Interaction with YAP underlies the species differences between humans and rodents in CAR-dependent hepatocyte proliferation.

Constitutive androstane receptor (CAR), a nuclear receptor predominantly expressed in the liver, is activated by diverse chemicals and induces hepatocyte proliferation and hepatocarcinogenesis in rodents. However, the underlying mechanism responsible for CAR-dependent hepatocyte proliferation remains unclear. Importantly, this phenomenon has not been observed in the human liver. This study aimed to investigate the molecular mechanism underlying CAR-induced hepatocyte proliferation and to explore the species differences in hepatocyte proliferation between humans and rodents. Treatment of mice with the CAR activator TCPOBOP induced hepatocyte proliferation and nuclear accumulation of yes-associated protein (YAP), a known liver cancer inducer. This induction was abolished in CAR-knockout mice. Exogenously expressed YAP in cultured cells was accumulated in the nucleus by the coexpression with mouse CAR but not human CAR. Pull-down analysis of recombinant proteins revealed that mouse CAR interacted with YAP, whereas human CAR did not. Further investigations using YAP deletion mutants identified the WW domain of YAP as essential for interacting with CAR and showed that the PY motif (PPAY) in mouse CAR was crucial for binding to the WW domain, whereas human CAR with its mutated motif (PPAH) failed to interact with YAP. A mouse model harboring the Y150H mutation (PPAY to PPAH) in CAR displayed drastically attenuated TCPOBOP-induced hepatocyte proliferation and nuclear accumulation of YAP. CAR induces the nuclear accumulation of YAP through the PY motif-WW domain interaction to promote hepatocyte proliferation. The absence of this interaction in human CAR contributes to the lack of CAR-dependent hepatocyte proliferation in human livers.

FDI-6, a FOXM1 inhibitor, activates the aryl hydrocarbon receptor and suppresses tumorsphere formation.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is activated by environmental contaminants such as dioxins and polycyclic aromatic hydrocarbons. Following ligand binding, AhR binds to xenobiotic responsive elements and modulates the transcription of AhR target genes. Multiple studies have shown that AhR plays important roles in a range of cancer cells and is attracting attention as a therapeutic target for cancer treatment. We have previously reported that AhR agonists inhibit tumorsphere formation in an AhR-dependent manner in the MCF-7 breast cancer cell line. In the present study, we found that FDI-6, an inhibitor of the transcription factor Forkhead Box M1 (FOXM1) induced the mRNA expression of AhR target genes, nuclear translocation of AhR, and transcriptional activity of AhR. In addition, FDI-6 dose-dependently reduced the mRNA expression of FOXM1-regulated genes in AhR-expressing MCF-7 cells, although not in AhR-deficient MCF-7 cells. Furthermore, FDI-6 was found to suppress tumorsphere formation via the AhR in MCF-7 cells and HepG2 human liver cancer cell line. On the basis of the findings of this study, we show that FDI-6, a FOXM1 inhibitor, functions as an AhR agonist, and suppresses tumorsphere formation via the AhR.

Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats.

Constitutive androstane receptor (CAR) is a nuclear receptor that plays a key role in drug metabolism and disposition and in the development of liver tumors in rodents. CAR is activated by ligands and indirect activators, which do not bind to the receptor but activate it through cellular signaling. In this study, we sought to identify direct and indirect activators of rat CAR (rCAR). Assessment of the influence of mutations on the transcriptional activity of rCAR identified a mutant termed rCAR-3A-G354Q that displays low constitutive activity and high ligand responsiveness. Reporter assays using the mutant were performed with compounds that increased the mRNA levels of Cyp2b1, a CAR target gene, in rat primary hepatocytes. Several compounds activated rCAR-3A-G354Q and were implicated as rCAR ligands. Since indirect CAR activators are considered to display little species differences, we then determined CYP2B6 mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increased Cyp2b1 mRNA levels in rat hepatocytes but did not activate rCAR-3A-G354Q. The results demonstrated six compounds as possible rCAR indirect activators. Taken together, the combined measurement of Cyp2b1 mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals.

Possible Involvement of the Upregulation of ΔNp63 Expression Mediated by HER2-Activated Aryl Hydrocarbon Receptor in Mammosphere Maintenance.

Cancer stem cells (CSCs) contribute to the drug resistance, recurrence, and metastasis of breast cancers. Recently, we demonstrated that HER2 overexpression increases mammosphere formation via the activation of aryl hydrocarbon receptor (AHR). In this study, the objective was to identify the mechanism underlying mammosphere maintenance mediated by HER2 signaling-activated AHR. We compared the chromatin structure of AHR-knockout (AHRKO) HER2-overexpressing MCF-7 (HER2-5) cells with that of wild-type HER2-5 cells; subsequently, we identified TP63, a stemness factor, as a potential target gene of AHR. ΔNp63 mRNA and protein levels were higher in HER2-5 cells than in HER2-5/AHRKO cells. Activation of HER2/HER3 signaling by heregulin treatment increased ΔNp63 mRNA levels, and its induction was decreased by AHR knockdown in HER2-5 cells. The results of the chromatin immunoprecipitation assay revealed an interaction between AHR and the intronic region of TP63, which encodes ΔNp63. A luciferase reporter gene assay with the intronic region of TP63 showed that AHR expression increased reporter activity. Collectively, our findings suggest that HER2-activated AHR upregulates ΔNp63 expression and that this signaling cascade is involved in CSC maintenance in HER2-expressing breast cancers.

Involvement of the CYP1A1 inhibition-mediated activation of aryl hydrocarbon receptor in drug-induced hepatotoxicity.

Hepatotoxicity is one of the most common toxicities observed in non-clinical safety studies of drug candidates, and it is important to understand the hepatotoxicity mechanism to assess the risk of drug-induced liver injury in humans. In this study, we investigated the mechanism of hepatotoxicity caused by 2-[2-Methyl-1-(oxan-4-yl)-1H-benzimidazol-5-yl]-1,3-benzoxazole (DSP-0640), a drug candidate that showed hepatotoxicity characterized by centrilobular hypertrophy and vacuolation of hepatocytes in a 4-week oral repeated-dose toxicity study in male rats. In the liver of rats treated with DSP-0640, the expression of aryl hydrocarbon receptor (AHR) target genes, including Cyp1a1, was upregulated. In in vitro reporter assays, however, DSP-0640 showed only minimal AHR-activating potency. Therefore, we investigated the possibility that DSP-0640 indirectly activated AHR by inhibiting the CYP1 enzyme-dependent clearance of endogenous AHR agonists. In in vitro assays, DSP-0640 showed inhibitory effects on both rat and human CYP1A1 and enhanced rat and human AHR-mediated reporter gene expression induced by 6-formylindolo[3,2-b]carbazole, a well-known endogenous AHR agonist. The possible involvement of CYP1A1 inhibition in AHR activation was also demonstrated with other hepatotoxic compounds tacrine and albendazole. These results suggest that CYP1A1 inhibition-mediated AHR activation is involved in the hepatotoxicity caused by DSP-0640 and that DSP-0640 might induce hepatotoxicity in humans as well. We propose that CYP1A1 inhibition-mediated AHR activation is a novel mechanism for drug-induced hepatotoxicity.

Differential DNA-binding and cofactor recruitment are possible determinants of the synthetic steroid YK11-dependent gene expression by androgen receptor in breast cancer MDA-MB 453 cells.

Recently, selective androgen receptor modulators (SARMs), which bind to AR and act in a tissue/effect-specific manner, have been developed, but the selective mechanism is not well understood. In this study, we investigated the selective mechanism using the synthetic steroid YK11, which showed AR-mediated gene-selective transactivation. In the AR-positive human breast cancer MDA-MB-453 cells, different patterns of AR-mediated target gene expression and AR recruitment to their enhancer regions were observed between DHT and YK11. A docking study suggested the helices 11 and 12 was moved by the sterically hindered C17-group of YK11. Furthermore, the mutational studies of AR Gln902 and mammalian two-hybrid assays suggested different cofactor recruitment between DHT and YK11. The results of this study suggest that gene selective regulation by SARMs results from differential DNA-binding and/or cofactor recruitment by ligands. These results provide novel insights into the mechanism of action of SARMs.

Aryl Hydrocarbon Receptor Directly Regulates VTCN1 Gene Expression in MCF-7 Cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.

Camalexin, an indole phytoalexin, inhibits cell proliferation, migration, and mammosphere formation in breast cancer cells via the aryl hydrocarbon receptor.

Breast cancer is the most commonly diagnosed cancer among women worldwide. Despite a variety of drugs available for the treatment of patients with breast cancer, drug resistance remains a significant clinical problem. Therefore, there is an urgent need to develop drugs with new mechanisms of action. Camalexin is the main indole phytoalexin in Arabidopsis thaliana and other crucifers. Camalexin inhibits the proliferation of various cancer cells. However, the mechanism by which camalexin inhibits cell proliferation remains unclear. In this study, we found that camalexin inhibited cell proliferation and migration of breast cancer cell lines. Furthermore, camalexin also suppressed breast cancer stem cell-derived mammosphere formation. We previously reported that the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) agonist suppresses mammosphere formation. Several compounds with indole structures are known to act as AhR agonists. Therefore, we hypothesized that the inhibition of mammosphere formation by camalexin may involve AhR activation. We found that camalexin increased the nuclear translocation of AhR, AhR-mediated transcriptional activation, and expression of AhR target genes. In addition, camalexin suppressed mammosphere formation in AhR-expressing breast cancer cells more than in the breast cancer cells that lacked AhR expression. Taken together, the data demonstrate that camalexin is a novel AhR agonist and that the inhibition of cell proliferation, migration, and mammosphere formation by camalexin involves the activation of AhR. Our findings suggest that camalexin, an AhR agonist, may be a novel therapeutic agent for breast cancer.

Chemical characterization of anemia-inducing aniline-related substances and their application to the construction of a decision tree-based anemia prediction model.

Anemia is a well-observed toxicity of chemical substances, and aniline is a typical anemia-inducing substance. However, it remains unclear whether all aniline-like substances with various substituents could induce anemia. We thus investigated the physicochemical characteristics of anemia-inducing substances by decision tree analyses. Training and validation substances were selected from a publicly available database of rat repeated-dose toxicity studies, and discrimination models were constructed by decision tree and bootstrapping methods with molecular descriptors as explanatory variables. To improve the accuracy of discrimination, we individually evaluated the explanatory variables to modify them, established "prerules" that were applied before subjecting a substance to a decision tree by considering metabolism, such as azo reduction and N-dealkylation, and introduced the idea of "partly negative" evaluation for substances having multiple aniline-like substructures. The final model obtained showed 79.2% and 77.5% accuracy for the training and validation dataset, respectively. In addition, we identified some chemical properties that reduce the anemia inducibility of aniline-like substances, including the addition of a sulfonate or carboxy functional group and/or a bulky multiring structure to anilines. In conclusion, the present findings will provide a novel insight into the mechanistic understanding of chemically induced anemia and help to develop a prediction system.

Activation of the aryl hydrocarbon receptor by 3-methylcholanthrene, but not by indirubin, suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates various toxicological and biological functions. We reported previously that 3-methylcholanthrene (3MC), an exogenous AhR agonist, inhibited tumorsphere (mammosphere) formation from breast cancer cell lines, while the endogenous AhR agonist, indirubin, very weakly inhibited this process. However, the difference in inhibition mechanism of mammosphere formation by 3MC or indirubin is still unknown. In this study, we established AhR-re-expressing (KOTR-AhR) cells from AhR knockout MCF-7 cells using the tetracycline (Tet)-inducible gene expression systems. To identify any difference in inhibition of mammosphere formation by 3MC or indirubin, RNA-sequencing (RNA-seq) experiments were performed using KOTR-AhR cells. RNA-seq experiments revealed that cell division cycle 20 (CDC20), which regulates the cell cycle and mitosis, was decreased by 3MC, but not by indirubin, in the presence of AhR expression. Furthermore, the mRNA and protein levels of CDC20 were decreased by 3MC in MCF-7 cells via the AhR. In addition, mammosphere formation was suppressed by small interfering RNA-mediated CDC20 knockdown compared to the negative control in MCF-7 cells. These results suggest that AhR activation by 3MC suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells. This study provides useful information for the development of AhR-targeted anti-cancer drugs.

Helix 12 stabilization contributes to basal transcriptional activity of PXR.

Pregnane X receptor (PXR) plays an important role in xenobiotic metabolism. While ligand binding induces PXR-dependent gene transcription, PXR shows constitutive transcriptional activity in the absence of ligands when expressed in cultured cells. This constitutive activity sometimes hampers investigation of PXR activation by compounds of interest. In this study, we investigated the molecular mechanism of PXR activation. In the reported crystal structures of unliganded PXR, helix 12 (H12), including a coactivator binding motif, was stabilized, while it is destabilized in the unliganded structures of other nuclear receptors, suggesting a role for H12 stabilization in the basal activity of PXR. Since Phe420, located in the loop between H11 and H12, is thought to interact with Leu411 and Ile414 to stabilize H12, we substituted alanine at Phe420 (PXR-F420A) and separately inserted three alanine residues directly after Phe420 (PXR-3A) and investigated their influence on PXR-mediated transcription. Reporter gene assays demonstrated that the mutants showed drastically reduced basal activity and enhanced responses to various ligands, which was further enhanced by coexpression of the coactivator peroxisome proliferator-activated receptor gamma coactivator 1α. Mutations of both Leu411 and Ile414 to alanine also suppressed basal activity. Mammalian two-hybrid assays showed that PXR-F420A and PXR-3A bound to corepressors and coactivators in the absence and presence of ligands, respectively. We conclude that the intramolecular interactions of Phe420 with Leu411 and Ile414 stabilize H12 to recruit coactivators even in the absence of ligands, contributing to the basal transcriptional activity of PXR. We propose that the generated mutants might be useful for PXR ligand screening.

The Differential Selectivity of Aryl Hydrocarbon Receptor (AHR) Agonists towards AHR-Dependent Suppression of Mammosphere Formation and Gene Transcription in Human Breast Cancer Cells.

We had previously reported that treatment with the aryl hydrocarbon receptor (AHR) agonist ß-naphthoflavone (ßNF) suppressed mammosphere formation derived from cancer stem cells in human breast cancer MCF-7 cells (Cancer Lett., 317, 2012, Zhao et al.). Here, using several AHR agonists, we have investigated the association of this suppression with the classical ability to induce AHR-mediated gene transcription in the xenobiotic response element (XRE). The mammosphere formation assays were performed using wild-type and AHR-knockout MCF-7 cells in the presence of AHR agonists including 3-methylcholanthrene (3MC), benzo[a]pyrene (BaP), 7,12-dimethylbenz[a]anthracene (DMBA), 6-formylindolo[3,2-b]carbazole (FICZ), indirubin, indole-3-carbinol (I3C), indole-3-acetic acid (IAA), and kynurenine (KYN), followed by the XRE-reporter gene assays of the agonists. We showed that treatments with 3MC, BaP, and DMBA strongly suppressed mammosphere formation of the stem cells in an AHR-dependent manner, while other agonists showed weaker suppression. In reporter gene assays, the strength or duration of AHR/XRE-mediated gene transcription was found to be dependent on the agonist. Although strong transcriptional activation was observed with 3MC, FICZ, indirubin, I3C, IAA, or KYN after 6 h of treatment, only weak activation was seen with BaP or DMBA. While transcriptional activation was sustained or increased at 24 h with 3MC, BaP, or DMBA, appreciable reduction was observed with the other agonists. In conclusions, the results demonstrated that the suppressive effects of AHR agonists on mammosphere formation do not necessarily correlate with their abilities to induce AHR-mediated gene transcription. Hence, different AHR functions may be differentially induced in an agonist-dependent manner.

Polycyclic aromatic hydrocarbons induce CYP3A5 gene expression via aryl hydrocarbon receptor in HepG2 cells.

The aryl hydrocarbon receptor (AhR) regulates expression of genes encoding drug/xenobiotic metabolizing enzymes. Cytochrome P450 (CYP) 3A5 is involved in drug metabolism. However, regulation of CYP3A5 gene expression is not yet well understood. In this study, we aimed to investigate the effect of the ligands of AhR on CYP3A5 gene expression. CYP3A5 mRNA expression was induced by the polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (3MC) and benzo[a]pyrene in HepG2 cells. To determine whether the PAHs induced CYP3A5 gene expression via AhR, we generated AhR knockout (AhR KO) HepG2 cells. CYP3A5 mRNA expression was not induced by 3MC treatment in AhR KO cells. In addition, we generated AhR rescue cells from AhR KO cells and evaluated CYP3A5 mRNA expression. We found that CYP3A5 mRNA expression was induced by 3MC treatment in AhR rescue cells. Taken together, these results demonstrated that CYP3A5 mRNA expression was induced by PAHs via AhR in HepG2 cells. Our findings suggest that ligand-activated AhR affects CYP3A5-mediated drug metabolism.

Antiepileptic Drug-Activated Constitutive Androstane Receptor Inhibits Peroxisome Proliferator-Activated Receptor α and Peroxisome Proliferator-Activated Receptor γ Coactivator 1α-Dependent Gene Expression to Increase Blood Triglyceride Levels.

Long-term administration of some antiepileptic drugs often increases blood lipid levels. In this study, we investigated its molecular mechanism by focusing on the nuclear receptors constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are key transcription factors for enzyme induction and lipid metabolism, respectively, in the liver. Treatment of mice with the CAR activator phenobarbital, an antiepileptic drug, increased plasma triglyceride levels and decreased the hepatic expression of PPARα target genes related to lipid metabolism. The increase in PPARα target gene expression induced by fenofibrate, a PPARα ligand, was inhibited by cotreatment with phenobarbital. CAR suppressed PPARα-dependent gene transcription in HepG2 cells but not in COS-1 cells. The mRNA level of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a coactivator for both CAR and PPARα, in COS-1 cells was much lower than in HepG2 cells. In reporter assays with COS-1 cells overexpressing PGC1α, CAR suppressed PPARα-dependent gene transcription, depending on the coactivator-binding motif. In mammalian two-hybrid assays, CAR attenuated the interaction between PGC1α and PPARα Chemical inhibition of PGC1α prevented phenobarbital-dependent increases in plasma triglyceride levels and the inhibition of PPARα target gene expression. These results suggest that CAR inhibits the interaction between PPARα and PGC1α, attenuating PPARα-dependent lipid metabolism. This might explain the antiepileptic drug-induced elevation of blood triglyceride levels. SIGNIFICANCE STATEMENT: Constitutive active/androstane receptor activated by antiepileptic drugs inhibits the peroxisome proliferator-activated receptor α-dependent transcription of genes related to lipid metabolism and upregulates blood triglyceride levels. The molecular mechanism of this inhibition involves competition between these nuclear receptors for coactivator peroxisome proliferator-activated receptor γ coactivator-1α binding.

Aryl hydrocarbon receptor counteracts pharmacological efficacy of doxorubicin via enhanced AKR1C3 expression in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) is associated with poor prognosis, because of no effective targeted therapy. In the present study, we demonstrated the crucial role of the aryl hydrocarbon receptor (AhR) in mediating the effects of the chemotherapeutic agent doxorubicin (DOX) in the chemotherapeutic sensitivity of TNBC. Firstly, we established AhR knockout (KO) MDA-MB 231 TNBC cells. The cytotoxic effects of DOX were more pronounced in AhR KO cells than in parental cells. In addition, our results indicated that AhR KO cells showed downregulated expression of DOX-metabolism enzyme, aldo-keto reductase (AKR) 1C3, relative to those of parental cells. Furthermore, AhR was found to enhance AKR1C3 promoter reporter activity, suggesting that AKR1C3 mRNA transcription is activated by AhR. Additionally, our findings confirmed that the downregulation of AKR1C3 expression enhanced DOX sensitivity in MDA-MB 231 cells. Finally, AhR and AKR1C3 expression were positively correlated in human breast cancer. Taken together, our results suggested that AhR is involved in DOX sensitivity by regulating AKR1C3 expression in TNBC cells.

Identification of 10-dehydrooxyglycyuralin E as a selective human estrogen receptor alpha partial agonist.

Selective estrogen receptor modulators (SERMs) act as either agonist or antagonist of estrogen receptor (ER) in a tissue selective manner and have been used in several diseases such as breast cancer, postmenopausal syndrome, osteoporosis, and cardiovascular diseases. However, current SERMs may also increase the risk of serious side effects and trigger drug resistance. Herein, a screening program, that was designed to search for novel SERMs, resulted in the identification of a series of 2-arylbenzofuran-containing compounds that are ligands for ERα, when applying the Gaussia-luciferase reporter assay. One of these compounds, 10-dehydrooxyglycyuralin E (T9) was chemically synthesized. T9 showed anti-estrogenic/proliferative activity in ERα-positive breast cancer cells. Pretreatment of T9 prevented the mRNA expression of GREB1, which is an estrogen response gene. Furthermore, by an in silico docking simulation study we demonstrated that T9 showed interactions directly to ERα. Taken together, these results demonstrated that T9 is a candidate of SERMs and a useful seed compound for the foundation of the selective activity of SERMs.

Cell Cycle and Apoptosis Regulator 1, CCAR1, Regulates Enhancer-Dependent Nuclear Receptor CAR Transactivation.

The constitutive active/androstane receptor (CAR) controls genes involved in xenochemical metabolism. Although numerous cofactors have been reported to be involved in CAR-mediated transactivation, unknown and poorly defined proteins recruited by CAR have yet to be characterized. In this study, a novel CAR-interacting protein, cell cycle and apoptosis regulator 1 (CCAR1), was identified by coimmunoprecipitation analysis using human hepatocarcinoma HepG2 cells expressing FLAG epitope-tagged CAR. We demonstrated that CCAR1 can act as an enhancer-dependent coactivator of CAR. First, we showed that overexpression of CCAR1 enhanced CAR-induced reporter gene activity with triplicate consensus direct repeat 4 motif (DR4-Luc), xenobiotic-responsive enhancer module (XREM)-enhancer of CYP3A4 (XREM-Luc), and phenobarbital-responsive enhancer module of UDP-glucuronosyltransferases 1A1 (UGT1A1) (gtPBREM)-enhancer of UGT1A1 (gtPBREM-Luc)-driven reporter plasmids but not PBREM-enhancer of CYP2B6 (PBREM-Luc)-driven reporter activity. Furthermore, we showed that knockdown of CCAR1 suppressed CAR-induced UGT1A1 mRNA expression but did not affect CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells. Moreover, CCAR1 could be recruited to the gtPBREM of the UGT1A1 enhancer by CAR but not to the PBREM of the CYP2B6 enhancer. Moreover, we showed that CCAR1 can act as a secondary coactivator by cooperating with the p160 family of steroid receptor coactivators (SRCs). These findings demonstrated CCAR1 to be a novel transcriptional cofactor for CAR and provided insight regarding the mechanism of CAR-mediated gene-selective transactivation.

Selective Androgen Receptor Modulator, YK11, Up-Regulates Osteoblastic Proliferation and Differentiation in MC3T3-E1 Cells.

Androgens are key regulators that play a critical role in the male reproductive system and have anabolic effects on bone mineral density and skeletal muscle mass. We have previously reported that YK11 is a novel selective androgen receptor modulator (SARM) and induces myogenic differentiation and selective gene regulation. In this study, we show that treatment of YK11 and dihydrotestosterone (DHT) accelerated cell proliferation and mineralization in MC3T3-E1 mouse osteoblast cells. Further, YK11-treated cells increased osteoblast specific differentiation markers, such as osteoprotegerin and osteocalcin, compared to untreated cells. These observations were attenuated by androgen receptor (AR) antagonist treatment. To clarify the effect of YK11, we investigated rapid non-genomic signaling by AR. The phosphorylated Akt protein level was increased by YK11 and DHT treatment, suggesting that YK11 activates Akt-signaling via non-genomic signaling of AR. Because it is known Akt-signaling is a key regulator of androgen-mediated osteoblast differentiation, YK11 has osteogenic activity as well as androgen.

Heregulin-induced cell migration is promoted by aryl hydrocarbon receptor in HER2-overexpressing breast cancer cells.

HER2 overexpression accounts for approximately 15-20% of all breast cancers. We have shown that HER2 overexpression leads to elevated expression of the aryl hydrocarbon receptor (AhR) in breast cancer cells. In this study, firstly, we showed that AhR expression was up-regulated by treatment with the HER3 ligand heregulin (HRG) in HER2-overexpressing breast cancer cell lines. Induction of AhR was mediated by transcriptional activation of the region of AhR promoter corresponding to - 190 to - 100 bp. In addition, HRG treatment elicited nuclear translocation of AhR. To investigate the role of AhR in HRG-HER2/HER3 signaling in HER2-overexpressing cells, we established AhR knockout (KO) HER2-overexpressing cells to perform wound-healing assays. HRG-induced cell migration was markedly attenuated by AhR KO. HRG-induced cell migration was associated with increased expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 in wild type cells, but not in AhR KO cells. These results elucidate that AhR is an important factor for the malignancy in HER2 overexpressing breast cancers.

Tripartite Motif Containing 24 Acts as a Novel Coactivator of the Constitutive Active/Androstane Receptor.

The constitutive androstane receptor (CAR) is a nuclear receptor that acts as a transcription factor for a variety of genes, including genes encoding xenobiotic, steroid, and drug-metabolizing enzymes and transporters. Transactivation of a target gene by a transcription factor is generally mediated through the concerted and stepwise recruitment of various proteins termed coregulators, including coactivators and corepressors. In this study, TRIM24 (also known as transcriptional intermediary factor 1 alpha) was found to interact with the CAR. TRIM24 enhanced the CAR-dependent transactivation in reporter assays using the direct repeat-4 motif, a binding site of the CAR. This enhancement was synergistically augmented in the presence of steroid receptor coactivator (SRC) 1 or SRC2, both of which are coactivators of the CAR. In addition, TRIM24 was recruited to the CAR-binding element of the CYP2B6 promoter together with the CAR. We also noted that knockdown of TRIM24 suppressed CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells and suppressed CAR-induced CYP3A4 mRNA expression in HepaRG cells but not HepTR/CAR cells. From these results, we suggest that TRIM24 is a novel coactivator of the CAR that is involved in cell- and/or promoter- selective transactivation.