HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/10/1308 04-3399fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hirsilä, M. Articles by Myllyharju, J. Search for Related Content PubMed PubMed Citation Articles by Hirsilä, M. Articles by Myllyharju, J.

Effect of desferrioxamine and metals on the hydroxylases in the oxygen sensing pathway

Maija Hirsilä^*, Peppi Koivunen^*, Leon Xu, Todd Seeley, Kari I. Kivirikko^* and Johanna Myllyharju^*,1

^* Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland;
FibroGen Inc., South San Francisco, California, USA

¹Correspondence: Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, P.O. Box 5000, University of Oulu, Oulu FIN-90014, Finland. E-mail: johanna.myllyharju{at}oulu.fi

SPECIFIC AIMS

Hypoxia-inducible transcription factor (HIF), a key regulator of O₂ homeostasis, is regulated by two oxygen-dependent events. Hydroxylation of specific prolines by HIF prolyl 4-hydroxylases (HIF-P4Hs) is a prerequisite for its proteasomal degradation, while hydroxylation of an asparagine by a hydroxylase known as factor inhibiting HIF (FIH) prevents its transcriptional activation. The HIF-P4Hs and FIH belong to 2-oxoglutarate dioxygenases and require Fe²⁺, 2-oxoglutarate, O₂, and ascorbate. Under hypoxia these hydroxylations are inhibited, HIF- is stabilized and gains its maximal transcriptional activity. The iron chelator desferrioxamine (DFO), cobalt, and nickel are well-known hypoxia mimics, but their exact mechanisms are largely unknown. Our aim was to purify the three recombinant human HIF-P4Hs, determine their specific activities and K_m values for iron and study the inhibition of these enzymes and FIH by DFO. We also studied the inhibition of the activities of the HIF-P4Hs and FIH by various metals and the effects of some of these metals on the stabilization of HIF-1 and the expression of VEGF in cultured cells.

PRINCIPAL FINDINGS

1. Purification and specific activities of HIF-P4Hs
Recombinant FLAGHis-tagged HIF-P4Hs 1–3 were expressed in insect cells and purified to near homogeneity by anti-FLAG chromatography. Their specific activities were at least 40–50 mol/mol/min, about one-third of that of purified recombinant human FIH and about one-tenth of that of the well-characterized human type I collagen P4H (C-P4H-I), but close to those of the three isoenzymes of lysyl hydroxylase (LH), another 2-oxoglutarate dioxygenase involved in collagen synthesis. As some HIF--like peptides give a higher V_max than our standard substrate, a 19-residue peptide corresponding to the C-terminal hydroxylation site of HIF-1, the specific activities measured with more effective substrates would reach at least 70 mol/mol/min. The HIF-P4Hs and FIH, like the C-P4Hs and LHs, also catalyzed in the presence of all cosubstrates, but in the absence of any peptide substrate an uncoupled decarboxylation of 2-oxoglutarate at rates that were somewhat lower than for the collagen hydroxylases.

2. Effect of Fe²⁺ on HIF-P4H activity
We previously showed that crude HIF-P4Hs retain partial activity even in the absence of added Fe²⁺. The same phenomenon was found here with purified HIF-P4Hs (e.g., HIF-P4H-3 retained 37% of its maximal activity in the absence of added Fe²⁺). K_m values for Fe²⁺ could therefore be determined only using HIF-P4Hs that already had considerable activity without any iron addition, and thus they do not represent true K_m values. The apparent K_m values for Fe²⁺ were very low (0.03 µM) in the case of HIF-P4Hs 1 and 2, and slightly higher (0.1 µM) in that of HIF-P4H-3, being 5- to 15-fold and 20- to 65-fold lower than those of FIH and C-P4H-I, respectively.

3. Effect of DFO on HIF-P4H and FIH activities
The effect of DFO on the in vitro activities of recombinant HIF-P4Hs and FIH was studied at a 5 µM Fe²⁺ concentration. Surprisingly, up to 1 mM DFO caused no inhibition of crude HIF-P4H-2 preparations and inhibited crude HIF-P4H-1 only by 10–20%. In contrast, the IC₅₀ of crude and purified HIF-P4H-3 was 10 µM and that of purified FIH 8 µM. HIF-P4H-3 was not completely inhibited at higher concentrations, however, as crude preparations retained 25% of the activity and purified preparations 10% even at 1 mM DFO, whereas FIH was totally inhibited at 25 µM DFO. Purified HIF-P4Hs 1 and 2 were inhibited by 40–50% with 10 µM DFO, the level of inhibition of HIF-P4H-1 increasing only to 60% and that of HIF-P4H-2 remaining steady 40–45% up to a 1 mM concentration. DFO produced a stronger inhibition in insect cells synthesizing recombinant HIF-P4H-2, by 75–80% with 100–300 µM DFO (activity expressed per unit enzyme protein), but complete inhibition was not achieved.

4. Inhibition of HIF-P4Hs and FIH by metals
Cobalt and nickel mimic hypoxia by stabilizing HIF-, but it is currently unknown to what extent this is due to inhibition of HIF-P4Hs, as cobalt and nickel are also reported to deplete intracellular ascorbate levels favoring iron oxidation and to become bound to HIF-, thereby inhibiting its degradation by VHL-dependent and VHL-independent pathways. Surprisingly, cobalt was found here to inhibit the HIF-P4Hs rather ineffectively (Table 1 ). Crude HIF-P4H-2 preparations were inhibited by only 20% with 100 µM cobalt and 40% even with a 2 mM concentration. Crude HIF-P4Hs 1 and 3 were inhibited slightly more effectively (Table 1) , but the extent of inhibition remained at 50–65% even with 500 µM cobalt. Purified HIF-P4Hs 1, 2, and 3 had IC₅₀ values of 40, 100, and 10 µM, respectively (Table 1) , but inhibition of HIF-P4Hs 1 and 2 increased only to slightly above 50% and 60% with a 500 µM concentration, whereas inhibition of HIF-P4H-3 increased to 90%. Cobalt was somewhat more effective in insect cells synthesizing recombinant HIF-P4H-2, 100-1000 µM cobalt leading to 40–70% inactivation. The activity of the enzyme synthesized in the presence of cobalt could not be increased by adding iron to the in vitro reaction mixture, indicating it was inactivated irreversibly.

Nickel was even less effective than cobalt (Table 1) , crude HIF-P4H-2 being inhibited by only 15% with 100 µM nickel and no more than 25% even with 1 mM. Crude HIF-P4H-1 was inhibited by 45% with 1 mM, while crude HIF-P4H-3 had an IC₅₀ of 300 µM, 70% inhibition being obtained with 1 mM nickel. Purified HIF-P4Hs 1, 2, and 3 were inhibited slightly more effectively than crude preparations, HIF-P4H-1 having an IC₅₀ of 130 µM, while 65% inhibition was reached with 1 mM, and HIF-P4H-2 was inhibited by 15–20% with a 100 µM concentration and 45% with 1 mM, HIF-P4H-3 having an IC₅₀ of 120 µM.

Among the other metals studied, zinc was the most efficient inhibitor of crude and purified HIF-P4H-3, with an IC₅₀ of 3-4 µM (Table 1) , and 100% inhibition was reached at 50 µM, whereas it was far less efficient for HIF-P4Hs 1 and 2 (Table 1) . Cadmium also inhibited all three HIF-P4Hs, magnesium and manganese being the least potent inhibitors (Table 1) .

Several metals were effective competitive inhibitors of FIH with respect to iron (as shown for zinc and cobalt in Fig. 1 ), most metals inhibiting FIH much more effectively than they did the HIF-P4Hs (Table 1) . Zinc, cobalt, and nickel were the most potent FIH inhibitors, with K_i values of 0.5, 1, and 4 µM, respectively, all much lower than those of the HIF-P4Hs (Table 1) . Cadmium and manganese were also effective FIH inhibitors, with a K_i of 10 µM for both metals (Table 1) , magnesium being the only metal tested that was a poor inhibitor of FIH (Table 1) .

View larger version (24K): [in this window] [in a new window]   Figure 1. Inhibition of FIH by zinc and cobalt with respect to iron. Inhibition of purified FIH by zinc and cobalt with respect to iron was studied by adding zinc (A) or cobalt (B) at 4–5 constant concentrations (indicated by symbols) while the concentration of iron was varied. Ki values were calculated from the plots of the slopes of the lines in panels A and B at the inhibitor concentrations studied (insets), the intercept on the X-axis being equal to -Ki.

5. Effect of cobalt, nickel, and zinc on the stabilization of HIF-1 and the production of VEGF in human HEK and Hep3B cells
HEK293 and Hep3B cells were cultured in the presence of increasing concentrations of cobalt, nickel, or zinc. As expected, cobalt and nickel led to stabilization of HIF-1, this being observed in both cell types with 100 µM cobalt and 200 µM nickel, whereas zinc caused no stabilization at concentrations up to 1 mM.

Cobalt and nickel were efficient inducers of VEGF expression in both cell types, but their effect was more marked in the HEK cells. Even 10 µM cobalt increased the VEGF production to 150% (P<0.05) of the control value in HEK cells, while 25 µM cobalt increased it to 190% (P<0.001) in HEK and 130% (P<0.05) in Hep3B cells. Expression increased further at higher concentrations, being 1040% and 380% with 300 µM cobalt in the HEK and Hep3B cells, respectively. No statistically significant increase in VEGF expression was achieved with nickel at concentrations below 100 µM, which increased the expression to 190% (P<0.01) in the HEK cells, whereas a 200 µM concentration was required to obtain an increase to 180% (P<0.001) in the Hep3B cells. Expression increased to 1400% and 290% with 500 µM nickel in HEK and Hep3B cells, respectively. Although zinc caused no detectable stabilization of HIF-1, it did cause a slight increase in VEGF production at very low concentrations, 10 µM zinc increasing it to 150% in both cell types (P<0.05), while 50 µM zinc gave levels of 170% (P<0.01) and 220% (P<0.001) in the HEK and Hep3B cells, respectively, no further increase being seen at 100 µM zinc. Higher concentrations caused detachment of the cells from the plates and abolished their VEGF expression.

CONCLUSIONS AND SIGNIFICANCE

The specific activities of the purified HIF-P4Hs were comparable to those of FIH and collagen hydroxylases, but the HIF-P4Hs differed from these enzymes in having much lower apparent K_m values for iron. In agreement with the firm iron binding, DFO was a much less efficient inhibitor of the HIF-P4Hs than of FIH or C-P4H-I. DFO may not be able to enter the catalytic site of any of these hydroxylases, the effective inhibition of FIH and the C-P4Hs probably being due to chelation of the free Fe²⁺ from the reaction mixture, whereas the HIF-P4Hs retained most of their in vitro activity even when all the free Fe²⁺ had been chelated, while the bound Fe²⁺ diffused out of the enzymes only to a low extent. In agreement with this suggestion, DFO was more effective in insect cells synthesizing a recombinant HIF-P4H, but complete inhibition was still not obtained.

Many metals were effective competitive inhibitors of FIH but, surprisingly, ineffective inhibitors of the HIF-P4Hs, especially of HIF-P4H-2, the most abundant and hence the most critical HIF-P4H in most cell types. An additional surprising finding was that the HIF-P4H synthesized in the presence of cobalt was inactivated irreversibly. The well-known stabilization of HIF- by cobalt and nickel is thus not due to a simple competitive inhibition of HIF-P4Hs, and the other reported stabilization mechanisms may at least contribute or even be more important than this (Fig. 2 ). The highly effective inhibition of FIH by these metals leads to full transcriptional activity of the low levels of HIF- that are present even in normoxic cells and also renders the stabilized HIF- transcriptionally fully active at higher metal concentrations (Fig. 2) . The minor increases seen in VEGF production by zinc are likewise probably due to the highly efficient inhibition of FIH, and thus expression of the full transcriptional activity of the small amounts of HIF- present in normoxic cells.

View larger version (31K): [in this window] [in a new window]   Figure 2. Possible mechanisms involved in the cobalt-induced up-regulation of HIF target genes in normoxic cells. A) Under normoxic conditions, HIF-1 is hydroxylated by the HIF-P4Hs and FIH. Proline hydroxylation leads to binding of HIF-1 to the VHL E3 ubiquitin ligase complex, followed by its rapid proteasomal degradation, while asparagine hydroxylation prevents binding of p300. B) Low cobalt concentrations cause no stabilization of HIF-1 and no inhibition of HIF-P4Hs, but FIH is effectively inhibited, which allows binding of p300 and thus expression of the full transcriptional activity of the small amounts of HIF-1 present even in normoxic cells. This leads to slight up-regulation of HIF target genes. C) Higher cobalt concentrations cause stabilization of HIF-1 and a marked up-regulation of its target genes. This may be due to at least three mechanisms: partial inhibition of HIF-P4Hs, depletion of ascorbate, which is required to maintain the HIF-P4Hs and FIH in an active state, and/or direct binding of cobalt to HIF-1, which may prevent its degradation by VHL-dependent and VHL-independent pathways.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3399fje;

This article has been cited by other articles:

				D. Magda, P. Lecane, Z. Wang, W. Hu, P. Thiemann, X. Ma, P. K. Dranchak, X. Wang, V. Lynch, W. Wei, et al. Synthesis and Anticancer Properties of Water-Soluble Zinc Ionophores Cancer Res., July 1, 2008; 68(13): 5318 - 5325. [Abstract] [Full Text] [PDF]

				Y.-K. Park, D.-R. Ahn, M. Oh, T. Lee, E. G. Yang, M. Son, and H. Park Nitric Oxide Donor, ({+/-})-S-Nitroso-N-acetylpenicillamine, Stabilizes Transactive Hypoxia-Inducible Factor-1{alpha} by Inhibiting von Hippel-Lindau Recruitment and Asparagine Hydroxylation Mol. Pharmacol., July 1, 2008; 74(1): 236 - 245. [Abstract] [Full Text] [PDF]

				M. K. Koski, R. Hieta, C. Bollner, K. I. Kivirikko, J. Myllyharju, and R. K. Wierenga The Active Site of an Algal Prolyl 4-Hydroxylase Has a Large Structural Plasticity J. Biol. Chem., December 21, 2007; 282(51): 37112 - 37123. [Abstract] [Full Text] [PDF]

				P. Pichiule, J. C. Chavez, A. M. Schmidt, and S. J. Vannucci Hypoxia-inducible Factor-1 Mediates Neuronal Expression of the Receptor for Advanced Glycation End Products following Hypoxia/Ischemia J. Biol. Chem., December 14, 2007; 282(50): 36330 - 36340. [Abstract] [Full Text] [PDF]

				E. I. Chang, S. A. Loh, D. J. Ceradini, E. I. Chang, S.-e Lin, N. Bastidas, S. Aarabi, D. A. Chan, M. L. Freedman, A. J. Giaccia, et al. Age Decreases Endothelial Progenitor Cell Recruitment Through Decreases in Hypoxia-Inducible Factor 1{alpha} Stabilization During Ischemia Circulation, December 11, 2007; 116(24): 2818 - 2829. [Abstract] [Full Text] [PDF]

				P. Koivunen, P. Tiainen, J. Hyvarinen, K. E. Williams, R. Sormunen, S. J. Klaus, K. I. Kivirikko, and J. Myllyharju An Endoplasmic Reticulum Transmembrane Prolyl 4-Hydroxylase Is Induced by Hypoxia and Acts on Hypoxia-inducible Factor {alpha} J. Biol. Chem., October 19, 2007; 282(42): 30544 - 30552. [Abstract] [Full Text] [PDF]

				G. Chachami, A. Hatziefthimiou, P. Liakos, M. G. Ioannou, G. K. Koukoulis, S. Bonanou, P.-A. Molyvdas, G. Simos, and E. Paraskeva Exposure of differentiated airway smooth muscle cells to serum stimulates both induction of hypoxia-inducible factor-1{alpha} and airway responsiveness to ACh Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L913 - L922. [Abstract] [Full Text] [PDF]

				F. A. Groenman, M. Rutter, J. Wang, I. Caniggia, D. Tibboel, and M. Post Effect of chemical stabilizers of hypoxia-inducible factors on early lung development Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L557 - L567. [Abstract] [Full Text] [PDF]

				A.J. Harvey, K.L. Kind, and J.G. Thompson Regulation of Gene Expression in Bovine Blastocysts in Response to Oxygen and the Iron Chelator Desferrioxamine Biol Reprod, July 1, 2007; 77(1): 93 - 101. [Abstract] [Full Text] [PDF]

				D. Viemann, M. Schmidt, K. Tenbrock, S. Schmid, V. Muller, K. Klimmek, S. Ludwig, J. Roth, and M. Goebeler The Contact Allergen Nickel Triggers a Unique Inflammatory and Proangiogenic Gene Expression Pattern via Activation of NF-{kappa}B and Hypoxia-Inducible Factor-1{alpha} J. Immunol., March 1, 2007; 178(5): 3198 - 3207. [Abstract] [Full Text] [PDF]

				P. Koivunen, M. Hirsila, A. M. Remes, I. E. Hassinen, K. I. Kivirikko, and J. Myllyharju Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates: POSSIBLE LINKS BETWEEN CELL METABOLISM AND STABILIZATION OF HIF J. Biol. Chem., February 16, 2007; 282(7): 4524 - 4532. [Abstract] [Full Text] [PDF]

				D. C. Birle and D. W. Hedley Suppression of the Hypoxia-Inducible Factor-1 Response in Cervical Carcinoma Xenografts by Proteasome Inhibitors Cancer Res., February 15, 2007; 67(4): 1735 - 1743. [Abstract] [Full Text] [PDF]

				P. Koivunen, M. Hirsila, K. I. Kivirikko, and J. Myllyharju The Length of Peptide Substrates Has a Marked Effect on Hydroxylation by the Hypoxia-inducible Factor Prolyl 4-Hydroxylases J. Biol. Chem., September 29, 2006; 281(39): 28712 - 28720. [Abstract] [Full Text] [PDF]

				J. Fandrey, T. A. Gorr, and M. Gassmann Regulating cellular oxygen sensing by hydroxylation Cardiovasc Res, September 1, 2006; 71(4): 642 - 651. [Abstract] [Full Text] [PDF]

				A. A. Qutub and A. S. Popel A computational model of intracellular oxygen sensing by hypoxia-inducible factor HIF1{alpha}. J. Cell Sci., August 15, 2006; 119(Pt 16): 3467 - 3480. [Abstract] [Full Text] [PDF]

				F. Kerendi, P. M. Kirshbom, M. E. Halkos, N.-P. Wang, H. Kin, R. Jiang, Z.-Q. Zhao, K. R. Kanter, R. A. Guyton, and J. Vinten-Johansen Cobalt Chloride Pretreatment Attenuates Myocardial Apoptosis After Hypothermic Circulatory Arrest Ann. Thorac. Surg., June 1, 2006; 81(6): 2055 - 2062. [Abstract] [Full Text] [PDF]

				P. S. Lecane, M. W. Karaman, M. Sirisawad, L. Naumovski, R. A. Miller, J. G. Hacia, and D. Magda Motexafin Gadolinium and Zinc Induce Oxidative Stress Responses and Apoptosis in B-Cell Lymphoma Lines Cancer Res., December 15, 2005; 65(24): 11676 - 11688. [Abstract] [Full Text] [PDF]

				R. H. Wenger, D. P. Stiehl, and G. Camenisch Integration of Oxygen Signaling at the Consensus HRE Sci. Signal., October 18, 2005; 2005(306): re12 - re12. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/10/1308 04-3399fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hirsilä, M. Articles by Myllyharju, J. Search for Related Content PubMed PubMed Citation Articles by Hirsilä, M. Articles by Myllyharju, J.