Expression and signaling of CD30 a tumor necrosis factor (TNF) receptor family member is upregulated in numerous lymphoid-derived neoplasias most notably anaplastic large cell lymphoma (ALCL) and Hodgkin’s lymphoma (HL). was potentiated resulting in augmented expression of these NF-κB-responsive genes. These findings indicate that ARNT functions in concert with RelB in a CD30-induced negative feedback mechanism. The activation of the transcription factor NF-κB as a consequence of signaling from the tumor necrosis factor (TNF) receptor family member CD30 accounts for many of the physiological phenomena regulated by CD30 (1 2 For example CD30 signaling contributes to tumorigenesis of Hodgkin’s lymphoma cells (HL) by increasing NF-κB activity (3 4 TNF receptor associated factor GSK2118436A (TRAF) proteins are important for CD30-mediated NF-κB activation (5-8) but other proteins may also participate in regulation of CD30 signaling. Furthermore aspects of NF-κB regulation remain enigmatic especially regarding the mechanisms that control NF-κB transactivation at the chromatin level (9). Therefore in an attempt to identify signaling intermediates between CD30 and NF-κB activity we conducted a biochemical affinity screen (10 11 in Karpas 299 a cell line exhibiting inducible CD30 signaling that was derived from a patient with anaplastic large cell lymphoma (ALCL) (12). Analysis of the biochemically purified products revealed the aryl hydrocarbon receptor nuclear translocator (ARNT; also known as hypoxiainducible factor 1-β) as a CD30 interacting protein (Fig. 1 S1). ARNT is a transcription factor that is integral for the regulation of xenobiotic and hypoxic responses (13-15). Fig. 1 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 … We cloned the coding sequence of ARNT from Karpas 299 cells and used it to derive nested GSK2118436A deletions of ARNT (Fig. S1C). We tested three major regions of ARNT the basic-helix-loop-helix (bHLH) domain the Period-ARNT-Single minded (PAS) domain (a protein-protein interface) and the transactivation domain for their ability to bind the cytoplasmic region of CD30. Precipitation analysis of the different ARNT constructs with a tagged form of CD30 in which the cytoplasmic domain of CD30 was fused downstream of glutathione-S-transferase (GST) confirmed that ARNT interacted with CD30 through the PAS and transactivation domains (Fig. 1A). Thus at least two regions of ARNT are responsible for its binding to the cytoplasmic tail of CD30 and other regions of ARNT appear to negatively regulate this binding (Fig. 1A). To explore the spatial and temporal co-localization of CD30 and ARNT Goserelin Acetate we surface labeled Karpas 299 cells expressing either GFP or ARNT-GFP with a CD30-phytoerythrin (PE) conjugated GSK2118436A antibody. Confocal microscopy showed that CD30 was predominantly localized to the plasma membrane three hours after stimulation with anti-CD30-PE (Fig. 1B and S2) whereas ARNT was localized to the nucleus as expected and no co-localization was detected with CD30 at the plasma membrane. In contrast twenty-four hours after stimulation all of the originally surface labeled CD30 was found in the cytoplasm consistent with a previous report (16). ARNT-GFP was also detected in the cytoplasm at this later time point where it co-localized with internalized CD30. A panel of classical NF-κB responsive genes showed increased transcription when CD30 was labeled with anti-CD30-PE to a level comparable to that in cells stimulated for 10 min with CD30 GSK2118436A ligand (CD30L; Fig. 1C and ?and2A) 2 ensuring that anti-CD30-PE induces CD30 signaling. Thus the association of ARNT with CD30 appears to occur in the cytoplasm late in CD30 signaling. Fig. 2 Augmented CD30-mediated activation of NF-κB after suppression of ARNT. Control siRNA (siControl) or ARNT siRNA GSK2118436A (siARNT) oligonucleotides were introduced into Karpas 299 cells followed by a 10-minute exposure to (A) CD30L or (B) continuous exposure … The interaction of ARNT with CD30 was greatly impaired if the last 36 amino acids of CD30 were deleted (Fig. 1C). The sequences for TRAF binding are within these 36 amino acids of CD30 and the ability to recruit the TRAF proteins is required for full NF-κB activation (8) suggesting that ARNT might influence CD30-mediated NF-κB activity. We investigated this possibility with two short interfering RNA (siRNA) oligonucleotides that suppress ARNT expression in Karpas 299 cells. After ARNT suppression Karpas 299 cells were exposed to either a 10 min CD30 stimulation by the.