Fyziologie působení farmak a toxických látek Přednáška č.5 Proteiny PAS a jejich úloha v organismu Per-Arnt-Sim - PAS superfamily of proteins environmental sensors, which mediate transcriptional responses to various types stimuli: */ circadian rhythms; */ oxygen sensing; */ sensing of toxicants; */ developmental role/cancer; These proteins enable adaptation to rapid changes in the environment* PAS proteiny jsou součástí širší rodiny bHLH proteinů: There are three main sub-families of bHLH proteins: (a) those containing only the bHLH domain; and those where the bHLH domain is contiguous with a second dimerisation domain, either (b) the leucine zipper (Zip) or (c) the PER/aryl hydrocarbon receptor nuclear translocator (ARNT)/single minded (SIM) (PAS) homology domain. helii-loop- receptor-interaction interaction domain helix dumain PAS proteiny (rodina transkripčních faktorů): E a* a 3 (/) in < Q. Dioxin.'Liver development ahr AR NT AHRR Hypo»a signaling hifio/mopi EPAS1/MQP2/HIF2a HIF3a Neurogenesis ARNT2 SIM1/SIM2 SIM2/SIM1 b H LH ZEHE n^rr M] J^VT MĽĹ M ML PAS Variable I lAl iBl I 14 23 23 21 1G 21 23 13 11 12 13 13 10 11 100 aa 15 E E n Orcadian Orphans Coactřvators MQP3/B MAL 1/C vele NPAS2/MQP4 Clock Per1,2a-id3 MOP9 NPAS1/MOP5 H PAS3/MOP6 SRC-1 TIF2 RAC3 WT \ML \ML 15 1G 18 1—iš- TL d rrrr 23 23 _23_ 23 12 14 15 13 http://mcardle.oncology.wisc.edu/bradfield/ PAS domain The PAS region consists of two adjacent degenerate repeats of ~130 amino acids, PAS A and PAS B. The domain is an ancient signalling device conserved through evolution, having been identified in proteins throughout the animal kingdom, in bacteria, fungi and yeast in addition to mammals and flies, where the most commonly studied bHLH/PAS proteins originate. Many bacteria contain PAS-like proteins that detect light and oxygen (Dos, Aer, FixL, PYP). Similar proteins sense light in plants (phytochromes PhyA-PhyE, NPH1; phytochrome interacting factor PIF3). Domain structure and function of PAS proteins: bimerization Ligand/HSP90/X AP2 binding AhR protein N ™ ^~ C A B Variable length NLS bHLH TAD PAS TAD A B NLS bimerization Arnt protein 100 aa (Gu et al., Annu Rev Pharmacol Toxicol. 2000;40:519-61.) The circadian response pathway PAS Protein Signal Transduction Pathways 113 mRNA of mPERs .... ,"1 AAAA Cytoplasm ^A ^ a-class ^f ^ ( r ß- ß-class sensor TJ (J partner Output (Comprehensive Toxicology, vol. 14) Daily changes in light/dark require physiological and behavioral adaptation: */ CLOCK/MOP3 heterodimer controls expression of circadian responsive gene products - PER, TIM; Nature Genetics 26, 23 - 27 (2000) The hypoxia response pathway HIF-a NORMOXIA 02 2-oxoglutarate co2 succinate Prolyl & Asparaginyl Hydroxylases Fa* Proteasomal Degradation HYPOXIA |02 ItolyLJř Aspacäginyl Hydroxylases ► Hypoxic response genes The International Journal of Biochemistry <& Cell Biology 36 (2004) 189-204 The ability to maintain 02 homeostasis is essential for survival of mammals. The hyperoxic state, or high 02 tension, can result in the generation of reactive oxygen intermediates and potentially lethal damage to membranes and DNA. The hypoxic state, or low 02 tension, can result in levels of ATP insufficient to maintain essential cellular functions. The hypoxic state occurs in a number of medical conditions, such as cancer and ischemias, inspiring research into understanding the cellular mechanisms for detecting and responding to low levels of oxygen. Responses to hypoxia are mediated by three bHLH/PAS proteins, HIF-la, HIF-2a (also known as Endothelial PAS domain protein 1, HIF-like factor and member of PAS family 2), and HIF-3a. hypoxia normoxia ^m ^b ^^h Dernand=supply V^"^^l m nul ii| | 1 Supply Demand # of blood vessels X Blood flow per vessel if of cells X 02 consumption per cell Acute response ^blood flow per vessel A02 use per cell Chronic response ^number of vessels AN u m be ľ of cells Fig. L Supply and demand governs oxygen availability. «A. .J..J.:. _i_ czr» /onnc\ 0-7 ir HIF subfamily HIF-1a HIF-2CC bHLH PAS za. ODD N-TAD C-TAD i----------1 i i ia <6 ifcAY« 826 ^m As /Cl 870 HIF-3a1 IPAS/HIF-3a2 HIF-3a3 HIF-3a4 HIF-3a5 HIF-3a6 r ia LZIP 11 J IB 667 632 648 663 Biochimica et Biophysica Acta 1755 (2005) 107 - 120 Hypoxia-inducible factor (HIF-la): Hypoxia-inducible factor-1 (HIF-1), composed of HIF-a and HIF-ß L (ARNT) subunits, is a heterodimeric transcriptional activator. In I response to hypoxia, stimulation of growth factors, and activation of oncogenes as well as carcinogens, HIF-la is overexpressed and/or activated and targets those genes which are required for angiogenesis, metabolic adaptation to low oxygen and survival of cells. HIF-1 is critical for both physiological and pathological processes. Several dozens of putative direct HIF-1 target genes have been identified on the basis of one or more cis-acting hypoxia-response elements that contain an HIF-1 binding site. A variety of regulators including growth factors, genetic alterations, stress activators, and some carcinogens have been documented for regulation of HIF-1 in which several signaling pathways are involved depending on the stimuli and cell types. Activation of HIF-1 in combination with activated signaling pathways and regulators is implicated in tumour progression and prognosis. N LS-N bHLH PAS VHL (OH)~(OH) P402 ODD P564 TAD-N ID Ä N 803 TAD-C 'P300/CBP) NLS-C Transcritiorial activity bHLH PAS Figure 1 Molecular structure of HlF-lfjand HIF-1 ß. bHLH domain mediates dimerization of the two subunits. PAS domain is responsible for DNA binding. Proline residues of 402 and 564 at ODD domain are hydroxylized by proline hydroxylase and recognized by VHL and then targeted to the ubiquitin proteasome pathway. Asn803 at the C-terminal transactivation domain (TAD-C) is hydroxylized by FIH-1 (factor inhibiting HIF-1) with a result of inhibition of HIF-la interaction with co-activator p300 and consequently inhibits transcriptional activity. The nuclear location signal at C-terminal functions in HIF-1 a translocation into nuclei. Normoxia Active I Active PHD FIH World J Gastroenterol 2004; 10(8): 1082-108 Hypoxia o2 ir 02 PHD-dependent I I FIH-dependent Prolyl hydroxylation XX Asparaginyl hydroxylation HO OH / \ VHL E3-mediated proteolysis Inactive C-TAD \ / Inactive PHD Inactive FIH HIF-a stable C-TAD active Inactive HIF Active HIF Fig. 1. Two independent hydroxylation pathways regulate HIF activity in response to cellular oxygen level. In normoxia, oxygen availability enables PHD-dependent prolyl hydroxylation of the HIF-a ODD. This prolyl hydroxylation allows binding of the VHL E3 ligase leading to ubiquitylation and degradation of HIF-a subunits;. Oxygen availability also enables FIH-dependent asparaginyl hydroxylation of the C-TAD, blocking interaction with the p300/CBP co-activator. In hypoxia, the PHD and FIH enzymes are inactive and the lack of hydroxylation results in stable HIF-a able to form a DNA-binding heterodimer with HIF-ß and recruit p300/CBP at the C-TAD. HIF-dependent responses to O 2 may be modulated by the cellular environment: 02 Induction of enzyme expression Iron t f Growth, \ Iron differentiation [metabolism . and apoptosis 1 Ascorbate 2-Oxogl utarate Intermediates and cofactors of energy metabolism reactions Graded HIF responses to O2 and cellular environment ODD Phosphorylation Acetylation Growth factors, oncogenes, tumour suppressor pathways Masson, N. et al. J Cell Sei 2003;116:3041-3049 Table 2. HIF-1 target genes. FunctioD Gene (abbreviation) Reference Erythropoietin (EPO) ;Semeuza et al. 1991) Eryľhrjpcdesis-' Transferrin (Tf) [Rolfs et al. 1597) iron metabolism Transferrin receptor {Tfi") [Bianchi et al, 1999) C'enuopiasniin [Lok and Pouka: 1999) Va&cukr erjdothelkl growth factor (VEGF) [Levy et al, 1995) Angiogenesis Endccrine-gküd-denved VEGF (E G-VEGF) [UCsuteretal, 2001) Leptm {LEP> [Grosfeld et al. 2002) Transforming growth factor-betai (TGF- \\i) [Scheid e: al, 2002) Mltíc Guide synthase (NOS2) (Melillo et al, 1995) Heme sxygenease 1 ;LeeetaL 1997) Vascular tone Endothelial CETI) (Hu et aL 1998) ^drenomedulin (ADM) [Neuven and Ckvcouib 1999) .; 13-a orežeme rece'xir [Eckhaitetal. 1997) Vlaľrix oetaUoproteniases. (VMPs) ;Ben-Yo5efetal..2002) Matrix metabolism Plasminogen activator receptors and inhibitors (PAK) ;Kie:zmann e: al, 1999) '.'. nlkien prclvL bvdroxvlase ;Takabashietal, 2000) ■\denvkte kinase-3 ;ORourke et al, 199 6) ŕ^ldokse-AC (ALDA:C) [Semeuza et al. 1996) Carbonic anhydrase-9 ;\Vykorfetal,2000) Euolase-1 (ENOl) [Semeuza et al, 1996) Glucose ttansporter-1.3 (GLU1,3) ;Chenetal, 2001) GrvceTaldehvde phosphate defrvdroeenase [Graven et al, 1999) Glucose metabolism ;gapdh) Hexokinase 1,2 (HK1,2) [Mathupala et al, 2001) Lactate dehydrogenase-A (LDHA) [Semeuza et al, 1996) Pyruvate kinase M (PKM) [Semeuza et al, 1994) Phosphs juctokuiise L (PFKL) ;Semeuza et al, 1994) Phosphcglycerate kinase 1 (PGK1) [Semeuza et al, 1994) S-püos.phofruc:5-2-kinase-'gnictos.e-2.ö- [MmchenkoecaL 2002) :is'jJ:o;phf.:í-.:' (P7KJB3) Insulin-like growth fáctor-2 {IGF2) [Feldseretal, 1999) Cell proliferation:' survival Trauslbrming growth factor-a (TGF- a) Airenomedullňi (ADM) 'Krishnaniacharv et al. 2003) 'Cormier-Resard et ai 199Ě) Bcl-2 ■'adenovirus EIB lQkD-interactiD5 prctein i ;C'aneroetal, 2000) Apoptosis ;BNip3) NÍLp3-]ikepro:eiuX(>JIX) ;B:uick 2000) Molecular Pharmacology Fast Forward. Published on August 3, 2006 as doi:10.1124/mol.l06.0 27029 ARNT - základní dimerizační partner: AhR liver development xenobiotie response Vaskularisation during organogenesis ARNT HIF-a signal responsive neural development SIM Fig. 4. ARNT is central to transcriptional regulation within the bHLH PAS family of proteins. ARNT forms both homodimers and heterodimers with the AhR. HIF-a and SIM which play roles both during mammalian development and in response to environmental stimuli in mammals. Symbol "?' indicates where these roles have yet to be characterised. The International Journal of Biochemistry <& Cell Biology 36 (2004) 189-204 Jak HIF-la, tak ARNT představují proteiny nezbytné pro přežití - KO myši odumírají již v průběhu embryonálního vývoje. The Ah receptor pathway Denlson et al., C hem. Biol. Interact. 141: 3 AhR = • I igand-activated transcription factor; • important mediator of toxicity of POPs; • regulator of xenobiotic metabolism and activation of promutagens. AhR discovery • different sensitivity of inbred mouse strains to TCDD and 3-MC inducers of CYP1A activity in liver microsomes; • autosomal dominant Mendel ian trait; • isolation of protein; cloning 3H (103cpm) s Br. Br O O N 3 V25 2-azido-3-[125l]-7,8-dibromo-dibenzo-p-dioxin 200 kDii 115 kDa m kDa 52 ki)u HjN bHLH PAS domain B Q-rleli COOH ligand and hsp90 binding AhR:Arnt:DRE complex formation Molecular Toxicology, 2nd ed. Overview of aryl hydrocarbon receptor and dioxin-like toxicity: • what is AhR; • evolution perspective; • activation of AhR; AhR-dependent genes • toxic effects associated with AhR activation; • AhR interactions • the role of AhR in cell cycle regulation AhR domain structure: bHLH i—i r PAS Domain NH2 1 ■ A B; 5 Q rich COOH ^4. MLS NES L J L J Ligand & hsp90 Transactivation Binding AhR:Arnt:DRE Complex Formation L Trans format ion Fig. 2. Domain structure of tlie AhR. Ďenison et al., Chem. Biol. Interact. 141: 3 970 MVA 100 I 100 100 100 100 Arthropods Nematoda Priapula Mollusca Annelida Platyhelmlnthes Brachiopoda Echlnodermata Hemichordata Chordata Cnidaria Po rife ra - Fundulus AHR2 - zebrafísh AHR2 - human AHR - mouse AHR - Fundulus AHR1 ■ zebrafísh AHR1 - mouse AHRR - human AHRR - Fundulus AHRR ■ Drosophila AHR - C. elegans AHR Ecdysozoa Lopholrochozoa Deuterostoma cvoiuTion OT miik: AHR1 AHR2 AHRR ARNT AHR2 clade AHR1 clade AHR repressor clade Invertebrate AHRs Invertebrate phyla Hahn, Chem. Biol. Interact. (2002) 141: 131 Evolution of AhR: Organism: Name: Ligand-binding: Physiological function: Nematodes: AHR-1 No Neuronal development; Caenorhabditis elegans Behavioral effects. Insects: Spineless (Ss) No Development; örosophila melanogasfer S^\ Regulation of homeobox genes and dendrite morphology. Vertebrates: AhR ( Yes \ ^Toxicity mechanisms^ (AhRl, AhR2) \Zs development; Neuronal differentiation? Circadian rhytms? Natural ligands of AhR??????????? */ light hypothesis - tryptophane derivatives ts.__:____o kí___. a____ r\_.. r»i_________I -r___• _l a Natural ligands of AhR??????????? */ lipid compounds and "Flavonoids Pflj c mew Lmflhflnf OCH Ö lanfcritiii CH J.Lnmin A4 CM3 -CH3 Bilirubin DOM l'r»«rtelflrwlinbii>liťDVI 2.3,7,«-letťř>chLorodibeiizoíurat] VlVfethvkholanthrene Beiizr>ŕa^p\T«>e li-lNanhrltoilavoiic řs.___:_____o Kl____. a r\_.. m_______ a n Toxic effects of dioxins: Epithelial hyperplasia Tumor promotion Induction of drug-metabolizing enzymes Altered ER signaling Porphyria Deregulated lipid metabolism Decreased serum thyroxine Wasting Metabolism of arachidonic acid to biologically active products Persistent thyroid hormone receptor activation EGF receptor down-regulation Lipid peroxidation Immunosuppression Inhibition of gluconeogenesis Teratogenesis/embryotoxicityl Utilization of brown adipose tissue Vitamin A depletion Cardiac dysfunction Figure I Biological responses to TCDD. A wide variety of cellular processes have been shown to be affected by TCDD. r-_i___:_Ij. o n____!.£• I_i a____ r\_.. /■• II ts.__. n:_ I 1 „Non-classical" AhR ligands M.S. Denison et al. i Chemko-Biologiccd Interactions 141 (2002) 3 24 2^,7,8 TctTflcMorodibcnzo p dioíin 24Melhvlmer».n^aniHnP CH3 CH2 CH3- CF, 0_N=N-irO 2-f4'-Chlorophenylíl>eiizotliiazole ■r^isi—N- CN SKF71739 CH3 CH2 Biliruhin NH2 1 ^THfliwiitoiiatththitlene OCONHCH 3 CH3° CH, CH, Carbarvl Omeprazole OCH< (Xf Cé-Methyienedioivbenzene H Trvptamine CH-, Physiological role for AhR - AhR-deficient mice: * significant growth retardation; v devective development of liver and immune system; v retinoid accummulation in liver; v abnormal kidney and hepatic vascular structures. v resistant to BaP-induced carcinogenesis and TCĎĎ- induced teratogenesis; S no inducible expression of CYP 1A1 and 2. +/+ Liver defects: ■ 1000 _5DO B o c. UXE E (0 — Sern CLO) äU Ž? C ) T * +/+ -/- +/+ -!- C012) < ao k— 5 2 0 E -/- +>+ -/- Flg. 1. Ah -/- mice have smaller hepatocytes than wild-type mice. Livers of 1-year-old mice were fixed in formalin, and 6-jim sections were examined after staining with hematoxylin/eosin. (A and B) Thin sections from wild-type ÍA) and age-matched Ah knockout {8) mice are shown, and results of mor-phornetric analyses follow. {Q There is a significant decrease in the total area of the hepatocytes of Ah -/- mice. (D and F) Whereas the cytoplasmic area □f Ah -/- hepatocytes is significantly decreased (D), the nuclear areas of Ah +/+ and Ah -/- hepatocytes are not different (£). Mean and standard errors generated from comparison of six 1-year-old male Ah +/+ and six age- and sex-matched ,4/? -/- mice areshown; asterisks indicate significance {P < 0.05). PNAS 2000 vol. 97:10447 +/+ -/- BqP není karcinogenní v AhR KO myších: Skin + - + - + Liver +/+ +/- -/--+■-+■ Genotype B[a]P Cyplal Cypta2 AhR ff -Actin Flg. 1. Cyplal, Cyplté, and AhR gene expression in the skin and liver of AhR( + / + ), AhR(+/-)f and AhR(-/-) mice, with and without B[a]P treatment One-microgram aliquots of RNA extracted from skin and live r of control and B[a]P-treated mice of the three genotypes we re reverse-transcribed and analyzed by PCR using specific primers for the Cypläl, Cyp1ä2, and AhR and ß-actin genes. I AhR<-/- ) AhR|+/-) AhR (+,'+) t \ 3WIV :.■!.■: ;ľt'9I>' Wrnkü Flg. 2. Subcutaneous tumor induction in wild-type (a) and AhR-deficient male mice (+/-, □, -/-, O) injected with B[e]P. Fig. 3. Gross appearance of flank skins in AhR-wiId-type mice (+/+), AhR-heterozygous mice (+/-), and AhR-deficient mice (-/-) injected subcutane-ously with B[a]P. r»K i a c f~tr\r\r\\ r\-r. -r-rr\ -re AhR je nezbytný pro imunotoxické účinky TCĎĎ: N.I. Kerkvhet f internatiorici! iininunophariTiacohgy 2 {2002} 277-291 AhR~'L Chimera AhR^+Chimera C57R1/6 10 100 10 100 10 100 Effector: Target CTL response Interactions of AhR with other proteins TABLE 1. Interactions Between Signal Transduction Pathways and AhR"-ŕ Interact btis References Direct interactions with All R HSP90 [79] XAP2 [80-82] ER, ERFfl [24] NFkB (RelA/p65) [39] Rb [44-46] RIP 140, p300/CBP [41,51,53] SRC-l,NCoA-2,pCIF [41,54] ERAP140, SMRT [49,50] COUP-TF1 [24] ppĚCF [70,71] tyrosine phosphorylation [69] Direct interactions with AhR comples proteins' HIF-la, PAS proteins (ARNT) [32,35] p300/CBP (ARNT) [52] SRC-l,NCoA-2(ARNT) 1=41 SHP (ARNT) [78] AhRR(ARNT) [20] ARNT Repressor ("ARNT") [21] CK2 (XAP2) 1741 p23 (HSP90) 17,-4 XAP2 (HSF90) [80] Indirect interactions (crosstalk) with AhR ER [8,25,29] hypoxia [33,36] NFkB [40-42] PKC [59-66] tyrosine kinases/phosplia lases [69,72,73] r-mj/r,AP-l,CK2 [72] TCF-ß [7] p27(Kipl) [43] NF-1 [27] C2-ceramide [47] JR. Petruifs. G.H. Perdew Chemico-Biological Interactions 141 í2002) 25-40 l»J*> Ä p2í 9> AhR XAP2 Fig. 4. Model for the arrangement of proteins found in the unliganded AhR complex O Htmkinstm I Archives of Biochemistry and Biophysics 433 (2005) 379 3B6 .\R1.= C CA AT TATA Fig. 3. Hypothetical model of ooactivator recruitment at the Cyphil gene. iochem Mol Toxicol 16:317-325, 2002; nMTCDD AP-1 1 Fold induction FIG. Z. Indu ŕ t km of ť-J mi mRNA by TCDD. Hernial cells were treated for 24 hr with TCDD in 0.05 M-ó DMSO tit tlie indicated concentrations, T o till RNA whs extracted from these eel Is. fractionated in agarose—formaldehyde gels, und transferred and hybridised to a mouse e-jun probe lis des e ri bed in the Methods See t ion. Fold indue t ion. determined by densitometry, is indi-tilted belo^v each Ume, Hours after TCDD AHR/ARNT NF-kB 0 2 5 9 24 48 72 V*"»»" «WWi* Hours after TCDD 24 48 72 12 24 36 ľ SO Hours after TCDD treatment n:_ i /_ en__r\r\-7 ir\r\iz o/w AhR-ERa crosstalk E2 HO-E2 CYP1A1 /CYP1B1 t Decreassd El $\ TM) MDrtE ■*■ Inhibition of E2-induccd genes Direct Inhibition of E2-iiidu€t3d gene« CoäCtivätor binding E2 ^ Limiting levels orcoactivators EZ ERa clo^nrogulíHŕn \^ (proteasomes ~~^ #2 #3 Limiting levels of ERa #4 #5 Figure 3, Proposed mechanisms of inhibitory AhR-Klíct crosstalk {123-í26}. /■•i_____ r\_- -r-___• _ w_ 1/. kl. -7 on/" Využitií AhR-ERa crosstalk v nádorové terapii? TABLE I Effects of 17jS-Esti adiol and TCDD on Cell Cycle Distribution of MCF-7 Human Breast Cancer Cells3 Treatment tím«, hi Cell cycle phase (%) C ().''( J ] S C, /M Control E2 (12) E2 + TCDD (12) TCDD (12) S9.9± 2.1 87.7 ± 2.1 87.2 ± 0.2 89.1 ± 0.8 4.9 ± 1.6 6.0 ± 1,1 7.9 ± 0.7 6.7 ± 0.8 5.2 ± 0.6 4.4 ± 0.7 4.9 ± 0.5 4.2 ± 0.2 :2(24) \2 + TCP U (24) 75.1 ± 0.eb 81.0 ± 1.3r 23.4 15 .S 1.7'J 1.8" 1.5 ± 1.2 3.2 ± 0.7 ľCDD (24) 90.8 ± 0.6 f).2 ± 0.5 4.0 ± 0.9 E2 r Time (h) u 6 12 24 E2+T I 12 24 12 24 cdk4 activity-*- cdk£ activity-*- GST-Rb 1200C | 10000 ^ 6000 > tt 4000 O I- B 2000 12000 1000 Q E. gj 8000 - 3 ^ 6000 > K 4000 □ 3 2000 12000 1COQ0 eooo UJ £ eoQO > K 4000 O i 2000 -•- Control -^TAM10Ofig/Kg ■*- TAM 50 ]xglkg -^- TAM 25 ^g/kg 3 5 7 -•- Control -*- 6-MCDF 100 (xg/kg -»- 6-MCDF 50 iíglkg -^6-MCDF 25 tig/kg T-----H------1------[------T^—I-----'T ' "I......I----"1------1-------1------1 9 11 13 15 17 19 21 9 11 13 15 17 19 21 -*-* Control -•-TAM 100 ng/kg + 6-MCDF 100 ^g/kg -"-TAM 50 )jg/kg *■ 6-MCDF 50 ^gŕkg -^- TAM 25 ng/kg +■ 6-MCDF 25 \igfkq 7 9 11 13 15 17 19 21 TREATMENT DAY ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 356, 239-248,1998; /•/kl; «i i z 1 onrko ^r\r\-7 onr>i Nhb O CHg "N^" "O" NH: Aminoflavone A 15° j--------------1--------------1________i________i____,____i________i__i 0 1e-10 1e-9 le-8 1e-7 1e-6 1e-5 Aminoflavone (M) 13 15 í8 21 26 28 32 35 40 42 47 49 54 57 Experimental Day *A_I /■•_______-i-i____o/Vk/i. o/V N.-71 c r Direct AhR-ER interaction? IB IP IgQ Anti-ER-a 1 1 1 1 Anti-ER-a ■ ■ ^" W Anti-AhR Mi Antl-AiTTt » * 1 2 3 4 5 6 7 8 E2 - - + + — - + + 3MC - + - + - + - + i- p * •* J P C Glandule epitheliui y «ť V ■ ■ ^^^^~ ^^^^™ ^^^^~ . F '/-* ■ ŕ *; f, * - dS T 4 ' ■L ■ .' ti " -■ * t ť 4 * » A* ■'.• r V ' .' ' ■ Vehicle 3MC Vehicle 3MC E2 31*? +E2 E2 3MC+E2 Glandular epithelium Luminal epithelium Vehicle 3MC E2 3MC +C2 /^ki-j._i-_ _j. _ o/VkO k i a -i-i ir\i— yio-5 ._______/___j Regulation of the eukaryotic cell cycle cycl in A/B + cdkl pRB-dephosphorylation 4 Č cyclin A + cdk2 D-cyclins + cdk4 / cdk6 pRB-phosphorylation p27- cyclin E + cdk2 pRB = retinoblastoma protein cdk = cycl in-dependent kinase (pRb) ÍAhR s> 1 l Cell cycle Progression 5L BP8 B Cell Growth rAriR- Western rAhR- RT-PCR GAPDH-» LJ ■ 5L D BP8 TCDD tigure 2.. TCDD induces growth inhibition in rat 5L hepatoma cells. Panel A, total protein from 5L and BP8 cells was fractionated by SDS-PAGĽ and probed for AhR protein with an anti-AhR antibody (Western). Analysis of AhR expression was also performed by RT-PCR on total RNA from 5L and BP8 cells using priniers specific for rat AhR (rÁhR) and GAPDH (as a control for RT-PCR). ' Panel B, 5L (solid bars) and BF8 (open bars) cells (2x10s) were grown in the presence of 10 iiM TCDD (+) or absence of TCDD (-) for 24h or 48h and counted. The values presented are the mean ± ft.D. of three independent experiments. r>__________-.-. .•„ •»//•»_/_ /*»__________/_ i/_ l c 0/.1 oz-7 f>r\r\- Puga, Elferink p53 cycllrt D1 J) \ cdK* T y ^CAK7 ^J pol Ě PCNA Cdc6 ore complex mem2,3A5,6,7 Dietrich c U l/S transition :_j f". C/í «•ll-cell contacts 000 P-T(]ůO> active DNA replication VONDRÁČEK ET AL. H isto ne H1 pRb cdk4 activity B # f ■5 o frj í*5 fo Go Ä» £? 8 Sf # J* ß ß ß ß C *S Q. 1 Q. 1 Historie H1 WB-F344 Úloha AhR v regulaci buněčného cyklu je pravdepodobné složitější MCF-7 Control Early S-phase (left): 2.6% Late S-phase {right): 3.7% Total BrdU positive: 6.6% BaA Early S-phase (left): 7.6% Late S-phase fright): 7.3% Total BrdU positive: 14.9% fold over control DMSO CoCL Dioxin CoClz/Dioxin ■:.' — : — f-. »' LU r. ■— pr ^ š7 r 'j J — : -! — = p - — la B — CD ■Y — J [1.05; r: S. '- ■■-■■"- 'k r. =■ í -■— '" ŕ~"s - _■ ' ■ *- V 3 \ ■z = zz '■- ■— 5' A -. >-á '-. ■-■ '— r. — = V - ■— í - v< = > rľ ~ ~ x . - — _, = ' -— : : = — ■Vi ^ľ 1= 3 t '-—■ ti1 A - y 1= x: ■-.• i ■:. =■ :— m: f". 3 f ■-'.-■ — £ " - i j > r. 5 ■■", '— .. 1 j 4-T T 1 — ri j j-n - —. ■ — T. -r - "- -'■ EX] — ■< • "S -~ U tu T. - ~ — O *' ^- > --—. E. n — Ľ. 3 — "*—' -í -- '". ■— : — —■ Z T-—. H — —' — ^ — Ě -> ■_ 3 Ě5 > < 3 Fo id Ex press io n C YP1A1 S E R 0 D Aciivi ty (pmol/ m i nŕ106 ceils) O fo *■ tn co o ro O o •o AhR-retinoid receptors crosstalk SOME SYNTHETIC RETINOIDS, Physiological and Pharmacological Levels RAR a RAR l i RAR y Ľ ATRA Pharmacological l-cvels CQ2H AhR AhR FIGURE 2 Schematic representation of the AhR/Arnt signaling pathway indicating the five steps (see text for descriptions} that have been shown to be modulated by specific retinoids. TCDD 01 naphthoflavone benzo[a]pyrene <5> CYP1A1 ^^ j * ^^ ^^ ^^' * t ki..j._ 1-5-5. o-7-7r ooie tr\r\ TABLE 2 Effects of Ah Receptor Ligands on Enzyme Activities Involved in Retinoid Metabolism1 Activity Effect Tissue RcFctence Retinole acid glucuronidation liver, kidney liver Bank et al. 1989 Sass ct al. 1994 Retinole acid oxidation 1 liver Ť liver liver Spear et al. 1988 Fiorella et al. 1995 Andreola et al. 1997 Retinol cstenfication t hepatic stellate cells kidney Nilsson ct »I. Nilsson et al. 1996 2000 Retiny 1 ester hydrolysis ±0 liver Nils sun et al. 2000 1 TCDD was used in all studies except Sass et al, 1994 (3-melľiylchulantľirene) and Spear et al. 1998 (3r3'A4",5r5'-he!(abriomobipher>yl}. All studies were on rats except Andreola et al. 1997 (mice). Fig. 9. Schematic depiction of the activation of MMP-1 inRNA levels by TCDD and atRA in NHKs. The data presented in this report suggest that TCDD is having an impact on MMP-1 expression in NHKs through at least two mechanisms: 1 j by inducing the binding of Fos and Jun proteins to the AP-1 elements in its promoter and thereby activating transcription; and 2) by altering the expression of RAR-y and RXRa expression, which leads to an enhancement of MMP-1 mRNA stability following exposure to atRA. ATTA J± y MMP-1 XRE JunTFos Tn/< f>r\r\A\ o-7r»/o/i\.ocoo a c\r