U2OS osteosarcoma and HEK (human embryonic kidney)-293 cells were grown in DMEM (Dulbecco's modified Eagle's medium) (Lonza) supplemented with 10% (v/v) fetal bovine serum (Gibco), 50 units/ml penicillin (Lonza) and 50 μg/ml streptomycin (Lonza) for no more than 30 passages. U2OS-NF-κB luciferase reporter cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum, 50 units/ml penicillin and 50 μg/ml streptomycin with 150 μg/ml Hygromycin B (Roche).

U2OS cells were transfected with 2 μg of NF-κB-reporter construct pGL4.32[luc2P/NF-κB-RE/Hygro] (Promega) using GeneJuice® (Merck Biosciences). After 48 h, cells were split and cultivated under 300 μg/ml Hygromycin B. Individual clones were picked and selected on the basis of their response to TNFα. Selected cell lines were grown in DMEM supplemented with 10% (v/v) fetal bovine serum, 50 units/ml penicillin and 50 μg/ml streptomycin with 150 μg/ml Hygromycin B.

The GFP (green fluorescent protein)–TfR1 expression construct was a gift from Professor Wolfhard Almers (Oregon Health and Sciences University, Portland, OR, U.S.A.). pCNA3-IKKβ-Flag and pCNA3-IKKα were gifts from Professor Ron Hay (Dundee University, Dundee, U.K.) pGL4.32[luc2P/NF-κB-RE/Hygro] was obtained from Promega. RSV (respiratory syncytial virus)-RelA was a gift from Professor Neil Perkins (Newcastle University, Newcastle upon Tyne, U.K.). pCMVTAG-IKKγ-Flag was a gift from Professor Jon Ashwell (National Cancer Institute, Bethesda, MD, U.S.A.) (Addgene plasmid 11970) [¹²].

siRNA duplex oligonucleotides were synthesized by MWG and transfected using Interferin (Polyplus) as per the manufacturer's instructions.

The siRNA sequences used were: Control, CAGUCGCGUUUGCGACUGG [¹³]; RelA, GCUGAUGUGCACCGACAAG [¹³]; TfR1_A, CCAAUACAGAGCAGACAUA; TfR1_B, ACUUGCUGUAGAUGAAGAA; TfR1_C, CUUCCAGACUAACAACAGA; and IKKγ, ACAGGAGGUGAUCGAUAAG [¹⁴].

Semi-quantitative RT–PCR and PCR was performed as described previously [¹⁴–¹⁶]. PCR products were resolved on 2% agarose gels and scanned using a PhosphoImager (FujiFilm FLA-5100) into TIFF format. Quantitative RT–PCR was performed using cDNA templates [cDNA synthesis was performed using Quantitect Reverse Transcription kit (Qiagen)] amplified using specific primer sets and the Stratagene Brilliant II SYBR® Green qPCR mix according to the manufacturer's instructions. Amplification and detection were performed using a Stratagene Mx3005P detection system. Sample values obtained with specific primer sets were normalized to β-actin primer set values.

The PCR primer sequences used were: actin, 5′-CTGGGAGTGGGTGGAGGC-3′ (forward) and 5′-TCAACTGGTCTCAAGTCAGTG-3′ (reverse); IAP2 (inhibitor of apoptosis protein 2), 5′-GTCAAATGTTGAAAAAGTGCCA-3′ (forward) and 5′-GGGAAGAGGAGAGAGAAAGAGC-3′ (reverse); p100, 5′-AGCCTGGTAGACACGTACCG-3′ (forward) and 5′-CCGTACGCACTGTCTTCCTT-3′ (reverse); and TfR1-Quantitect primer assay (Qiagen). ChIP (chromatin immunoprecipitation) primers used were: p100 κB, 5′-CACTCCGAGGAGGAGACACT-3′ (forward) and 5′-GGAGCAACCTTGGGATTTTC-3′ (reverse); and p100 control, 5′-TATGGGGGATTGAAGCAGAG-3′ (forward) and 5′-TGGGCCAAATGGAATACTGT-3′ (reverse).

Proteins were cross-linked with formaldehyde for 10 min. Glycine (0.125 mM) was added, and cells were washed with PBS. Cells were lysed with lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris/HCl, pH 8.1, 1 mM PMSF, 1 mg/ml leupeptin and 1 mg/ml aprotonin), followed by sonication and centrifugation. The supernatant was pre-cleared with sheared salmon sperm DNA and Protein G–Sepharose beads (Sigma). The supernatant was incubated with specific antibodies overnight, and then with Protein G–Sepharose beads for 1 h. After an extensive wash step, the complexes were eluted with buffer (100 mM NaHCO₃ and 1% SDS) and incubated with proteinase K. DNA was purified using QIAquick® PCR purification kit (Qiagen). PCR was performed on the purified immunoprecipitated DNA using specific primers.

Antibodies used were: anti-TfR1 (sc-51829, Santa Cruz Biotechnology), anti-phospho-RelA (Ser⁵³⁶) (3031, Cell Signaling Technology), anti-RelA (sc-372, Santa Cruz Biotechnology), anti-PCNA (proliferating-cell nuclear antigen) (P8825, Sigma), anti-β-actin (A5441, Sigma), anti-IκBα (sc-371, Santa Cruz Biotechnology; 4812, Cell Signaling Technology), anti-phospho-IκBα (Ser³²/Ser³⁶) (9246, Cell Signaling Technology), anti-phospho-IKKα/β (2681, Cell Signaling Technology), anti-IKKγ (sc-8330, Santa Cruz Biotechnology), anti-IKKβ (2678, Cell Signaling Technology), anti-IKKα (2682, Cell Signaling Technology; sc-7182, Santa Cruz Biotechnology), anti-GFP (Roche), anti-FLAG (Sigma), anti-IAP2 (3130, Cell Signaling Technology), anti-p100/p52 (05-361, Millipore) and anti-(cleaved PARP) [poly(ADP-ribose) polymerase] (9541, Cell Signaling Technology).

DFX mesylate (Sigma) was added for 2 h at a final concentration of 200 μM, ammonium iron(III) sulfate (Sigma) and ascorbate (Sigma) were also added for 2 h at final concentrations of 300 and 2 μM respectively.

Nuclear RelA blots were quantified using ImageJ (NIH), normalized with PCNA loading control and untreated control levels set to 1. All other conditions are compared with untreated non-targeting siRNA control levels.

Whole-cell protein extracts, nuclear extracts, EMSAs (electrophoretic mobility-shift assays) and luciferase assays were performed as described previously ([¹⁵] and references therein).

The IKK complex is central in the activation pathway of the transcription factor NF-κB [²]. As such, identification of a novel control mechanism would be of great interest for the understanding of the pathway, as well as for future therapeutic intervention. To unveil novel interacting proteins and potential novel regulators we employed the TAP (tandem affinity purification) technique followed by quantitative MS, utilizing SILAC (stable isotope labelling of amino acids in culture). We reconstituted IKKβ^−/− MEFs (mouse embryonic fibroblasts) with TAP–IKKβ or TAP alone (Supplementary Figure S1A at http://www.BiochemJ.org/bj/449/bj4490275add.htm). The TAP-tag consists of Protein A and a calmodulin-binding peptide separated by the recognition motif for the TEV (tobacco etch virus) protease. The functionality of the TAP–IKKβ-reconstituted MEFs was tested using NF-κB DNA binding following TNFα treatment. It was possible to see a rescue of IKK function in these cells, as measured by EMSA using an NF-κB consensus probe, to a level comparable with that of wild-type MEFs (Supplementary Figure S1B).

Having established the cellular system, the TAP–IKKβ- or TAP-reconstituted IKKβ^−/− MEFs were cultured in either ‘light’ (i.e. non-labelled) L-lysine and L-arginine or heavy-isotope-labelled amino acids L-[¹³C₆]lysine hydrochloride and L-[¹³C₆]arginine hydrochloride medium. Lysates were obtained from TAP–IKKβ cells (heavy) and TAP cells (light) mixed at an equal ratio, before being subjected to a sequential purification procedure as outlined in Supplementary Figure S1(C). The purified material was separated by SDS/PAGE (4–12% gels), and gel fragments were excised. A proteomic analysis was performed to identify and quantify the proteins that interact with TAP–IKKβ. Peptide ratios were quantified using MSQuant software to identify proteins that interact more with IKKβ. Comparison of the obtained peptide sequences with protein databases identified both IKKα and IKKγ, confirming that our purification technique pulls down known IKKβ-interacting proteins (Table 1). In addition, we also identified HSP90 as one of the IKKβ-associated proteins (Table 1). However, we did find a novel interaction partner for IKKβ: TfR1 (Table 1). We confirmed this interaction by Western blotting, using a specific antibody against TfR1 (Figure 1A).

To confirm that the interaction between TfR1 and IKK proteins is conserved in human cells, co-immunoprecipitation experiments between exogenous IKKα, IKKβ, IKKγ and TfR1 were performed. Our results demonstrate that TfR1 interacts with IKKα, IKKβ or IKKγ, but not with the negative controls (Figure 1B and Supplementary Figure S1D). To validate further our analysis, we performed co-immunoprecipitation experiments analysing endogenous proteins. This analysis confirmed that endogenous TfR1 interacts with all members of the IKK complex (Figure 1C). Finally, we tested whether under conditions of activation of the IKK complex, such as treatment with TNFα, TfR1 was still present in the complex. Our analysis revealed that TfR1 association with IKKβ or IKKγ does not change significantly with treatment with TNFα (Figure 1D).

To analyse the extent of association between TfR1 and the IKK complex, we analysed size-exclusion chromatography fractions for the presence of these proteins (Figure 2A). We found that endogenous TfR1 co-fractionates with the IKK complex subunits over several fractions, ranging from high-molecular-mass (1 MDa) to smaller complexes (Figure 2A). Since the bulk of TfR1 seems to co-fractionate mostly with IKKγ, we determined whether TfR1 was still able to associate with IKKα or IKKβ in the absence of IKKγ. Our analysis revealed that TfR1 remained associated with the catalytic IKKs, when IKKγ was depleted using siRNA (Figure 2B). These results indicate that TfR1 binds to the IKK complex in cells.

TfR1 is a single-pass type II membrane protein [¹⁷–¹⁹]. However, it does have a small intracellular portion, and it is internalized when bound to transferrin and subsequently recycled to the membrane again [¹⁷–¹⁹]. To test whether the intracellular portion of TfR1 was binding directly to IKK, we cloned the intracellular 64 residues of TfR1 into a bacterial expression construct. Protein fragments expressed in bacteria were purified and used in an in vitro binding assay with in vitro transcribed and translated IKKβ. Interestingly, we could not detect any specific binding of IKKβ to the small intracellular portion of TfR1 (Supplementary Figure S1E). These results suggest that the interaction is mediated by post-translational modifications on either of the proteins, requiring full-length TfR1, or it is indirect, via additional protein partners.

To address whether TfR1 influences IKK activity, we first sought to address whether IKK levels were altered by depleting TfR1 using RNAi (RNA interference). Relative to a non-targeting control, levels of the TfR1 protein were specifically decreased when U2OS cells were transfected with siRNAs against the TfR1 mRNA (Figure 3A). In contrast, TfR1 siRNA did not diminish the levels of the two IKK catalytic subunits IKKα and IKKβ or the regulatory subunit IKKγ.

The association between the regulatory subunit IKKγ, and the kinase subunits IKKα or IKKβ, is absolutely critical for the activity of the IKK complex [⁹]. To assess whether TfR1 was involved in IKK complex assembly, co-immunoprecipitation assays were used to investigate whether the integrity of the endogenous IKK complex is altered in the absence of TfR1. To this end, we depleted TfR1 using two different siRNAs, and immunoprecipitated IKKγ or IKKβ. Levels of associated IKKs were analysed by Western blotting (Figure 3B). Although expression of the IKK subunits is not altered by TfR1 depletion, association between the regulatory subunit IKKγ and the kinase subunit IKKβ is diminished in TfR1-compromised cells (Figure 3B). This is seen when either IKKβ or IKKγ are used to immunoprecitate the complex. Interestingly, IKKα interaction with IKKγ is still preserved, suggesting that TfR1 facilitates the IKKγ–IKKβ interaction, but has minimal effect on IKKγ–IKKα binding. These data indicate that TfR1 is required for the integrity of the full IKK complex.

The IKK complex responds to a number of stress stimuli, including cytokines such as TNFα [³]. Activation of the IKK complex can be assessed by phosphorylation in the T-loop, and by analysis of the phosphorylation status and levels of its substrate IκBα. To determine whether TfR1 is important for IKK activation following TNFα, TfR1 was depleted by siRNA and cells were treated with TNFα for different periods of time (Figure 4A). TNFα-induced IKK phosphorylation and activity were severely delayed and impaired when TfR1 was depleted (Figure 4A). As the IKK complex is the upstream kinase for the NF-κB family of transcription factors, we tested whether NF-κB transcriptional activity is altered in cells lacking TfR1. We created a U2OS cell line stably transfected with a NF-κB luciferase reporter construct. We initially tested NF-κB transcriptional activity in response to TNFα in a time-dependent manner (Supplementary Figure S2 at http://www.BiochemJ.org/bj/449/bj4490275add.htm). Luciferase activity was increased following 2 h of TNFα treatment and reached maximal levels following 6 h of treatment (Supplementary Figure S2). These results are in agreement with the NF-κB-activation pattern in most cells, which is cyclical in nature [²⁰,²¹]. Using the NF-κB reporter cells, we depleted TfR1 using siRNA, and stimulated for 5 h with TNFα before harvest and luciferase assay. In cells depleted of TfR1, both basal and TNFα-induced NF-κB activity were reduced compared with control cells (Figure 4B). Indeed, the fold decrease in NF-κB activity observed in cells lacking TfR1 is comparable with that observed with depletion of RelA, the NF-κB family member that is most responsive to TNFα (Figure 4B).

Our results based on siRNA-mediated depletion indicate that TfR1 is required for IKK complex formation and activation in response to TNFα. To assess whether increased TfR1 levels increase IKK activity, we overexpressed TfR1 and assessed the NF-κB transcriptional response to TNFα stimulation (Figure 4C). Our analysis revealed that increased TfR1 levels result in a slight increase in NF-κB transcriptional activity, supporting further the hypothesis that TfR1 modulates IKK–NF-κB activity. In addition, we tested whether NF-κB activity could be modulated by iron levels. As such, we treated cells with either an iron chelator or increased iron, and analysed NF-κB luciferase activity following TNFα treatment. We observed that depletion of iron resulted in lower luciferase activity, whereas addition of extra iron to the cells potentiated NF-κB activity (Figure 4D). This could also be observed at the level of two endogenous NF-κB targets: IAP2 and p100 (Figure 4E).

Activation of the canonical NF-κB signalling pathway by TNFα requires the translocation of the RelA subunit from the cytoplasm to the nucleus where it binds target gene promoters and enhancers. To determine the role of TfR1 in this process, we transfected U2OS cells with a control or TfR1 siRNA oligonucleotides, treated with TNFα for different periods of time and isolated nuclear fractions. In control cells, we observed robust translocation of RelA from the cytoplasm to the nuclear compartment within 30 min of TNFα stimulation, whereas in cells depleted of TfR1, the levels of nuclear RelA were significantly decreased (Figure 5A). No change in total levels of RelA was observed; however, when TfR1 was depleted, there was a reduction in the levels of phosphorylated Ser⁵³⁶ on RelA (Figure 5A). These results are consistent with impairment of IKK function as this residue can be phosphorylated by IKK [²²].

We investigated further the functional significance of this reduced RelA translocation/activation by analysing the effects of TfR1 depletion on the levels of mRNA from endogenous target genes by quantitative RT–PCR (Figure 5B). Induction of NF-κB target genes c-IAP2 (cellular IAP2) and p100 in response to stimulation with TNFα was compromised in cells depleted of TfR1 (Figure 5B). We also noted an induction of TfR1 mRNA by TNFα treatment at 4 h (Supplementary Figure S3A at http://www.BiochemJ.org/bj/449/bj4490275add.htm). This is in agreement with previous studies that have found that HIF-1 (hypoxia-inducible factor-1) and NF-κB can control TfR1 gene expression in response to inflammatory signals [²³].

We further validated our results with Western blot analysis, where we observed that TNFα induction of IAP2 and p100 protein is impaired in the absence of TfR1 in U2OS cells (Figure 5C) and MDA-MB-231 cells (Supplementary Figure S3B). Importantly, in the absence of TfR1, RelA recruitment to the p100 promoter following TNFα treatment is visibly decreased (Figure 5D). Taken together, these results demonstrate that TfR1 is required for TNFα-mediated activation of endogenous IKK and NF-κB in the cell.

NF-κB signalling pathway in response to TNFα is to transactivate anti-apoptotic genes, in order to protect the cell against programmed cell death [¹]. As we have demonstrated that depletion of TfR1 interferes with the IKK–NF-κB signalling pathway, we sought to determine whether loss of TfR1 could interfere with the anti-apoptotic functions associated with the IKK–NF-κB axis. We depleted U2OS cells of TfR1 and then treated them with increasing concentrations of TNFα for 24 h before lysis. Western blot analysis was performed to determine the levels of the known apoptosis marker cleaved PARP. In control cells, TNFα does not lead to significant levels of cell death owing to NF-κB anti-apoptotic function (Figure 6A). However, in cells lacking TfR1, TNFα-induced apoptosis is readily observed even with low concentrations of TNFα, as these cells display much higher levels of cleaved PARP when compared with control cells (Figure 6A).

To rule out the possibility that the effects of TfR1 depletion on TNFα-induced apoptosis are not dependent on modulation of IKK–NF-κB, we performed rescue experiments. Cells were depleted of TfR1, but, in addition, we expressed control or a plasmid for the NF-κB subunit RelA, treated with TNFα and analysed the levels of cleaved PARP. Expression of exogenous RelA visibly reduced the levels of cleaved PARP observed in cells depleted of TfR1 and treated with TNFα (Figure 6B). The results confirm the role of TfR1 in the modulation of IKK–NF-κB function in cells.