Vpliv estrogenov, oksidativnega stresa in hipoksije na epigenetske procese v osteoblastih : doktorska disertacija

Vrtačnik, Peter (author), Ostanek, Barbara (mentor)

Estrogeni, oksidativni stres in hipoksija so pomembni regulatorji kostne prenove in homeostaze. Poleg pomanjkanja estrogenov, ki so najpogosteje opisan patološki dejavnik, odgovoren za izgubo kostne mase in spremembe v mikroarhitekturi kostnine, ki vodijo v razvoj osteoporozo, postaja vedno bolj jasen tudi prispevek staranja in z njim povezanega oksidativnega stresa. Dobra vaskularizacija kostnega tkiva in s tem ustrezna preskrbljenost s kisikom prav tako igra pomembno vlogo pri normalnem razvoju skeleta in učinkoviti regeneraciji poškodb kostnine. Poleg omenjenih regulatorjev kostne homeostaze igrajo pomembno regulatorno vlogo v kosteh tudi epigenetski mehanizmi. Epigenetika je veda, ki proučuje stabilne in potencialno dedne spremembe v izražanju genov, za katere niso odgovorne spremembe v zaporedju DNA. Med najpomembnejše epigenetske mehanizme uvrščamo posttranslacijske spremembe histonov, posttranskripcijsko uravnavanje z mikroRNA in metilacijo DNA. Najbolj proučevana posttranslacijska sprememba histonov je acetilacija, za katero so odgovorne histonske acetilaze in deacetilaze. Medtem ko acetilacija histonov omogoči in spodbudi izražanje tarčnih genov, deacetilacija histonov transkripcijo zavre. Slednja pogosto predstavlja le prehodno inhibicijo izražanja tarčnih genov, ki pa lahko preide v bolj trajno obliko z metilacijo DNA. To nalogo opravljajo DNA-metiltransferaze, pri čemer pa je demetilacija lahko pasivna ali aktivna. Podobno kot deacetilacija histonov in metilacija DNA igrajo tudi mikroRNA predvsem inhibitorno regulatorno vlogo. Epigenetske spremembe so postale pomemben del raziskav številnih fizioloških in patoloških procesov tudi zato, ker lahko starost, mehanske obremenitve in okoljski dejavniki ravno preko njih vplivajo na izražanje tarčnih genov. Ker je povezava med znanimi regulatorji kostne homeostaze na eni strani in epigenetskimi procesi na drugi strani še vedno slabo raziskana, je bil namen doktorskega dela proučiti, kako estrogeni, oksidativni stres in hipoksija vplivajo na izražanje genov za histonske acetilaze, histonske deacetilaze, DNA-metiltransferaze in mikroRNA ter s tem na epigenetske procese v osteoblastih. V ta namen smo si najprej pripravili primerni eksperimentalni in vitro model, pri čemer smo uporabili osteosarkomsko celično linijo HOS, v katero smo vnesli kodirajoči del gena za estrogenski receptor α. Za proučevanje vpliva estrogenov smo uporabili 17β-estradiol, oksidativni stres smo izzvali z vodikovim peroksidom, hipoksijo pa z dodatkom mimetika hipoksije deferoksamina. Analizo izražanja genov, povezanih z epigenetskimi mehanizmi, smo izvedli dvostopenjsko. Na osnovi poznavanja epigenetskih mehanizmov in kostne biologije smo za presejalno testiranje izbrali 13 genov, ki kodirajo histonske acetilaze, 18 genov, ki kodirajo histonske deacetilaze, 9 genov, ki kodirajo DNA- metiltransferaze, in druge proteine, povezane z metilacijo DNA, 5 genov, značilnih za osteoblastno celično vrsto, in 3 hišne gene ter njihovo izražanje izmerili s pomočjo mrež, ki temeljijo na kvantitativni verižni reakciji s polimerazo v realnem času (qPCR). V potrditveni stopnji smo tiste gene, pri katerih smo zaznali spremembe v izražanju, analizirali še s posameznimi qPCR-testi na vzorcih večjega števila bioloških ponovitev. Rezultate, pridobljene na eksperimentalnem in vitro modelu, smo nato nadgradili in jim dodali poseben pomen z analizo izražanja najbolj obetavnih genov v obsežni zbirki kostnih vzorcev, pridobljenih od bolnikov z osteoporozo ali osteoartrozo, ter kontrol, brez znakov kostnih bolezni. Proučili smo tudi vpliv estrogenov, oksidativnega stresa in hipoksije na izražanje mikroRNA v osteoblastih, pri čemer smo uporabili zelo zmogljivo tehnologijo barvnih kod. V času razvoja in optimizacije našega eksperimentalnega in vitro modela smo se soočili z več izzivi, katerih pomen presega okvire naše študije. Tako smo ugotovili, da tripsinski reagent, pridobljen iz živalskega pankreasa, ki se sicer rutinsko uporablja pri presajanju adherentnih celic, vsebuje RNaze, ki vstopajo v celice z endocitozo in razgradijo njihovo RNA, ki je tako neprimerna za nadaljnje analize. Rešitev smo našli v neposredni lizi celic z reagentom, ki vsebuje fenol in gvanidin, tik pred samo izolacijo RNA ali v uporabi rekombinantnih tripsinskih nadomestkov, ki niso živalskega izvora. Prav tako se je izkazalo, da v osteosarkomski celični liniji HOS estrogenski receptor α ni prisoten niti na ravni mRNA niti na ravni proteinov. Najizraziteje se je izražal z G proteinom sklopljen estrogenski receptor 1, prisotno pa je bilo tudi šibko izražanje estrogenskega receptorja β. V našem eksperimentalnem in vitro modelu, z vnesenim estrogenskim receptorjem α, estrogeni niso vplivali na izražanje izbranih genov, povezanih z epigenetskimi mehanizmi. Izjema je bilo izražanje mikroRNA miR-338-3p. V skladu s tem estrogeni prav tako niso preprečili sprememb, ki sta jih v osteoblastih povzročila oksidativni stres in hipoksija. Oksidativni stres in hipoksija sta značilno vplivala na izražanje HDAC6, HDAC9, SIRT1 in MBD1, hipoksija pa še dodatno na izražanje HAT1, KAT5, HDAC7 in DNMT3A. Oksidativni stres in hipoksija sta prav tako značilno vplivala na izražanje miR-27, miR-34c, miR-133a, miR-148a, miR-155, miR-214-3p, miR-335, miR-503, miR-630 in miR-1246. Kljub temu, da sta imela zelo blag učinek na aktivnost signalne poti WNT/β-katenin, sta jo oba zavrla. Močnejši, vendar nasproten učinek sta imela tudi na sistem RANK/RANKL/OPG. Naši rezultati razkrivajo potencialno povezanost omenjenih signalnih poti z epigenetskimi mehanizmi v osteoblastih. Analiza izražanja genov, povezanih z epigenetskimi mehanizmi, v kostnih vzorcih je še dodatno potrdila izjemen pomen identificiranih genov v fiziologiji in patologiji kostnih celic. HAT1, KAT5, HDAC6, HDAC9, MBD1 in DNMT3A so tako novi kandidatni geni z zelo verjetno vlogo pri osteoporozi in osteoartrozi, na katere bi se morale osredotočiti prihodnje študije. Omenjeni geni prav tako predstavljajo potencialne tarče v osteoblastih, na katerih bi lahko temeljila prihodnja generacija zdravilnih učinkovin z anabolnim delovanjem na skelet.

• Language: Slovenian
• Keywords: estrogeni, oksidativni stres, hipoksija, kostnina, homeostaza, regulatorji, osteoblasti, epigenetika, gensko izražanje, histonske acetilaze, histonske deacetilaze, DNA-metiltransferaze, mikroRNA, disertacije
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [P. Vrtačnik]
• Year: 2015
• Number of pages: VIII, 189 str.
• PID: 20.500.12556/RUL-143726
• UDC: 577.21:611.018.4(043.3)
• COBISS.SI-ID: 278090496
• Publication date in RUL: 11.01.2023

Secondary language

• Language: English
• Title: The influence of estrogens, oxidative stress and hypoxia on epigenetic processes in osteoblasts

Abstract

Estrogens, oxidative stress and hypoxia are important regulators of bone remodeling and homeostasis. In addition to estrogen deficiency, the most often described factor responsible for osteoporosis-associated loss of bone mass and changes in bone microarchitecture, the contribution of aging and associated oxidative stress is becoming increasingly accepted. Sufficient vascularization and oxygen supply to bone tissues are also important for proper skeletal development and efficient bone regeneration and repair. In addition to the described regulators of bone homeostasis, epigenetic mechanisms also play an important regulatory role in bone. Epigenetics is the study of stable and potentially heritable changes in gene expression not associated with changes in DNA sequence. The most important epigenetic mechanisms include posttranslational histone modifications, posttranscriptional regulation through microRNA and DNA methylation. Acetylation is the most studied posttranslational histone modification mediated by histone acetylases and deacetylases. While histone acetylation enables and stimulates target gene expression, histone deacetylation inhibits gene transcription. The latter often represents only a transient inhibition of target gene transcription that can become more permanent through DNA methylation, mediated by DNA methyltransferases. Of note, DNA demethylation can be either passive or active. Similarly to histone deacetylation and DNA methylation, microRNAs also play mostly inhibitory regulatory role. Epigenetic changes have become an important part of research in several physiological and pathological processes, since they represent a mean by which aging, mechanical stress and environmental factors influence target gene transcription. The association between known regulators of bone homeostasis and epigenetic processes is still poorly understood, therefore the aim of the thesis was to examine how estrogens, oxidative stress and hypoxia influence the expression of genes encoding histone acetylases, histone deacetylases, DNA methyltransferases and microRNAs and through that epigenetic processes in osteoblasts. To this end a suitable experimental in vitro model was prepared by transfecting an osteosarcoma cell line HOS with the gene encoding estrogen receptor α. 17β-estradiol was used to study the influence of estrogens, hydrogen peroxide to induce oxidative stress and hypoxia mimetic deferoxamine to establish hypoxia. The expression of genes associated with epigenetic mechanisms was analyzed in a stepwise manner. Based on our knowledge of epigenetic mechanisms and bone regulation, 13 genes encoding histone acetylases, 18 genes encoding histone deacetylases, 9 genes encoding proteins associated with DNA methylation, 5 osteoblast lineage-associated genes and 3 housekeeping genes were selected for gene expression screening using qPCR arrays. In the confirmation step, the most affected genes were analyzed in individual qPCR analyses on samples from several biological replicates. The results obtained in our experimental in vitro model were further substantiated with the expression analysis of the most promising candidate genes on our extensive collection of bone tissue samples obtained from patients with osteoporosis and osteoarthritis as well as controls with no signs of skeletal pathologies. The influence of estrogens, oxidative stress and hypoxia on expression of microRNAs in osteoblasts was also analyzed by using a very powerful technology of color codes. During the development and optimization of our experimental in vitro model we were faced with several challenges, the impact of which exceeds the extent of our study. Our work revealed the presence of contaminating ribonucleases in the animal pancreas-derived trypsin reagent, used routinely for subculturing of adherent cell lines. Ribonucleases entered the cells through endocytosis causing degradation of RNA rendering it useless for further analysis. Our solution was cell lysis with a phenol-guanidine-based reagent directly in the cell culture vessel prior to RNA isolation, or the use of an animal-origin free recombinant trypsin replacement reagent. In addition, we observed a complete lack of estrogen receptor α mRNA and protein in the osteosarcoma cell line HOS. Interestingly, G protein-coupled estrogen receptor 1 gene showed the highest level of expression accompanied by a weak expression of estrogen receptor β. In our experimental in vitro model transfected with estrogen receptor α estrogens had no influence on expression of selected genes associated with epigenetic mechanisms. Expression of microRNA miR-338-3p was the only exception. In accordance with these observations estrogens were not able to prevent changes induced by oxidative stress and hypoxia in osteoblasts. Oxidative stress and hypoxia significantly affected expression of HDAC6, HDAC9, SIRT1 and MBD1, while hypoxia additionally affected expression of HAT1, KAT5, HDAC7 and DNMT3A. Oxidative stress and hypoxia also significantly affected the expression of miR-27, miR-34c, miR-133a, miR-148a, miR-155, miR-214-3p, miR-335, miR-503, miR-630 and miR-1246. Despite having only a mild effect on the activity of WNT/β-catenin signaling pathway, both stimuli exhibited an inhibitory effect. They exhibited a more substantial although opposite effect on RANK/RANKL/OPG system. Our results revealed a potential link between the two signaling pathways and epigenetic mechanisms in osteoblasts. Expression analysis of genes associated with epigenetic mechanisms in bone tissue samples further underlined the importance of identified genes in the physiology and pathology of bone cells. HAT1, KAT5, HDAC6, HDAC9, MBD1 and DNMT3A represent new candidate genes with a very likely role in osteoporosis and osteoarthritis and should be the focus of studies in the future. These genes also represent potential drug targets in osteoblasts that could form the basis for the next generation of active substances with an anabolic effect on the skeleton.