Augmented CD30-mediated activation of NF-κB after suppression of ARNT. Control siRNA (siControl) or ARNT siRNA (siARNT) oligonucleotides were introduced into Karpas 299 cells followed by a 10-minute exposure to (A) CD30L or (B) continuous exposure to a suboptimal concentration of agonistic TNFR2 antibody. Cells were collected at the times indicated, total RNA was isolated and subjected to reverse transcription followed by quantitative real-time PCR analysis for the indicated genes. All samples were normalized to control unstimulated cells. The results represent the mean +/− SD of three independent experiments performed in triplicate. Statistical analyses were performed using a two-way analysis of variance (ANOVA) followed by Student-Newman-Keuls post hoc analysis. (A) The expression of the NF-κB responsive genes ICAM1, RELB, and NFKBIA (IκBα) was increased in ARNT-suppressed cells compared to that in siControl cells at each time point after CD30 stimulation (*p<0.01). (B) ARNT suppression increased basal and TNFR-induced expression of ICAM1 and NFKBIA at each time point (*p<0.01). Basal expression of RELB was not increased, but expression was increased at 3 to 9 hours after TNFR2 stimulation in cells in which ARNT was depleted (*p<0.01). In ARNT-depleted cells, expression of ICAM1 and RELB was increased at 3 and 9 hours after TNFR2 stimulation as compared to that in ARNT-suppressed cells at time 0 (#p<0.01). Similarly, expression of NFKBIA in ARNT-suppressed cells was increased at 3, 9 and 12 hours after TNFR2 stimulation as compared to that in ARNT-suppressed cells at time 0 (#p<0.01). (A) and (B) The basal expression of the ARNT responsive genes CYP1A1 and SLC2A1 was significantly reduced in the absence of ARNT (*p<0.01). To confirm suppression of ARNT, cDNA was analyzed (ARNT graph; *p<0.01) and lysates from unstimulated samples were probed for ARNT and β-actin.

Effects of ARNT on RelB DNA binding. (A–D) Decreased RelB DNA binding in cells depleted of ARNT. Karpas 299 cells were transfected with control siRNA (siControl) or ARNT siRNA (siARNT) followed by CD30 stimulation. At the indicated time points the cells were collected and ChIP was performed with the specified antibodies. RELB (A, B) and NFKBIA (C, D) promoter binding was analyzed using conventional and quantitative PCR. Data (A) and (C) are representative of one experiment that was performed twice. Samples for quantitative PCR (B) and (D) were normalized to the applicable input samples and the results represent percent input of two experiments performed in triplicate.

Association of ARNT with CD30. (A) Two regions of ARNT mediate the interaction with CD30. HEK293 cells were transfected with plasmids for the expression of combinations of GST-CD30 and HA-tagged versions of the mutants depicted in Supplementary Fig. S1C. CD30 pulldowns (PD) were performed with glutathione (GSH)-sepharose and the eluates were probed for ARNT (HA) and CD30 (GST). (B) Localization of CD30 with ARNT in the cytoplasm late in CD30 signaling. Karpas 299 cells expressing ARNT-GFP were labeled with anti-CD30-PE and analyzed by confocal microscopy at 3 and 24 hours. (C) Anti-CD30-PE is an agonistic antibody that stimulates CD30-mediated NF-κB activation. Karpas 299 cells were treated as indicated for 3 hours. Total RNA was isolated and subjected to reverse transcription followed by analysis by real-time PCR. All samples were normalized to control unstimulated cells. (D) CD30 associates with ARNT through the cytoplasmic TRAF binding domain. HEK293 cells were transfected with plasmids for the expression of ARNT-HA and either GST, GST-CD30, or GST-CD30Δ36. Lysates were precipitated with glutathione-sepharose and the eluates were probed for ARNT (HA) and CD30 (GST). A, B, and D are representative of one individual experiment that was repeated 3 times. C represents two independent experiments performed in triplicate.

ARNT as a NF-κB-associated co-regulator. (A) ARNT modulates NF-κB DNA binding potential. Karpas 299 cells were transfected with either control siRNA (siControl) or ARNT siRNA (siARNT). Nuclear extracts were probed for ARNT to ensure suppression and analyzed by EMSA. The asterisk denotes altered migration of the NF-κB-DNA complex. (B) and (C) Binding of ARNT to the NF-κB subunit, RelB. (B) Lysates from HEK293 cells transfected with the indicated expression plasmids were precipitated with glutathione (GSH)-sepharose. Inputs were probed for ARNT (HA) or the NF-κB subunits (GST) and the elutions were probed for ARNT. (C) Nuclear extracts from Karpas 299 cells were prepared at the indicated times following a 10-min CD30L exposure. Immunoprecipitations were performed with a RelB or control antibody and eluates were probed for ARNT. (D) Effect of ARNT on NF-κB subunit binding to DNA. Nuclear extracts prepared from HEK293 cells transfected with the indicated plasmids were analyzed by EMSA. The binding of RelA-p50 complexes was 1.07-fold greater in the presence of ARNT whereas the binding of RelB-p50 complexes was 1.3-fold greater in the presence of ARNT as determined by densitometry analysis. (E) Effect of ARNT on RelB-p50 binding to DNA. RelB and p50 were expressed with control vector, 0.5 µg or 1 µg of vector encoding ARNT in HEK293 cells. Nuclear extracts were prepared and analyzed by EMSA. The expression of ARNT increased the binding of RelB-p50 complexes by 1.5- (0.5 µg of ARNT) and 2-fold (1 µg of ARNT) as determined by densitometry analysis. All experiments were performed at least 3 times, and representative data are shown.

Proposed model of ARNT regulation of CD30-mediated NF-κB activity. Before CD30 stimulation RelB is present at classical RelA-p50 regulated target promoters. An initial CD30 signal results in RelA-p50 translocation to the nucleus, which replaces the RelB complex, and transcription ensues. As the CD30 signal progresses, RelB translocates to the nucleus where it associates with ARNT, enhancing the binding of RelB to DNA, and negatively regulating RelA-p50 complexes. Furthermore, ARNT associates with the TRAF binding region of internalized CD30, which may assist in the negative feedback loop of CD30 signaling by blocking TRAF binding.