ET-1 Aşırı Anlatımının Bir Sonucu Olan, Sinyal Çapraz Etkileşimi ‘Crosstalk’, Fare Meme Bezlerinde Proliferatif Lezyonların Oluşumunu Teşvik Eder

Abstract

Amaç: Farklı bir hücresel sinyalleşme modeli olarak, G proteini ile eşleşmiş reseptörler (GPCR) ve epidermal growth faktör reseptör (EGFR) arasındaki reseptör çapraz etkileşimi (crosstalk), çeşitli in vit-ro çalışmalarda gösterilmiştir. Ayrıca, son dönem in vitro çalışmalar, endotelin-1 (ET-1) sinyal yolağının kanser patofizyolojisi üzerinde-ki ilişkisine odaklanmıştır. Bu bağlamda, ET-1 reseptörleri ve EGFR arasındaki reseptör çapraz etkileşim analizine ve EGFR aktivasyo-nunun fonksiyonel sonuçlarından proliferatif hastalıklara olan ar-tan ilgi, bize bu fenomeni in vivo analiz etme fikrini oluşturmuştur.

Gereç ve Yöntem:ET-1 transgenik fareler ve kontrol fareleri arasın-da geç gebelik dönemi (n=7 ), erken laktasyon dönemi (n=6 ) orta dönem laktasyon (n=10) ve involusyon 14. gün (n=7) sırasında kar-şılaştırmalı bir çalışma gerçekleştirildi. Meme bezlerinden hema-toksilen-eozin (HE) ile boyanmış paralel kesitler mikroskobik olarak incelendi. EGFR'nin transaktivasyonunda görev alan anahtar sinyal proteinleri (ETAR, ETBR, ERK1/2, pEGFR), Western blot teknikleri kulla-nılarak analiz edildi. Bu aktivitelerde önemli bir rol oynadığı bilinen genler (amfiregulin, TGFa, EGF, HB-EGF, ADAM 17), gercek zamanlı po-limeraz zincir reaksiyonu (RT-PCR) teknikleri kullanılarak analiz edildi.

Bulgular:ET-1 transgenik fareler, laktasyon döneminin ortasında hiper-proliferatif lezyonlar (laktasyonel hiperplazi) geliştirdi. RT-PCRanalizlerimiz, transgenik farelerde amfiregulin ve ADAM 17 gen anlatımlarında belirgin bir artış olduğunu göstermektedir. Ayrıca transgenik meme bezlerinde daha yüksek EGFR ve ERKs aktivas-yonları tespit edilmiştir.

Sonuç: Bu çalışma, yüksek seviyede ET-1 gen anlatımının EGFR yi transaktive etme yoluyla meme bezlerinde proliferatif lezyonları tetiklediğini göstermektedir. Ayrıca, ET-1 aşırı anlatımı, transgen meme bezlerinde amfiregulin ve ADAM17 anlatımlarının belirgin bir şekilde yukarı regülasyonuna neden olmaktadır. Bu sonuçlar bize ET-1 gen ekspresyonunu ve reseptörlerinin EGFR aktivasyonu-nu sağlayarak proliferatif hastalıklarda önemli bir role sahip oldu-ğunu göstermektedir.

Keywords

• Endothelin 1
• EGFR transaktivasyonu
• laktasyonel hiperplazi
• Transgenik fareler
• Amfiregülin

Signal Crosstalk Promoted Proliferative Lesions in Mouse Mammary Glands As a Consequence of ET-1 Overexpression

Abstract

Objective: As a distinct cellular signaling model, a receptor cross-talk between G protein-coupled receptors (GPCR) and epidermal growth factor receptor (EGFR) has been demonstrated in various in vitro studies. In addition, recent in vitro studies had focused on the signaling pathways of endothelin-1 (ET-1) in the pathophysiology of cancer. Accordingly, a growing interest in the analysis of the re-ceptor crosstalk between ET-1 receptors and EGFR and functional consequences of EGFR activation of proliferative diseases evoked us to the analysis of this phenomenon in vivo.

Materials and Methods: We performed a comparative study between ET-1 transgenic mice and control mice during the late pregnancy (n=7), early lactation (n=6) the mid of lactation (n=10) and involution day 14 (n=7) periods. Hematoxylin and eosin (HE)-stained parallel sections from mammary glands were microscop-ically examined. The key signal proteins (ETAR, ETBR, ERK1/2, pEG-FR) in transactivation of EGFR were analyzed employing Western blot techniques. Genes (amphiregulin, TGFa, EGF, HB-EGF, ADAM 17) known to play an important role in these activities were analyzed using real-time PCR (RT-PCR) techniques.

Results:ET-1 transgenic mice exhibited hyperproliferative lesions (lactational hyperplasia) during the middle of the lactation period. Our RT-PCR analyses showed a prominent up regulation of amphi-regulin and ADAM17 in ET-1 transgenic mice. Moreover, we found higher EGFR and ERKs activations in the transgenic mammary glands.

Conclusion: This study highlights a causative effect of upregulated ET-1 gene expression on the induction of proliferative lesions via EGFR transactivation in mammary glands. Further, ET-1 overexpres-sion induced an upregulation of amphiregulin and ADAM17 expres-sions in the transgenic mammary glands. These results suggests that the enhanced ET-1 gene expression and its receptors might have a crucial role in proliferative diseases maintaining EGFR activation.

Keywords

• Endothelin 1
• EGFR Transactivation
• Lactational hyperplasia
• Transgenic mice
• Amphiregulin

Supporting Institution

Charite Universitätsmedizin Berlin

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