Báo cáo khoa học: Inhibitor of nuclear factor-kappaB alpha derepresses hypoxia-inducible factor-1 during moderate hypoxia by sequestering factor inhibiting hypoxia-inducible factor from hypoxia-inducible factor 1a ppt

Inhibitor of nuclear factor-kappaB alpha derepresses hypoxia-inducible factor-1 during moderate hypoxia by sequestering factor inhibiting hypoxia-inducible factor from hypoxia-inducible factor 1a Dong Hoon Shin 1, *, Shan Hua Li 1, *, Seung-Won Yang 1 , Byung Lan Lee 2 , Myung Kyu Lee 3 and Jong-Wan Park 1 1 Department of Pharmacology Ischemic ⁄ Hypoxic Disease Institute, Seoul National University College of Medicine, Korea 2 Department of Anatomy, Ischemic ⁄ Hypoxic Disease Institute, Seoul National University College of Medicine, Korea 3 Omics and Integration Research Center, KRIBB, Yuseong, Daejeon, Korea Hypoxia-inducible factor (HIF)-1 plays a crucial role in tumor promotion by switching on many genes that are required to overcome oxygen and nutrient defi- ciency. HIF-1 is composed of HIF-1a and HIF-1b (HIF-1b is also called ARNT) [1]. HIF-1a enables mRNA synthesis through its own transactivation domains (TADs), whereas ARNT assists the stabiliza- tion and binding between HIF-1a and DNA [2]. Under aerobic conditions, HIF-1a is hydroxylated by dioxygenase members [the HIF-prolyl-hydroxylases Keywords factor inhibiting hypoxia-inducible factor (FIH); hypoxia-inducible factor-1 (HIF-1); IjBa; nuclear factor-kappaB (NF-jB); protein interaction Correspondence J W. Park, Department of Pharmacology, Seoul National University College of Medicine, Seoul, Korea Fax: +82 2 7457996 Tel: +82 2 7408289 E-mail: parkjw@snu.ac.kr *These authors contributed equally to this work (Received 11 January 2009, revised 17 March 2009, accepted 21 April 2009) doi:10.1111/j.1742-4658.2009.07069.x Hypoxia and inflammation often develop concurrently in numerous diseases, and both hypoxia-inducible factor (HIF)-1a and nuclear factor- kappaB (NF-jB) are key transcription factors of stress response genes. An NF-jB inhibitor, inhibitor of NF-jBa (IjBa), was found to interact with factor inhibiting HIF (FIH) and to be hydroxylated by FIH. How- ever, FIH did not functionally regulate IjBa, and the consequence of the FIH–IjBa interaction thus remains uncertain. In the present study, we tested the possibility that IjBa regulates FIH. FIH–IjBa binding was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. Functionally, IjBa expression further enhanced the transcriptional activity of HIF-1a under hypoxic conditions. Furthermore, IjBa knockdown repressed HIF-1a activity. Mechanistically, IjBa derepressed HIF-1a activity by inhibiting the FIH-mediated Asn803 hydroxylation of HIF-1a. It was also found that IjBa activated HIF-1a by sequestering FIH from HIF-1a. However, the effect of IjBa on HIF-1a activity was only observed in atmospheres containing 1% or more of oxygen. After tumor necrosis factor-a treatment, IjBa downregulation, Asn803 hydroxylation and HIF-1a inactivation all occurred up to 8 h, but subsided later. On the basis of these results, we propose that IjBa plays a positive regula- tory role during HIF-1-mediated gene expression. Therefore, IjBa, owing to its interactions with NF-jB and HIF-1a , may play a pivotal role in the crosstalk between the molecular events that underlie inflammatory and hypoxic responses. Abbreviations ARD, ankyrin repeat domain; CAD, C-terminal transactivation domain; EPO, erythropoietin; FIH, factor inhibiting hypoxia-inducible factor; GFP, green fluorescent protein; HA, hemagglutinin; HIF, hypoxia-inducible factor; IjBa, inhibitor of nuclear factor-kappaB alpha; NF-jB, nuclear factor-kappaB; PHD, prolyl-hydroxylase; SD, standard deviation; sHIF-1a, stable hypoxia-inducible factor-1a; siRNA, short interfering RNA; TAD, transactivation domain; TNF-a, tumor necrosis factor-a; b-gal, b-galactosidase. 3470 FEBS Journal 276 (2009) 3470–3480 ª 2009 The Authors Journal compilation ª 2009 FEBS (PHD1–3)], ubiquitinated by von Hippel–Lindau pro- tein-containing E3-ubiquitin ligase complex, and conse- quently degraded by 26S proteasomes [3]. In addition, the transcriptional activity of HIF-1a is oxygen-depen- dently regulated by another dioxygenase member, fac- tor inhibiting HIF (FIH) [4]. HIF-1a harbors two TADs, an N-terminal TAD (amino acids 531–575) and a C-terminal TAD (CAD, amino acids 786–826) [5]. Furthermore, CAD predominantly affects gene expres- sion in an oxygen tension-dependent manner. How- ever, when Asn803 of CAD is hydroxylated by FIH, CAD cannot recruit p300⁄ CBP coactivator, and thus loses its transcriptional activity [4]. Furthermore, as both proline and asparagine hydroxylases require molecular oxygen to function, HIF-1a becomes stabi- lized and activated under oxygen-deficient conditions. FIH was first shown to interact with HIF-1a by yeast two-hybrid screening [6]. However, FIH has also been found to bind and hydroxylate asparagines in several proteins other than HIF-1a. For instance, immunoprecipitation and MS analyses revealed that SOCS Box protein 4 (ASB4) and Notch-1 ⁄ 2 were tar- geted and hydroxylated by FIH [7,8]. Ankyrin repeat domains (ARDs) in these proteins have been found to be FIH-binding sites. Moreover, Asn246 of ASB4 and Asn1945 ⁄ 2012 of Notch-1 have been identified as the amino acids hydroxylated by FIH. In terms of the bio- logical significance of FIH interaction, the FIH-medi- ated asparagine hydroxylation promoted vascular differentiation under aerobic conditions by activating ASB4 or accelerated myogenic differentiation by inhib- iting Notch [7,8]. However, the role of FIH in Notch signaling (FIH fi Notch) is controversial, because there was another report demonstrating that FIH had no significant effect on Notch activity [9]. Interestingly, in both reports, it was also examined whether Notch-1 affects FIH activity (Notch fi FIH), which is the reverse of the former mechanism. It was found that the intracellular domain of Notch-1 blocked the FIH- mediated HIF-1 repression by sequestering FIH through its ARD. Taken together, these findings sug- gest that FIH mediates cross-coupling between the Notch and HIF-1 signaling pathways. The FIH interaction with inhibitor of nuclear fac- tor-kappaB alpha (IjBa) or p105 [the precursor of p50 nuclear factor-kappaB (NF-jB)] was discovered before its interactions with ASB4 and Notch-1. By using yeast two-hybrid screening, Ratcliffe et al. identified two ARD-containing proteins, p105 and uveal autoantigen with coiled-coil domains and ankyrin repeats. They also found that ARD-containing IjBa was targeted by FIH and hydroxylated at Asn210 ⁄ 244 [10]. However, as FIH expression and knockdown both failed to affect IjBa binding to NF-jB or NF-jB activity, the biological significance of the FIH–IjBa interaction was not identified. In the present study, we investi- gated the consequence of the FIH–IjBa interaction in the opposite direction, namely, IjBa fi FIH. As FIH controls HIF-1a CAD activity, we focused on the roles of IjBa in HIF-1 activation and hypoxic gene regulation. Results IjBa associates with FIH To search for FIH-interacting proteins, we screened a HeLa cDNA library by using the yeast two-hybrid method, using full-length FIH as bait. Of 22 positive clones representing four different cDNAs, IjBa cDNA alone was shown to be fused to Gal4–TAD in the right frame, whereas Notch cDNAs were not fished out (Fig. S1). Hemagglutinin (HA)–FIH and Flag–IjBa were immunoprecipitated from HEK293 cells coex- pressing HA–FIH and Flag–IjBa, and Flag–IjB a and HA–FIH were then copurified in the precipitates (Fig. S2A). To determine whether FIH and IjBa asso- ciate endogenously, FIH or IjBa was immunoprecipi- tated in untransfected HEK293 cells. It was found that endogenous FIH and IjBa were coimmunoprecipitated by either anti-FIH or anti-IjBa serum (Fig. S2B). In agreement with what has been reported previously [10], binding of FIH to IjBa was confirmed in our experi- mental setting. FIH does not affect IjBa expression or NF-jB activity We first examined whether or not FIH regulates IjBa expression by binding IjBa. However, IjBa levels were not changed by FIH overexpression or knock- down (Fig. 1A). Furthermore, when IjBa was over- expressed or knocked down, FIH levels were unchanged (Fig. 1B). Next, we checked the possibility that FIH regulates NF-jB activity by interacting with IjBa. However, both basal and stimulated activities of NF-jB were unaffected by FIH expression or knock- down (Fig. 1C). On the basis of these results, we suggest that FIH does not affect the expression or the NF-jB-inhibitory function of IjBa. Ij Ba positively regulates HIF-1 activity by antagonizing FIH As the FIH–IjBa interaction did not affect NF-jB activity, we checked the possibility that this interaction D. H. Shin et al. HIF-1 activation by IjBa FEBS Journal 276 (2009) 3470–3480 ª 2009 The Authors Journal compilation ª 2009 FEBS 3471 affects HIF-1 activity, which is known to be regulated by FIH. HIF-1 activities were evaluated using a lucif- erase reporter plasmid containing the HIF-1-targeting erythropoietin (EPO)-enhancer segment. Reporter activity was found to increase under 5% O 2 hypoxia, and this activity was markedly enhanced by IjBa expression (Fig. 2A). Furthermore, the effect of IjBa on HIF-1 activity was found to be attenuated by FIH expression in a gene dose-dependent manner. In addi- tion, when endogenous IjBa was knocked down, the hypoxic activation of HIF-1 was significantly inhibited, but this was rescued by FIH inhibition (Fig. 2A). To verify the HIF-1 dependencies of the effect of IjBa or FIH on the reporter activity, we analyzed the activity of a reporter containing EPO-enhancer segment lack- ing the HIF-1-binding site, and confirmed that IjBa and FIH did not affect the reporter lacking the HIF- targeted segment (Fig. 2B). As FIH is known to repress the activity of HIF-1a CAD by hydroxylating Asn803, we examined whether CAD activity is regu- lated by the FIH–IjBa interaction using the Gal4– CAD ⁄ Gal4–Luc reporter system. Figure 2C shows that IjBa noticeably increased the hypoxic activation of wild-type CAD, but that it failed to increase the activ- ity of the CAD N803A mutant. IjBa is likely to acti- vate HIF-1a CAD in an Asn803-dependent manner. As Asn803 of CAD plays a critical role in the interac- tion between HIF-1a and p300 ⁄ CBP coactivator, we examined whether IjBa regulates HIF-1a and p300 binding. As expected, p300 was found to associate with HIF-1a under hypoxic conditions, whereas this binding did not occur under normoxic conditions. Moreover, HIF-1a and p300 binding was enhanced by IjBa expression and inhibited by IjBa knockdown (Fig. 2D). This result indicates that IjBa positively regulates the p300 recruitment by HIF-1a. HIF-1 derepression by IjBa occurs in 1–5% O 2 but not under more severe hypoxia Because molecular oxygen is an essential substrate of PHDs and FIH, the activities of PHDs and FIH should be restricted under oxygen-deficient conditions, although FIH has a much higher affinity for oxygen than PHDs, and thus can retain its activity under moderate hypoxia [11]. Furthermore, although HIF-1a is stabilized in moderately hypoxic conditions, it is not fully activated, owing to hydroxylation by FIH, which suggests that the IjBa-dependent regulation of HIF-1a activity, which depends on FIH, might occur only under moderate hypoxia, when sufficient oxygen is still available for FIH. To confirm the oxygen dependency of the activity of IjBa, we incubated HEK293 cells in 1% or 0.5% oxygen. In the EPO-enhancer reporter system, HIF-1 activity was modulated by IjBa expres- sion or knockdown in 1% oxygen (Fig. 3A, left panel), as was observed in 5% oxygen. However, IjBa did not regulate HIF-1 activity at an oxygen level of 0.5% (Fig. 3A, right panel). To rule out the possibility that the effect of IjBa on HIF-1 activity is related to the oxygen-dependent regulation of HIF-1a, we analyzed the transcriptional activity of stable HIF-1a (sHIF-1a), which is stably expressed oxygen-independently, owing A C B Fig. 1. FIH does not affect IjBa expression and NF-jB activity. (A) FIH does not affect IjBa expression. HEK293 cells were transfect- ed with the HA–FIH plasmid (0.2 or 0.4 lg) or FIH siRNA (40 or 80 n M). The empty vector (Mock) and green fluorescent protein (GFP) siRNA (80 n M) were transfected as transfection and siRNA controls, respectively. After being stabilized for 48 h, the cells were prepared for the analysis of cellular levels of IjBa and FIH by immunoblotting. b-Tubulin levels were analyzed as loading controls. (B) IjBa does not affect FIH expression. HEK293 cells were trans- fected with the Flag–IjBa plasmid (0.2 or 0.4 lg) or IjBa siRNA (40 or 80 n M). After being stabilized for 48 h, the cells were pre- pared for the analysis of cellular levels of FIH and IjBa by immuno- blotting. (C) NF-jB activity was not regulated by FIH. NF-jB reporter plasmid (0.02 lg) and b-gal plasmid (0.1 lg) were cotrans- fected with FIH plasmid (0.2 lg) or siRNA (40 n M). Flag–IjBa plas- mid was transfected to test the NF-jB-specific expression of the reporter luciferase. To stimulate NF-jB, cells were also stimulated with 10 ngÆmL )1 TNF-a or 2 ngÆmL )1 interleukin-1b (IL-1b) for 8 h before harvesting. Luciferase activities were measured using a Biocounter LB960 luminometer, and transfection efficiencies were normalized by b-gal activity. Each bar represents the mean ± SD (n = 4); NS, not statistically significant. HIF-1 activation by IjBa D. H. Shin et al. 3472 FEBS Journal 276 (2009) 3470–3480 ª 2009 The Authors Journal compilation ª 2009 FEBS to the deletion of both PHD-targeted and ARD1-tar- geted motifs. Expression of sHIF-1a stimulated EPO- enhancer activity even under normoxia, and further enhanced the reporter activity under hypoxia. More importantly, the sHIF-1a activity was also positively regulated by IjBa in 1% oxygen but not in 0.5% oxy- gen (Fig. S3). Also in the Gal4 reporter system, the HIF-1a CAD activity was modulated by IjBa expres- sion or knockdown in 1% oxygen but not in 0.5% oxygen (Fig. 3B). These results suggest that IjBa AB C D Fig. 2. IjBa enhances the hypoxic activity of HIF-1 by antagonizing FIH. (A) IjBa is required for the hypoxic activation of HIF-1. EPO-enhan- cer–luciferase plasmid (0.1 lg) and b-gal plasmid (0.1 lg) were cotransfected with Flag–IjBa plasmid (0.2 lg), HA–FIH plasmid (0.2 lg), IjBa siRNA (40 n M, si-IjBa), or FIH siRNA (40 nM, si-FIH). After being stabilized for 48 h, cells were incubated under normoxia or hypoxia for 16 h, and then harvested for the analysis of luciferase activity. (B) IjBa enhances the EPO reporter activity HIF-1-dependently. Mutated reporter plasmid (0.1 lg) lacking the HIF-1 target sequence and b-gal plasmid (0.1 lg) were cotransfected with Flag–IjBa plasmid (0.2 lg) or HA–FIH plasmid (0.2 lg). After 16 h of normoxic or hypoxic incubation, luciferase activity was measured. (C) IjBa stimulates the transcrip- tional activity of HIF-1a CAD in an Asn803-dependent manner. Wild-type or N803A Gal4–CAD (amino acids 776–826) plasmids (0.1 lg) and Gal4–luciferase reporter plasmid (0.1 lg) were cotransfected with b-gal plasmid (0.1 lg) or ⁄ and Flag–IjBa plasmid (0.2 lg) into HEK293 cells. After 16 h of normoxic or hypoxic incubation, luciferase activity was measured. Each bar represents the mean ± SD from four indepen- dent experiments. *P < 0.05, NS, no significance. (D) IjBa positively regulates the interaction between HIF-1a and p300. The HIF-1a–p300 interaction was examined using a coimmunoprecipitation assay. HEK293 cells were cotransfected with HA-tagged stable HIF-1a plasmid (HA–sHIF-1a,1lg) and p300 plasmid (1 lg), Flag–IjBa plasmid, and ⁄ or IjBa siRNA (80 n M). Cells were incubated under normoxic or hypoxic conditions for 8 h, and then homogenized. p300 was immunoprecipitated with anti-p300 serum (a-p300) and protein G ⁄ A beads (IP), and coprecipitated HA–sHIF-1a was identified by immunoblotting (IB). Input levels were measured by immunoblotting using specific antibodies. D. H. Shin et al. HIF-1 activation by IjBa FEBS Journal 276 (2009) 3470–3480 ª 2009 The Authors Journal compilation ª 2009 FEBS 3473 functionally stimulates the transcriptional activity of HIF-1a at moderate levels of hypoxia (1–5% O 2 ) but not at a more severe level (0.5% O 2 ). IjBa inhibits FIH-mediated Asn803 hydroxylation under moderate hypoxia As shown in Fig. 2C, Asn803 was found to be required for CAD activation by IjBa, which suggests that FIH- dependent Asn803 hydroxylation is related to the activity of IjBa. To test this possibility, we analyzed Asn803 hydroxylation in CAD using a specific anti- body against hydroxylated Asn803 in HEK293 cells. Under normoxic conditions, Asn803 hydroxylation could be detected, although HIF-1a was negligibly expressed. In 1% oxygen, HIF-1a was robustly expressed and Asn803 was partially hydroxylated. IjBa overexpression noticeably inhibited the Asn803 hydroxylation, which was rescued by FIH coexpression (Fig. 4A, left panel). When IjBa was knocked down, Asn803 hydroxylation was enhanced, and this was attenuated by FIH knockdown (Fig. 4A, right panel). On the other hand, in 0.5% oxygen, Asn803 was negli- gibly hydroxylated, and IjBa or FIH overexpres- sion ⁄ knockdown failed to affect Asn803 hydroxylation (Fig. 4B). Next, we checked the expression of three HIF-1-targeted genes, namely, those encoding vascular endothelial growth factor-A, aldolase-A, and lactate A B Fig. 3. The IjBa-mediated activation of HIF-1 occurs in moderate hypoxia. (A) IjBa stimulation of HIF-1 activity occurs only in 1% oxygen. EPO-enhancer–luciferase plasmid (0.1 lg) and b-gal plasmid (0.1 lg) were cotransfected into HEK293 cells with Flag–IjBa plasmid (0.2 lg), IjBa siR- NA(80 n M, si-IjBa), or GFP siRNA (80 nM, si-GFP). (B) IjBa stimulates HIF-1a CAD activity only in 1% oxygen. Wild-type Gal4– CAD plasmid (0.1 lg), Gal4–luciferase reporter plasmid (0.1 lg) and b-gal plasmid (0.1 lg) were cotransfected into HEK293 cells with Flag–IjBa plasmid (0.2 lg), IjBa siRNA (80 n M, si-IjBa), or GFP siRNA (80 n M, si-GFP). After cells were incubated in 1% or 0.5% oxygen for 16 h, luciferase activities (mean ± SD, n = 4) were analyzed. *P < 0.05, NS, no significance. HIF-1 activation by IjBa D. H. Shin et al. 3474 FEBS Journal 276 (2009) 3470–3480 ª 2009 The Authors Journal compilation ª 2009 FEBS dehydrogenase-A. In 1% oxygen, the hypoxic expres- sion of these genes was regulated positively by IjBa but negatively by FIH (Fig. 4C). However, the expres- sion of these genes was unaffected by IjBa or FIH in 0.5% oxygen (Fig. 4D). These results suggest that IjBa inhibits FIH-dependent Asn803 hydroxylation under moderate hypoxia, but not under severe hypoxia. IjBa competes with HIF-1a in FIH binding To understand the interplay between HIF-1a, FIH, and IjBa, we coexpressed these proteins in HEK293 cells and examined protein interactions by performing immunoprecipitation and immunoblotting assays. Figure 5A shows the effect of IjBa on the interaction between HIF-1a and FIH. FIH and HIF-1a proteins were coimmunoprecipitated, but this was noticeably reduced by IjBa expression, which suggests that IjBa interferes with FIH binding to HIF-1 a. Next, we checked whether IjBa–FIH binding is disrupted by HIF-1a. It was found that HIF-1a inhibited IjBa–FIH binding (Fig. 5B). As FIH binding affinities for HIF-1a and IjBa are likely to be similar, it appears that HIF-1a and IjBa compete for FIH binding. AB CD Fig. 4. IjBa stimulates HIF-1-dependent gene expression by inhibiting Asn803 hydroxylation. (A, B). IjBa inhibits FIH-mediated Asn803 hydroxylation. HEK293 cells were transfected with Flag–IjBa plasmid (0.2 lg), HA–FIH plasmid (0.2 lg), IjBa siRNA (80 n M, si-IjBa), and ⁄ or FIH siRNA (80 n M, si-FIH). pcDNA (0.2 lg) and GFP siRNA (80 nM, si-GFP) were used as transfection controls. After cells had been incu- bated in 1% or 0.5% oxygen for 16 h, protein levels were analyzed by immunoblotting. Asn803-hydroxylated HIF-1a was detected using an antibody against hydroxylated asparagine (N803-OH). (C, D) HEK293 cells were transfected as described above and then incubated in 1% or 0.5% oxygen for 16 h. Vascular endothelial growth factor (VEGF), aldolase, lactate dehydrogenase (LDH) and b-actin mRNA levels were analyzed by semiquantitative RT-PCR and autoradiography. D. H. Shin et al. HIF-1 activation by IjBa FEBS Journal 276 (2009) 3470–3480 ª 2009 The Authors Journal compilation ª 2009 FEBS 3475 HIF-1a CAD becomes inactivated shortly after tumor necrosis factor-a (TNF-a) stimulation What is the biological significance of the IjBa-medi- ated HIF-1 activation? If FIH-mediated HIF-1a inac- tivation is antagonized by IjBa, the HIF-1a CAD activity might be regulated IjBa-dependently during inflammation. As IjBa is dynamically regulated dur- ing inflammation, the CAD activity needs to be exam- ined in the time course of inflammation. Therefore, we analyzed CAD activity and Asn803 hydroxylation 4, 8 and 16 h after TNF-a stimulation. CAD activity was increased by hypoxia in a time-dependent man- ner, and was generally inhibited by TNF-a treatment. The inhibitory effect of TNF-a on hypoxic CAD activity was more significant after 4 h (42.1% inhibi- tion) or 8 h (30.5% inhibition) of incubation than after 16 h of incubation (6.9% inhibition) (Fig. 6A, upper panel). Also, CAD activity was inhibited by TNF-a even under normoxic conditions. Under the same conditions, IjBa expression was found to be noticeably downregulated 4 and 8 h after TNF-a stimulation, but to be somewhat restored after 16 h of incubation (Fig. 6A, lower panel). However, Asn803 hydroxylation of Gal4–CAD was stimulated 4 h after TNF-a treatment and was abolished in a time-dependent manner. Asn803 hydroxylation appears to be inversely correlated with IjBa expres- sion. On the basis of these results, the HIF-1-medi- ated hypoxic responses are likely to be repressed at the early phase of inflammation, and this may be attributed to IjBa downregulation and subsequent activation of FIH. Discussion In the present study, we tested the possibility that the interaction between FIH (HIF-1 inhibitor) and IjBa (NF-jB inhibitor) participates in crosstalk between the HIF-1 and NF-jB signaling pathways. FIH–IjBa binding was confirmed by yeast two-hybrid assays and coimmunoprecipitation analyses. Functionally, IjBa was found to regulate the transcriptional activity of HIF-1 positively, whereas FIH did not affect NF-jB activity. Mechanistically, IjBa derepressed HIF-1a CAD activity by inhibiting the FIH-mediated Asn803 hydroxylation of CAD. It was also found that IjBa inhibited FIH by competing with FIH for HIF-1a binding. Furthermore, IjBa affected HIF-1a activity only in moderately hypoxic conditions, under which FIH remains functional. When cells were treated with TNF-a,IjBa downregulation, Asn803 hydroxylation and HIF-1a inactivation all occurred up to 8 h, but subsided after 16 h. On the basis of these results, we propose that IjBa plays a pivotal role in the crosstalk between the HIF-1 and NF-jB signaling pathways. This hypothesis is summarized in Fig. 6B. Hypoxia and inflammation often codevelop in immunological, ischemic and cancerous diseases. Inflammation stimulates oxygen consumption, which AB Fig. 5. IjBa and HIF-1a compete with each other in FIH binding. (A) IjBa inhibits the FIH–HIF-1a interaction. HEK293 cells were cotransfect- ed with HA–HIF-1a plasmid (0.7 lg), HA–FIH plasmid (0.3 lg), and ⁄ or Flag–IjBa plasmid (0.2 or 0.4 lg). HIF-1a or FIH protein was precipi- tated using their specific antibodies, and then FIH or HIF-1a coprecipitation was identified by immunoblotting. (B) HIF-1a inhibits the IjBa–FIH interaction. HEK293 cells were cotransfected with Flag–IjBa plasmid (0.3 lg), HA–FIH plasmid (0.3 lg), and ⁄ or HA–HIF-1a plasmid (0.2 or 0.4 lg). FIH or IjBa protein was precipitated using specific antibodies, and then IjBa or FIH coprecipitation was identified by immunoblotting. HIF-1 activation by IjBa D. H. Shin et al. 3476 FEBS Journal 276 (2009) 3470–3480 ª 2009 The Authors Journal compilation ª 2009 FEBS results from increased energy metabolism of resident cells and from respiratory bursting of infiltrating phagocytes. These events lead to the establishment of a hypoxic microenvironment around inflamed tissue [12]. Conversely, hypoxia stimulates inflammation, because immune cells gather around hypoxia-injured tissues [13]. Given that HIF-1a and NF-jB are key factors of the hypoxic and inflammatory responses, respectively, the possibility of crosstalk between two factors is of growing interest. Many lines of evidence support the notion that sub- stantial crosstalk occurs between the HIF-1a and NF-jB pathways under pathological circumstances associated with hypoxia and inflammation [14]. For example, HIF-1 has been reported to be activated under nonhypoxic conditions by a number of proin- flammatory cytokines, such as TNF-a, interleukins, lipopolysaccharide, and reactive oxygen ⁄ nitrogen species [15]. In terms of mechanisms underlying HIF- 1a induction by inflammatory stimuli, Frede et al. [16] previously demonstrated that NF-jB mediated the transcription of the HIF1A gene during lipopolysac- charide stimulation of human monocytes. Recently, the role of NF-jB in HIF-1a mRNA expression was further investigated, using the HIF-1a promoter– luciferase reporter and electrophoretic mobility-shift analyses [17]. Rius et al. [18] also showed, in macro- phages of IkB kinase-b knockout mice, that NF-jB plays a critical role in transactivation of the HIF1A gene and that basal activity of NF-jB is required for hypoxic induction of HIF-1a. In addition to its posi- tive role in HIF-1 regulation, NF-jB can negatively control HIF-1a expression by inducing an HIF-1a- silencing micro-RNA, miR-155 [19]. Although HIF-1a is under the control of NF-jB, HIF-1a is also required for NF-kB activation. Walmsley et al. [20] demon- strated that NF-jB induction in response to hypoxia was significantly attenuated in neutrophils of HIF-1a knockout mice and that NF-jB signaling activated by HIF-1a contributed to neutrophil survival in hypoxia. In addition, HIF-1a overexpression in keratinocytes was found to activate NF-jB in the mouse skin and to augment the skin inflammation in response to 12-O- tetradecanoylphorbol-13-acetate. Mechanistically, it was suggested that HIF-1a is involved in IjB a degra- dation and NF-jB activation by extracellular signal- related kinase-mediated phosphorylation [21]. Given these reports, HIF-1a is likely to regulate NF-jB-med- iated inflammatory responses positively. However, we did not observe the reciprocal regulation between HIF- 1a and NF-jB. Even when IjBa was overexpressed or knocked down, HIF-1a protein levels were not signifi- cantly changed (Figs 4 and 5A). Also, HIF-1a overex- pression did not affect IjBa expression (Fig. 5B). We here transiently expressed or silenced HIF-1a or IjBa and performed all experiments within 2 days after transfection. A distinct change in HIF-1a or IjBa expression may require a longer incubation time than 2 days. In the present study, we found that an NF-jB inhi- bitor, IjBa, activated HIF-1a by sequestering an HIF-1a inhibitor, FIH. During inflammation, IjBa is phosphorylated at Ser32 and Ser36 by IkB kinase complex and then degraded by proteasomes [22]. Our results suggest that IjBa degradation may reinforce A B Fig. 6. HIF-1a is functionally inhibited by inflammatory stimulation. (A) HIF-1a CAD is inactivated shortly after TNF-a stimulation. Wild- type Gal4–CAD plasmid (0.1 lg), Gal4–luciferase reporter plasmid (0.05 lg) and b-gal plasmid (0.1 lg) were cotransfected into HEK293 cells. Cells were treated with 10 ngÆmL )1 TNF- and incu- bated under normoxic or hypoxic conditions for 4, 8, or 16 h. Each bar represents the mean ± SD (n = 4), and the percentage inhibi- tion by TNF-a is marked above the corresponding bars. (B) Sum- mary of IjBa roles in inflammatory and hypoxic responses. As IjBa inhibits NF-jB and FIH through direct interaction, it inhibits inflam- matory responses but stimulates hypoxic responses. D. H. Shin et al. HIF-1 activation by IjBa FEBS Journal 276 (2009) 3470–3480 ª 2009 The Authors Journal compilation ª 2009 FEBS 3477 the FIH-mediated repression of HIF-1a, which would negatively affect cellular adaptation to hypoxia. Indeed, we found that IjBa was noticeably downregulated 4 and 8 h after TNF-a stimulation (Fig. 6A). However, its expression recovered after 16 h of incubation, which suggests that IjBa is resynthesized by NF-jB activated by TNF-a [22]. Likewise, both HIF-1a CAD inactiva- tion by TNF-a and Asn803 hydroxylation showed a similar time course (Fig. 6A). Therefore, HIF-1a inacti- vation by IjBa suppression seems to occur transiently during the early stage of inflammation, and HIF-1a activity may subsequently resume, owing to IjBa resynthesis. ARD, which consists of 30–34 amino acids, is one of the most common motifs in nature, and functions exclu- sively to mediate protein–protein interactions [23]. Three thousand six hundred and eight proteins have been identified as containing ARD sequences in the non- redundant smart protein database [24], but only Notch and IjBa have been found to regulate HIF-1a by inter- acting with FIH. However, the high affinity of FIH for ARD suggests that other ARD-containing proteins also affect HIF-1-mediated hypoxic responses by interacting with FIH. Furthermore, given the diverse functions of ARD-containing proteins, such as transcriptional regu- lation, cell cycle regulation, differentiation, cytoskeletal organization, and stress response [23], it is possible that the ARD–FIH interaction could lead to various hypoxic responses other than inflammation. Here, we suggest that IjBa derepresses HIF-1a activ- ity by inhibiting FIH. If this is the case, this action of IjBa can only occur when FIH is functional, and this functionality requires molecular oxygen. To determine the minimal oxygen tension required for FIH activity, we analyzed cellular levels of Asn803-hydroxylated HIF-1a in 1% or 0.5% oxygen (Fig. 5). In 1% oxygen, HIF-1a was found to be stabilized but to be still hydroxylated at Asn803. Moreover, ectopically expressed FIH increased the amount of Asn803 hydroxylation. These results suggest that FIH remains functional in 1% oxygen. However, in 0.5% oxygen, Asn803 hydroxylation was found to be almost com- pletely inhibited, and under this condition, FIH overex- pression failed to increase Asn803 hydroxylation levels, which suggests that FIH is inactivated in 0.5% oxygen. Furthermore, the oxygen-dependent action of IjBa was also confirmed by our reporter and mRNA analyses. Does IjBa, then, regulate HIF-1a in hypoxic and inflammatory tissues? Given that the oxygen tension in tissues rarely falls below 1% (7.6 mmHg), even in hyp- oxic or inflammatory diseases [25], we believe that IjBa may positively regulate HIF-1a by inhibiting FIH in most hypoxia-related diseases. We conclude that IjBa is likely to function as a positive regulator of HIF-1a under moderately hypoxic conditions, and this action of IjBa appears to be attributable to its ability to sequester FIH from HIF-1a. However, little is known of the significance of the IjBa– FIH interaction during the progress of diseases associated with hypoxia and inflammation. On the basis of our results, we suggest that IjBa plays a pivotal role in shifting the cell response from NF-jB-mediated inflammation to HIF-1-mediated hypoxic adaptation. Experimental procedures Reagents and antibodies Culture media and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA, USA). Anti-HIF-1a serum was raised in rabbits against glutathione S-transferase-tagged human HIF-1a (amino acids 418–698), and a monoclonal antibody against hydroxylated Asn803 of HIF-1a was raised in mice immunized with a peptide containing a hydroxylated asparagine residue, as previously described [26,27]. Anti-IjBa and anti-FIH sera were purchased from SantaCruz Biotech (Santa Cruz, CA, USA). Other chemi- cals were purchased from Sigma-Aldrich (St Louis, MO, USA). Cell culture and hypoxic incubation The HEK293 cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and cultured in DMEM, supplemented with 10% heat-inac- tivated fetal bovine serum in a humidified 5% CO 2 incuba- tor. For hypoxic incubation, cells were incubated in a hypoxic chamber (5% CO 2 and 5% ⁄ 1% ⁄ 0.5% O 2 ). Preparation of short interfering RNAs (siRNAs) and plasmids To knock down IjBa and FIH, siRNA duplexes were designed on the basis of cDNA sequences provided by the NCBI (NM_020529 for IjBa, and NM_017902 for FIH), and synthesized by Samchully Pharm. (Seoul, Korea). The sequences for IjBa siRNA duplex are 5¢-GAGUACGA GCAGAUGGUCA-3¢ and 5¢-UGACCAUCUGCUCGUA CUC-3¢, and those of FIH siRNA duplex are 5¢-GAAUC CCAGUUGCGCAGUUAUAGCU-3¢ and 5¢-AGCUAUA ACUGCGCAACUGGGAUUC-3¢. cDNAs of IjBa, FIH and HIF-1a were cloned by RT-PCR using Pfu DNA poly- merase and inserted into pcDNA–Flag or pcDNA–HA plasmid by blunt-end ligation. The plasmid for sHIF-1a mutant was made by deleting three degradation motifs (amino acids 397–405, 513–553, and 554–595), using a HIF-1 activation by IjBa D. H. Shin et al. 3478 FEBS Journal 276 (2009) 3470–3480 ª 2009 The Authors Journal compilation ª 2009 FEBS PCR-based mutagenesis kit (Stratagene, Cedar Creek, TX, USA), as previously described [28]. Reporter assays Luciferase reporter genes containing EPO enhancer, hypoxia-response element-mutated EPO enhancer or Gal4- binding promoter were constructed as previously described [29]. An NF-jB reporter plasmid, which contains 5· NF- jB response elements fused to luciferase, was purchased from Stratagene (La Jolla, CA, USA). HEK293 cells were cotransfected with 1 lg each of reporter gene and plasmid cytomegalovirus–b-gal and ⁄ or plasmid of HIF-1 or Gal4– CAD, using the calcium phosphate method. pcDNA was added to ensure that the final DNA concentrations in both the control and experimental groups were similar. After being stabilized for 24 h, the cells were incubated under normoxic or hypoxic conditions, and then lysed to deter- mine luciferase and b-galactosidase (b-gal) activities. Lucif- erase activity was analyzed using a Lumat LB960 luminometer (Berthold Technologies, Bad Wildbad, Germany), and a b-gal assay was performed to normalize transfection efficiency. Immunoprecipitation and immunoblotting assay HEK293 cell lysates were incubated with specific antibodies or nonimmunized serum at 4 °C for 4 h. The immune com- plexes were further incubated with protein G–Sepharose beads (GE Healthcare Bio-science, Piscataway, NJ. USA) at 4 °C for 4 h. After washing of the beads, immunocom- plexes were eluted with an SDS buffer containing 200 mm Tris ⁄ HCl (pH 6.8), 40% glycerol, and 10% b-mercaptoeth- anol. For immunoblotting assays, cells were lysed with an SDS buffer, separated on SDS ⁄ polyacrylamide gels, and transferred to an Immobilon-P membrane (Millipore, Bed- ford, MA, USA). Membranes were blocked with 5% nonfat milk in NaCl ⁄ Tris containing 0.1% Tween-20 at room tem- perature for 30 min, and then incubated overnight at 4 °C with a primary antibody. b-Tubulin was used as a loading control. Semiquantitative RT-PCR Total RNAs were isolated from HEK293 cells by Trizol reagent (Invitrogen). RNA was quantified by measuring absorbance at 260 nm. RNAs (1 lg) were reverse tran- scribed at 48 °C, and the resulting cDNAs were amplified over 18–23 PCR cycles in 20 lL of reaction mixture con- taining 0.185 MBq [ 32 P]dCTP[aP] and 250 nm each primer set. The PCR products were electrophoresed on a 4% poly- acrylamide gel, and dried gels were autoradiographed. The nucleotide sequences of primer pairs have been described previously [26]. Statistical analysis All data [means and standard deviations (SDs)] were ana- lyzed using excel 2003 software (Microsoft, Redmond, WA, USA). Two-sided, Student’s t-tests were used for two- group comparisons, and P < 0.05 was considered to be significant. Acknowledgements This work was supported by a Korea Research Foundation Grant (2008-E00054) and by a Bone Metabolism Research Center Grant (R11-2008-023- 02001-0) provided by the Korea Science and Engi- neering Foundation. We thank E. Huang (University of Utah) for kindly providing us with gene constructs. References 1 Wang GL & Semenza GL (1995) Purification and char- acterization of hypoxia-inducible factor 1. J Biol Chem 270, 1230–1237. 2 Ke Q & Costa M (2006) Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 70, 1469–1480. 3 Schofield CJ & Ratcliffe PJ (2004) Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 5, 343–354. 4 Lando D, Peet DJ, Whelan DA, Gorman JJ & White- law ML (2002) Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch. 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Inhibitor of nuclear factor- kappaB alpha derepresses hypoxia- inducible factor- 1 during moderate hypoxia by sequestering factor inhibiting hypoxia- inducible factor from hypoxia- inducible factor. FIH, factor inhibiting hypoxia- inducible factor; GFP, green fluorescent protein; HA, hemagglutinin; HIF, hypoxia- inducible factor; IjBa, inhibitor of nuclear factor- kappaB alpha; NF-jB, nuclear factor- kappaB; . is hydroxylated by dioxygenase members [the HIF-prolyl-hydroxylases Keywords factor inhibiting hypoxia- inducible factor (FIH); hypoxia- inducible factor- 1 (HIF-1); IjBa; nuclear factor- kappaB (NF-jB);