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Vpliv izbranih transkripcijskih dejavnikov iz družine SOX in c-MYB na uravnavanje izražanja gena RANKL v kostnih celicah pri osteoporozi : doktorska disertacija
ID Kodrič, Klemen (Avtor), ID Marc, Janja (Mentor) Več o mentorju... Povezava se odpre v novem oknu

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Izvleček
Kostna remodelacija je skrbno uravnavan proces za ohranjanje homeostaze kalcija in fosfata v krvnem obtoku. Istočasno ta mehanizem skrbi tudi za strukturno integriteto kosti. Remodelacija poteka sklopljeno. Najprej osteoklasti kostnino razgradijo, nato jo osteoblasti znova izgradijo. Po fazi mirovanja se krog po nekaj letih ponovi. Da je sklopitev uravnotežena skrbijo mnogi lokalni in sistemski regulatorji, med katerimi so poglavitnega pomena PTH, vitamin D in triada proteinov RANKL/RANK/OPG. Protein RANKL je citokin, ki ga v kosteh sproščajo celice osteoblastne linije. RANKL deluje preko vezave na svoj receptor RANK, izražen na osteoklastih. RANKL aktivira ostaoklaste in kostno razgradnjo, ter deluje kot ena izmed sklopitvenih molekul med osteoblasti in osteoklasti. OPG je protein, ki delovanju proteina RANKL nasprotuje tako, da se veže nanj in s tem prepreči njegovo vezavo na RANK. Delovanje proteina RANKL in njegovo fiziološko vlogo ter vpletenost v kostna obolenja dobro poznamo, medtem ko je uravnavanje njegovega izražanja slabše preučeno. Poznamo nekaj transkripcijskih dejavnikov, ki na izražanje gena RANKL vplivajo preko distalnih regulatornih regij, npr. vitamin D, PTH in Runx2. Po drugi strani pa ostaja uravnavanje izražanja gena RANKL preko proksimalnega promotorja gena, ki je po navadi poglavitna regulatorna regija genov, v veliki meri nepoznano. Tako je bil namen te doktorske naloge preučiti proksimalni promotor gena RANKL in identificirati nove transkripcijske dejavnike, ki bi lahko vplivali na izražanje gena RANKL, ter bi lahko bili vpleteni v patogenezo najpogostejše kostne metabolne bolezni, osteoporoze. Osteoporoza je multifaktorska bolezen na katero vplivajo številni genetski in okoljski dejavniki. Mnogi avtorji so preiskovali z osteoporozo povezane polimorfizme, spremembe v izražanju genov ter spremembe v proteomu in metabolomu. Ugotovili so, da na bolezen vpliva veliko število pogostih sprememb v mnogih različnih genih. Danes še ne poznamo veliko takih sprememb, zato znamo pojasniti zgolj manjši del variance v mineralni kostni gostoti, ki predstavlja glavni kazalec bolezni. V prvem poglavju smo naredili pregled študij na vsegenomskem nivoju ter na osnovi rezultatov pripravili izhodišče za nadaljnje delo. V drugem poglavju smo opisali računalniško analizo proksimalnega promotorja gena s programom TF Search. Program je odkril potencialna vezavna mesta za različne transkripcijske dejavnike. Na podlagi podatkov iz literature smo se odločili, da se bomo osredotočili na transkripcijska dejavnika Sry in c-Myb. Preverili smo njun vpliv na aktivnost promotorja gena RANKL in odkrili, da c-Myb aktivira, Sry pa zavira aktivnost proksimalnega promotorja gena RANKL. Nadaljevali smo z čezmernim izražanjem in utišanjem genov c-Myb in Sry ter potrdili, da c-Myb poveča izražanje gena RANKL v celičnem modelu in vitro, medtem ko ga Sry zmanjša. Vpletenost teh transkripcijskih dejavnikov v uravnavanje izražanja RANKL in osteoporozo smo želeli potrditi tudi v pogojih in vivo, zato smo njuno izražanje in izražanje gena RANKL izmerili v vzorcih kosti preiskovancev z osteoporozo in brez bolezni. Odkrili smo, da je izražanje gena RANKL v kosteh osteoporotičnih moških povečano za 17-krat, v primerjavi s kontrolami. Prav tako smo odkrili, da je pri osteoporotičnih moških kostno izražanje za moške specifičnega gena Sry kar 105-krat manjše kot pri kontrolah. To nakazuje, da je pri moških z osteoporozo povečanje izražanja gena RANKL posledica zmanjšanega izražanja gena Sry, ter da so razlike med spoloma v izražanju gena RANKL in v kvaliteti kostnine posledica delovanja za moške specifičnega transkripcijskega dejavnika Sry. Kostno izražanje gena c-Myb je bilo pri osebah z osteoporozo zmanjšano, zato ga v patogenezo osteoporoze ne moremo vplesti. Smo pa potrdili njegovo vlogo v kostni biologiji. V tretjem poglavju smo preverjali, ali tudi drugi člani družine transkripcijskih dejavnikov SOX (poleg Sry) uravnavajo izražanje gena RANKL preko proksimalnega promotorja, ter ali so vpleteni v patogenezo osteoporoze. Z uporabo celičnih modelov in vitro smo preverili ali lahko SOX4, SOX5, SOX6 in SOX9 uravnavajo aktivnost promotorja gena RANKL, ter ali lahko uravnavajo njegovo izražanje. Z uporabo človeškega kostnega tkiva smo testirali relevantnost naših rezultatov in vivo. Dokazali smo, da SOX5 in SOX9 aktivirata promotor gena RANKL ter povečata njegovo izražanje v osteoblastnih celičnih modelih. Poleg tega smo pokazali, da je v kostnem tkivu moških z osteoporozo izražanje gena RANKL povečano, izražanje gena SOX9 pa zmanjšano, v primerjavi s kontrolami, ter da bi izražanje gena SOX9 lahko imelo napovedno vrednost za osteoporozo. Osteoporotična in kontrolna skupina preiskovancev sta bili potrjeni z uporabo slikovne tehnike mikro-CT, trenutno najboljše tehnike za analizo strukture in kvalitete kostnega tkiva. V dodatnem poglavju smo preverjali, ali transkripcijski dejavniki iz družine SOX vplivajo na izražanje gena RANKL in na kostne spremembe tudi pri osteoartrozi. Avtorji mnogih študij namreč opisujejo kostni fenotip pri tej bolezni kot nasproten tistemu pri osteoporozi, vendar rezultati niso enotni. Naši rezultati so pokazali razgrajeno strukturo subhondralne kostnine pri osteoartrozi in povečano izražanje genov RANKL in SOX5 ter zmanjšano izražanje gena SOX9, v primerjavi s kontrolnimi kostnimi vzorci. Nadalje smo odkrili, da mezenhimske matične celice, izolirane iz vzorcev kosti osteoartroznih bolnikov, izražajo več SOX5 in RANKL kakor mezenhimske matične celice kontrolnih bolnikov po osteogeni diferenciaciji. S tem smo potrdili, da bi transkripcijski dejavniki iz družine SOX lahko prispevali h kostnim spremembam, ki smo jih potrdili pri bolnikih z osteoartrozo, in sicer preko poveča izražanja gena RANKL. V doktorski nalogi smo odkrili nove transkricpijske dejavnike, ki na izražanje gena RANKL vplivajo preko vezave na proksimalni promotor gena, ter so vpleteni v kostne spremembe pri osteoporozi (c-Myb, Sry, SOX5 in SOX9) in tudi osteoartrozi (SOX5 in SOX9). Transkripcijski dejavniki gena RANKL predstavljajo nove tarče za napoved, diagnozo in zdravljenje kostnih bolezni.

Jezik:Slovenski jezik
Ključne besede:osteoblasti, osteoklasti, beljakovine, citokini, RANKL, gensko izražanje, uravnavanje, promotorji, transkripcijski dejavniki, patogeneza
Vrsta gradiva:Doktorska disertacija
Tipologija:2.08 - Doktorska disertacija
Organizacija:FFA - Fakulteta za farmacijo
Kraj izida:Ljubljana
Založnik:[K. Kodrič]
Leto izida:2019
Št. strani:191 str.
PID:20.500.12556/RUL-137327 Povezava se odpre v novem oknu
UDK:577.21:616.71-007.23(043.3)
COBISS.SI-ID:302178816 Povezava se odpre v novem oknu
Datum objave v RUL:10.06.2022
Število ogledov:463
Število prenosov:19
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Sekundarni jezik

Jezik:Angleški jezik
Naslov:Influence of selected transcription factors from the SOX family and c-MYB in regulation of RANKL gene expression in bone cells in osteoporosis
Izvleček:
Bone remodeling is a carefully regulated mechanism, responsible for maintaining calcium and phosphate homeostasis in the bloodstream. This mechanism also guaranties the structural integrity of bones. The process of remodeling is coupled. First, the osteoclasts degrade bone tissue, which is later rebuilt by the osteoblasts. After the resting phase, the cycle is repeated after a couple of years. Many local and systemic regulators exist in order to keep the bone remodeling balanced, of which PTH, vitamin D and the RANKL/RANK/OPG triad are vital. RANKL protein is a cytokine which is, in bone, mainly produced by the osteoblast lineage cells. RANKL functions by binding to its receptor RANK, expressed on osteoclasts, causing their activation. Besides bone degradation, RANKL also functions as one of coupling molecules between osteoblasts and osteoclasts. OPG protein acts as a decoy receptor for RANKL. It opposes the function of RANKL by binding to it, therefore preventing its effect on osteoclasts via RANKL-RANK binding. The functions of RANKL, its physiological role and its involvement in bone diseases are well understood; however, the regulation of its expression remains mostly unknown. There are some transcription factors known to influence the expression of the RANKL gene via its distal regulatory regions, for example, vitamin D, PTH and Runx2. On the other hand, the regulation of the RANKL gene expression through its proximal promoter remains unexplored. Thus, the aim of our doctoral thesis was to study the proximal promoter of the RANKL gene and identify novel transcription factors that influence the expression of the RANKL gene, and could be involved in the pathogenesis of the most common bone metabolic disease - osteoporosis. Osteoporosis is a multifactorial disease influenced by a number of genetic and environmental factors. Many authors investigated osteoporosis-related polymorphisms, changes in gene expressions, and changes in proteome and metabolome. They discovered that a large number of frequent changes in many different genes affect the onset of the disease. To date, many such changes remain undiscovered and we can only explain a minor part of the bone mineral density variance, which is the most commonly used indicator of the disease. In the first chapter we performed a literature review of genome-wide studies. The results served as foundation for our following work. In the second chapter, we first conducted a computational analysis of the RANKL gene proximal promoter using the program TF Search. The program revealed potential transcription factors binding sites. Based on data from the literature review we decided to focus on the transcription factors Sry and c-Myb. We tested their effect on the activity of the RANKL gene promoter and discovered that c-Myb activates, while Sry reduces the activity of the proximal promoter of the RANKL gene. We continued with overexpression and silencing of the c-Myb and Sry genes. We confirmed that c-Myb increases the expression of the RANKL gene in the cell model in vitro, while Sry decreases it. The involvement of these transcription factors in the regulation of RANKL gene expression and in osteoporosis was also tested in vivo. We measured their expressions and the expression of the RANKL gene in bone tissue samples of donors with osteoporosis and controls. We discovered that in the osteoporotic bone tissue samples the expression of the RANKL gene was increased 17-times, compared to controls. We also discovered that, in male osteoporotic bone tissue, the expression of male specific Sry gene was 105-times decreased compared to controls. These results suggest that, in osteoporotic men, the increased expression of RANKL comes as a result of the immense decrease of Sry expression, and that the gender differences in RANKL gene expression and in quality of bones are caused by the actions of the male specific transcription factor Sry. The expression of c-Myb was also reduced in osteoporotic bone tissue samples. Therefore, we cannot argue that c-Myb is involved in the pathogenesis of osteoporosis. We can, however, confirm its role in bone biology. In the third chapter we tested if other members from the SOX family of transcription factors (besides Sry) regulate the expression of RANKL gene through its proximal promoter, and if they are involved in the pathogenesis of osteoporosis. Using in vitro cell models we tested if SOX4, SOX5, SOX6, and SOX9 can regulate the activity of the RANKL gene promoter, and if they can regulate its expression. Using human bone tissue, we tested the in vivo relevance of our results. We have demonstrated that SOX5 and SOX9 activate the RANKL gene promoter and increase the expression of RANKL gene in osteoblast cell model. In addition, we have shown that the expression of the RANKL gene in the bone tissue of osteoporotic men is increased and the expression of the SOX9 gene is decreased compared to controls. Also, the expression of the SOX9 gene may have a predictive value for osteoporosis. The osteoporotic and control group of donors were confirmed using micro-CT, currently the best method for bone tissue structure and quality evaluation. In the additional chapter, we examined whether the transcription factors from the SOX family affect the expression of the RANKL gene and are responsible for the bone changes also in osteoarthritis. Authors of many studies describe the bone phenotype of this disease as the opposite of that found in osteoporosis; however, the results are not consistent. Our results revealed degraded structure of the subchondral bone in osteoarthritis, increased expressions of RANKL and SOX5 genes and decreased expression of SOX9 gene, as compared to controls. Furthermore, we discovered that mesenchymal stem cells isolated from bone samples of osteoarthritic patients express more SOX5 and RANKL than the mesenchymal stem cells of the control patients after osteogenic differentiation. This confirmed that the transcription factors from the SOX family could contribute to the bone changes, which we discovered in patients with osteoarthritis, by increasing the expression of RANKL. In the doctoral thesis, we discovered novel transcription factors that impact the expression of the RANKL gene through binding to its proximal promoter, and are involved in bone changes in osteoporosis (c-Myb, Sry, SOX5 and SOX) and in osteoarthritis (SOX5 and SOX9). Transcriptional regulators of the RANKL gene represent novel targets for prediction, diagnosis and treatment of bone diseases.

Ključne besede:osteoporosis, RANKL gene promoter, SOX, c-Myb, regulation of RANKL gene expression, micro-CT

Projekti

Financer:ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
Številka projekta:P3-0298
Naslov:Geni, hormonske in osebnostne spremembe pri metabolnih motnjah

Financer:ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
Številka projekta:J3-7245
Naslov:Odkrivanje novih regulatorjev izražanja RANKL, ključne molekule ne samo v kostni prenovi

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