miRNA profiling of granulosa cell-derived exosomes reveals their role in promoting follicle development.

To explore whether granulosa cell (GC)-derived exosomes (GC-Exos) and follicular fluid-derived exosomes (FF-Exos) have functional similarities in follicle development and to establish relevant experiments to validate whether GC-Exos could serve as a potential substitute for follicular fluid-derived exosomes to improve folliculogenesis. GC-Exos were characterized. MicroRNA (miRNA) profiles of exosomes from human GCs and follicular fluid were analyzed in depth. The signature was associated with folliculogenesis, such as phosphatidylinositol 3 kinases-protein kinase B signal pathway, mammalian target of rapamycin signal pathway, mitogen-activated protein kinase signal pathway, Wnt signal pathway, and cyclic adenosine monophosphate signal pathway. A total of five prominent miRNAs were found to regulate the above five signaling pathways. These miRNAs include miRNA-486-5p, miRNA-10b-5p, miRNA-100-5p, miRNA-99a-5p, and miRNA-21-5p. The exosomes from GCs and follicular fluid were investigated to explore the effect on folliculogenesis by injecting exosomes into older mice. The proportion of follicles at each stage is counted to help us understand folliculogenesis. Exosomes derived from GCs were isolated successfully. miRNA profiles demonstrated a remarkable overlap between the miRNA profiles of FF-Exos and GC-Exos. The shared miRNA signature exhibited a positive influence on follicle development and activation. Furthermore, exosomes derived from GCs and follicular fluid promoted folliculogenesis in older female mice. Exosomes derived from GCs had similar miRNA profiles and follicle-promoting functions as follicular fluid exosomes. Consequently, GC-Exos are promising for replacing FF-Exos and developing new commercial reagents to improve female fertility.

Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.

Effects of serum cholesterol on cholesterol content in the retina are currently unknown. It is also unclear how cholesterol levels are controlled in the retina. High-cholesterol diet and oral administrations of simvastatin were used to modulate serum cholesterol in mice. These treatments only modestly affected cholesterol content in the retina and had no significant effect on retinal expression of the major cholesterol- and vision-related genes; the sterol-regulatory element binding protein pathway of transcriptional regulation does not seem to be operative in the retina under the experimental conditions used. Evidence is obtained that posttranslational mechanisms play a role in the control of retinal cholesterol. Retinal genes were only upregulated by oral administrations of TO901317 activating liver X receptors. Three of the upregulated genes could be of particular importance (apoD, Idol, and Rpe65) and have not yet been considered in the context of cholesterol homeostasis in the retina. Collectively, the data obtained identify specific features of retinal cholesterol maintenance and suggest additional therapies for age-related macular degeneration, a blinding disease characterized by cholesterol and lipid accumulations in chorioretinal tissues.

Retinal and nonocular abnormalities in Cyp27a1(-/-)Cyp46a1(-/-) mice with dysfunctional metabolism of cholesterol.

Cholesterol elimination from nonhepatic cells involves metabolism to side-chain oxysterols, which serve as transport forms of cholesterol and bioactive molecules modulating a variety of cellular processes. Cholesterol metabolism is tissue specific, and its significance has not yet been established for the retina, where cytochromes P450 (CYP27A1 and CYP46A1) are the major cholesterol-metabolizing enzymes. We generated Cyp27a1(-/-)Cyp46a1(-/-) mice, which were lean and had normal serum cholesterol and glucose levels. These animals, however, had changes in the retinal vasculature, retina, and several nonocular organs (lungs, liver, and spleen). Changes in the retinal vasculature included structural abnormalities (retinal-choroidal anastomoses, arteriovenous shunts, increased permeability, dilation, nonperfusion, and capillary degeneration) and cholesterol deposition and oxidation in the vascular wall, which also exhibited increased adhesion of leukocytes and activation of the complement pathway. Changes in the retina included increased content of cholesterol and its metabolite, cholestanol, which were focally deposited at the apical and basal sides of the retinal pigment epithelium. Retinal macrophages of Cyp27a1(-/-)Cyp46a1(-/-) mice were activated, and oxidative stress was noted in their photoreceptor inner segments. Our findings demonstrate the importance of retinal cholesterol metabolism for maintenance of the normal retina, and suggest new targets for diseases affecting the retinal vasculature.

Binding of a cyano- and fluoro-containing drug bicalutamide to cytochrome P450 46A1: unusual features and spectral response.

Cytochrome P450 46A1 (CYP46A1) is the cholesterol 24-hydroxylase initiating the major pathways of cholesterol removal from the brain, and bicalutamide (BIC) is a drug of choice for the treatment of progressive androgen-dependent prostate cancer. We evaluated the interactions of BIC with CYP46A1 by x-ray crystallography and by conducting solution and mutagenesis studies. Because BIC is administered to patients as a racemic mixture of the S and R isomers, we studied all three, racemic BIC as well as the S and R isomers. Co-crystallization of CYP46A1 with racemic BIC led to structure determinations at 2.1 Å resolution with the drug complexes exhibiting novel properties. Both BIC isomers bind to the CYP46A1 active site in very similar single orientation and adopt an energetically unfavorable folded conformation. This folded BIC conformation is correlated with drug-induced localized shifts of amino acid side chains in CYP46A1 and unusual interactions in the CYP46A1-BIC complex. One of these interactions involves a water molecule that is coordinated to the P450 heme iron and also hydrogen-bonded to the BIC nitrile. Due to polarization of the water in this environment, the heme elicits previously unreported types of P450 spectral responses. We also observed that access to the P450 active site was affected by differential recognition of S versus R isomers at the CYP46A1 surface arising from BIC conformational polymorphism.

Widespread photosynthesis reaction centre barrel proteins are necessary for haloarchaeal cell division.

Cell division is fundamental to all cellular life. Most archaea depend on either the prokaryotic tubulin homologue FtsZ or the endosomal sorting complex required for transport for division but neither system has been robustly characterized. Here, we show that three of the four photosynthesis reaction centre barrel domain proteins of Haloferax volcanii (renamed cell division proteins B1/2/3 (CdpB1/2/3)) play important roles in cell division. CdpB1 interacts directly with the FtsZ membrane anchor SepF and is essential for cell division, whereas deletion of cdpB2 and cdpB3 causes a major and a minor division defect, respectively. Orthologues of CdpB proteins are also involved in cell division in other haloarchaea, indicating a conserved function of these proteins. Phylogenetic analysis shows that photosynthetic reaction centre barrel proteins are widely distributed among archaea and appear to be central to cell division in most if not all archaea.

Antifungal Azoles: Structural Insights into Undesired Tight Binding to Cholesterol-Metabolizing CYP46A1.

Although there are currently three generations of antifungal azoles on the market, even the third-generation agents show undesirable interactions with human cytochrome P450 (P450) enzymes. CYP46A1 is a cholesterol-metabolizing P450 in the brain that tightly binds a number of structurally distinct azoles. Previously, we determined the crystal structures of CYP46A1 in complex with voriconazole and clotrimazole, and in the present work we cocrystallized the P450 with posaconazole at 2.5 Å resolution. This long antifungal drug coordinates the P450 heme iron with the nitrogen atom of its terminal azole ring and adopts a linear configuration occupying the whole length of the substrate access channel and extending beyond the protein surface. Numerous drug-protein interactions determine the submicromolar Kd of posaconazole for CYP46A1. We compared the crystal structure of posaconazole-bound CYP46A1 with those of the P450 in complex with other drugs, including the antifungal voriconazole and clotrimazole. We also analyzed the accommodation of posaconazole in the active site of the target enzymes, CYPs 51, from several pathogenic species. These and the solution studies with different marketed azoles, collectively, allowed us to identify the determinants of tight azole binding to CYP46A1 and generate an overall picture of azole binding to this important P450. The data obtained suggest that development of CYP51-specific antifungal agents will continue to be a challenge. Therefore, structural understanding of the azole binding not only to CYPs 51 from the pathogenic species but also to different human P450s is required to deal efficiently with this challenge.

Stimulation of mouse Cyp1b1 during adipogenesis: characterization of promoter activation by the transcription factor Pax6.

Cytochrome P4501B1 (Cyp1b1) is expressed specifically in certain neural crest (NC) cells during embryogenesis. Mesenchymal progenitor cells that develop from NC cells are modeled here by mouse C3H10T1/2 and 3T3-L1 cells. Dexamethasone in combination with methylisobutylxanthine (DM) induces Cyp1b1 and a 6.7 kb mouse Cyp1b1 promoter-luciferase reporter in each cell type prior to adipogenesis. An 18 base sequence (at -6.11 kb) (PaxE) which was essential for this reporter stimulation in 3T3-L1 cells bound the transcription factor Pax6. This is shown by gel mobility shifts and sequence mutations. Heterologous vector expression of Pax6 in 3T3-L1 cells enhanced DM stimulated Cyp1b1 promoter activity through cooperation with two Sp1 sites in the proximal promoter region. Chromatin immunoprecipitation showed that DM stimulated binding of Pax6 adjacent to Sp1 in the proximal promoter more than in the PaxE region. The Cyp1b1 induction by DM in C3H10T1/2 cells was more rapid but independent of Pax6. The far upstream enhancer region (FUER) found in rat Cyp1b1 responded to DM but was inactive in the mouse promoter due to key sequence changes. The expression patterns of Pax6 and Cyp1b1 frequently overlap during mouse embryogenesis. The relationship between Pax6 and Cyp1b1 expression warrants further investigation, particularly in the NC.

Widespread PRC barrel proteins play critical roles in archaeal cell division.

Cell division is fundamental to all cellular life. Most of the archaea employ one of two alternative division machineries, one centered around the prokaryotic tubulin homolog FtsZ and the other around the endosomal sorting complex required for transport (ESCRT). However, neither of these mechanisms has been thoroughly characterized in archaea. Here, we show that three of the four PRC (Photosynthetic Reaction Center) barrel domain proteins of Haloferax volcanii (renamed Cell division proteins B1/2/3 (CdpB1/2/3)), play important roles in division. CdpB1 interacts directly with the FtsZ membrane anchor SepF and is essential for division, whereas deletion of cdpB2 and cdpB3 causes a major and a minor division defect, respectively. Orthologs of CdpB proteins are also involved in cell division in other haloarchaea. Phylogenetic analysis shows that PRC barrel proteins are widely distributed among archaea, including the highly conserved CdvA protein of the crenarchaeal ESCRT-based division system. Thus, diverse PRC barrel proteins appear to be central to cell division in most if not all archaea. Further study of these proteins is expected to elucidate the division mechanisms in archaea and their evolution.

Marked variability in hepatic expression of cytochromes CYP7A1 and CYP27A1 as compared to cerebral CYP46A1. Lessons from a dietary study with omega 3 fatty acids in hamsters.

Two diets simulating the recommendations of the American Heart Association to increase the intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) were tested on Golden Syrian hamsters and compared to the diet simulating the current estimated consumption of fat in the United States. N-3 PUFAs were evaluated for their effects on serum and brain lipids and on the three cytochrome P450 enzymes (CYPs 7A1, 27A1, and 46A1) that play key roles in cholesterol elimination from different organs. Hamsters on the highest concentration of n-3 PUFAs had a statistically significant decrease in LDL and HDL cholesterol and no change in serum total cholesterol and triglycerides levels. CYP27A1 and CYP46A1 mRNA levels were increased in the liver and brain, respectively, whereas possible effects on CYP7A1 were obscured by a marked intergroup variability at mRNA, protein, and sterol product levels. Increased levels of CYP46A1 mRNA in the brain did not lead to significant changes in the levels of lathosterol, 24S-hydroxycholesterol or cholesterol in this organ. The data obtained are discussed in relation to inconsistent effects of n-3 PUFAs on serum lipids in human trials and reported positive effects of fish oil on cognitive function.

Coinfection with influenza A virus enhances SARS-CoV-2 infectivity.

The upcoming flu season in the Northern Hemisphere merging with the current COVID-19 pandemic raises a potentially severe threat to public health. Through experimental coinfection with influenza A virus (IAV) and either pseudotyped or live SARS-CoV-2 virus, we found that IAV preinfection significantly promoted the infectivity of SARS-CoV-2 in a broad range of cell types. Remarkably, in vivo, increased SARS-CoV-2 viral load and more severe lung damage were observed in mice coinfected with IAV. Moreover, such enhancement of SARS-CoV-2 infectivity was not observed with several other respiratory viruses, likely due to a unique feature of IAV to elevate ACE2 expression. This study illustrates that IAV has a unique ability to aggravate SARS-CoV-2 infection, and thus, prevention of IAV infection is of great significance during the COVID-19 pandemic.

Binding of steroidogenic factor-1 to the regulatory region might not be critical for transcriptional regulation of the human CYP1B1 gene.

Cytochrome P450 (CYP) 1B1, which catalyzes 17beta-estradiol 4-hydroxylation, is expressed in steroid-related tissues including ovary, testis, and adrenal gland. Generally, the expressions of steroidogenic CYPs are transcriptionally regulated by steroidogenic factor-1 (SF-1) and cAMP response element (CRE) binding protein (CREB). In the present study, we examined the possibility that the human CYP1B1 gene might be regulated by SF-1 and CREB. Gel shift analyses revealed that in vitro translated SF-1 can bind to the putative SF-1 binding sites, SF-1a (at -1722) and SF-1b (at -2474), on the CYP1B1 gene. In vitro translated CREB barely binds to the putative SF-1 binding sites. Luciferase analysis revealed that a reporter plasmid, pGL3 (-2623/+25), containing the SF-1a and SF-1b elements is transactivated by the concomitant co-expression of SF-1 and protein kinase A (PKA). However, the transcriptional activity is induced by PKA alone. Mutations in the SF-1a and SF-1b elements did not affect the luciferase activity. Thus, the binding of SF-1 to the putative SF-1 binding sites of the human CYP1B1 gene might not be essential for transcriptional regulation. Interestingly, deletion and mutation analyses indicated that the PKA signaling pathway is involved in the xenobiotic responsive element (XRE)-mediated transactivation of the human CYP1B1 gene.

tRNA-embedded Hammerhead Ribozymes Mediate Destruction of HBV (Subtype adr) in vitro.

Two hammerhead ribozymes, RS3 and RC2, targeting the HBsAg and HBcAg regions of HBV (adr) RNA respectively, have been designed. To improve the stability of ribozymes, two tRNA-embedded ribozymes, RtS3 and RtC2, were obtained by inserting RS3 and RC2 into the anticodon region of tRNA. Although the catalytic activity of the tRNA-embedded ribozymes was lower than that of their naked counterparts, the stability of the former in fetal bovine serum or cytoplasmic extract of HepG2 cells was much higher than that of the naked ribozymes. These results suggest that the antivirus effect of tRNA-embedded ribozymes may be greater in vivo in comparison with naked ribozymes.

Hammerhead Ribozymes Suppress HBV(adr) in HepG2 Cells.

Three hammerhead ribozymes (RS3, RC2 and RC1) targeting to the HBV genome have been designed. Plasmids were constructed by inserting the genes of naked and tRNA-embedded ribozymes into RNA trimming vector pRG523 and then were transferred to eukaryotic expression vector. By the similar cloning method the shotgun-type plasmids carrying homogeneous RS3 or RtS3 unitconnected in tandem were obtained. After co-transfecting the above plasmids and HBV genome containing plasmid into human hepatoma cell line HepG2 respectively and selection by G418, the HBV-inhibiting activity of different kinds of ribozyme in G418-resistant cells was achieved by measuring the decrease of HBV-RNA, progeny DNA and the antigens expressed. The results showed that all the ribozymes were active with more than 70% inhibition activity against the HBV and that tRNA-embedded ribozymes had higher activity than naked ribozymes. It is worth particular interest that shotgun-type ribozymes with the connected unit in tandem with 8 and 12 units constructed in the plasmid revealed the highest activity, reaching >90% inhibition.

Abnormal vascularization in mouse retina with dysregulated retinal cholesterol homeostasis.

Several lines of evidence suggest a link between age-related macular degeneration and retinal cholesterol maintenance. Cytochrome P450 27A1 (CYP27A1) is a ubiquitously expressed mitochondrial sterol 27-hydroxylase that plays an important role in the metabolism of cholesterol and cholesterol-related compounds. We conducted a comprehensive ophthalmic evaluation of mice lacking CYP27A1. We found that the loss of CYP27A1 led to dysregulation of retinal cholesterol homeostasis, including unexpected upregulation of retinal cholesterol biosynthesis. Cyp27a1-/- mice developed retinal lesions characterized by cholesterol deposition beneath the retinal pigment epithelium. Further, Cyp27a1-null mice showed pathological neovascularization, which likely arose from both the retina and the choroid, that led to the formation of retinal-choroidal anastomosis. Blood flow alterations and blood vessel leakage were noted in the areas of pathology. The Cyp27a1-/- retina was hypoxic and had activated Müller cells. We suggest a mechanism whereby abolished sterol 27-hydroxylase activity leads to vascular changes and identify Cyp27a1-/- mice as a model for one of the variants of type 3 retinal neovascularization occurring in some patients with age-related macular degeneration.

Spatial distribution of the pathways of cholesterol homeostasis in human retina.

BACKGROUND: The retina is a light-sensitive tissue lining the inner surface of the eye and one of the few human organs whose cholesterol maintenance is still poorly understood. Challenges in studies of the retina include its complex multicellular and multilayered structure; unique cell types and functions; and specific physico-chemical environment. METHODOLOGY/PRINCIPAL FINDINGS: We isolated specimens of the neural retina (NR) and underlying retinal pigment epithelium (RPE)/choroid from six deceased human donors and evaluated them for expression of genes and proteins representing the major pathways of cholesterol input, output and regulation. Eighty-four genes were studied by PCR array, 16 genes were assessed by quantitative real time PCR, and 13 proteins were characterized by immunohistochemistry. Cholesterol distribution among different retinal layers was analyzed as well by histochemical staining with filipin. Our major findings pertain to two adjacent retinal layers: the photoreceptor outer segments of NR and the RPE. We demonstrate that in the photoreceptor outer segments, cholesterol biosynthesis, catabolism and regulation via LXR and SREBP are weak or absent and cholesterol content is the lowest of all retinal layers. Cholesterol maintenance in the RPE is different, yet the gene expression also does not appear to be regulated by the SREBPs and varies significantly among different individuals. CONCLUSIONS/SIGNIFICANCE: This comprehensive investigation provides important insights into the relationship and spatial distribution of different pathways of cholesterol input, output and regulation in the NR-RPE region. The data obtained are important for deciphering the putative link between cholesterol and age-related macular degeneration, a major cause of irreversible vision loss in the elderly.

Steroidogenic factor-1 interacts with cAMP response element-binding protein to mediate cAMP stimulation of CYP1B1 via a far upstream enhancer.

CYP1B1 activates polycyclic aromatic hydrocarbon carcinogens in cAMP-regulated tissues such as the adrenal, ovary, and testis. A 27-fold cAMP stimulation of the CYP1B1-luciferase reporter in Y-1 adrenal cells depends entirely on a far upstream enhancer region (FUER; -5298 to -5110). Cooperative participation of multiple steroidogenic factor 1 (SF-1) elements with the downstream cAMP response element (CRE) in FUER is essential for both basal and cAMP-stimulated activities of FUER. Basal and induced activities were similarly lowered by DAX-1, an SF-1 suppressor, and raised by steroid receptor coactivator 1, an SF-1 coactivator. cAMP response element-binding protein (CREB)-binding protein (CBP) that interacts preferentially with the phosphorylated-CREB increased the cAMP-induced FUER. 10T1/2 cells and human embryonic kidney (HEK)293 cells do not express SF-1. Introduction of exogenous SF-1 generated cAMP stimulation of the FUER in 10T1/2 fibroblasts. The same transfection only increased basal activity of FUER in HEK293 cells, despite presence of active CREB in cells. HEK293 cells therefore remain deficient in additional factor(s) critical to the cAMP stimulation of CYP1B1. Mutations of the protein kinase A (PKA) and the mitogen-activated protein kinase phosphorylation sites (Ser-430 and Ser-203) on SF-1 had no effect on the SF-1-dependent FUER stimulation in Y-1 and 10T1/2 cells. This contrasts with loss of activity with mutation of CREB at PKA phosphorylation site (Ser-133). SF-1 phosphorylation at these sites is therefore not essential for the cAMP stimulation and the cooperation with CREB. cAMP-enhanced activation protein 1 (AP-1) and stimulatory protein 1 (Sp1) complexes in the proximal promoter region contributed substantially to both basal and cAMP-stimulated FUER activity. Chromatin immunoprecipitation from primary rat adrenal cells demonstrated cAMP stimulation of histone acetylation proximal to, respectively, the FUER and AP-1 sites of CYP1B1.

Identification of novel TCDD-regulated genes by microarray analysis.

TCDD exposure of multipotential C3H10T1/2 fibroblasts for 72 h altered the expression of over 1000 genes, including coordinated changes across large functionally similar gene clusters. TCDD coordinately induced 23 cell cycle-related genes similar to epidermal growth factor (EGF)-induced levels but without any affect on the major mitogenic signaling pathway (extracellular signal-regulated kinase, ERK). TCDD treatment also decreased glycolytic and ribosomal clusters. Most of these TCDD-induced changes were attenuated by the presence of EGF or an adipogenic stimulus, each added during the final 24 h. TCDD prevented 10% of EGF-induced gene responses and 40% of adipogenic responses. Over 100 other genes responded to TCDD during adipogenesis. This group of responses included complete suppression of three proliferins and stimulations of several cytokine receptors. Despite these varied secondary effects of TCDD, direct AhR activation measured by integrated AhR-responsive luciferase reporters was similar under quiescent, EGF-stimulated or adipogenic conditions. Only 23 genes were similarly induced by TCDD regardless of conditions and 10 were suppressed. These 23 genes include: 4 genes previously recognized to contain AhR response elements (cytochrome P450 (CYP) 1B1, CYP1A1, NAD(P)H quinone reductase 1 (NQO1), and aldehyde dehydrogenase 3A1); two novel oxidative genes (alcohol dehydrogenase 3 and superoxide dismutase 3); and glypican 1, a plasma membrane proteoglycan that affects cell signaling. Further experiments demonstrated that TCDD maximally induced NQO1, glypican 1 and alcohol dehydrogenase 3 by 6 h. Glypican 1 activates the actions of many growth factors and therefore may contribute to secondary effects on gene expression.

Differentiation of pluripotent C3H10T1/2 cells rapidly elevates CYP1B1 through a novel process that overcomes a loss of Ah Receptor.

Stimulation of C3H10T1/2 cells by an adipogenic hormonal mixture (IDM) consisting of insulin (I), dexamethasone (D), and methylisobutylxanthine (M) substantially induces cytochrome P450 (CYP) 1B1 expression. This stimulation represents up to 40% of the level produced by maximum activation of the arylhydrocarbon receptor (AhR) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dexamethasone and methylisobutylxanthine in combination produced near maximum elevation of CYP1B1 along with a subsequent decline in AhR that paralleled the rise in peroxisome proliferator-activated receptorgamma1 (PPARgamma1). Inhibitors of AhR activity, which block TCDD induction, did not affect this increase of CYP1B1 expression, which was, therefore, independent of AhR activity. These responses were unaffected by inhibition of DNA synthesis, which was required for PPARgamma1 induction and terminal differentiation. Induction of CYP1B1 mRNA was paralleled by increased CYP1B1 promoter-luciferase reporter activity. The initial 0.8kb of promoter region, which was sufficient for 24h near maximum stimulation, did not contain either the key AhR-responsive elements that mediate the TCDD response or CREB and SF1 elements that mediate cAMP stimulation of rat CYP1B1 in steroidogenic cells. This reporter response to IDM stimulation, but not to TCDD, was maintained in AhR-null fibroblasts. CYP1B1 expression, unlike TCDD induction, was stimulated by IDM in only about half the cells. CYP1B1 expression partially overlapped with PPARgamma expression, which was also inversely related in clonal sub-lines. CYP1B1 expression may, therefore, represent an early stage of differentiation that requires factors associated with DNA synthesis to subsequently generate PPARgamma1.

Disruption of cell-cell contact maximally but transiently activates AhR-mediated transcription in 10T1/2 fibroblasts.

The aryl hydrocarbon receptor (AhR) is activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but activation without an exogenous ligand also occurs when normal cell-cell contact is prevented. Suspension of several C3H10T1/2 fibroblast clonal sub-lines that contain an integrated AhR-responsive reporter produced a time course and level of reporter activation and CYP1B1 induction that paralleled TCDD stimulation in confluent monolayer culture. Suspension activation was, however, more transient. Loss of cell-cell contact at low density also activated these reporters independent of cell cycle changes to levels comparable to TCDD stimulation of confluent cells. Loss of cell-cell contact may, therefore, activate AhR. Suspension and TCDD activations exhibited comparable nuclear translocation of AhR and then AhR/ARNT complex formation. Each AhR activation process was equally attenuated by inhibition of, respectively, HSP90 ATPase, the 26S proteosome, and by depletion of intracellular Ca2+. By contrast, the AhR antagonist alpha-naphthoflavone (alphaNF) blocked ligand-stimulated AhR activity, but not activation through loss of cell-cell contact. Suspension-induced reporter activation was selectively enhanced by LiCl, which prevented GSK-3beta effects on the simultaneously released beta-catenin. The effects of suspension and LiCl on reporters were reversed by Ro-31-8220, which did not affect beta-catenin, TCDD-activation processes, or AhR turnover. Neither LiCl nor Ro-31-8220 altered suspension-induced AhR/ARNT complex formation. Loss of cell-cell contact permits nuclear translocation and AhR activation that is largely replicated after TCDD binding, but with activity differences due to contact-sensitive factors functioning after AhR/ARNT complex formation.

Ah receptor regulation of mouse Cyp1B1 is additionally modulated by a second novel complex that forms at two AhR response elements.

Cyp1B1 is expressed constitutively in many extrahepatic cells and is induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). An enhancer region (AhER-810 to -1075 of the mouse Cyp1B1 promoter), which mediates aryl hydrocarbon receptor (AhR) regulation of transcription, contains three consensus XRE sequences (designated XRE1, XRE4, and XRE5) and a central Ebox. XRE5 is essential for both basal and induced activity in C3H10T1/2 cells. AhR/ARNT binding to XRE1, XRE4, and the Ebox complex function in combination to support the AhR/ARNT complex at XRE5. The identical 12 base cores of XRE1 and XRE4 differ from the core of XRE5 by two bases outside of the consensus XRE. These sites bind a constitutive complex slightly smaller than AhR/ARNT (anomalous complex; anC), which is not formed at XRE5 or six Cyp1A1 XREs. Exchange of these bases (m3 mutations) restores selective AhR/ARNT binding at XRE1/XRE4 and introduces anC binding at XRE5. The activities of multimeric XRE1 and XRE5 luciferase reporters responded in parallel to the extent of AhR/ARNT binding. The consensus anC binding sequence ((C/T)GCG(C/T)GCGC(C/A)GC) overlaps the XRE1/XRE4 AhR/ARNT element. Gel mobility analyses show that anC binds to XRE1/XRE4 under basal conditions, while AhR/ARNT partially displaces anC following TCDD induction. Selective depletion of anC with biotin-oligonucleotides increases AhR/ARNT binding. M3-mutations at, respectively, XRE1 and XRE4 of the AhER sequence, had opposite effects on luciferase reporters. Activities increased for the XRE1 mutation and decreased for the XRE4 mutation, but also depended on the level of AhR transfected into AhR -/- fibroblasts. AnC compete with AhR at XRE1 while playing an activating role at XRE4. This positive effect of constitutive anC binding at XRE4 may contribute to the characteristic basal Cyp1B1 expression in embryo fibroblasts, which is mediated by low constitutive activities of AhR.