Effects of Overexpression of ET-1 in Mammary Gland Physiology

Year 2021, , 120 - 129, 25.08.2021

Nadir Gül , Franz Theuring

https://doi.org/10.26650/experimed.2021.864772

Abstract

Objective: Alongside its role as a potent vasoconstrictor in the cardio-vascular system, endothelin-1 (ET-1) is involved in numerous physio-logical processes. Its expression is thought to be an important function for both breastfeeding and newborns since it occurs especially during pregnancy and lactation. In this study, we projected the effects of ET-1 on mammary gland physiology employing ET-1 transgenic mice.

Material and Method: In this study, we compared the mammary glands of ET-1 transgenic mice with the control mice in different physiological states (adolescence, pregnant, lactation and involution). Parallel sections were prepared from mammary glands and microscopic examinations were performed with hematoxylin and eosin (HE)-staining. Western blot techniques were used in the analysis of signal proteins that have important functions in mam-mary gland development and the metabolism (STAT5, AKT, STAT3). Genes known to play a key function in metabolic activities (WAP, beta casein, GLUT-1, SRBF-1 Claudin 8, IL-6, LIF) were analyzed em-ploying real-time PCR techniques.

Results: It was determined that ET-1 transgenic mice developed pathological conditions such as secretory damage, lactational failure and early involution, respectively. Our Western blot studies showed that STAT5 activation was not affected by this process in the trans-genic group, but a decrease in AKT protein level was detected. We detected STAT3 activation seen in the involution process in trans-genic mice only on the 3rd day of lactation. In addition, we found that WAP was suppressed due to high ET-1 expression and that LIF protein expression increased significantly, by real-time PCR analysis.

Conclusion: ET-1 expression is thought to be an important func-tion for both lactation and newborns, especially during pregnancy and lactation. In this study, it was found that when ET-1 expression increases, it causes lactation failure in the mammary gland and directly affects the synthesis of genes known to be important in mammary physiology. Hereby, it was determined that the regulation of ET-1 plays a critical role in the development and secretory function of the mammary gland.

Keywords

Endothelin, Lactational incompetence, WAP, LIF, Precoicious involution, Transgenic mice

Supporting Institution

Charite Universitätsmedizin Berlin

Effects of Overexpression of ET-1 in Mammary Gland Physiology

Abstract

Amaç: Endotelin (ET-1), kardiyovasküler sistemde güçlü bir vazo-konstrüktör rolü olmasının yanı sıra birçok fizyolojik olayda kendini göstermektedir. ET-1 ekspresyonunun özellikle gebelik ve emzirme döneminde ortaya çıkması nedeniyle hem emzirme hem de yeni doğan için önemli bir işlevi olduğu düşünülmektedir. Bu çalışmada ET-1 transgenik fare modeli kullanarak meme bezi üstünde ET-1'in etkilerinin incelenmesi amaçlanmıştır.

Gereç ve Yöntem: Meme dokusunun farklı fizyolojik durumlarında (ergenlik, gebelik, laktasyon ve involusyon) ET-1 in etkisini incelemek üzere karşılaştırmalı bir çalışma gerçekleştirildi. Meme dokularından paralel kesitler hazırlanıp hematoksilen-eozin (HE) boyama ile mik-roskobik inceleme yapıldı. Meme bezi gelişimi ve metabolizmasın-da önemli görevleri olan sinyal proteinlerinin (STAT5, AKT, STAT3) analizlerinde Western blot tekniği kullanıldı. Metabolik faaliyetlerde anahtar rolü olduğu bilinen genler (WAP, beta kazein, GLUT-1, SRBF-1 Claudin 8, IL-6, LIF) gercek zamanlı polimeraz zincir reaksiyonu (RT-PCR) tekniği ile analiz edildi.

Bulgular: ET-1 transgenik farelerde sırasıyla sekresyonel hasar, laktasyonel yetmezlik ve erken involusyon gibi patolojik bulgular tespit edildi. Transgenik grupta STAT5’in aktivasyonunun bu durumdan etkilenmediği ancak AKT protein seviyesinde düşüş oldu-ğu saptandı. İnvolusyonda görülen STAT3 aktivasyonu transgenik farelerde laktasyonun 3. gününde tespit edildi. Ayrıca, RT-PCR analizleri ile transgenik farelerde yüksek ET-1 ifadesine bağlı ola-rak WAP'ın baskılandığını LIF protein ifadesinin belirgin bir şekilde arttığı tespit edildi.

Sonuç: Sadece gebelik ve laktasyon döneminde ifade edilen ET-1'in laktasyon ve yeni doğan için önemli bir işlevi olduğu düşünül-mektedir. Yüksek ET-1 ifadesi meme fizyolojisinde önemli olduğu bilinen genlerin sentezini doğrudan etkileyerek laktasyonel yeter-sizliğe sebep olmuştur. Bu çalışmada ET-1 regülasyonunun meme bezi gelişiminde ve süt sekresyonu fonksiyonunda kritik bir rol oynadığı gosterilmiştir.

Keywords

Endothelin, lactational incompetence, WAP, LIF, preco-cious involution, transgenic mice

References

• 1. Muldoon TG. Prolactin mediation of estrogen-induced changes in mammary tissue estrogen and progesterone receptors. Endocri-nology 1987; 121: 141-9. [CrossRef] google scholar
• 2. Robinson GW, Johnson PF, Hennighausen L, Sterneck E. The C/ EBPbeta transcription factor regulates epithelial cell proliferation and differentiation in the mammary gland. Genes Dev 1998; 12: 1907-16. [CrossRef] google scholar
• 3. Naylor MJ, Oakes SR, Gardiner-Garden M, Harris J, Blazek K, Ho TWC, et al Transcriptional changes underlying the secretory ac-tivation phase of mammary gland development. Mol Endocrinol 2005; 19: 1868-83. [CrossRef] google scholar
• 4. Yang ZZ, Tschopp O, Baudry A, Dümmler B, Hynx D, Hemmings BA. "Physiological functions of protein kinase B/Akt". Biochem Soc Trans 2004; 32(Pt 2): 350-4. [CrossRef] google scholar
• 5. Green KA & Streuli CH. Apoptosis regulation in the mammary gland. Cell Mol Life Sci 2004; 61(15): 1867-83. [CrossRef] google scholar
• 6. Watson CJ. Involution: apoptosis and tissue remodelling that con-vert the mammary gland from milk factory to a quiescent organ. Breast Cancer Res 2006; 8(2): 203. Epub 2006 Apr 10. [CrossRef] google scholar
• 7. Kritikou EA, Sharkey A, Abell K, Came PJ, Anderson E, Clarkson RW, et al. A dual, non-redundant, role for LIF as a regulator of develop-ment and STAT3-mediated cell death in mammary gland. Deve-lopment 2003; 130(15): 3459-68. [CrossRef] google scholar
• 8. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y., et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332(6163): 411-5. [CrossRef] google scholar
• 9. Itoh Y, Kimura C, Onda H, Fujino M. Canine endothelin-2: cDNA sequence for the mature peptide. Nucleic Acids Res 1989; 11; 17(13): 5389. [CrossRef] google scholar
• 10. Lam HC, Takahashi K, Ghatei MA, Bloom SR. Presence of immuno-reactive endothelin in human milk. FEBS Lett 1990; 261(1): 184-6. [CrossRef] google scholar
• 11. Baley PA, Resink TJ, Eppenberger U, Hahn AW. Endothelin mes-senger RNA and receptors are differentially expressed in cultured human breast epithelial and stromal cells. J Clin Invest 1990; 85(4): 1320-3. [CrossRef] google scholar
• 12. Kozakai T, Sakate M, Masuo Y, Uchide T, Saida K. Increased gene expression of endothelin-1 and vasoactive intestinal contractor/ endothelin-2 in the mammary gland of lactating mice. Biochem Biophys Res Commun 2002; 297(5): 1339-43. [CrossRef] google scholar
• 13. Neville MC. Introduction: alpha-lactalbumin, a multifunctional protein that specifies lactose synthesis in the Golgi. J Mammary Gland Biol Neoplasia 2009; 14(3): 211-2. [CrossRef] google scholar
• 14. Rudolph MC, McManaman JL, Phang T, Russell T, Kominsky DJ, Serkova NJ, et al. Metabolic regulation in the lactating mammary gland: a lipid synthesizing machine. Physiol Genomics 2007; 28(3): 323-36. [CrossRef] google scholar
• 15. Blackman B, Russell T, Nordeen SK, Medina D, Neville MC. Clau-din 7 expression and localization in the normal murine mammary gland and murine mammary tumors. Breast Cancer Res 2005; 7(2): R248-55. [CrossRef] google scholar
• 16. Seagroves TN, Hadsell D, McManaman J, Palmer C, Liao D, McNulty W, et al. HIF1 alpha is a critical regulator of secretory differentiati-on and activation, but not vascular expansion, in the mouse mam-mary gland. Development 2003; 130(8): 1713-24. [CrossRef] google scholar
• 17. Camps M, Vilaro S, Testar X, Paladn M Zorzano A. High and pola-rized expression of GLUT1 glucose transporters in epithelial cells from mammary gland: acute down-regulation of GLUT1 carriers by weaning. Endocrinology 1994; 134(2): 924-34. [CrossRef] google scholar
• 18. Maroulakou IG, Oemler W, Naber SP, Klebba I, Kuperwasser C, Tsichlis PN. Distinct roles of the three Akt isoforms in lactogenic differentiation and involution. J Cell Physiol 2008; 217(2): 468-77. [CrossRef] google scholar
• 19. Schwertfeger KL, Richert MM, Anderson SM. Mammary gland in-volution is delayed by activated Akt in transgenic mice. Mol Endo 2001; 15: 867-81. [CrossRef] google scholar
• 20. Clarkson RW, Wayland MT, Lee J, Freeman T, Watson CJ. Gene exp-ression profiling of mammary gland development reveals putati-ve roles for death receptors and immune mediators in postlactati-onal regression. Breast Cancer Res 2004; 6(2): 92-109. [CrossRef] google scholar
• 21. Akira S, Nishio Y, Inoue M, Wang XJ, Wei S, Matsusaka T, et al. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91related transcription factor involved in the gp130-mediated Signaling pathway. Cell 1994; 77(1): 63-71. [CrossRef] google scholar
• 22. Anderson SM, Rudolph MC, McManaman JL, Neville MC. Secretory activation in the mammary gland: its not just about milk protein synthesis. Breast cancer research 2007; 9: 204. [CrossRef] google scholar
• 23. Triplett AA, Sakamoto K, Matulka LA, Shen L, Smith GH, Wagner KU. Expression of the whey acidic protein (WAP) is necessary for adequate nourishment of the offspring but not functional diffe-rentiation of mammary epithelial cells. Genesis 2005; 43(1): 1-11. [CrossRef] google scholar
• 24. Iavnilovitch E, Groner B, Barash I. Overexpression and forced ac-tivation of stat5 in mammary gland of transgenic mice promotes cellular proliferation, enhances differentiation, and delays postla-ctational apoptosis. Mol Cancer Res 2002; 1(1): 32-47. google scholar
• 25. Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, et al. Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 2002; 156(6): 1099-111. [CrossRef] google scholar
• 26. Creamer BA, Sakamoto K, Schmidt JW, Triplett AA, Moriggl R, Wagner KU. Stat5 promotes survival of mammary epithelial cells through transcriptional activation of a distinct promoter in Akt1. Mol Cell Biol 2010; 30(12): 2957-70. [CrossRef] google scholar
• 27. Lund LR, Bjorn SF, Sternlicht MD, Nielsen BS, Solberg H, Usher PA, et al. Lactational competence and involution of the mouse mam-mary gland require plasminogen. Development 2000; 127: 448192. google scholar
• 28. Zhao L, Hart S, Cheng J, Melenhorst JJ, Bierie B, Ernst M, et al. Mammary gland remodeling depends on gp130 signaling through Stat3 and MAPK. J Biol Chem 2004; 279(42): 44093-100. [CrossRef] google scholar
• 29. Pfaffl MW, Horghan GW, Dempfle L. Relative expression software tool (REST) for group- wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Resear-ch 2002; 30(9): e36. [CrossRef] google scholar