HIF-1 caused increased expression of a (24). A very weak but distinct stabilization of HIF-1 was seen HIF target gene, we assayed the amount of VEGF in medium in extracts from these cells cultured under normoxia in three samples collected from cells cultured in the presence of increas- independent experiments, whereas no HIF-1 was detected in ing diethyl fumarate concentrations in 3 to 5 independent control fibroblasts (Fig. 4). HIF-1 was stabilized to an equal experiments. Dose-dependent increases were found in VEGF extent in the FH mutant cells and in control fibroblasts cultured production, the highest mean values being 640% with 80 M under hypoxia (Fig. 4). diethyl fumarate in HEK293 cells, 250% with 100 M in Hep3B 4528 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 •FEBRUARY 16, 2007 Inhibition of HIF Prolyl 4-Hydroxylases cells, 240% with 20 M in Kelly cells, and 210% with 100 M in No increase in VEGF production was found with diethyl succi- fibroblasts as compared with that in nontreated cells (as shown nate or dimethyl succinate when studied in a concentration for HEK293, Hep3B, and Kelly cells in Fig. 6, A–C). The highest range of 50 M to 20 mM (details not shown). mean values seen in various cells range from about 30 to 100% To study the temporal relation of cell viability, HIF-1 stabi- of those obtained under 1% O in the same cells, the increases in lization, and VEGF production in the presence of increasing VEGF production by diethyl fumarate thus being much larger concentrations of diethyl fumarate, a time course analysis was than those seen in the extent of stabilization of HIF-1 (Fig. 2). performed with HEK293 cells. The viability of cells treated with A similar effect was seen with dimethyl fumarate, although the 40 M diethyl fumarate was 93–95% at 12 and 20 h, no differ- magnitude of the increase was smaller (as shown for HEK293 ence in viability being seen between the two time points (data cells in Fig. 6D). The different levels obtained are probably due not shown). The viability of cells treated with 100 M diethyl in part to differences in the uptake of diethyl fumarate and fumarate was likewise 94% at 12 h, but decreased to 83% at 20 h. dimethyl fumarate and their conversion to fumarate in various Stabilization of HIF-1 was seen in total extracts from cells cell types, in part to differences in the detachment of cells at cultured in the presence of 10–100 M diethyl fumarate for 8 h, high diethyl and dimethyl fumarate concentrations (see the the amount of stabilized HIF-1 increasing at 12 and 20 h (Fig. 7, decreased staining for  tubulin in Fig. 2) and in part to differ- A–C). The amount of VEGF secreted into the culture medium ences in the abilities of various cell types to produce VEGF (22). increased in a time- and dose-dependent manner (Fig. 7D). Additional experiments demonstrated that the stabilized HIF-1 also caused increased VEGF production in the fibro- blasts from the patient with autosomal recessive FH deficiency and a small increase in HEK293 cells transfected with FH siRNA (above). The amount of VEGF in the medium of FH fibroblasts was 170% (p 0.05) of that in the medium of control fibroblasts (Fig. 6F), and the amount in the medium of the siRNA-transfected HEK293 cells was 140% of that in the medium of the non-transfected cells, although the last mentioned increase was not statistically significant (data not shown). FIGURE 5. Stabilization of HIF-1 in cells transfected with FH siRNA. Increased Fumarate Concentrations in HEK293 Cells Cul- HEK293 cells were transfected with FH-specific (lanes 4), glyceraldehyde-3- tured with Diethyl Fumarate and FH Mutant Skin Fibro- phosphate dehydrogenase (GAPDH)-specific (lanes 2), and negative control blasts—Cellular fumarate concentration was measured in (lanes 3) siRNAs. Non-transfected controls are shown in lanes 1. mRNA levels of FH (upper panel) and GAPDH (lower panel) were analyzed by reverse tran- HEK293 cells cultured with 40 M diethyl fumarate for 20 h and scriptase-PCR (A). Total cell extracts were analyzed by 8% SDS-PAGE under in cultured skin fibroblasts from a patient with autosomal reducing conditions followed by Western blotting with HIF-1 and  tubulin antibodies and ECL immunodetection (B). recessive FH deficiency. Control experiments indicated that the FIGURE 6. Increased VEGF and Epo production by diethyl fumarate, dimethyl fumarate, and FH deficiency in cultured cells. Production of VEGF by HEK293 (A), Hep3B (B), and Kelly cells (C) incubated for 20 h with increasing concentrations of diethyl fumarate (indicated below the columns), HEK293 cells incubated with dimethyl fumarate (D), and fibroblasts from a patient with autosomal recessive FH deficiency (F). Production of Epo was also measured from the Hep3B cells incubated with diethyl fumarate (E). In control experiments the cells were subjected to hypoxia (H,1%O ) or incubated with 300 or 500 M CoCl . 2 2 The amounts of VEGF and Epo were measured in medium samples by immunoassay. The values are mean S.D. from at least three individual experiments, and their statistical significance in comparison to the controls in a one-tailed one sample t test are indicated by asterisks;*, p 0.05; **, p 0.01; and ***, p 0.005. FEBRUARY 16, 2007• VOLUME 282 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4529 Inhibition of HIF Prolyl 4-Hydroxylases TABLE 2 Fumarate concentrations in HEK293 cells cultured in the presence of diethyl fumarate for 20 h and in fibroblasts from a patient with autosomal recessive FH deficiency Cellular fumarate concentration Cells 6 b pmol/10 cells % Control Micromolar HEK293 123  21 100 37 HEK293  40 M 219  83 178 66 diethyl fumarate FH fibroblasts 108  28 100 22 / c FH fibroblasts 205  6 190 42 Values are from three to six measurements. The approximate M cellular concentrations were calculated using the reported 12 12 volumes of 3.3 10 and 4.9 10 liters for HEK293 cells (30) and fibroblasts (31), respectively. Statistical significance versus corresponding control cells (Student’s t test) was p 0.02. and in the latter cells by FH deficiency (Table 2). The cellular fumarate levels were converted to micromolar concentrations by using reported (30, 31) values for the volumes of the two cell types. Such calculations suggested that the concentration in cultured HEK293 cells was about 40 M and increased to about 70 M by incubation with 40 M diethyl fumarate, whereas the concentration in cultured control fibroblasts was about 20 M, and that in the FH-deficient fibroblasts about 40 M (Table 2). All these values are within concentrations that inhibited the activities of the three purified HIF-P4Hs, the concentrations in the diethyl fumarate-treated cells even exceeding the K values of HIF-P4Hs 2 and 3 (Table 1 and Fig. 1). Increased Epo Production by Diethyl Fumarate in Cultured Hep3B Cells—Production of Epo was also measured in Hep3B cell medium samples in the above experiments with diethyl fumarate. An increase of 220% was found with the lowest diethyl fumarate concentration that led to a detectable stabili- zation of HIF-1 in Hep3B cells (Fig. 6E), this value being only about 25% of that obtained under 1% O . Interestingly, the value decreased to control level or even below it with concen- trations that led to the highest increases in VEGF production (compare with Fig. 5B). DISCUSSION Our data demonstrate that three citric acid cycle intermedi- ates, fumarate, succinate, and oxaloacetate, inhibit all three human HIF-P4Hs in vitro, fumarate being markedly more FIGURE 7. Time course analysis of HIF-1 stabilization and VEGF produc- effective than the two others, its K for the most abundant and tion in HEK293 cells at increasing diethyl fumarate concentrations. Total extracts from HEK293 cells incubated with increasing concentrations (M, hence most important HIF-P4H, isoenzyme 2 (32, 33), being 60 indicated below the lanes) of diethyl fumarate for 8 (A), 12 (B), and 20 h (C) M, i.e. about 13% of that of succinate, whereas oxaloacetate were analyzed by 8% SDS-PAGE under reducing conditions followed by West- was the weakest inhibitor, its K for isoenzyme 2 being 1 mM. ern blotting with HIF-1 and tubulin antibodies and ECL immunodetection. i Similar results were obtained in at least three independent experiments. The Citrate was an effective inhibitor of HIF-P4H-3 but not of the amount of VEGF was measured in the corresponding medium samples by two others. The K values determined here for fumarate and immunoassay (D). In control experiments the cells were subjected to hypoxia succinate under initial velocity conditions and using four inhib- (H,1%O ). The values are mean  S.D. from at least three independent experiments. itor concentrations are in reasonable agreement with the IC of about 0.5 mM reported for succinate in assays of nonspecified enzymatic assay used here did not utilize diethyl fumarate as a total HIF-P4H activity in crude cell extracts in vitro (18) but the substrate, and thus the increased concentration measured in K for fumarate is about 20-fold and succinate 50-fold when the HEK293 cells incubated with this compound represents compared with the apparent K values reported for these two that of intracellular fumarate produced from the membrane- compounds using purified HIF-P4H-2 and an in vitro VHL cap- permeable ester. The fumarate concentrations, when expressed ture assay performed under nonsaturating substrate concentra- per 10 cells, were highly similar in the non-treated HEK293 tions and in the presence of only one inhibitor concentration cells and control fibroblasts and increased to almost 2-fold by (19). Although oxaloacetate and pyruvate have been reported to the incubation of the former cells with 40 M diethyl fumarate stabilize HIF-1 in cultured cancer cells and to inhibit the HIF- 4530 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 •FEBRUARY 16, 2007 Inhibition of HIF Prolyl 4-Hydroxylases P4Hs in an in vitro VHL capture assay in the presence of non- diethyl fumarate concentrations than the increases in VEGF saturating ascorbate levels (20, 21), we found only a weak inhi- production, and the Epo levels returned to baseline values or bition by oxaloacetate, especially in the case of HIF-P4H-2, and even below them at concentrations that gave the highest no inhibition by pyruvate in the presence of 2 mM ascorbate and increases in VEGF production. This finding agrees with data no increase in the level of inhibition in the presence of lower indicating that hypoxic conditions can show selectivity with ascorbate concentrations. respect to stimulation of Epo versus VEGF (34). One possible Membrane-permeable diethyl and dimethyl ester derivatives explanation for such differences is that Epo has been found to of fumarate produced stabilization of HIF-1 in all the cell be regulated in Hep3B cells unequivocally by HIF-2, whereas types studied, and a very low extent of HIF-1 stabilization was VEGF is regulated by HIF-1 (35). additionally demonstrated in cultured fibroblasts from a Fumarate levels assayed in cultured HEK293 cells and human patient with autosomal recessive FH deficiency and in cells skin fibroblasts were about 40 and 20 M, respectively, being transfected with FH siRNA. Interestingly, by contrast with our about 70 and 30% of the K of the purified HIF-P4H-2 for fuma- findings, culturing with monoethyl fumarate stabilized HIF-1 rate. The concentration in HEK293 cells incubated with 40 M in human A549 lung carcinoma cells only when added in com- diethyl fumarate increased to about 70 M, i.e. slightly above the bination with 3-nitropropionic acid, a dual SDH and FH inhib- K of purified HIF-P4H-2, whereas the value in the FH-deficient itor (19), and the concentrations of monoethyl fumarate (0.5 fibroblasts was about 70% of this K . These concentrations mM) needed to obtain this effect in the presence of the inhibitor could thus be expected to lead to HIF-P4H inhibition, as sug- were much higher than those of diethyl and dimethyl fumarate gested by our data. needed in our study. This difference agrees with findings indi- Distinct VHL mutations that prevent the binding of HIF-s, cating that monoethyl esters of dicarboxylic acids are much less and thus their subsequent pVHL-mediated degradation, can efficient in passing through cell membranes than diethyl and lead to HIF-dependent Epo production and a hereditary poly- dimethyl esters, and our finding that monoethyl fumarate gave cythemia known as Chuvash polycythemia (36), or a cancer a detectable stabilization of HIF-1 only ata5mM concentra- syndrome (2, 37) that is characterized by highly vascular tumors tion. In agreement with our data, a very weak stabilization of that overproduce hypoxia-inducible mRNAs such as those for HIF-1 was seen in the A549 cells upon transfection with FH VEGF. SDH and FH mutations likewise predispose to domi- siRNA, whereas a much stronger stimulation was obtained in nantly inherited highly vascular tumors, findings in SDH muta- the presence of 3-nitropropionic acid (19). tions including pheochromocytoma, paraganglioma, renal cell The stabilized HIF-1 produced in our study by incubation carcinoma, and papillary thyroid cancer (18, 38–40) and those with diethyl or dimethyl fumarate or by the lack of FH activity in FH mutations oncluding uterine fibroids, skin leiomyomata, was transcriptionally active, as an increased production of a and papillary renal cell cancer (19, 38, 41). The fumarate levels HIF-1 target protein VEGF was found in all these cases, a in FH-deficient uterine fibroids were 200-fold relative to the small increase in the VEGF production having also been level in normal tissue, whereas the succinate levels in normal reported recently in FH siRNA-transfected cells even in the tissue were about 3-fold relative to the fumarate levels and absence of 3-nitropropionic acid (19). Time course experi- increased in tumors with germline SDH mutations up to about ments with diethyl fumarate indicated that stabilization of 13-fold relative to the level in corresponding tumors without HIF-1 was detectable already at 8 h and that the VEGF SDH mutations (19). It is very clear from our data that the production increased between 8 and 20 h in a time- and fumarate levels in FH-deficient tumors, and probably also the concentration-dependent manner indicating that the two succinate levels in SDH-deficient tumors, would strongly events were coupled. inhibit the HIF-P4Hs. Our data thus support recent suggestions Although transfection of HEK293 cells with SDH siRNA or that such tumors accumulate fumarate or succinate to levels incubation with 20 mM succinate dimethyl ester for 48 h has that inhibit the HIF-P4Hs and stabilize HIF-1, leading to been reported to cause stabilization of HIF-1 (18), we found expression of HIF target proteins such as VEGF (18, 19, 38). In no stabilization in cells treated with 5 mM succinate dimethyl or agreement with this hypothesis, both FH- and SDH-deficient diethyl ester for 20 h or in the VEGF production even with 20 tumors, and also tumors arising from pVHL mutations associ- mM concentrations of either of these compounds. Others have ated with the VHL syndrome, have been shown to overproduce likewise seen no stabilization of HIF-1 with succinate dimeth- HIF-1 and VEGF and to have a high microvessel density (18, yl ester in the absence of 3-nitropropionic acid (19). These find- 19, 37, 38, 42). It should be noted, however, that although the ings are explained at least in part by our data indicating that the increased HIF-1 levels are likely to play an important role in IC and K values of the three HIF-P4Hs for succinate are 50 i the pathology of these tumors, such as the high microvessel much higher than those for fumarate and possibly additionally density, there is no evidence that HIF is sufficient to induce in part by a less efficient cellular uptake of diethyl and dimethyl tumorigenesis, and there is indeed some evidence that it is not succinate and their cleavage by cellular esterases than those of (37). The very high cellular fumarate or succinate levels are diethyl and dimethyl fumarate. likely to have various additional effects on cellular metabolism, Diethyl fumarate also increased Epo production by cultured Hep3B cells. Interestingly, this effect was seen at much lower some of which may play a crucial role in tumor formation. Although overproduction of reactive oxygen species has been speculated as a mechanism by which FH and SDH mutations V. Gu¨ nzler, personal communication. may mediate the induction of HIF-1, two very recent studies FEBRUARY 16, 2007• VOLUME 282 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4531 Inhibition of HIF Prolyl 4-Hydroxylases 17. Koivunen, P., Hirsila¨, M., Gu¨nzler, V., Kivirikko, K. I., and Myllyharju, J. found no evidence for the generation of these species in FH- or (2004) J. Biol. Chem. 279, 9899–9904 SDH-deficient cells (18, 38). 18. Selak, M. A., Armour, S. M., MacKenzie, E. D., Boulahbel, H., Watson, The IC and K values of FIH for fumarate and succinate 50 i D. G., Mansfield, K. D., Pan, Y., Simon, M. C., Thompson, C. B., and were found to be more than 10 mM, our data thus indicating Gottlieb, E. (2005) Cancer Cell 7, 77–85 that inhibition of FIH is not likely to contribute to the expres- 19. Isaacs, J. S., Jung, Y. J., Mole, D. R., Lee, S., Torres-Cabala, C., Chung, Y.-L., sion of HIF target genes in tumors from patients with FH or Merino, M., Trepel, J., Zbar, B., Toro, J., Ratcliffe P. J., Linehan, M. W., and SDH mutations. Despite the lack of FIH inhibition, we found Neckers, L. (2005) Cancer Cell 8, 143–153 20. Dalgard, C. L., Lu, H., Mohyeldin, A., and Verma, A. (2004) Biochem. J. increases in VEGF production in response to diethyl and di- 380, 419–424 methyl fumarate in all the cell types studied and in the FH 21. Lu, H., Dalgard, C. L., Mohyeldin, A., McFate, T., Tait, A. S., and Verma, A. fibroblasts and the HEK293 cells transfected with FH siRNA as (2005) J. Biol. Chem. 280, 41928–41939 well as in Epo production in Hep3B cells. These findings agree 22. Hirsila¨, M., Koivunen, P., Xu, L., Seeley, T., Kivirikko, K. I., and Myllyharju, with previous data demonstrating that silencing of HIF-P4Hs J. (2005) FASEB J. 19, 1308–1310 alone with siRNA is sufficient to lead to marked increases in the 23. 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(1997) EMBO J. 16, 1173–1180 4532 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 •FEBRUARY 16, 2007 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates: POSSIBLE LINKS BETWEEN CELL METABOLISM AND STABILIZATION OF HIF *

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DOI
10.1074/jbc.m610415200
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 7, pp. 4524 –4532, February 16, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates POSSIBLE LINKS BETWEEN CELL METABOLISM AND STABILIZATION OF HIF Received for publication, November 8, 2006 Published, JBC Papers in Press, December 19, 2006, DOI 10.1074/jbc.M610415200 ‡§¶ ‡§¶  ¶ ‡§¶ Peppi Koivunen , Maija Hirsila¨ , Anne M. Remes , Ilmo E. Hassinen , Kari I. Kivirikko , ‡§¶1 and Johanna Myllyharju ‡ § ¶ From the Collagen Research Unit, Biocenter Oulu, Departments of Medical Biochemistry and Molecular Biology and Neurology, University of Oulu, FIN-90014 Oulu, Finland The stability and transcriptional activity of the hypoxia-induc- get genes have important roles in many physiological and path- ible factors (HIFs) are regulated by two oxygen-dependent events ological events such as angiogenesis, vascular remodeling, that are catalyzed by three HIF prolyl 4-hydroxylases (HIF-P4Hs) erythropoiesis, glucose utilization, iron transport, cell prolifer- and one HIF asparaginyl hydroxylase (FIH). We have studied pos- ation, survival and apoptosis, and tumor progression (1–3). sible links between metabolic pathways and HIF hydroxylases by HIFs are  heterodimers, the human  subunit having three analyzing the abilities of citric acid cycle intermediates to inhibit isoforms, HIF-1 to HIF-3 (1–3). HIF-1 and HIF-2 are syn- purified human HIF-P4Hs and FIH. Fumarate and succinate were thesized constitutively, and hydroxylation of at least one of two identifiedas in vitroinhibitorsofallthreeHIF-P4Hs,fumaratehav- critical proline residues mediates their interaction with the von ing K values of 50–80 M and succinate 350–460 M, whereas Hippel-Lindau (VHL) E3 ubiquitin-ligase complex, which tar- neither inhibited FIH. Oxaloacetate was an additional inhibitor of gets them for rapid proteasomal degradation under normoxic all three HIF-P4Hs with K values of 400–1000 M and citrate of conditions (4–6). This hydroxylation is catalyzed in humans by HIF-P4H-3, citrate being the most effective inhibitor of FIH with a three cytoplasmic and nuclear HIF prolyl 4-hydroxylases (HIF- K of 110 M. Culturing of cells with fumarate diethyl or dimethyl P4Hs, also known as PHDs 1–3, HPHs 3-1, and EGLNs 2, 1, and ester, or a high concentration of monoethyl ester, stabilized 3, respectively) (7–9), which are distinct from the well charac- HIF-1 and increased production of vascular endothelial growth terized endoplasmic reticulum lumenal collagen P4Hs factor and erythropoietin. Similar, although much smaller, (C-P4Hs) (10, 11). All P4Hs are 2-oxoglutarate dioxygenases changes were found in cultured fibroblasts from a patient with and require Fe , 2-oxoglutarate, O , and ascorbate (1, 10). fumarate hydratase (FH) deficiency and upon silencing FH using Even small decreases in the O concentration (12, 13) will small interfering RNA. No such effects were seen upon culturing of inhibit the activities of the HIF-P4Hs so that HIF-1 and cells with succinate diethyl or dimethyl ester. As FIH was not inhib- HIF-2 escape degradation and dimerize with HIF-, this ited by fumarate, our data indicate that the transcriptional activity dimer then recognizing the HIF-responsive elements in numer- of HIF is quite high even when binding of the coactivator p300 is ous hypoxia-regulated target genes such as those for vascular prevented. Our data also support recent suggestions that the endothelial growth factor (VEGF), erythropoietin (Epo), glu- increased fumarate and succinate levels present in the FH and suc- cose transporters, and glycolytic enzymes (1–5). cinate dehydrogenase-deficient tumors, respectively, can inhibit Transcriptional activation in an oxygen-dependent manner the HIF-P4Hs with consequent stabilization of HIF-s and effects is another key event that regulates HIF activity. Hydroxylation on tumor pathology. of a critical asparagine residue in the C-terminal transactivation domain of HIF- blocks its interaction with the transcriptional coactivator p300 (14). The asparaginyl hydroxylase catalyzing The hypoxia-inducible transcription factors (HIFs) play a this modification is identical to a protein known as factor inhib- central role in the regulation of oxygen homeostasis. Their tar- iting HIF (FIH) (15, 16). It is also a 2-oxoglutarate dioxygenase and requires the same cosubstrates as the P4Hs (15) but its * This work was supported by Grants 200471 and 202469 from the Health catalytic and inhibitory properties are distinct from those of the Science Council and 44843 from the Finnish Centre of Excellence Pro- HIF-P4Hs (17). gramme 2000 –2005 of the Academy of Finland, the S. Juselius Foundation, and FibroGen Inc. (South San Francisco, CA). The costs of publication of this Certain citric acid cycle intermediates and related com- article were defrayed in part by the payment of page charges. This article pounds have recently been reported to inhibit activities of HIF- must therefore be hereby marked “advertisement” in accordance with 18 P4Hs, whereas no data are available on their effects on FIH. U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: P. O. Box 5000, University Succinate inhibited nonspecified total HIF-P4H activity in of Oulu, FIN-90014 Oulu, Finland. Tel.: 358-8-537-5740; Fax: 358-8-537- crude cell extracts in vitro with an apparent IC of 0.5 mM (18) 5811; E-mail: [email protected]. and purified HIF-P4H-2 in an in vitro VHL capture assay per- The abbreviations used are: HIF, hypoxia-inducible transcription factor; VHL, von Hippel-Lindau; HIF-P4H, HIF prolyl 4-hydroxylase; C-P4H, collagen formed under nonsaturating substrate concentrations and in prolyl 4-hydroxylase; VEGF, vascular endothelial growth factor; Epo, eryth- the presence of one succinate concentration with an apparent ropoietin; FIH, HIF asparaginyl hydroxylase; SDH, succinate dehydrogen- K of 9 M (19), whereas fumarate had in a similar assay an ase; FH, fumarate hydratase; siRNA, small interfering RNA; HEK293, human embryonic kidney 293. apparent K of 3 M (19). In agreement with these data, cultur- This is an Open Access article under the CC BY license. 4524 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 •FEBRUARY 16, 2007 Inhibition of HIF Prolyl 4-Hydroxylases ing of cancer cells with 20 mM succinate dimethyl ester for 48 h ues obtained are often somewhat different from those obtained (18) or with 2.5 mM succinate diethyl ester or 0.5 mM fumarate in assays involving capture of hydroxylated HIF- or a fragment monoethyl ester together with 3-nitropropionic acid (19), a of this by pVHL. (Pro-Pro-Gly) was used as the substrate for a dual inhibitor of succinate dehydrogenase (SDH) and fumarate purified recombinant human type I C-P4H (23). The K values hydratase (FH), led to stabilization of HIF-1, and a similar were determined by adding the 2-oxoglutarate analogues at effect was seen upon silencing of SDH or FH by the correspond- four constant concentrations while varying the concentration ing siRNA (18, 19). Oxaloacetate and pyruvate have likewise of 2-oxoglutarate or the peptide substrate. been reported to stabilize HIF-1 in cultured cancer cell lines Stabilization of HIF-1 by Diethyl Fumarate and Dimethyl and inactivate HIF-P4Hs in a manner reversible by ascorbate Fumarate in Cultured Cells—Human embryonic kidney (20, 21). (HEK293), hepatocellular carcinoma (Hep3B), and neuroblas- We have studied possible links between metabolic pathways toma (Kelly) cells, and fibroblasts (CRL-2086) were obtained and HIF hydroxylases by systematically analyzing the abilities from the American Type Culture Collection and cultured in of citric acid cycle intermediates and other aliphatic 2-oxoglut- Dulbecco’s (Biochrom) or Eagle’s (Sigma) minimal essential arate analogues to inhibit purified human HIF-P4Hs and FIH medium or in RPMI 1640 (Sigma) medium with 10% fetal under initial velocity conditions and in the presence of saturat- bovine serum (BioClear) and 50 g/ml ascorbate. They were 5 4 2 ing peptide substrate concentrations in vitro. Fumarate and then subcultured at a density of 1  10 or 1.6  10 cells/cm succinate were identified as inhibitors of all three HIF-P4Hs, in the case of fibroblasts, grown overnight, and incubated for fumarate having K values of 50–80 M and succinate 350–460 8–20 h with increasing concentrations of diethyl fumarate, di- M, whereas neither inhibited FIH to any significant extent methyl fumarate, monoethyl fumarate, diethyl succinate, or di- even at a 10 mM concentration. Oxaloacetate was an additional methyl succinate (Sigma). In control experiments the cells were inhibitor of all three HIF-P4Hs with K values of 400–1000 M treated with 300–500 M cobalt or subjected to hypoxia by and citrate was an inhibitor of HIF-P4H-3 with a K of 180 M, culturing in an airtight incubator (Billups-Rothenberg) under whereas pyruvate and many other 2-oxoglutarate analogues 1% O balanced with N . The viability of cells was determined 2 2 showed no inhibition of any of the HIF-P4Hs even at a 10 mM using trypan blue dye exclusion. Skin fibroblasts from a patient concentration, variations in ascorbate levels having no effect on with a homozygous loss of function mutation in the FH gene inhibition by oxaloacetate or pyruvate. FIH was inhibited most (24) and fibroblasts from a healthy control were cultured in effectively by citrate and oxaloacetate with K values of 110 and Dulbecco’s medium with 10% fetal bovine serum. Medium 400 M, respectively. Our further experiments using culturing samples were collected for analysis of the production of VEGF of cells with fumarate diethyl ester or dimethyl ester, which pass and Epo (see below) and total cell extracts were obtained by through cell membranes, led to stabilization of HIF-1 and to lysing the cells in 150 mM NaCl, 0.1% SDS, and 20 mM Tris-HCl, increases in VEGF production in all the cell types studied and in pH 6.8. Nuclear extracts were prepared by the NE-PER nuclear Epo production in human hepatocellular carcinoma Hep3B extraction reagent (Pierce). The extracts were analyzed by 8% cells. Similar, although much smaller, changes were found in SDS-PAGE under reducing conditions followed by Western cultured skin fibroblasts from a patient with autosomal reces- blotting with a monoclonal antibody against human HIF-1 TM sive FH deficiency and in cells transfected with FH siRNA. No (BD Biosciences) and ECL immunodetection. Antibodies to such effects were seen upon culturing of cells with succinate  tubulin (total extracts, Sigma) and  actin (nuclear extracts, diethyl ester or dimethyl ester. Novus Biologicals) were used as loading controls. RNA Interference—Four siRNAs of the FH coding sequence TM MATERIALS AND METHODS (set of 4 siGENOME duplexes, Dharmacon) were trans- Expression, Purification, and Activity Assays of Recombinant fected at 75 nM concentration into HEK293 cells using Lipo- TM HIF-P4Hs and FIH—FLAG His-tagged HIF-P4Hs 1–3 and FIH fectamine 2000 (Invitrogen) according to the manufactur- TM (17, 22) were expressed using recombinant baculoviruses in H5 er’s instructions. The Silencer GAPDH and Silencer insect cells cultured in suspension or on plates in Sf900IISFM negative control siRNAs (Ambion) were used as controls. serum-free medium (Invitrogen). The cells, seeded at a density Medium samples were collected 48 h after transfection, and of 1 10 /ml, were infected at a multiplicity of 5 and harvested total RNA and protein were isolated from the cells using the TM 72 h after infection, washed with a solution of 0.15 M NaCl and PARIS kit (Ambion). The ability of FH siRNAs to down- 0.02 M phosphate, pH 7.4, and homogenized in a 0.1 M NaCl, 0.1 regulate FH mRNA was studied by semiquantitative reverse TM M glycine, 10 M dithiothreitol, 0.1% Triton X-100, and 0.01 M transcriptase-PCR using the SMART PCR cDNA synthesis Tris buffer, pH 7.8. The soluble fractions were subjected to kit (BD Biosciences). PCR was carried out under the following purification with an anti-FLAG M2 affinity gel (Sigma) (17, 22). conditions: 1 min at 94 °C followed by 22 cycles of 1 min at HIF-P4H and FIH activities were assayed by methods based on 94 °C, 1 min at 60 °C, and 1 min at 72 °C. The primers used for measurement of the hydroxylation-coupled stoichiometric amplification were: 5-GGAGGTGTGACAGAACGCATGC- 14 14 release of CO from 2-oxo-[1- C]glutarate with synthetic CACC-3 and 5-GCTGCCTTGTCATACCCTATATGAGG-3. HIF-1 peptides as substrates and the K and IC values of the Stabilization of HIF-1 was analyzed by Western blotting of the m 50 purified enzymes were determined as described previously (12, protein samples as described above. 17, 22). This method makes it possible to measure HIF-P4H Analysis of the Production of VEGF and Epo—The VEGF and activities under true initial velocity conditions and using satu- Epo concentrations in the medium samples were measured rating peptide substrate concentrations, and therefore the val- using the Quantikine human VEGF and Epo immunoassays FEBRUARY 16, 2007• VOLUME 282 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4525 Inhibition of HIF Prolyl 4-Hydroxylases TABLE 1 K values of HIF-P4Hs, FIH, and C-P4H-I for 2-oxoglutarate and IC and K values for fumarate, succinate, oxaloacetate, and citrate m 50 i a,b a,b a,b c Compound Constant HIF-P4H-1 HIF-P4H-2 HIF-P4H-3 C-P4H-I FIH d e 2-Oxoglutarate K 2  0.4 1  0.2 12 420 25 Fumarate IC 120  10 80  30 60  30 ND 10000 K 80  10 60  20 50  15 190  30 10000 Succinate IC 830  540 510  310 570  170 ND 10000 K 350  20 460  70 430  200 400 10000 Oxaloacetate IC 1000  420 3800  1400 1200  350 ND 1400  370 K 400  130 1000  50 590  120 100 400  40 Citrate IC 6300  1300 4800  1600 550  200 ND 850  190 K ND 1800  320 180  80 450 110  30 Values are mean  S.D. from three to ten independent experiments. The IC values of HIF-P4Hs 1, 2, and 3 for pyruvate, malate, isocitrate, 2-oxoadipinate, 2-oxovalerate, 2-oxobutyrate, adipinate, glutarate, malonate, 3-oxoglutarate, levulinate, and lactate were at least 10 mM, except those of HIF-P4H-3 for glutarate and HIF-P4H-2 for 3-oxoglutarate, which were 6 and 5 mM, respectively. Similar values were obtained from at least two independent experiments. The IC value of FIH for isocitrate was about 5 mM, those for all the other compounds being at least 10 mM. Similar values were obtained from at least two independent experiments. Ref. 43. Ref. 17. ND, not determined. Ref. 26. Ref. 27. (R&D Systems) according to the manufacturer’s instructions. Inhibition of the purified recombinant HIF-P4Hs and FIH Statistical analyses were performed with SPSS 14.0 for was studied using six citric acid cycle intermediates and 10 Windows. other aliphatic 2-oxoglutarate analogues (Table 1). The IC Determination of Cellular Fumarate Concentrations—Equal values of the HIF-P4Hs and FIH were first determined in the numbers of HEK293 cells were incubated in the absence and presence of 10 and 50–100 M 2-oxoglutarate, respectively. presence of 40 M diethyl fumarate for 20 h, the culture Fumarate was found to be the most efficient inhibitor of the medium was carefully removed, and the cell pellets were imme- three HIF-P4Hs, with IC values of 120, 80, and 60 M for diately cooled in liquid N . The frozen pellets were lysed in 8% 2 HIF-P4Hs 1, 2, and 3, respectively, whereas succinate had IC cold perchloric acid and centrifuged. The supernatant was col- values of 830, 510, and 570 M (Table 1). The IC values of lected and the remaining pellet was washed and extracted with HIF-P4Hs 1, 2, and 3 for oxaloacetate were 1, 3.8, and 1.2 mM, 6% perchloric acid and centrifuged. The supernatants were respectively, and those for citrate 6.3, 4.8, and 0.55 mM, whereas pooled and neutralized by K CO and used to determine the 2 3 the IC values for pyruvate, malate, isocitrate, 2-oxoadipinate, amount of fumarate with fumarase, malate dehydrogenase, and 2-oxovalerate, 2-oxobutyrate, adipinate, glutarate, malonate, aspartate aminotransferase (Sigma) with a Fluoromax-2 spec- 3-oxoglutarate, levulinate, and lactate were at least 10 mM for all trofluorometer (HORIBA Jobin Yvon, Inc., Edison, NJ) as three isoenzymes, except that glutarate had an IC of about 6 described previously (25) with the exception that glycyl glycine, mM for HIF-P4H-3 and 3-oxoglutarate had an IC of about 5 pH 9, supplemented with glutamic acid was used as a buffer. mM for HIF-P4H-2 (Table 1). The maleate present in the commercial fumarase preparation FIH was inhibited most effectively by citrate, with an IC of was removed by gel filtration in a PD-10 column (GE Health- 850 M (Table 1). The IC values of FIH for oxaloacetate and care). The excitation wavelength was 340 nm and emission was isocitrate were about 1.4 and 5 mM, respectively, whereas those recorded at 460 nm. Samples of skin fibroblasts from a patient of all the other compounds tested, including fumarate, succi- with a homozygous FH mutation and from a healthy control nate, and pyruvate were at least 10 mM (Table 1). were prepared and analyzed as above. Inhibition of the HIF-P4Hs by fumarate, succinate, oxaloac- etate, and citrate, the compounds with the lowest IC values RESULTS among those tested, and of FIH by citrate and oxaloacetate, its Inhibition of HIF-P4Hs and FIH by Citric Acid Cycle Intermedi- most effective inhibitors, was analyzed in more detail. The ates and Certain Other Aliphatic 2-Oxoglutarate Analogues— mode of inhibition and K values were determined by adding FLAG His-tagged human HIF-P4Hs 1–3 and FIH were fumarate, succinate, oxaloacetate, or citrate at four constant expressed in insect cells, which were harvested 72 h after infec- concentrations while varying the concentration of 2-oxoglut- tion, homogenized in a buffer containing Triton X-100, and arate or the peptide substrate. The inhibition was found to be centrifuged. The recombinant enzymes were purified from the competitive with respect to 2-oxoglutarate (as shown for fuma- soluble fraction by anti-FLAG affinity chromatography and rate with HIF-P4Hs 2 and 3, succinate with HIF-P4H-1, and their activities were assayed by methods based on measurement citrate with FIH in Fig. 1, A–D) and noncompetitive with of the hydroxylation-coupled stoichiometric release of CO respect to the peptide substrate (data not shown). The K values from 2-oxo-[1- C]glutarate (12, 17, 22). The K values of the of HIF-P4Hs 1, 2, and 3 for fumarate were 80, 60, and 50 M, purified HIF-P4Hs for 2-oxoglutarate were found to be 1–2 M for isoenzymes 1 and 2, and 12 M for isoenzyme 3 (Table 1), respectively, and those for succinate 350, 460, and 430 M, these values being distinctly lower than those previously whereas the values for oxaloacetate ranged from 400 to 1000 reported for the HIF-P4Hs in crude insect cell extracts (12). M and the K values of HIF-P4H-2 and HIF-P4H-3 for citrate 4526 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 •FEBRUARY 16, 2007 Inhibition of HIF Prolyl 4-Hydroxylases FIGURE 1. Competitive inhibition of HIF-P4H-2 (A) and HIF-P4H-3 (B) by fumarate, HIF-P4H-1 by succinate (C), and FIH by citrate (D) with respect to 2-oxoglutarate. Inhibition of purified HIF hydroxylases was studied by adding fumarate, succinate, or citrate at 4 constant concentrations (indicated with the symbols given in the figures) while varying the concentration of 2-oxoglutarate. K values were calculated from the plots of the slopes of the lines at the inhibitor concentrations studied (insets), the intercept on the x axis being equal to K . Essentially identical K values were measured in at least three independent i i experiments (see Table 1). were 1800 and 180 M, respectively (Table 1). The K values of cinate diethyl or dimethyl ester, all of which are converted to FIH for citrate and oxaloacetate were 110 and 400 M, respec- fumarate or succinate by the action of cellular esterases (28, 29). tively, whereas those for fumarate and succinate were more These compounds gave no inhibition of purified HIF-P4Hs in than 10 mM (Table 1). vitro even at 10 mM concentrations (details not shown). In con- Succinate, oxaloacetate, and citrate have also been reported trol experiments, cells were treated with cobalt or cultured (26, 27) to inhibit the C-P4Hs, their K values for the C-P4H under hypoxia. Total cell extracts and nuclear extracts were isoenzyme I being 400, 100, and 450 M, respectively (Table 1). prepared and analyzed by 8% SDS-PAGE under reducing con- Our current data indicate that fumarate is an additional ditions followed by Western blotting with a HIF-1 antibody C-P4H-I inhibitor, with a K of 190 M (Table 1). and an  tubulin antibody as a loading control. Stabilization of As oxaloacetate and pyruvate have been reported to inhibit HIF-1 was seen in total extracts from the HEK293, Hep3B, the HIF-P4Hs in an in vitro VHL capture assay in the presence and Kelly cells and fibroblasts with 20–100, 10–100, 40–120, of nonsaturating ascorbate levels (30–100 M) (20, 21), we also and 100–150 M diethyl fumarate, respectively (Fig. 2, A–D), studied inhibition of the HIF-P4Hs by these two compounds in higher concentrations leading to cell detachment (as shown by the presence of varying ascorbate concentrations. However, the decreased staining for  tubulin). Similar results were obtained IC values obtained under these conditions were identical to with dimethyl fumarate (as shown for HEK293 cells in Fig. 2E), those determined in the presence of 2 mM ascorbate (details not whereas no stabilization of HIF-1 was seen with succinate shown). diethyl or dimethyl ester in a concentration range of 50 M to 5 Stabilization of HIF-1 by Fumarate Diethyl and Dimethyl mM (as shown for succinate dimethyl ester in HEK293 cells in Esters in Cultured Cells—To study whether fumarate and suc- Fig. 2F). Stabilization of HIF-1 by diethyl fumarate was also cinate stabilize HIF-1, we cultured human embryonic kidney seen in nuclear extracts (as shown for the HEK293, Hep3B, and (HEK293), hepatocellular carcinoma (Hep3B), and neuroblas- Kelly cells in Fig. 3, A–C). Fumarate monoethyl ester also sta- toma (Kelly) cells and fibroblasts (CRL-2086) for 20 h in the bilized HIF-1 (as shown for nuclear extracts from HEK293 presence of increasing concentrations of the membrane-per- cells in Fig. 3D), but the concentration required for a detectable meable fumarate diethyl, dimethyl or monoethyl ester, or suc- stabilization was 5 mM, being thus about 2 orders of magnitude FEBRUARY 16, 2007• VOLUME 282 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4527 Inhibition of HIF Prolyl 4-Hydroxylases FIGURE 3. Stabilization of HIF-1 by fumarate diethyl and monoethyl esters in cultured cells. HEK293 (A), Hep3B (B), and Kelly cells (C) were incu- bated for 20 h with increasing concentrations (M, indicated below the lanes) of fumarate diethyl ester. HEK293 cells were also incubated with increasing concentrations of fumarate monoethyl ester (D). Stabilization of HIF-1 in cells cultured under hypoxic conditions (lanes H,1%O ) is also shown. Nuclear extracts from cells were analyzed by 8% SDS-PAGE under reducing conditions followed by Western blotting with HIF-1 and  actin antibodies and ECL immunodetection. The exposure times of lanes H are 12, 12, and 6 times shorter than those of other lanes in A, C, and D, respectively. Lanes 5000 and H in D were run in a parallel gel at the same time as lanes 0 –1000. Similar results were obtained from at least three independent experiments. FIGURE 2. Stabilization of HIF-1 by fumarate diethyl and dimethyl esters and succinate dimethyl ester in cultured cells. HEK293 (A), Hep3B (B) and Kelly cells (C), and fibroblasts (D) were incubated for 20 h with increasing concentrations (M, indicated below the lanes) of membrane-permeable FIGURE 4. Stabilization of HIF-1 in cultured fibroblasts from a patient fumarate diethyl ester. HEK293 cells were also incubated with increasing con- with autosomal recessive FH deficiency. Skin fibroblasts from a patient centrations of fumarate dimethyl ester (E) and succinate dimethyl ester (F). with a homozygous Q376P mutation in the FH gene (FH ) and control fibro- Stabilization of HIF-1 in cells cultured under hypoxic conditions (lanes H,1% blasts (FH ) were cultured under normoxia (N) and hypoxia (H,1%O ). Total O ) is also shown. Total cell extracts were analyzed by 8% SDS-PAGE under cell extracts were analyzed by 8% SDS-PAGE under reducing conditions fol- reducing conditions followed by Western blotting with HIF-1 and  tubulin lowed by Western blotting with HIF-1 and  tubulin antibodies and ECL antibodies and ECL immunodetection. The exposure times of lanes H are 10, 4, immunodetection. The exposure times of lanes H detected by the HIF-1 12, and 20 times shorter than those of other lanes in A–D, respectively. Similar antibody are 10 times shorter than those of lanes N. Similar results were results were obtained from at least three independent experiments. obtained from at least three independent experiments. higher than the concentration of fumarate diethyl ester required In further experiments endogenous FH was silenced by for a similar extent of stabilization (compare with Fig. 3A). transfecting HEK293 cells with FH-specific siRNAs. FH mRNA Stabilization of HIF-1 in Cultured Fibroblasts from a level, analyzed by reverse transcriptase-PCR, was found to be Patient with Autosomal Recessive FH Deficiency and in HEK293 lower in the FH siRNA-transfected cells than control-trans- Cells Transfected with FH siRNA—To study whether lack of fected ones (Fig. 5A), and a very weak but reproducible stabili- endogenous FH leads to stabilization of HIF-1, skin fibroblasts zation of the HIF-1 was seen in the FH siRNA-transfected from a patient with a homozygous Q376P mutation in the FH cells (Fig. 5B). gene (24) were cultured and analyzed as above. The level of FH Increased VEGF Production by Diethyl Fumarate, Dimethyl activity in the mitochondrial and cytosolic fractions of cultured Fumarate, and FH Deficiency in Cultured Cells—To study fibroblasts from this patient is only 0.5% of the control mean whether the stabilized HIF-1 caused increased expression of a (24). A very weak but distinct stabilization of HIF-1 was seen HIF target gene, we assayed the amount of VEGF in medium in extracts from these cells cultured under normoxia in three samples collected from cells cultured in the presence of increas- independent experiments, whereas no HIF-1 was detected in ing diethyl fumarate concentrations in 3 to 5 independent control fibroblasts (Fig. 4). HIF-1 was stabilized to an equal experiments. Dose-dependent increases were found in VEGF extent in the FH mutant cells and in control fibroblasts cultured production, the highest mean values being 640% with 80 M under hypoxia (Fig. 4). diethyl fumarate in HEK293 cells, 250% with 100 M in Hep3B 4528 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 •FEBRUARY 16, 2007 Inhibition of HIF Prolyl 4-Hydroxylases cells, 240% with 20 M in Kelly cells, and 210% with 100 M in No increase in VEGF production was found with diethyl succi- fibroblasts as compared with that in nontreated cells (as shown nate or dimethyl succinate when studied in a concentration for HEK293, Hep3B, and Kelly cells in Fig. 6, A–C). The highest range of 50 M to 20 mM (details not shown). mean values seen in various cells range from about 30 to 100% To study the temporal relation of cell viability, HIF-1 stabi- of those obtained under 1% O in the same cells, the increases in lization, and VEGF production in the presence of increasing VEGF production by diethyl fumarate thus being much larger concentrations of diethyl fumarate, a time course analysis was than those seen in the extent of stabilization of HIF-1 (Fig. 2). performed with HEK293 cells. The viability of cells treated with A similar effect was seen with dimethyl fumarate, although the 40 M diethyl fumarate was 93–95% at 12 and 20 h, no differ- magnitude of the increase was smaller (as shown for HEK293 ence in viability being seen between the two time points (data cells in Fig. 6D). The different levels obtained are probably due not shown). The viability of cells treated with 100 M diethyl in part to differences in the uptake of diethyl fumarate and fumarate was likewise 94% at 12 h, but decreased to 83% at 20 h. dimethyl fumarate and their conversion to fumarate in various Stabilization of HIF-1 was seen in total extracts from cells cell types, in part to differences in the detachment of cells at cultured in the presence of 10–100 M diethyl fumarate for 8 h, high diethyl and dimethyl fumarate concentrations (see the the amount of stabilized HIF-1 increasing at 12 and 20 h (Fig. 7, decreased staining for  tubulin in Fig. 2) and in part to differ- A–C). The amount of VEGF secreted into the culture medium ences in the abilities of various cell types to produce VEGF (22). increased in a time- and dose-dependent manner (Fig. 7D). Additional experiments demonstrated that the stabilized HIF-1 also caused increased VEGF production in the fibro- blasts from the patient with autosomal recessive FH deficiency and a small increase in HEK293 cells transfected with FH siRNA (above). The amount of VEGF in the medium of FH fibroblasts was 170% (p 0.05) of that in the medium of control fibroblasts (Fig. 6F), and the amount in the medium of the siRNA-transfected HEK293 cells was 140% of that in the medium of the non-transfected cells, although the last mentioned increase was not statistically significant (data not shown). FIGURE 5. Stabilization of HIF-1 in cells transfected with FH siRNA. Increased Fumarate Concentrations in HEK293 Cells Cul- HEK293 cells were transfected with FH-specific (lanes 4), glyceraldehyde-3- tured with Diethyl Fumarate and FH Mutant Skin Fibro- phosphate dehydrogenase (GAPDH)-specific (lanes 2), and negative control blasts—Cellular fumarate concentration was measured in (lanes 3) siRNAs. Non-transfected controls are shown in lanes 1. mRNA levels of FH (upper panel) and GAPDH (lower panel) were analyzed by reverse tran- HEK293 cells cultured with 40 M diethyl fumarate for 20 h and scriptase-PCR (A). Total cell extracts were analyzed by 8% SDS-PAGE under in cultured skin fibroblasts from a patient with autosomal reducing conditions followed by Western blotting with HIF-1 and  tubulin antibodies and ECL immunodetection (B). recessive FH deficiency. Control experiments indicated that the FIGURE 6. Increased VEGF and Epo production by diethyl fumarate, dimethyl fumarate, and FH deficiency in cultured cells. Production of VEGF by HEK293 (A), Hep3B (B), and Kelly cells (C) incubated for 20 h with increasing concentrations of diethyl fumarate (indicated below the columns), HEK293 cells incubated with dimethyl fumarate (D), and fibroblasts from a patient with autosomal recessive FH deficiency (F). Production of Epo was also measured from the Hep3B cells incubated with diethyl fumarate (E). In control experiments the cells were subjected to hypoxia (H,1%O ) or incubated with 300 or 500 M CoCl . 2 2 The amounts of VEGF and Epo were measured in medium samples by immunoassay. The values are mean S.D. from at least three individual experiments, and their statistical significance in comparison to the controls in a one-tailed one sample t test are indicated by asterisks;*, p 0.05; **, p 0.01; and ***, p 0.005. FEBRUARY 16, 2007• VOLUME 282 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4529 Inhibition of HIF Prolyl 4-Hydroxylases TABLE 2 Fumarate concentrations in HEK293 cells cultured in the presence of diethyl fumarate for 20 h and in fibroblasts from a patient with autosomal recessive FH deficiency Cellular fumarate concentration Cells 6 b pmol/10 cells % Control Micromolar HEK293 123  21 100 37 HEK293  40 M 219  83 178 66 diethyl fumarate FH fibroblasts 108  28 100 22 / c FH fibroblasts 205  6 190 42 Values are from three to six measurements. The approximate M cellular concentrations were calculated using the reported 12 12 volumes of 3.3 10 and 4.9 10 liters for HEK293 cells (30) and fibroblasts (31), respectively. Statistical significance versus corresponding control cells (Student’s t test) was p 0.02. and in the latter cells by FH deficiency (Table 2). The cellular fumarate levels were converted to micromolar concentrations by using reported (30, 31) values for the volumes of the two cell types. Such calculations suggested that the concentration in cultured HEK293 cells was about 40 M and increased to about 70 M by incubation with 40 M diethyl fumarate, whereas the concentration in cultured control fibroblasts was about 20 M, and that in the FH-deficient fibroblasts about 40 M (Table 2). All these values are within concentrations that inhibited the activities of the three purified HIF-P4Hs, the concentrations in the diethyl fumarate-treated cells even exceeding the K values of HIF-P4Hs 2 and 3 (Table 1 and Fig. 1). Increased Epo Production by Diethyl Fumarate in Cultured Hep3B Cells—Production of Epo was also measured in Hep3B cell medium samples in the above experiments with diethyl fumarate. An increase of 220% was found with the lowest diethyl fumarate concentration that led to a detectable stabili- zation of HIF-1 in Hep3B cells (Fig. 6E), this value being only about 25% of that obtained under 1% O . Interestingly, the value decreased to control level or even below it with concen- trations that led to the highest increases in VEGF production (compare with Fig. 5B). DISCUSSION Our data demonstrate that three citric acid cycle intermedi- ates, fumarate, succinate, and oxaloacetate, inhibit all three human HIF-P4Hs in vitro, fumarate being markedly more FIGURE 7. Time course analysis of HIF-1 stabilization and VEGF produc- effective than the two others, its K for the most abundant and tion in HEK293 cells at increasing diethyl fumarate concentrations. Total extracts from HEK293 cells incubated with increasing concentrations (M, hence most important HIF-P4H, isoenzyme 2 (32, 33), being 60 indicated below the lanes) of diethyl fumarate for 8 (A), 12 (B), and 20 h (C) M, i.e. about 13% of that of succinate, whereas oxaloacetate were analyzed by 8% SDS-PAGE under reducing conditions followed by West- was the weakest inhibitor, its K for isoenzyme 2 being 1 mM. ern blotting with HIF-1 and tubulin antibodies and ECL immunodetection. i Similar results were obtained in at least three independent experiments. The Citrate was an effective inhibitor of HIF-P4H-3 but not of the amount of VEGF was measured in the corresponding medium samples by two others. The K values determined here for fumarate and immunoassay (D). In control experiments the cells were subjected to hypoxia succinate under initial velocity conditions and using four inhib- (H,1%O ). The values are mean  S.D. from at least three independent experiments. itor concentrations are in reasonable agreement with the IC of about 0.5 mM reported for succinate in assays of nonspecified enzymatic assay used here did not utilize diethyl fumarate as a total HIF-P4H activity in crude cell extracts in vitro (18) but the substrate, and thus the increased concentration measured in K for fumarate is about 20-fold and succinate 50-fold when the HEK293 cells incubated with this compound represents compared with the apparent K values reported for these two that of intracellular fumarate produced from the membrane- compounds using purified HIF-P4H-2 and an in vitro VHL cap- permeable ester. The fumarate concentrations, when expressed ture assay performed under nonsaturating substrate concentra- per 10 cells, were highly similar in the non-treated HEK293 tions and in the presence of only one inhibitor concentration cells and control fibroblasts and increased to almost 2-fold by (19). Although oxaloacetate and pyruvate have been reported to the incubation of the former cells with 40 M diethyl fumarate stabilize HIF-1 in cultured cancer cells and to inhibit the HIF- 4530 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 •FEBRUARY 16, 2007 Inhibition of HIF Prolyl 4-Hydroxylases P4Hs in an in vitro VHL capture assay in the presence of non- diethyl fumarate concentrations than the increases in VEGF saturating ascorbate levels (20, 21), we found only a weak inhi- production, and the Epo levels returned to baseline values or bition by oxaloacetate, especially in the case of HIF-P4H-2, and even below them at concentrations that gave the highest no inhibition by pyruvate in the presence of 2 mM ascorbate and increases in VEGF production. This finding agrees with data no increase in the level of inhibition in the presence of lower indicating that hypoxic conditions can show selectivity with ascorbate concentrations. respect to stimulation of Epo versus VEGF (34). One possible Membrane-permeable diethyl and dimethyl ester derivatives explanation for such differences is that Epo has been found to of fumarate produced stabilization of HIF-1 in all the cell be regulated in Hep3B cells unequivocally by HIF-2, whereas types studied, and a very low extent of HIF-1 stabilization was VEGF is regulated by HIF-1 (35). additionally demonstrated in cultured fibroblasts from a Fumarate levels assayed in cultured HEK293 cells and human patient with autosomal recessive FH deficiency and in cells skin fibroblasts were about 40 and 20 M, respectively, being transfected with FH siRNA. Interestingly, by contrast with our about 70 and 30% of the K of the purified HIF-P4H-2 for fuma- findings, culturing with monoethyl fumarate stabilized HIF-1 rate. The concentration in HEK293 cells incubated with 40 M in human A549 lung carcinoma cells only when added in com- diethyl fumarate increased to about 70 M, i.e. slightly above the bination with 3-nitropropionic acid, a dual SDH and FH inhib- K of purified HIF-P4H-2, whereas the value in the FH-deficient itor (19), and the concentrations of monoethyl fumarate (0.5 fibroblasts was about 70% of this K . These concentrations mM) needed to obtain this effect in the presence of the inhibitor could thus be expected to lead to HIF-P4H inhibition, as sug- were much higher than those of diethyl and dimethyl fumarate gested by our data. needed in our study. This difference agrees with findings indi- Distinct VHL mutations that prevent the binding of HIF-s, cating that monoethyl esters of dicarboxylic acids are much less and thus their subsequent pVHL-mediated degradation, can efficient in passing through cell membranes than diethyl and lead to HIF-dependent Epo production and a hereditary poly- dimethyl esters, and our finding that monoethyl fumarate gave cythemia known as Chuvash polycythemia (36), or a cancer a detectable stabilization of HIF-1 only ata5mM concentra- syndrome (2, 37) that is characterized by highly vascular tumors tion. In agreement with our data, a very weak stabilization of that overproduce hypoxia-inducible mRNAs such as those for HIF-1 was seen in the A549 cells upon transfection with FH VEGF. SDH and FH mutations likewise predispose to domi- siRNA, whereas a much stronger stimulation was obtained in nantly inherited highly vascular tumors, findings in SDH muta- the presence of 3-nitropropionic acid (19). tions including pheochromocytoma, paraganglioma, renal cell The stabilized HIF-1 produced in our study by incubation carcinoma, and papillary thyroid cancer (18, 38–40) and those with diethyl or dimethyl fumarate or by the lack of FH activity in FH mutations oncluding uterine fibroids, skin leiomyomata, was transcriptionally active, as an increased production of a and papillary renal cell cancer (19, 38, 41). The fumarate levels HIF-1 target protein VEGF was found in all these cases, a in FH-deficient uterine fibroids were 200-fold relative to the small increase in the VEGF production having also been level in normal tissue, whereas the succinate levels in normal reported recently in FH siRNA-transfected cells even in the tissue were about 3-fold relative to the fumarate levels and absence of 3-nitropropionic acid (19). Time course experi- increased in tumors with germline SDH mutations up to about ments with diethyl fumarate indicated that stabilization of 13-fold relative to the level in corresponding tumors without HIF-1 was detectable already at 8 h and that the VEGF SDH mutations (19). It is very clear from our data that the production increased between 8 and 20 h in a time- and fumarate levels in FH-deficient tumors, and probably also the concentration-dependent manner indicating that the two succinate levels in SDH-deficient tumors, would strongly events were coupled. inhibit the HIF-P4Hs. Our data thus support recent suggestions Although transfection of HEK293 cells with SDH siRNA or that such tumors accumulate fumarate or succinate to levels incubation with 20 mM succinate dimethyl ester for 48 h has that inhibit the HIF-P4Hs and stabilize HIF-1, leading to been reported to cause stabilization of HIF-1 (18), we found expression of HIF target proteins such as VEGF (18, 19, 38). In no stabilization in cells treated with 5 mM succinate dimethyl or agreement with this hypothesis, both FH- and SDH-deficient diethyl ester for 20 h or in the VEGF production even with 20 tumors, and also tumors arising from pVHL mutations associ- mM concentrations of either of these compounds. Others have ated with the VHL syndrome, have been shown to overproduce likewise seen no stabilization of HIF-1 with succinate dimeth- HIF-1 and VEGF and to have a high microvessel density (18, yl ester in the absence of 3-nitropropionic acid (19). These find- 19, 37, 38, 42). It should be noted, however, that although the ings are explained at least in part by our data indicating that the increased HIF-1 levels are likely to play an important role in IC and K values of the three HIF-P4Hs for succinate are 50 i the pathology of these tumors, such as the high microvessel much higher than those for fumarate and possibly additionally density, there is no evidence that HIF is sufficient to induce in part by a less efficient cellular uptake of diethyl and dimethyl tumorigenesis, and there is indeed some evidence that it is not succinate and their cleavage by cellular esterases than those of (37). The very high cellular fumarate or succinate levels are diethyl and dimethyl fumarate. likely to have various additional effects on cellular metabolism, Diethyl fumarate also increased Epo production by cultured Hep3B cells. Interestingly, this effect was seen at much lower some of which may play a crucial role in tumor formation. Although overproduction of reactive oxygen species has been speculated as a mechanism by which FH and SDH mutations V. Gu¨ nzler, personal communication. may mediate the induction of HIF-1, two very recent studies FEBRUARY 16, 2007• VOLUME 282 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4531 Inhibition of HIF Prolyl 4-Hydroxylases 17. Koivunen, P., Hirsila¨, M., Gu¨nzler, V., Kivirikko, K. I., and Myllyharju, J. found no evidence for the generation of these species in FH- or (2004) J. Biol. Chem. 279, 9899–9904 SDH-deficient cells (18, 38). 18. Selak, M. A., Armour, S. M., MacKenzie, E. D., Boulahbel, H., Watson, The IC and K values of FIH for fumarate and succinate 50 i D. G., Mansfield, K. D., Pan, Y., Simon, M. C., Thompson, C. B., and were found to be more than 10 mM, our data thus indicating Gottlieb, E. (2005) Cancer Cell 7, 77–85 that inhibition of FIH is not likely to contribute to the expres- 19. Isaacs, J. S., Jung, Y. J., Mole, D. R., Lee, S., Torres-Cabala, C., Chung, Y.-L., sion of HIF target genes in tumors from patients with FH or Merino, M., Trepel, J., Zbar, B., Toro, J., Ratcliffe P. J., Linehan, M. W., and SDH mutations. Despite the lack of FIH inhibition, we found Neckers, L. (2005) Cancer Cell 8, 143–153 20. Dalgard, C. L., Lu, H., Mohyeldin, A., and Verma, A. (2004) Biochem. J. increases in VEGF production in response to diethyl and di- 380, 419–424 methyl fumarate in all the cell types studied and in the FH 21. Lu, H., Dalgard, C. L., Mohyeldin, A., McFate, T., Tait, A. S., and Verma, A. fibroblasts and the HEK293 cells transfected with FH siRNA as (2005) J. Biol. Chem. 280, 41928–41939 well as in Epo production in Hep3B cells. These findings agree 22. Hirsila¨, M., Koivunen, P., Xu, L., Seeley, T., Kivirikko, K. I., and Myllyharju, with previous data demonstrating that silencing of HIF-P4Hs J. (2005) FASEB J. 19, 1308–1310 alone with siRNA is sufficient to lead to marked increases in the 23. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Feb 16, 2007

References