İnsan ADAMTS-3 gen promotorunun fonksiyonel analizi
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Tarih
2016
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info:eu-repo/semantics/openAccess
Özet
ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) ailesi memelilerde ve omurgasızlarda bulunan ekstraselüler proteinazlardır. ADAMTS’ler prokollajenin amino ucunun işlenmesi, matriks proteoglikanlarından agrekan, versikan ve brevikanın degredasyonu, anjiyogenezin inhibisyonu, kan pıhtılaşma homoeostazisinde önemli rollere sahiptir. ADAMTS-3 temel olarak eklem kıkırdağının spesifik bileşeni olan tip II kollajenin işlenmesini sağlar. Ayrıca osteoartirit, miyokard enfarktüs ve göğüs kanseri gibi bazı patolojik durumlarda da bağlantılı olduğu düşünülmektedir. ADAMTS-3’ün fonksiyonel özellikleri detaylı bir şekilde çalışılmış olmasına rağmen genin transkripsiyonel regülasyonunun nasıl olduğu bilinmemektedir. Bu proje ADAMTS-3 geninin transkripsiyonel regülasyonunu ve bu regülasyonda SP1, USF ve C/EBP? transkripsiyon faktörlerinin rolünü aydınlatmayı hedeflemektedir. Bu kapsamda, transkripsiyonel olarak en aktif bölgenin belirlenmesi amacıyla dört farklı uzunlukta ADAMTS-3 promotor parçası [-1340/+40] [-899/+40], [-576/+40], [-131/+40] lusiferaz vektörüne klonlandı. Saos-2 ve MG-63 hücrelerinde yapılan geçici transfeksiyon analizleri ile bütün parçaların aktif olduğu ve transkripsiyonel aktivite için en küçük promotor parçasının yeterli olduğu belirlendi. Her iki hücre hattında da 616 bç’lik promotor parçasının [-576/+40] en kuvvetli bazal aktiviteye sahip olduğu belirlendi. 1380 [1340/+40] ve 916 bç’lik [-899/+40] promotor parçalarının Saos-2 hücrelerindeki aktivitesinin MG-63 hücre hattına göre daha düşük olduğu tespit edildi. Biyoinformatik analizlere göre ADAMTS3 promotoru 40bç’lik 5’UTR bölgesi içermektedir ve TATA kutusu bulundurmamaktadır. Saos-2 ve MG-63 hücrelerinde yapılan ko-transfeksiyon deneyleriyle ADAMTS-3 promotor bölgesinde potansiyel bağlanma motifleri bulunan SP1, USF ve C/EBP? gibi bazı transkripsiyon faktörlerinin ADAMTS-3 promotor aktivitesini azaltıcı yönde etki gösterdiği belirlendi. Çalışmanın diğer bir kısmında SP1, USF ve C/EBP?’nın Saos-2 ve MG-63 hücrelerinde geçici aşırı ifadesi sağlanarak ADAMTS-3 ve kollajenlerin (tip I,II III) mRNA seviyelerine olan etkisi qRT-PCR, protein seviyesine olan etkisi de westernblot yöntemiyle belirlendi. Bahsedilen transkripsiyon faktörlerinin ADAMTS-3 mRNA ve protein düzeyini azalttığı belirlendi.Kollajen tiplerinin ekspresyonlarına olan etkisinin hücre tipine göre farklılık gösterdiği belirlendi. DNA-protein etkileşim çalışmalarında ise, SP1,C/EBP?,USF,AP1,E2F,NF?? ve STAT transkripsiyon faktörlerinin ADAMTS-3 promotor bölgesine fonksiyonel olarak bağlandığı EMSA deneyleri ile belirlendi. Ayrıca Saos, MG-63, Panc, HUVEC, PC-3 ve DU-145 hücre hatlarında ADAMTS-3 mRNA ekspresyon seviyeleri de RT-PCR ile belirlendi. Sonuç olarak bu çalışma ile insan ADAMTS-3 promotoru ilk kez klonlandı ve fonksiyonel olarak analizi yapıldı.SP1, USF ve C/EBP? transkripsiyon faktörlerinin ADAMTS3’ün ve kollajenlerin (tip I, II, III) transkripsiyonel regülasyonuna olan etkisi belirlenerek literatüre önemli katkılar sağlanmış oldu.
ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a family of extracellular proteases found in both mammals and invertebrates. ADAMTSs have significant roles in processing amino terminus of the procollagen molecules, cleavage of the matrix proteoglycans aggrecan, versican and brevican; inhibition of angiogenesis; blood coagulation homoeostasis. ADAMTS-3 plays a key role in the processing of type II fibrillar collagen, specific component of the articular cartilage. It was also thought that there was a connection with some pathological conditions such as osteoarthritis, myocardial infarction, breast cancer and ADAMTS-3. Although functional features of the ADAMTS-3 gene was studied in detail it wasn’t known how its own transcriptional regulation. This project was aimed to elucidate transcriptional regulation of the ADAMTS-3 gene and the roles of SP1, USF and C/EBP? transcription factors in this regulation. Within this scope, to determine transcriptionally most active promoter region, four ADAMTS-3 promoter fragments [-1340/+40] [-899/+40], [-576/+40], [-131/+40] were cloned into luciferase vector with different size. It was determined with transient transfection experiments that all promoter fragments were active and the smallest construct was enough to exhibit the transcriptional activity in Saos-2 and MG-63 cells. It was also determined that 616 bp promoter fragment [- 576/+40] has the strongest basal activity. It was detected that the activities of the 1380bp [1340/+40] and 916 bp [-899/+40] promoter constructs was lower in Saos-2cells compared to MG-63.According to the bioinformatic analyses ADAMTS-3 promoter has a 40bp 5’UTR region and doesn’t include a TATA box. The co-transfection experiments determined that some transcription factors such as USF,SP1 and C/EBP? decreased the ADAMTS3 promoter activity having potential binding motifs in ADAMTS-3 promoter region. Another part of the study SP1, USF and C/EBP? were overexpressed in Saos-2 and MG-63 cells. Effect of these transcription factors on ADAMTS-3 and collagen (type I, II and III) mRNA and protein levels was determined by qRT-PCR and western blot method. It was determined that these transcription factors were decreased ADAMTS-3 mRNA and protein levels. As for the collagen expression levels it was varied depending on the cell type. In DNA-protein interaction experiments, functional binding of SP1,C/EBP?,USF,AP1,E2F,NF?? and STAT transcription factors to ADAMTS3 promoter was determined by EMSA studies. ADAMTS-3 mRNA expression levels in Saos, MG-63, Panc, HUVEC, PC-3 ve DU145 cell lines were also determined by RT-PCR. As a result, human ADAMTS-3 promoter region was cloned and analysed functionally for the first time in this study. Determining the effects of SP1, USF ve C/EBP? on ADAMTS3 and collagen (type I, II, III) transcriptional regulations important contributions to the literature were provided.
ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a family of extracellular proteases found in both mammals and invertebrates. ADAMTSs have significant roles in processing amino terminus of the procollagen molecules, cleavage of the matrix proteoglycans aggrecan, versican and brevican; inhibition of angiogenesis; blood coagulation homoeostasis. ADAMTS-3 plays a key role in the processing of type II fibrillar collagen, specific component of the articular cartilage. It was also thought that there was a connection with some pathological conditions such as osteoarthritis, myocardial infarction, breast cancer and ADAMTS-3. Although functional features of the ADAMTS-3 gene was studied in detail it wasn’t known how its own transcriptional regulation. This project was aimed to elucidate transcriptional regulation of the ADAMTS-3 gene and the roles of SP1, USF and C/EBP? transcription factors in this regulation. Within this scope, to determine transcriptionally most active promoter region, four ADAMTS-3 promoter fragments [-1340/+40] [-899/+40], [-576/+40], [-131/+40] were cloned into luciferase vector with different size. It was determined with transient transfection experiments that all promoter fragments were active and the smallest construct was enough to exhibit the transcriptional activity in Saos-2 and MG-63 cells. It was also determined that 616 bp promoter fragment [- 576/+40] has the strongest basal activity. It was detected that the activities of the 1380bp [1340/+40] and 916 bp [-899/+40] promoter constructs was lower in Saos-2cells compared to MG-63.According to the bioinformatic analyses ADAMTS-3 promoter has a 40bp 5’UTR region and doesn’t include a TATA box. The co-transfection experiments determined that some transcription factors such as USF,SP1 and C/EBP? decreased the ADAMTS3 promoter activity having potential binding motifs in ADAMTS-3 promoter region. Another part of the study SP1, USF and C/EBP? were overexpressed in Saos-2 and MG-63 cells. Effect of these transcription factors on ADAMTS-3 and collagen (type I, II and III) mRNA and protein levels was determined by qRT-PCR and western blot method. It was determined that these transcription factors were decreased ADAMTS-3 mRNA and protein levels. As for the collagen expression levels it was varied depending on the cell type. In DNA-protein interaction experiments, functional binding of SP1,C/EBP?,USF,AP1,E2F,NF?? and STAT transcription factors to ADAMTS3 promoter was determined by EMSA studies. ADAMTS-3 mRNA expression levels in Saos, MG-63, Panc, HUVEC, PC-3 ve DU145 cell lines were also determined by RT-PCR. As a result, human ADAMTS-3 promoter region was cloned and analysed functionally for the first time in this study. Determining the effects of SP1, USF ve C/EBP? on ADAMTS3 and collagen (type I, II, III) transcriptional regulations important contributions to the literature were provided.
Açıklama
Anahtar Kelimeler
ADAMTS3, SP1, USF, C/EBP?, Cartilage, MG-63, Transcriptional Regulation, Kıkırdak, Transkripsiyonel Regülasyon
