FIH-1-Mint3 Axis Does Not Control HIF-1α Transcriptional Activity in Nucleus Pulposus Cells*

Background: Cells of the hypoxic nucleus pulposus (NP) require tightly regulated HIF-1 signaling for proper function. Results: Although overexpressed FIH-1 can regulate HIF-1 activity in NP cells, silencing endogenous FIH-1 results in no change in HIF-1 target gene expression. Conclusion: FIH-1 does not represent a major mechanism of controlling HIF-1-dependent transcription in NP cells. Significance: This study describes a physiological adaptation of NP cells. The objective of this study was to determine the role of FIH-1 in regulating HIF-1 activity in the nucleus pulposus (NP) cells and the control of this regulation by binding and sequestration of FIH-1 by Mint3. FIH-1 and Mint3 were both expressed in the NP and were shown to strongly co-localize within the cell nucleus. Although both mRNA and protein expression of FIH-1 decreased in hypoxia, only Mint3 protein levels were hypoxia-sensitive. Overexpression of FIH-1 was able to reduce HIF-1 function, as seen by changes in activities of hypoxia response element-luciferase reporter and HIF-1α-C-TAD and HIF-2α-TAD. Moreover, co-transfection of either full-length Mint3 or the N terminus of Mint3 abrogated FIH-1-dependent reduction in HIF-1 activity under both normoxia and hypoxia. Nuclear levels of FIH-1 and Mint3 decreased in hypoxia, and the use of specific nuclear import and export inhibitors clearly showed that cellular compartmentalization of overexpressed FIH-1 was critical for its regulation of HIF-1 activity in NP cells. Interestingly, microarray results after stable silencing of FIH-1 showed no significant changes in transcripts of classical HIF-1 target genes. However, expression of several other transcripts, including those of the Notch pathway, changed in FIH-1-silenced cells. Moreover, co-transfection of Notch-ICD could restore suppression of HIF-1-TAD activity by exogenous FIH-1. Taken together, these results suggest that, possibly due to low endogenous levels and/or preferential association with substrates such as Notch, FIH-1 activity does not represent a major mechanism by which NP cells control HIF-1-dependent transcription, a testament to their adaptation to a unique hypoxic niche.

tial transcriptional co-activators p300/CBP (16 -18). An asparagine residue within the C-TAD of HIF-1␣ (Asn 803 ) and of HIF-2␣ (Asn 851 ) is a target for hydroxylation by FIH-1, a nonredundant asparaginyl hydroxylase that, similar to PHD, requires molecular oxygen, ␣-ketogluratate, and ascorbate as its substrates. This post-translational modification of a specific Asn residue prevents binding to p300/CBP, thereby suppressing HIF-1/2 transcriptional activity (18,19). Noteworthy, because the K m of FIH-1 for oxygen is significantly lower than that of PHD1-3, even under conditions of moderate hypoxia, such as those present in the NP, FIH-1 activity is preserved (20). Thus, controlling expression/activity of FIH-1 is one of the important ways cells control HIF transcriptional activity. For example, in macrophages, FIH-1 activity is suppressed by an X11 protein family member, Mint3/APBA3 (21)(22)(23), through its N-terminal domain that binds and sequesters FIH-1. This interaction limits the ability of FIH-1 to hydroxylate and block HIF-1 function (23); as a consequence of this high HIF-1 activity, macrophages generate most of their ATP through glycolysis.
Although the PHD-dependent regulation of the activity of HIF in NP cells has received some attention, the role of FIH-1 and Mint3 in NP cells is completely unknown. Therefore, the major goal of this study is to delineate the role of FIH-1 and Mint3 in regulating HIF activity in NP cells. Our results clearly show that, although the Mint3 or FIH-1 system is capable of controlling HIF-1 function, due to the low endogenous levels of both of these proteins and/or preferential binding of FIH-1 with substrates such as Notch, they are likely to play a limited role in controlling HIF-1 transcriptional activity in physiologically hypoxic NP cells.
Isolation of NP Cells, Cell Treatments, and Hypoxic Culture-Rat and human NP cells were isolated and characterized as reported earlier (5). T/C-28, a human chondrocyte line, was kindly provided by Dr. Mary Goldring (Hospital for Special Surgery, Weill Cornell Medical College, New York) (24). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS) supplemented with antibiotics. To investigate the effect of nuclear transport inhibitors, cells were treated with ivermectin (12.5 M) or leptomycin B (10 ng/ml) for 16 or 24 h, respectively. Cells were cultured in a hypoxia work station (Invivo 2 300, Ruskinn, Bridgend, UK) with a mixture of 1% O 2 , 5% CO 2 , and 94% N 2 for 24 -72 h.
Immunohistological Studies-Freshly isolated rat spines were immediately fixed in 10% paraformaldehyde in PBS and then embedded in paraffin. Sagittal sections, 6 -8 m in thickness, were deparaffinized in xylene and rehydrated through graded ethanol and some sections were stained with Alcian blue, eosin, and hematoxylin. For localizing FIH-1 and Mint3, sections were incubated with either the anti-FIH-1 (1:100; Novus) or anti-Mint3 (1:50; Novus) antibodies in 5% goat serum in PBS at 4°C overnight. After thoroughly washing the sections, the bound primary antibodies were incubated with Alexa Fluor-488-conjugated anti-rabbit (Invitrogen) secondary antibody at a dilution of 1:200 for 1 h at room temperature. Sections were visualized using a fluorescence microscope (Nikon, Japan).
Real-time RT-PCR Analysis-Total RNA was extracted from NP cells using RNAeasy minicolumns (Qiagen). Before elution from the column, RNA was treated with RNase-free DNase I (Qiagen). The purified, DNA-free RNA was converted to cDNA using EcoDry TM Premix (Clontech). Template cDNA and gene-specific primers were added to the SYBR Green master mixture (Applied Biosystems), and mRNA expression was quantified using the Step One Plus real-time PCR system (Applied Biosystems). Hprt1 and ␤-actin were used to normalize gene expression. Melting curves were analyzed to verify the specificity of the RT-PCR and the absence of primer dimer formation. Each sample was analyzed in duplicate and included a template-free control. All primers used were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).
Transfections and Dual Luciferase Assay-Cells were transferred to 48-well plates at a density of 2 ϫ 10 4 cells/well 1 day before transfection. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent. For each transfection, plasmids were premixed with the transfection reagent. For measuring the effect of hypoxia or ivermectin and leptomycin B treatment on HRE-Luc activity, 24 h after transfection, the cells in some wells were treated with ivermectin or leptomycin B or moved to the hypoxia work station. The next day, the cells were harvested, and a Dual-Luciferase TM reporter assay system (Promega) was used for sequential measurements of firefly and Renilla luciferase activities. Quantification of luciferase activities and calculation of relative ratios were carried out using a luminometer (TD-20/20, Turner Designs, CA).
Lentiviral Production and Transduction-HEK 293T cells were seeded in 10-cm plates (1.0 ϫ 10 6 cells/plate) in DMEM with 10% heat-inactivated FBS 1 day before transfection. Cells were transfected with 12 g of Sh-FIH-1, Sh-Mint3, or FL-Mint3 plasmids along with 2 g of pMD2G, 2 g of pMDLg/ pRRE, and 2 g of pRSV/Rev using calcium phosphate solution. After 16 h, transfection medium was removed and replaced with DMEM with 10% heat-inactivated FBS and penicillinstreptomycin. Lentiviral particles were harvested at 48 and 60 h post-transfection. NP cells were plated in DMEM with 10% heat-inactivated FBS 1 day before transduction. Cells in 10-cm plates were transduced with 8 ml of conditioned medium containing viral particles along with 6 g/ml Polybrene. After 24 h, conditioned medium was removed and replaced with DMEM with 10% heat-inactivated FBS. Cells were harvested for mRNA or protein 5 days after viral transduction.
Microarray Analysis-Amplification of cDNA was performed using the Ovation Pico WTA-system V2 RNA amplification system (NuGen Technologies, Inc.). Briefly, 50 ng of total RNA was reverse transcribed using a chimeric cDNA/mRNA primer, and a second complementary cDNA strand was synthesized. Purified cDNA was then amplified with ribo-SPIA enzyme and SPIA DNA/RNA primers (NuGEN Technologies, Inc.). Amplified ST-DNA was purified with Qiagen MinElute reaction cleanup kit. The concentration of puri-fied ST-cDNA was measured using the nanodrop. 2.5 g of ST-cDNAs were fragmented and chemically labeled with biotin to generate biotinylated ST-cDNA using FL-Ovation cDNA biotin module V2 (NuGen Technologies, Inc.). Affymetrix gene chips (human gene 1.0 ST array (Affymetrix, Santa Clara, CA)) were hybridized with fragmented and biotin-labeled target (2.5 g) in 110 l of hybridization mixture. Target denaturation was performed at 99°C for 2 min and then 45°C for 5 min, followed by hybridization for 18 h. Arrays were then washed and stained using a Gene Chip Fluidic Station 450, and hybridization signals were amplified using antibody amplification with goat IgG and anti-streptavidin biotinylated antibody. Chips were scanned on an Affymetrix Gene Chip Scanner 3000, using Command Console software. Background correction and normalization were done using Iterative Plier 16 with GeneSpring version 12.0 software (Agilent, Palo Alto, CA). A 1.5-fold differentially expressed gene list was generated.
Statistical Analysis-All measurements were performed at least in triplicate, and data are presented as mean Ϯ S.E. Differences between groups were analyzed by Student's t test and one-way analysis of variance (*, p Ͻ 0.05).

FIH-1 and Mint3 Are Expressed in Both Rat and Human NP
Tissues and Are Co-localized-To investigate expression of FIH-1 and Mint3 in the intervertebral disc, we stained rat NP tissues with antibodies against FIH-1 and Mint3 (Fig. 1A). Fig.  1A shows that FIH-1 and Mint3 proteins are expressed in both NP and AF tissues. Real-time RT-PCR analysis shows lower expression of both Mint3 and Fih-1 mRNAs in AF tissue than in NP (Fig. 1B). We also measured expression of FIH-1 and Mint3 protein in NP tissues isolated from three rats by Western blot analysis (Fig. 1C). All three samples expressed both FIH-1 and Mint3. Then to investigate the cellular localization of FIH-1 and Mint3 in NP cells, we immunostained NP cells with anti-Mint3 and anti-FIH-1 along with antibodies directed against RCAS1, a Golgi marker, or EEA1, an early endosome marker. Surprisingly, Mint3 was strongly co-localized with FIH-1 in the cell nucleus. On the other hand, although some localization of Mint3 was seen in Golgi, little or no staining was seen in early endosomes (Fig. 1D).
Hypoxia Represses FIH-1 and Mint3 Expression in HIF-1-independent Fashion in NP Cells-To evaluate the effect of hypoxia on FIH-1 and Mint3 levels in NP cells, we measured their expression in NP cells under hypoxia using real-time RT-PCR and Western blot analysis. Our results show that although mRNA expression of Fih-1 was suppressed by hypoxia, Mint3 expression was not affected (Fig. 2, A and B). Interestingly, Western blot and subsequent densitometric analysis shows that protein expressions of both FIH-1 and Mint3 are significantly down-regulated by 72 h in hypoxia (Fig. 2, C-E); hypoxic decrease in FIH-1 levels is evident even at 24 h. We then investigated if HIF-1 played a role in hypoxic suppression of FIH-1 mRNA. For this purpose, we silenced HIF-1␣ in NP cells by lentiviral delivery of shRNA and measured FIH-1 and Mint3 expression. Real-time RT-PCR analysis shows that knockdown of HIF-1␣ does not affect mRNA levels of FIH-1 or MINT3 in NP cells (Fig. 2F).

HIF-1␣ Function in NP Cells Is Not Controlled by FIH-1-Mint3
Both HIF-1 and HIF-2 Activity Is Controlled by Forced Expression of FIH-1 in NP Cells-Next, we examined the effect of forced expression of FIH-1 on HIF transactivation in rat NP cells by measuring the ability of HIF-1␣-C-TAD and HIF-2␣-TAD to recruit co-factors and initiate transcription using a GAL4 binary system (see schematic in Fig. 3, A and B). Fig. 3, C and D, shows that the transactivation of HIF-1␣/HIF-2␣-TAD, which contain crucial Asn 803 and Asn 851 (19), respectively, is significantly suppressed by exogenous FIH-1. Moreover, overexpression of FIH-1 results in decreased activity of the HIFresponsive HRE reporter (PGK1-3xHRE-Luc) under both normoxia and hypoxia (Fig. 3, E and F). Although HRE reporter activity in hypoxia was ϳ3-fold higher than basal activity in normoxia, it failed to reach the normoxic baseline following forced expression of FIH-1 (Fig. 3F).
Decrease in HIF Activity by Exogenous FIH-1 Is Rescued by Mint3 Overexpression-Next, we evaluated if Mint3 modulates HIF function by measuring the activity of HRE-Luc reporter under normoxic and hypoxic conditions. Results show that overexpression of Mint3 increased HRE-Luc reporter activity under normoxia (Fig. 4A). However, to our surprise, overexpression of Mint3 failed to modulate HRE reporter under hypoxia. Similar to full-length Mint3, when the N-terminal of protein, which is shown to bind and sequester FIH-1, was transfected into nucleus pulpous cells, HRE-Luc reporter activity was significantly induced only under normoxia (Fig. 4, C and D). In contrast, overexpression of the C-terminal portion of Mint3 failed to induce HRE reporter activity; rather, a small but significant decrease in activity irrespective of oxemic tension

JOURNAL OF BIOLOGICAL CHEMISTRY 20597
was seen. To delineate whether Mint3 mediated its action on HIF function through controlling FIH-1, we overexpressed FIH-1 in the presence or absence of exogenous Mint3 and measured HRE reporter activity. Fig. 4, E and F, clearly shows that, irrespective of pO 2 , exogenous Mint3 can restore the decrease in HRE reporter activity mediated by exogenous FIH-1, suggesting interaction between these two proteins (Fig. 4, E and F). To confirm whether FIH-1 binds to Mint3 in NP cells, we performed immunoprecipitation using FIH-1 antibody. Fig. 4G shows that FIH-1 interacts with Mint3 under both normoxia and hypoxia with similar affinity (Fig.  4G). Ability of the FIH-1/Mint3 System to Regulate HIF Activity Depends on Subcellular Localization-To further clarify the mechanism of FIH-1/Mint3 action in NP cells, we first examined cellular localization of these two proteins. Immunofluorescence of human NP cells shows that both FIH-1 and Mint3 are localized in both the nucleus and cytoplasm (Fig. 5A). Next, using Western blot analysis, we evaluated the expression of FIH-1 and Mint3 in cytoplasmic and nuclear fractions of NP cells following 72 h of culture under normoxia or hypoxia (Fig.  5B). Densitometric analysis of multiple Western blots revealed that whereas FIH-1 and Mint3 levels in the cytoplasmic fraction remained unchanged, hypoxia decreased their levels in the nuclear fraction (Fig. 5, C and D). This clearly suggests that the hypoxic decrease seen in the levels of both of these proteins in total cell lysates (Fig. 2, C-E) is attributable to their reduction in the nuclear fraction. To investigate the importance of intracellular localization of FIH-1 on HIF-1 activity, we performed transfection experiments in the presence of ivermectin, a nuclear import inhibitor, or leptomycin B, a nuclear export inhibitor. As shown in Fig. 5, E and F, FIH-1-mediated suppression of HRE-Luc reporter activity was abolished by ivermectin treatment. In contrast, the reporter activity further decreased by leptomycin B under both normoxia and hypoxia, suggesting that in NP cells, FIH-1 is only able to regulate HIF activity in the nuclear compartment.
Endogenous FIH-1/Mint3 Has Minimal Effect on HIF Transcriptional Activity-To assess the contribution of endogenous FIH-1 and Mint3 on HIF-1 activity in NP cells, we transduced cells with lentivirus expressing Sh-FIH-1, Sh-Mint3, and fulllength Mint3 under normoxia. Because FIH-1 and Mint3 levels are comparatively higher in normoxia, and given the constitutive presence of HIF-1 protein in NP cells, silencing of FIH-1 and Mint3 in normoxia was more likely to evince changes in HIF-1 transcriptional activity, if any. Real-time RT-PCR analysis indicates that shRNA selectively suppressed the expression of either FIH-1 or MINT3 without affecting the expression of the other (Fig. 6, A and B). Western blot and densitometric analysis confirmed that shRNAs and overexpression of Mint3 significantly affected expression of the respective protein levels (Fig. 6, C and D). Next, to determine the changes in global gene expression profile following FIH-1 silencing, we performed microarray analysis on RNA isolated from cells transduced with Sh-FIH-1. The values were plotted in a volcano plot analyzing expression patterns in FIH-1-silenced NP cells versus control using the Ϫlog 10 (p value) and Ϫlog 2 (-fold change) (Fig. 6E). Knockdown of FIH-1 in NP cells induced 1.5-fold or greater change in expression of 535 gene transcripts (p Ͻ 0.1), of which 289 were up-regulated and 246 were down-regulated. Fig. 6F shows the heat map and dendrogram of the 535 gene transcripts. Surprisingly, as shown in Table 1, the expression of several known HIF-1 target genes, including those regulating glycolytic metabolism (PGK-1, PFKFB2, ENO1, SLC2A1, and SLC2A3), HIF-1 stability and function (EGLN1, EGLN3, and HSPA1A), and NP cell physiology (CTGF, LGALS3, B3GAT3, and VEGFA), were not affected by FIH-1 silencing in NP cells. A partial list of the maximally up-regulated and down-regulated transcripts following FIH-1 silencing is given in Table  2. Finally, to validate microarray results, we performed realtime RT-PCR analysis of known HIF target genes in FIH-1silenced cells. Fig. 7 clearly shows that mRNA expression of HIF target genes in NP cells, such as EGLN1 (PHD2) (Fig.  7A), EGLN3 (Fig. 7B), VEGFA (Fig. 7C), and ENO1 (Fig. 7D), remained unaffected by silencing of either FIH-1 or Mint3. Likewise, overexpression of Mint3 had no effect on any of these target genes.
To ascertain whether the seeming inability of FIH-1 to regulate HIF is a trait shared by other physiologically hypoxic tissues, we investigated the effect of FIH-1 and Mint3 knockdown in T/C-28 cells, a human chondrocyte line. Interestingly, following transfection with shRNA targeting FIH-1 as well as MINT3, HRE reporter activity did not change in the chondro- cytes at 3% oxygen, which is known to stabilize HIF-1␣ (data not shown), suggesting that, similar to NP cells, HIF activity in chondrocytes is not regulated by endogenous FIH-1. Furthermore, we examined the expression of HIF target genes in T/C-28 cells transduced with shRNA targeting FIH-1 and MINT3. Results clearly show that VEGFA, PHD2, and ENO1 expression was unaltered in FIH-1-or Mint3-silenced T/C28 cells (data not shown).

Notch1-ICD and Notch2-ICD Restores Suppression of HIF-␣-TAD Activity Mediated by Exogenous FIH-1-Due to known functional interactions between
Notch receptors and FIH-1, we first determined whether FIH-1 knockdown in NP cells affects the expression of genes concerned with the Notch signaling pathway. Fig. 8A shows that FIH-1 silencing results in induction of several known Notch signaling pathway genes, including HELT (HES/HEY-like transcription factor), a basic helix-loop-helix-containing transcriptional repressor, and neuralized homolog (Drosophila) (NEURL), an activator of JAG1 signaling, whereas expression of reelin (RELN), a large secreted extracellular matrix protein involved in controlling cell-cell interactions, was suppressed. Next, we investigated whether co-expression of Notch1-ICD or Notch2-ICD can rescue FIH-1 mediated inhibition of HIF-␣ transcriptional activity. Fig. 8, B-E, clearly shows that a decrease in HIF-1␣-C-TAD (Fig. 8, B and D) and HIF-2␣-TAD (Fig. 8, C and E)   . C and D, the overexpression of the N terminus (Mint3-NT) (C) but not the C terminus (Mint3-CT) (D) of Mint3 is necessary and sufficient for increasing HRE reporter activity in normoxia. No increase in HRE activity was seen by either of the shorter Mint3 constructs under hypoxia. HRE activity is represented as relative change compared with the respective controls in normoxia or hypoxia. E and F, overexpression of Mint3 can rescue loss of HRE reporter activity induced by exogenous FIH-1 irrespective of pO 2 , suggesting that the level of endogenous FIH-1 in hypoxia is low and thus has little effect on HIF activity. Data represent mean Ϯ S.E. of three independent experiments, each performed in triplicate (*, p Ͻ 0.05). G, immunoprecipitation (IP) of FIH-1 from rat NP cells in normoxia or hypoxia followed by Western blot analysis (IB) using Mint3 antibody indicated association between FIH-1 and Mint3. When an isotype IgG was used in place of FIH-1 antibody in normoxia, precipitation of either FIH-1 or Mint3 was absent. ns, not significant.

DISCUSSION
The cells of the NP reside in a physiologically hypoxic niche. The cellular processes through which the cells survive in hypoxia are not wholly understood. We have previously shown that the cells of the NP robustly express HIF-1␣ and HIF-2␣, and these isoforms play an important role in regulating glycolytic metabolism, survival, and synthesis of extracellular matrix (4 -9). It is noteworthy that in NP cells, HIF-1 activity is uniquely regulated in that PHDs play a limited role in its proteasomal degradation. Relevant to this study, we have also shown that PHD2 is capable of modulating HIF-1␣ levels in NP cells even under hypoxia, indicating that even at low oxemic tension, molecular oxygen is not a limiting substrate for PHD2mediated hydroxylation of HIF-1␣ (10). In light of these observations, it was important to investigate the role of FIH-1, an oxygen-dependent asparginyl hydroxylase in the control of HIF-1 activity in the NP. This is the first study to our knowledge that investigates the expression and role of the FIH-1/Mint3 system in the intervertebral disc.
Results of our studies show that NP cells express both FIH-1 and Mint3. Interestingly, although some localization of Mint3 was seen in Golgi, consistent with other reports (25), unlike other cell types, Mint3 showed a strong nuclear localization in NP cells. Furthermore, in contrast to previous findings in macrophages that showed a prominent perinuclear co-localization between Mint3 and FIH-1 (23), these proteins showed a strong nuclear staining and co-localization in NP cells. This raised a possibility that in NP, they interact in this subcellular compartment, a notion further confirmed by their co-immunoprecipitation. These results also suggest that the subcellular location of interaction between FIH-1 and Mint3 is cell type-specific. This hypothesis is further validated by our finding that in NP cells, the ability of exogenously expressed FIH-1 to repress HIF-1 activity was dependent on its nuclear localization. Thus, when exogenous FIH-1 was restricted to the cytoplasm by ivermectin, which targets importin ␣/␤, its ability to modulate HIF activity was abrogated, whereas when it was retained in the N, nucleus. C and D, multiple blots were quantified by densitometric analysis. Expression of GAPDH for cytoplasmic and Lamin A/C for nuclear protein was used as a loading control and to calculate relative expression levels. Hypoxia decreased nuclear levels of both FIH-1 and Mint3. E and F, reduction of HRE-Luc activity mediated by exogenous FIH-1 was abolished by treatment with ivermectin, a nuclear import inhibitor. In contrast, treatment with leptomycin B, a nuclear export inhibitor, further reduced FIH-1-dependent HRE reporter activity under both normoxia (Nx) and hypoxia (Hx). LMB, leptomycin B. Data represent mean Ϯ S.E. (error bars) of three independent experiments. *, p Ͻ 0.05.

HIF-1␣ Function in NP Cells Is Not Controlled by FIH-1-Mint3
nucleus by the CRM1 inhibitor, leptomycin B, a further decrease in FIH-1-mediated HIF-1 function was seen. These results are consistent with those seen in breast cancer (26), pancreatic endocrine tumors (27), and clear cell renal cell carcinoma (28), where low nuclear FIH-1 expression is correlated with poor prognosis, sug-gesting that although FIH-1 expression may be predominantly cytoplasmic (29), it is only the nuclear fraction that has an active role in the regulation of HIF activity.
Because NP is an avascular and hypoxic tissue, it was important to determine the effect of hypoxia on FIH-1 and Mint3 In the dendrogram, a shorter arm indicates higher similarity, whereas a longer arm indicates lower similarity. The resulting heat map of the dendrogram tree reveals groups of genes with high expression levels (red), low expression levels (blue), or background expression levels (yellow).

HIF-1␣ Function in NP Cells Is Not Controlled by FIH-1-Mint3
expression and activity. Prolonged hypoxia resulted in a decrease in FIH-1 at both the protein and the transcript level and a decrease in Mint3 at the protein level. Because in NP cells, HIF-1 is a major regulator of hypoxia-dependent transcription, and considering that hypoxic expression of PHD1-3 is under the control of HIF-1 (15), it was surprising that the decrease in FIH-1 transcript in hypoxia was independent of HIF-1␣. The repression is possibly mediated by HIF-2 but more likely is through a HIF-independent hypoxic pathway (e.g. through PKC, which is both activated by hypoxia (30) and capable of modulating FIH-1 expression through Cut-like homeodomain protein (CDP/Cut) (31)).
Concerning FIH-1 function in NP cells, our results show that exogenously overexpressed FIH-1 is capable of repressing the activity of both HIF-1␣-C-TAD and HIF-2␣-TAD as well as an HRE-luciferase reporter. It is noteworthy that overexpression of FIH-1 results in a decrease in HIF-1␣ activity even under hypoxic (1% O 2 ) conditions, similar to what was seen previously for PHD2 activity in NP cells (10). Moreover, these results are consistent with previous findings that the in vitro K m for O 2 of FIH-1 is much smaller (90 M) than that of PHD2 (250 M) (32,33) and that substantial levels of hydroxylated Asn 803 can be detected even at 0.1% O 2 (20) in RCC4 and MCF7 cells, suggesting preservation of FIH-1 enzymatic function under hypoxia. Furthermore, a very recent report that FIH-1 can still suppress HIF-dependent GLUT1 and VEGF-A expression in hypoxic glioblastoma in vitro and also in vivo in a xenograft model supports this idea (34). Because in NP Mint3 and FIH-1 colocalize in the cell nucleus and interact, overexpression of Mint3 would be expected to increase HIF-1 activity, through inhibition of FIH-1. Of note, forced overexpression of Mint3 was able to increase HRE-luc activity only under normoxic conditions; in hypoxia, overexpression of exogenous Mint3 had no effect on HIF-1 activity. Importantly, consistent with studies of macrophages, the N terminal portion of the protein shown to interact with FIH-1 was important for its action on HIF-1 function (23). Taken together, these results suggest that endogenous FIH-1 is not present in a concentration sufficient to repress HIF-1 activity in the hypoxic NP cells and are consistent with our observation that the level of FIH-1 protein is decreased in hypoxia.
Because our initial results suggested a limited role of FIH-1 in regulation of HIF-1 activity, we decided to apply an unbiased approach of microarray analysis after FIH-1 silencing in primary human NP cells. Consistent with our transfection results, knockdown of FIH-1 did not significantly increase expression of any of the classically regulated HIF target genes, which include enzymes of glycolysis (PFK2, PGK1, and ENO1), glucose transporters (SLC2A1 and SLC2A3), and genes important for NP physiology and function (VEGFA, EGLN1, EGLN3, CTGF, and LGALS3). These results were confirmed by qPCR  for validated HIF-1 target genes in NP cells. It is noteworthy that knockdown of FIH-1 via lentivirally delivered shRNA in T/C-28 cells, a human chondrocyte cell line, showed similar results; there was no increase in HRE reporter activity and no increase in HIF-1 target gene expression with FIH-1 knockdown. Together, these results clearly suggest that in physiologically hypoxic tissues, like NP and cartilage, where HIF activity is crucial for cell survival, FIH-1 does not play a major role in regulating HIF activity, possibly due to its low levels. Our results are similar to what was reported in glomerular podocytes of the kidney (35). The fact that podocytes are prone to hypoxic insult during glomerular injury (36) and that activation of HIF target genes provides renal protection in acute and certain chronic conditions (37, 38) underscores a compelling argument that in cell types prone to experience hypoxia, FIH-1 activity does not represent a major mechanism of regulating HIF-1 transcriptional activity. Based on our findings, we conclude that FIH-1 does not play a major role in regulating HIF-1 activity in physiologically hypoxic NP tissue. This inability to regulate HIF-1␣ is probably due to stoichiometry and a low level of expression, especially under hypoxic conditions, and not due to substrate limitation of molecular oxygen. Thus, the preservation of enzymatic func-tion of FIH-1 in hypoxia raises the question of its physiological role in NP tissue. Although apparent deformity of the spinal column has not been reported in FIH-1-null mice, a careful analysis of the intervertebral disc phenotype has not been performed (39). Given the number of genes affected by FIH-1 silencing in NP cells, including those concerned with the Notch pathway, and in recognition of the fact that FIH-1 is known to hydroxylate many proteins with ankyrin repeats, including p105, IB␣ (40), and members of the Notch receptor family (41), it is very plausible that morphological and functional deficits may exist in discs of FIH-1 null mice with aging. Of note, the in vitro K m of FIH-1 for Notch1 is much lower (ϳ250-fold) than it is for HIF-1␣ (42), although in vivo analyses suggest that hydroxylation of ankyrin repeat domain proteins and HIF-1␣ occurs within the same range (43). It is noteworthy that Notch1 and Notch2 are highly expressed in the NP, and their expression is hypoxia-sensitive (44). Importantly, our data suggest that both Notch1-and Notch2-ICD can rescue FIH-1-mediated suppression of HIF-1␣ and HIF-2␣ transactivation, raising the possibility that the high Notch expression in the NP may serve as a preferential substrate and outcompete HIF-1␣ for binding with FIH-1, contributing to the seeming lack of regulation of HIF-1 signaling by FIH-1. Moreover, FIH-1 has also recently been reported to interact with and inhibit the proapoptotic protein Bax, independent of its hydroxylase activity (45). In light of our recent finding of PHD3 activating p65 signaling in NP cells independent of proline hydroxylation (46), it is important to also consider the potential role of FIH-1 independent of asparagine hydroxylation in NP physiology.