The role of thymosin β-4 in vitro and in vivo based on primary cilia formation = 섬모세포소기관 형성에 기초한 티모신 베타-4의 시험관 및 생체 내 역할 연구

다국어 초록 (Multilingual Abstract)

Non-motile primary cilia are antenna-like organelles protruding from the cortical cell surface in most eukaryotic cells. And primary cilia is enriched with diverse signaling receptors and transduce signals from outside to inside. Since their diverse function and distribution, dysfunction of them results in severe developmental defects and organ homeostasis failure. Therefore, studying of primary cilia formation is important to understand pathological mechanisms of ciliopathies such as cancer and obesity. Thymosin beta-4 (Tβ4) is an actin-sequestering protein and involved in several cellular functions, such as cell migration, cell proliferation and tissue repair. Furthermore, it is over-expressed in highly invasive and aggressive cancer cells. When yeast two-hybrid screening was implemented by using Tβ4 as a bait, nephrocystin-3 (NPHP3), one of ciliary protein, was physically interacted. I examined effect of Tβ4 in vitro and in vivo on primary cilia formation and ciliopathies, especially cancer and metabolic disorders.
For the first, I investigated whether Tβ4 regulates cilia formation by the association with NPHP3 in HeLa cervical cancer cells. I confirmed the direct binding of Tβ4 with NPHP3 by GST-pulldown assay and they were co-localized at the edge of cell peripheral surface. HeLa cells expressed noticeable number of primary cilia compared to that in many other cancer cells. Remarkably, inhibition of Tβ4 reduced primary cilia formation. Also, the ciliated cell number increased in Tβ4-GFP positive cells. The number of ciliated cells was reduced by the inhibition of NPHP3. In addition, the frequency of primary cilia in NPHP3-overexpressed cells was reduced by knockdown of Tβ4. In the point of molecular correlation, NPHP3 was decreased by the inhibition of Tβ4, but it was increased by Tβ4 overexpression. In contrast, Tβ4 expression was not affected by neither the interference nor overexpression of NPHP3. Taken together, the results demonstrate that primary cilium formation could be affected by Tβ4 through the regulation of NPHP3. It suggests that tumorigenesis could be associated with the perversion in Tβ4 and/or NPHP3 expression to maintain primary cilia formation normally.
Reactive oxygen species (ROS) are continuously generated during metabolic process and involved in cellular signaling and homeostasis. The production of ROS is also stimulated by cellular stress, such as growth factor deprivation. Serum starvation increased total ROS level in HeLa cells. And the frequency of ciliated cells were decreased by ROS scavenger, NAC, under the serum starve condition. We examined whether ROS are associated in Tβ4-mediated primary cilia formation. Inhibition of Tβ4 reduced ROS generation whereas Tβ4 expression was not affected by serum deprivation. Also, Hydrogen peroxide (H2O2), the one of ROS, increased in serum starvation. Treatment of H2O2 increased ciliated cell number that was attenuated by catalase, the H2O2 degrading enzyme. Moreover, the serum starvation-increased H2O2 was declined by knockdown of Tβ4. The H2O2–induced ciliogenesis was also reduced by knockdown of Tβ4. These results suggest that Tβ4 could affect ciliogenesis through the generation of ROS.
Based on the findings that primary cilia could be increased by Tβ4 in cancer cells, I investigated whether Tβ4-induced primary cilia leads malignant phenotypes in cancer cells. Vinblastine, an anti-tumor drug,-induced cell death decreased in serum staved HeLa cells. In contrast, Inhibition of primary cilia by ciliobrevin A (cilioA) increased vinblastine-induced cell death. Furthermore, Tβ4-overexpression reduced anti-tumor activity of vinblastine and the drug resistance was lessened by inhibition of primary cilia. These findings suggest Tβ4 could lead resistance to vinblastine via induction of primary cilia.
Type2 diabetes mellitus (T2DM) is caused by genetics and/or environmental factors such as eating high-fat foods. Tβ4 has an anti-fibrotic and anti-inflammatory effect on several T2DM complications. In addition, it regulated ciliogenesis. And primary cilia is involved in regulation of energy balance and metabolic process. I investigated the effects of Tβ4 on High fat diet (HFD)-induced obesity and T2DM with the association of primary cilia in vivo. Weight increase by HFD was significantly reduced in Tβ4-transgenic mouse group compared to C57BL6 control group although food consumption was even higher in Tβ4 group. Adipose tissue amounts and adipocytes size was considerably reduced in Tβ4 group. Lower level of plasma leptin was also showed in Tβ4 group. In addition, fatty liver was noticeably inhibited in Tβ4 group. Furthermore, Tβ4 group improved glucose control and insulin sensitivity with the prevention of hyperinsulinemia. Therefore, data demonstrated Tβ4 prevented HFD-induced lipogenesis and obesity and improved glucose and insulin intolerance. Next, to see the effect of primary cilia on metabolic disorder, I treated cilioA to dampen cilia number in mouse. Weight gain, fatty liver and glucose regulation was not affected by cilioA in HFD-WT group. The lipid accumulation in adipose tissue rather slightly decreased. Nevertheless, the fasting insulin level was worsen by cilioA. Since the number of primary cilia was well maintained in HFD-Tβ4-Tg mouse compared to that in HFD-WT mouse, when the maintained primary cilia was inhibited by cilioA in HFD-Tβ4-Tg mouse, glucose intolerance and insulin sensitivity was aggravated. Taken together, Tβ4 might have anti-diabetic properties correlated with maintaining primary cilia in obese state. It suggests that Tβ4 likely be a novel restrainer of morbid obesity and T2DM caused by bad eating habits.
Collectively, Tβ4 may regulate primary cilia formation through the control of NPHP3 or ROS production in HeLa cervical cancer cells. Cancer cells with primary cilia may acquire the malignant properties by showing resistance to vinblastine anticancer drugs. In addition, the expression of Tβ4 in the metabolic disorder showed the improvement effect of obesity and diabetes mellitus, which could be seen as the effect of in vivo energy and metabolism control through maintenance of primary cilia.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• 1. History of primary cilia 1
• 2. Structure of primary cilia 2
• 3. Process of ciliogenesis 3
• 4. Functional aspect of primary cilia 4
• 5. Primary cilia in cancer 5
• 6. Effect of the actin cytoskeleton on the primary cilia 6
• 7. Thymosin beta-4 in cancer 8
• 8. Nephrocystin-3 9
• 9. Reactive oxygen species 10
• 10. Thymosin beta-4 & Reactive oxygen species 12
• 11. Diabetes 13
• 12. Primary cilia and the metabolism 14
• 13. Role of actin dynamics in diabetes 16
• 14. Thymosin beta-4 in diabetes 17
• 15. Ac-SDKP with diabetic complications 18
• Ⅱ. Materials and methods 19
• 1. Reagents 19
• 2. Cell culture 19
• 3. Transfection of nucleic acids 20
• 4. Immunostaining 20
• 5. Reverse transcriptase polymerase chain reaction (RT-PCR) 21
• 6. Quantitative polymerase chain reaction (qPCR) 21
• 7. Western blot analysis 22
• 8. GST-pulldown assay 23
• 9. Gaussia luciferase assay for promoter activity 24
• 10. Measurement of reactive oxygen species (ROS) 24
• 11. Measurement of Hydrogen peroxide (H2O2) 24
• 12. MTT assay 25
• 13. Mice care and maintenance 26
• 14. Glucose tolerance test 26
• 15. Insulin tolerance test 26
• 16. Insulin ELISA assay 27
• 17. Leptin ELISA assay 27
• 18. Statistical analysis 28
• Ⅲ. Results 29
• Chapter.1. Tβ4 positively regulates primary cilia formation through the interaction with NPHP3
• in HeLa human cervical cancer cells
• 1. Identification of primary cilia-expressing cells 31
• 2. Primary cilia formation increased with time under serum starved condition in HeLa cells 34
• 3. NPHP3 is a component of primary cilia 37
• 4. NPHP3 is localized around the apical cell surface with the interaction of Tβ4 along F-actin in
• HeLa 40
• 5. Primary cilia is positively regulated by the alteration of Tβ4 expression in HeLa 44
• 6. NPHP3 normalizes the reduced primary cilia formation by knockdown of Tβ4 49
• 7. NPHP3 expression is controlled by alteration of Tβ4 52
• 8. Tβ4 expression is not changed by alteration of NPHP3 expression 55
• Chapter.2. Tβ4 mediates hydrogen peroxide-induced ciliogenesis in HeLa cervical cancer cells
• 9. Reactive oxygen species increased in serum starve condition 59
• 10. Increased primary cilia by serum starvation is reduced by ROS scavengers 61
• 11. Knock-down of Tβ4 down-regulated low-serum-induced ROS level 64
• 12. Hydrogen peroxide increased primary cilia formation 67
• 13. Knockdown of Tβ4 decreased the frequency of primary cilia via down-regulation of
• Hydrogen peroxide 72
• 14. Primary cilia formation was effectively inhibited by hedgehog signaling inhibitor, ciliobrevin
• A 74
• 15. Tβ4-mediated ciliogenesis attenuated vinblastine-induced tumor cell death 76
• Chapter.3. Tβ4-transgenic mice prevents high fat diet-induced Obesity and T2DM
• 16. Tβ4-transgenic mice prevented high fat diet-induced obesity 80
• 17. Lipogenesis in adipose tissue was suppressed in Tβ4-Tg mice 83
• 18. Lipogenesis in liver tissue was blocked in Tβ4-Tg mice 87
• 19. Tβ4-transgenic mice improved glucose tolerance and insulin tolerance 89
• 20. Tβ4-transgenic mice prevented hyperinsulinemia 92
• 21. Inhibition of primary cilia had no effect on weight gain and food consumption 94
• 22. Inhibition of primary cilia slightly reduced lipid accumulation in adipose tissue, but not in l
• iver tissue 96
• 23. Glucose regulation was not affected by the inhibition of primary cilia 100
• 24. Administration of ciliobrevin A resulted in increase of basal insulin level 102
• 25. Improved glucose regulation by thymosin beta-4 was aggravated by the inhibition of
• primary cilia 104
• Ⅳ. Discussion 106
• Ⅴ. Reference 112
• 국문초록 132