Activation of Chaperone-Mediated Autophagy Inhibits the Aryl Hydrocarbon Receptor Function by Degrading This Receptor in Human Lung Epithelial Carcinoma A549 Cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and a substrate protein of a Cullin 4B E3 ligase complex responsible for diverse cellular processes. In the lung, this receptor is responsible for the bioactivation of benzo[a]pyrene during tumorigenesis. Realizing that the AHR function is affected by its expression level, we are interested in the degradation mechanism of AHR in the lung. Here, we have investigated the mechanism responsible for AHR degradation using human lung epithelial A549 cells. We have observed that the AHR protein levels increase in the presence of chloroquine (CQ), an autophagy inhibitor, in a dose-dependent manner. Treatment with 6-aminonicotinamide (6-AN), a chaperone-mediated autophagy (CMA) activator, decreases AHR protein levels in a concentration-dependent and time-dependent manner. This decrease suppresses the ligand-dependent activation of the AHR target gene transcription, and can be reversed by CQ but not MG132. Knockdown of lysosome-associated membrane protein 2 (LAMP2), but not autophagy-related 5 (ATG5), suppresses the chloroquine-mediated increase in the AHR protein. AHR is resistant to CMA when its CMA motif is mutated. Suppression of the epithelial-to-mesenchymal transition in A549 cells is observed when the AHR gene is knocked out or the AHR protein level is reduced by 6-AN. Collectively, we have provided evidence supporting that AHR is continuously undergoing CMA and activation of CMA suppresses the AHR function in A549 cells.

Glycogen Synthase Kinase 3 Beta Regulates the Human Aryl Hydrocarbon Receptor Cellular Content and Activity.

The aryl hydrocarbon receptor (AHR) is a cytosolic receptor which is involved in diverse cellular events in humans. The most well-characterized function of AHR is its ability to upregulate gene transcription after exposure to its ligands, such as environmental toxicants, dietary antioxidants, drugs, and endogenous ligands. The cellular content of AHR is partly controlled by its degradation via the ubiquitin-proteasome system and the lysosome-dependent autophagy. We used human cervical cancer (HeLa) cells to investigate how AHR undergoes protein degradation and how its activity is modulated. Since the glycogen synthase kinase 3 beta (GSK3ß)-mediated phosphorylation can trigger protein degradation and substrates of GSK3ß contain stretches of serine/threonine residues which can be found in AHR, we examined whether degradation and activity of AHR can be controlled by GSK3ß. We observed that AHR undergoes the GSK3ß-dependent, LC3-mediated lysosomal degradation without ligand treatment. The AHR can be phosphorylated in a GSK3ß-dependent manner at three putative sites (S436/S440/S444, S689/S693/T697, and S723/S727/T731), which leads to lysosomal degradation of the AHR protein. Inhibition of the GSK3ß activity suppresses the ligand-activated transcription of an AHR target gene in HeLa, human liver cancer (Hep3B), and human breast cancer (MCF-7) cells. Collectively, our findings support that phosphorylation of AHR by GSK3ß is essential for the optimal activation of its target gene transcription and this phosphorylation may partake as an "off" switch by subjecting the receptor to lysosomal degradation.

The Aryl Hydrocarbon Receptor Undergoes Chaperone-Mediated Autophagy in Triple-Negative Breast Cancer Cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule expressed in many cell types, including triple-negative and non-triple-negative breast cancer cells. It affects breast cancer growth and crosstalk with estrogen receptor signaling. Normally, this receptor is degraded shortly after ligand activation via the 26S proteasome. Here, we report that AHR undergoes chaperone-mediated autophagy in MDA-MB-468 triple-negative breast cancer cells. This lysosomal degradation of AHR exhibits the following characteristics: (1) it is triggered by 6 amino-nicotinamide, starvation, and piperazinylpyrimidine compound Q18; (2) it is not observed in non-triple-negative breast cancer cells (MCF-7, T47D, and MDA-MB-361); (3) it can be inhibited by progesterone receptor B but not estrogen receptor alpha; (4) it can be reversed by chloroquine but not MG132; (5) it requires LAMP2A; and (6) it involves AHR-HSC70 and AHR-LAMP2A interactions. The NEKFF sequence localized at amino acid 558 of human AHR appears to be a KFERQ-like motif of chaperone-mediated autophagy, responsible for the LAMP2A-mediated AHR protein degradation.

Selective Autophagy Maintains the Aryl Hydrocarbon Receptor Levels in HeLa Cells: A Mechanism That Is Dependent on the p23 Co-Chaperone.

The aryl hydrocarbon receptor (AHR) is an environmental sensing molecule which impacts diverse cellular functions such as immune responses, cell growth, respiratory function, and hematopoietic stem cell differentiation. It is widely accepted that the degradation of AHR by 26S proteasome occurs after ligand activation. Recently, we discovered that HeLa cells can modulate the AHR levels via protein degradation without exogenous treatment of a ligand, and this degradation is particularly apparent when the p23 content is down-regulated. Inhibition of autophagy by a chemical agent (such as chloroquine, bafilomycin A1, or 3-methyladenine) increases the AHR protein levels in HeLa cells whereas activation of autophagy by short-term nutrition deprivation reduces its levels. Treatment of chloroquine retards the degradation of AHR and triggers physical interaction between AHR and LC3B. Knockdown of LC3B suppresses the chloroquine-mediated increase of AHR. Down-regulation of p23 promotes AHR degradation via autophagy with no change of the autophagy-related gene expression. Although most data in this study were derived from HeLa cells, human lung (A549), liver (Hep3B), and breast (T-47D and MDA-MB-468) cells also exhibit AHR levels sensitive to chloroquine treatment and AHR-p62/LC3 interactions. Here we provide evidence supporting that AHR undergoes the p62/LC3-mediated selective autophagy in HeLa cells.

Aryl hydrocarbon receptor acts as a tumor suppressor in a syngeneic MC38 colon carcinoma tumor model.

Background: Aryl hydrocarbon receptor (AHR), commonly known as an environmental sensor involved in the metabolism and elimination of xenobiotic substances, is also an important modulator in the development and functioning of the immune system. AHR expression is varied in the T cell subsets with the highest expression in T-helper 17 and T regulatory cells. It has been reported that AHR can act as a tumor promoter or a tumor suppressor, depending on the tumor type. Methods: In an effort to understand the role played by AHR in tumor growth, the MC38 syngeneic colon carcinoma tumor model was used on C57BL/6 or ahr knockout (KO, -/-) mice with or without AHR antagonist (CH223191) treatment. Tumor sizes were measured, and biomarkers were quantified in tumor microenvironment and draining lymph nodes using flow cytometry. Enzyme-linked immunosorbent assay was used to determine the amount of cytokines in tumors. Results: In ahr deficient mice, MC38 tumors progress more rapidly than in wild-type mice, accompanied by an increase in tumor-associated macrophages and M2 macrophages and a decrease in CD8a positive cytotoxic lymphocytes. Analysis of cytokines in the tumor microenvironment reveals a pro-inflammatory phenotype. Similar changes were observed by pharmacologic blockade of the receptor using CH223191. Conclusion: AHR acts as a tumor suppressor in mice implanted with MC38 colon carcinoma cells as evidenced by either a blockade or deficiency of AHR.

Down-Regulation of p23 in Normal Lung Epithelial Cells Reduces Toxicities From Exposure to Benzo[a]pyrene and Cigarette Smoke Condensate via an Aryl Hydrocarbon Receptor-Dependent Mechanism.

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule which controls tumor growth and metastasis, T cell differentiation, and liver development. Expression levels of this receptor protein is sensitive to the cellular p23 protein levels in immortalized cancer cell lines. As little as 30% reduction of the p23 cellular content can suppress the AHR function. Here we reported that down-regulation of the p23 protein content in normal, untransformed human bronchial/tracheal epithelial cells to 48% of its content also suppresses the AHR protein levels to 54% of its content. This p23-mediated suppression of AHR is responsible for the suppression of (1) the ligand-dependent induction of the cyp1a1 gene transcription; (2) the benzo[a]pyrene- or cigarette smoke condensate-induced CYP1A1 enzyme activity, and (3) the benzo[a]pyrene and cigarette smoke condensate-mediated production of reactive oxygen species. Reduction of the p23 content does not alter expression of oxidative stress genes and production of PGE2. Down regulation of p23 suppresses the AHR protein levels in two other untransformed cell types, namely human breast MCF-10A and mouse immune regulatory Tr1 cells. Collectively, down-regulation of p23 suppresses the AHR protein levels in normal and untransformed cells and can in principle protect our lung epithelial cells from AHR-dependent oxidative damage caused by exposure to agents from environment and cigarette smoking.

Single Versus Dual Lead Atrioventricular Sequential Pacing for Acquired Atrioventricular Block During Transcatheter Aortic Valve Implantation Procedures.

Atrioventricular block (AVB) after transcatheter aortic valve implantation (TAVI) is common. Pacing platforms that preserve atrioventricular (AV) synchrony may be beneficial in these patients. Single lead AV sequential pacemakers (VDD) allow AV synchrony and, by virtue of implanting a single lead, can be advantageous when compared with traditional dual lead (DDD) pacemakers. The objective of this retrospective case-controlled study was to compare the strategy of AV sequential pacing with a VDD versus DDD pacemaker in consecutive TAVI patients (n = 120) with acquired AVB. Patients were classified as receiving a VDD (n = 73) or DDD (n = 47) pacemaker. Procedure characteristics, complications, and the need for long-term ventricular pacing reported. Pacemaker implantation time (51 vs 66 minutes and p <0.001), fluoroscopy time (3.7 vs 7.8 minutes and p = 0.004), and radiation exposure (dose area product: 6.5 vs 15.3 mGy/cm2 and p = 0.006) were lower in patients receiving VDD pacemakers. Procedural complications were similar in the 2 groups. Overall, 59% of the cohort required ≥50% ventricular pacing at 1-year follow-up. In conclusion, VDD pacemaker implantation was associated with shorter procedure times, lower radiation exposure, and similar safety outcomes when compared with DDD pacemaker implantation in TAVI patients with acquired AVB.

p23 protects the human aryl hydrocarbon receptor from degradation via a heat shock protein 90-independent mechanism.

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule which is involved in diverse biological functions ranging from cancer metastasis to immune regulation. This receptor forms a cytoplasmic complex with Hsp90, p23, and XAP2. We have previously reported that down-regulation of p23 triggers degradation of the AHR protein, uncovering a potentially dynamic event which controls the cellular AHR levels without ligand treatment. Here we investigate the underlying mechanisms for this p23 effect using wild-type HeLa and the p23 knockdown HeLa cells. Reduction of the Hsp90 and XAP2 contents, however, did not affect the AHR protein levels, implying that this p23 effect on AHR is more than just alteration of the cytoplasmic complex dynamics. Association of p23 with Hsp90 is not important for the modulation of the AHR levels since exogenous expression of p23 mutants with modest Hsp90-binding affinity effectively restored the AHR message and protein levels. The protein folding property of p23 which resides at the terminal 50-amino acid region is not involved for this p23 effect. Results from our interaction study using the affinity purified thioredoxin fusion proteins and GST fusion proteins showed that p23 directly interacts with AHR and the interaction surface lies within AHR amino acid 1-216 and p23 amino acid 1-110. Down-regulation of the p23 protein content promotes the ubiquitination of AHR, indicating that p23 protects AHR from the ubiquitin-meditated protein degradation.

Selective suppression of the human aryl hydrocarbon receptor function can be mediated through binding interference at the C-terminal half of the receptor.

The human aryl hydrocarbon receptor is a cytosolic signaling molecule which affects immune response and aberrant cell growth. Canonical signaling of the receptor requires the recruitment of coactivators to the promoter region to remodel local chromatin structure. We predicted that interference of this recruitment would block the aryl hydrocarbon receptor function. To prove that, we employed phage display to identify nine peptides of twelve-amino-acid in length which target the C-terminal half of the human aryl hydrocarbon receptor, including the region where coactivators bind. Eight 12mer peptides, in the form of GFP fusion, suppressed the ligand-dependent transcription of six AHR target genes (cyp1a1, cyp1a2, cyp1b1, ugt1a1, nqo1, and ahrr) in different patterns in Hep3B cells, whereas the AHR antagonist CH-223191 suppressed all these target genes similarly. Three of the 12mer peptides (namely 11-3, 1-7, and 7-3) suppressed the 3MC-induced, CYP1A1-dependent EROD activity and the ROS production caused by benzo[a]pyrene. These 12mer peptides suppressed the AHR function synergistically with CH-223191. In conclusion, we provide evidence that targeting the C-terminal half of the human aryl hydrocarbon receptor is a viable, new approach to selectively block the receptor function.

Genetic ablation of PRAS40 improves glucose homeostasis via linking the AKT and mTOR pathways.

Alterations in PI3K-AKT-mTOR signaling have been implicated in diabetes. This study assessed whether disruption of PRAS40, a substrate of AKT and component of mTORC1, would alter glucose homeostasis and prevent hyperglycemia in the streptozotocin (STZ)-induced diabetes mouse model. PRAS40 ablation resulted in a mild lowering of blood glucose levels and glycated hemoglobin (HbA1C), a lowered insulin requirement, and improved glucose tolerance in untreated PRAS40 gene knockout (PRAS40(-/-)) as compared to wild-type (PRAS40(+/+)) mice. Diabetes was then induced in these mice using STZ at 50mg/kg/day over five days. Following STZ-treatment, PRAS40(-/-) mice exhibited significantly lower blood glucose and HbA1C levels than PRAS40(+/+) mice. Liver tissue of PRAS40(-/-) mice and shPRAS40 Hep3B cells showed increased activation of AKT (p-AKT T308) and mTORC1 (p-p70S6K) signaling as well as decreased p-AKT (S473) and increased p-IRS1 (S612) protein levels. Altered tissue gene expression of several glucose transporters (GLUT) and increased hepatic GLUT4 protein levels were observed in PRAS40(-/-) as compared to PRAS40(+/+) mice. In summary, PRAS40 deletion significantly attenuates hyperglycemia in STZ-induced PRAS40(-/-) mice through increased hepatic AKT and mTORC1 signaling, a lowered serum insulin requirement, and altered hepatic GLUT4 levels.

Differential suppression of the aryl hydrocarbon receptor nuclear translocator-dependent function by an aryl hydrocarbon receptor PAS-A-derived inhibitory molecule.

The aryl hydrocarbon receptor (AhR) heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) for transcriptional regulation. We generated three N-terminal deletion constructs of the human AhR of 12-24 kDa in size--namely D1, D2, and D3--to suppress the Arnt function. We observed that all three deletions interact with the human Arnt with similar affinities. D2, which contains part of the AhR PAS-A domain and interacts with the PAS-A domain of Arnt, inhibits the formation of the AhR gel shift complex. D2 suppresses the 3-methylcholanthrene-induced, dioxin response element (DRE)-driven luciferase activity in Hep3B cells and exogenous Arnt reverses this D2 suppression. D2 suppresses the induction of CYP1A1 at both the message and protein levels in Hep3B cells; however, the CYP1B1 induction is not affected. D2 suppresses the recruitment of Arnt to the cyp1a1 promoter but not to the cyp1b1 promoter, partly because the AhR/Arnt heterodimer binds better to the cyp1b1 DRE than to the cyp1a1 DRE. Interestingly, D2 has no effect on the cobalt chloride-induced, hypoxia inducible factor-1 (HIF-1)-dependent expression of vegf, aldolase c, and ldh-a messages. Our data reveal that the flanking sequences of the DRE contribute to the binding affinity of the AhR/Arnt heterodimer to its endogenous enhancers and the function of AhR and HIF-1 can be differentially suppressed by the D2 inhibitory molecule.

AG10 inhibits amyloidogenesis and cellular toxicity of the familial amyloid cardiomyopathy-associated V122I transthyretin.

The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Ig light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African American subjects, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 y causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the development of AG10, a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I- and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10, combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy.

A cell-penetrating peptide suppresses the hypoxia inducible factor-1 function by binding to the helix-loop-helix domain of the aryl hydrocarbon receptor nuclear translocator.

The heterodimeric hypoxia inducible factor-1 (HIF-1) complex is composed of the hypoxia inducible factor-1 alpha (HIF-1α) and the aryl hydrocarbon receptor nuclear translocator (ARNT). Activation of the HIF-1 function is essential for tumor growth and metastasis. We previously showed that transfection of a plasmid containing an ARNT-interacting peptide (Ainp1) cDNA suppresses the HIF-1 signaling in Hep3B cells. Here we generated TAT fusion of the Ainp1 peptide (6His-TAT-Ainp1) to determine whether and how the Ainp1 peptide suppresses the HIF-1 function. The bacterially expressed 6His-TAT-Ainp1 was purified under denatured condition and then refolded by limited dialysis. The refolded 6His-TAT-Ainp1 interacts with the helix-loop-helix (HLH) domain of ARNT in a similar fashion as the native 6His-Ainp1. 6His-TAT-Ainp1 colocalizes with ARNT in the nucleus of HeLa and Hep3B cells after protein transduction. The transduced protein reaches the maximum intracellular levels within 2 h while remains detectable up to 96 h in HeLa cells. At 2 µM concentration, 6His-TAT-Ainp1 is not cytotoxic in HeLa cells but suppresses the cobalt chloride-activated, hypoxia responsive enhancer-driven luciferase expression in a dose-dependent manner. In addition, it decreases the cobalt chloride-dependent induction of the HIF-1 target genes at both the message (vascular endothelial growth factor and aldolase C) and protein (carbonic anhydrase IX and glucose transporter 1) levels. The protein levels of HIF-1α and ARNT are not altered in the presence of 6His-TAT-Ainp1. In summary, we provided evidence to support that the Ainp1 peptide directly suppresses the HIF-1 function by interacting with the ARNT HLH domain, and in turn interfering with the heterodimerization of HIF-1α and ARNT.

p23 co-chaperone protects the aryl hydrocarbon receptor from degradation in mouse and human cell lines.

The aryl hydrocarbon receptor (AhR) is a ligand-sensitive transcription factor which is responsible for most 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicities. Without ligand, the AhR complex is cytoplasmic and contains p23. Our objective was to investigate whether the wild type p23 levels are important for the AhR function. We generated eight p23-specific knockdown stable cell lines via either electroporation or lentiviral infection. Five of these stable cell lines were generated from a mouse hepatoma cell line (Hepa1c1c7) and three were from human hepatoma and cervical cell lines (Hep3B and HeLa). All of them expressed lower AhR protein levels, leading to reduced ligand-induced, DRE-driven downstream activity. The AhR protein levels in p23-specific knockdown stable cells were reversed back to wild type levels after exogenous p23 was introduced. Reduction of the AhR protein levels in these stable cells was caused by a decrease in the AhR message levels and an increase of the AhR protein degradation in the absence of ligand. This ligand-independent degradation of AhR was not reversed by MG132, suggesting that the 26S proteasome was not responsible for the degradation. In addition, MG132 could not protect AhR from the ligand-induced degradation in both mouse and human p23-knockdown stable cells.

Suppression of the hypoxia inducible factor-1 function by redistributing the aryl hydrocarbon receptor nuclear translocator from nucleus to cytoplasm.

The aryl hydrocarbon receptor nuclear translocator (ARNT) heterodimerizes with hypoxia inducible factor-1α (HIF-1α), followed by upregulation of genes that are essential for carcinogenesis. We utilized a novel peptide (Ainp1) to address whether the HIF-1α signaling could be suppressed by an ARNT-mediated mechanism. Ainp1 suppresses the HIF-1α-dependent luciferase expression in Hep3B cells and this suppression can be reversed by ARNT. Ainp1 reduces the interaction between ARNT and HIF-1α, suppresses the formation of the HIF-1 gel shift complex, and suppresses the ARNT recruitment to the vegf promoter. These effects are partly mediated by redistribution of the nuclear ARNT contents to the cytoplasm.

Identification of cyclophilin-40-interacting proteins reveals potential cellular function of cyclophilin-40.

Cyclophilin-40 (CyP40) is part of the immunophilin family and is found in Hsp90-containing protein complexes. We were interested in identifying proteins that interact with CyP40. CyP40-interacting proteins in HeLa cells were identified using the tandem affinity purification approach. Adenovirus expressing human CyP40 protein (Ad-CyP40), fused with streptavidin and calmodulin binding peptides at the N terminus, was generated. Proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel after tandem affinity purification. Here 10 silver-stained protein bands that were enriched in the Ad-CyP40-infected lysate and the corresponding regions in the control lysate were excised, digested by trypsin, and identified by tandem mass spectrometric analysis. Of 11 interacting proteins that were identified, 4 (RACK1, Ku70, RPS3, and NF45) were expressed in rabbit reticulocyte lysate, bacteria, and MCF-7 cells. We confirmed that these proteins interact with CyP40. We observed that RACK1 suppressed the cobalt chloride-induced, hypoxia response element-dependent luciferase activity in MCF-7 cells but not in MCF-7 stable cells expressing approximately 10% of the cellular CyP40 content. In addition, RACK1 reduced the HIF-1α protein accumulation after cobalt chloride treatment, which was not observed when the CyP40 content was down-regulated. Collectively, we conclude that reduction of the HIF-1 α protein by RACK1 is CyP40-mediated.

The aryl hydrocarbon receptor nuclear translocator-interacting protein 2 suppresses the estrogen receptor signaling via an Arnt-dependent mechanism.

We explored whether modulation of the estrogen receptor (ER) signaling is possible through an aryl hydrocarbon receptor nuclear translocator (Arnt)-dependent mechanism. We utilized the Arnt-interacting protein 2 (Ainp2) to examine whether the presence of Ainp2 in MCF-7 cells would interfere with the Arnt-mediated ER signaling. We found that Arnt increased the 17 beta-estradiol (E2)-dependent luciferase activity and Ainp2 significantly suppressed this Arnt-mediated luciferase activity. Ainp2 significantly suppressed 25% of the E2- and Arnt-dependent up-regulation of the GREB1 message. No suppression of the ER target gene expression by Ainp2 was detected in Arnt-knockdown MCF-7 cells and in Arnt-independent ER signaling. Although Ainp2 did not interact with ER alpha and ER beta, it suppressed the ER alpha::Arnt interaction and reduced the E2-driven recruitment of Arnt to the GREB1 promoter. We concluded that Ainp2 suppresses the ER signaling by not allowing Arnt to participate in the ER-dependent, Arnt-mediated activation of gene transcription.

A truncated human Ah receptor suppresses growth of human cervical tumor xenografts by interfering with hypoxia signaling.

We used a xenograft model to investigate whether the aryl hydrocarbon receptor deletion construct CDelta553 suppresses tumor growth. HeLa cells that were infected with CDelta553 expressing adenovirus (Ad553) formed very small tumors whereas the control adenovirus-infected cells formed large tumors at day 15. CDelta553 inhibited the formation of the HIF-1 DNA complex and suppressed the induction of the HIF-1alpha target proteins CAIX and GLUT1. The Ad553 tumors had less HIF-1 function since they showed reduced microvessel formation and lesser amounts of HIF-1alpha, Arnt, phospho-Akt, CAIX, and GLUT1. Proteasome-mediated Arnt degradation was enhanced in Ad553-infected HeLa cells and tumors.

Cyclophilin-40 has a cellular role in the aryl hydrocarbon receptor signaling.

Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidyl-prolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.

A truncated Ah receptor blocks the hypoxia and estrogen receptor signaling pathways: a viable approach for breast cancer treatment.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which requires heterodimerization with the Ah receptor nuclear translocator (Arnt) for function. Arnt is also a dimerization partner of the hypoxia inducible factor 1alpha (HIF-1alpha) for the hypoxia signaling. Additionally, Arnt is found to be a potent coactivator of the estrogen receptor (ER) signaling. Thus we examined whether the presence of an increased amount of AhR may suppress both the HIF-1alpha and ER signaling pathways by sequestering Arnt. We tested our hypothesis using a human AhR construct C Delta553 which is capable of heterodimerizing with Arnt in the absence of a ligand. Transient transfection studies using a corresponding luciferase reporter plasmid in MCF-7 cells showed that C Delta553 effectively suppressed the AhR, HIF-1alpha, and ER signaling pathways. Reverse transcription/real-time QPCR data showed that C Delta553 blocked the up-regulation of the target genes controlled by AhR (CYP1A1), HIF-1alpha (VEGF, aldolase C, and LDH-A), and ER (GREB1, pS2, and c-myc) in MCF-7 cells. Since both HIF-1alpha and ER are highly active in the ER-positive breast cancer, C Delta553 has the potential to be developed as a protein drug to treat breast cancer by blocking these two signaling pathways.