The unliganded aryl hydrocarbon receptor (AHR) is found in a complex with other proteins including the 90-kDa heat shock protein (Hsp90) and a 37-kDa protein we refer to as ARA9. We found that the three tetratricopeptide repeats found in the COOH terminus of ARA9 are necessary and sufficient for interaction with the AHR complex. Conversely, the AHR's "repressor"/Hsp90 binding domain is required for interaction with ARA9. Because ARA9 closely resembles the 52-kDa FK506-binding protein (FKBP52), found in the unliganded glucocorticoid receptor (GR) complex, we compared the binding specificities of ARA9 and FKBP52 for AHR and GR. In co-immunoprecipitation experiments, ARA9 specifically associated with AHR-Hsp90 complex but not with GRHsp90 complexes. In addition, ARA9 showed a greater capacity than FKBP52 to associate with AHR-Hsp90 complexes. The biological importance of this interaction was suggested by the observation that in a yeast expression system ARA9 expression enhanced the response of AHR to the agonist ␤-napthoflavone, decreasing the EC 50 by greater than 5-fold and increasing the maximal response 2.5-fold. In contrast, co-expression of FKBP52 had no effect on AHR signaling. In addition, although ARA9 contains a domain similar to that found in other FK506-binding proteins, ARA9 binding to 3 H-FK506 could not be detected. Finally, we have characterized the developmental and expression pattern of ARA9 in the developing mouse embryo and mapped the ARA9 locus to human chromosome 11q13.3.

SnoN is an important negative regulator of transforming growth factor ␤ signaling through its ability to interact with and repress the activity of Smad proteins. It was originally identified as an oncoprotein based on its ability to induce anchorage-independent growth in chicken embryo fibroblasts. However, the roles of SnoN in mammalian epithelial carcinogenesis have not been well defined. Here we show for the first time that SnoN plays an important but complex role in human cancer. SnoN expression is highly elevated in many human cancer cell lines, and this high level of SnoN promotes mitogenic transformation of breast and lung cancer cell lines in vitro and tumor growth in vivo, consistent with its proposed prooncogenic role. However, this high level of SnoN expression also inhibits epithelial-to-mesenchymal transdifferentiation. Breast and lung cancer cells expressing the shRNA for SnoN exhibited an increase in cell motility, actin stress fiber formation, metalloprotease activity, and extracellular matrix production as well as a reduction in adherens junction proteins. Supporting this observation, in an in vivo breast cancer metastasis model, reducing SnoN expression was found to moderately enhance metastasis of human breast cancer cells to bone and lung. Thus, SnoN plays both protumorigenic and antitumorigenic roles at different stages of mammalian malignant progression. The growth-promoting activity of SnoN appears to require its ability to bind to and repress the Smad proteins, while the antitumorigenic activity can be mediated by both Smad-dependent and Smad-independent pathways and requires the activity of small GTPase RhoA. Our study has established the importance of SnoN in mammalian epithelial carcinogenesis and revealed a novel aspect of SnoN function in malignant progression.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of agonists like 2,3,7,8-tetrachlorodibenzo-p-dioxin. In the current model for AHR signaling, the unliganded receptor is found in the cytosol as part of a complex with a dimer of the 90-kDa heat shock protein and an immunophilin-like molecule, ARA9. In yeast, expression of ARA9 results in an increase in the maximal agonist response and a leftward shift in the AHR dose-response curve. To better understand the mechanism by which ARA9 modifies AHR signal transduction, we performed a series of coexpression experiments in yeast and mammalian cells. Our results demonstrate that ARA9's influence on AHR signaling is not due to inhibition of a membrane pump or modification of the receptor's transactivation properties. Using receptor photoaffinity labeling experiments, we were able to show that ARA9 enhances AHR signal transduction by increasing the available AHR binding sites within the cytosolic compartment of the cell. Our evidence suggests that ARA9's effects are related to its role as a cellular chaperone; i.e. we observed that expression of ARA9 increases the fraction of AHR in the cytosol and also stabilized the receptor under heat stress. The AHR1 is a ligand-activated transcription factor that mediates the biological effects of halogenated dioxins and related compounds (1). In a widely held model of dioxin signal transduction, the AHR is found in the cytosol, in a complex with Hsp90 and a newly discovered protein known as ARA9 (2-4).2 In the presence of agonist, the AHR translocates to the nucleus, where it binds to its nuclear partner, ARNT. This AHR⅐ARNT heterodimer is capable of binding DNA and promotes the transcription of a battery of responsive genes including those encoding a number of xenobiotic-metabolizing enzymes (5). Hsp90 has been shown to play a role in maintaining AHR in a conformation that can bind ligand with high affinity (6 -8). Although ARA9 has been shown to increase AHR function in yeast and mammalian cells, its role in AHR signaling is not understood (7-10).ARA9 was initially identified in yeast two-hybrid screens, in which the AHR or the hepatitis B virus protein X were used as bait (9 -11). Later, it was purified from monkey cells in a complex with the AHR (12). ARA9 contains two notable structural motifs. In its amino terminus, ARA9 contains an FKBP homology domain. This domain shares 28% amino acid sequence identity to FKBP12 (9,11,12). However, ARA9 is unable to bind FK506 and does not appear to have peptidyl prolyl isomerase activity 3 (13). In its carboxyl terminus, ARA9 contains three TPRs. TPRs are defined by strings of 34 amino acids that have been shown to play roles in protein-protein interaction (14). This domain structure is similar to that found in the GR-associated immunophilin, FKBP52, which contains two FKBP domains in its amino terminus and three TPRs in its carboxyl terminus (15,16). In addition to their structural similarities, ARA9 and FKBP52 are found associated to...

We tested the hypothesis that early alterations in calcium influx induced by an imposed 60 Hz magnetic field are propagated down the signal transduction (ST) cascade to alter c‐MYC mRNA induction. To test this we measured both ST parameters in the same cells following 60 Hz magnetic field exposures in a specialized annular ring device (220 G (22 mT), 1.7 maximal E induced, 37°C, 60 min). Ca2+ influx is a very early ST marker that precedes the specific induction of mRNA transcripts for the proto‐oncogene c‐MYC, an immediate early response gene. In three experiments influx of 45Ca2+ in the absence of mitogen was similar to that in cells treated with suboptimal levels of Con‐A (1 ). However, calcium influx was elevated 1.5‐fold when lymphocytes were exposed to Con‐A plus magnetic fields; this co‐stimulatory effect is consistent with previous reports from our laboratory [FEBS Lett. 301 (1992) 53‐59; FEBS Lett. 271 (1990) 157‐160; Ann. N.Y. Acad. Sci. 649 (1992) 74‐95]. The level of c‐MYC mRNA transcript copies in non‐activated cells and in suboptimally‐activated cells was also similar, which is consistent with the above calcium influx findings. Significantly, lymphocytes exposed to the combination of magnetic fields plus suboptimal Con‐A responded with an approximate 3.0‐fold increase in band intensity of c‐MYC mRNA transcripts. Importantly, transcripts for the housekeeping gene GAPDH were not influenced by mitogen or magnetic fields. We also observed that lymphocytes that failed to exhibit increased calcium influx in response to magnetic fields plus Con‐A, also failed to exhibit an increase in total copies of c‐MYC mRNA. Thus, calcium influx and c‐MYC mRNA expression, which are sequentially linked via the signal transduction cascade in contrast to GAPDH, were both increased by magnetic fields. These findings support the above ST hypothesis and provide experimental evidence for a general biological framework for understanding magnetic field interactions with the cell through signal transduction. In addition, these findings indicate that magnetic fields can act as a co‐stimulus at suboptimal levels of mitogen; pronounced physiological changes in lymphocytes such as calcium influx and c‐MYC mRNA induction were not triggered by a weak mitogenic signal unless accompanied by a magnetic field. Magnetic fields, thus, have the ability to potentiate or amplify cell signaling.