청각세포 사멸화 과정에서의 Connexin 43과 간극결합 세포간 신호전달의 역할
The Role of Connexin 43 and Gap Junctional Intercellular Communication in the Apoptotic Process of Auditory Cells
- 주제(키워드) Gap junction , Hemichannel , Connexin 43 , Cisplatin , Ototoxicity , HEI-OC1
- 발행기관 아주대학교
- 지도교수 정연훈
- 발행년도 2014
- 학위수여년월 2014. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000017809
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Cis-diamminedichloroplatinum (cisplatin) is one of effective chemotherapeutic drugs for cancer therapy. Nevertheless, most patients treated with cisplatin are exposed to high risk of ototoxicity such as hearing loss or dizziness. It has been reported that ototoxicity occurs in about 75-100 % of patients treated with cisplatin and shows irreversible, cumulative and bilateral characteristics. However, cisplatin-induced ototoxic mechanisms have not been clearly identified yet even through many studies. Histochemical studies on the distribution of cisplatin have shown that spiral ganglions, lateral wall cells, and organ of Cortis are major targets, where many connexins (Cxs) are expressed. Cxs are a family of membrane proteins and these are expressed abundantly in mammalian inner ears. Cxs oligomerize into a hexameric connexon, also known as a hemichannel, and transport to plasma membranes along microtubules finally forming a gap junction between cells. In the inner ear, gap junctions play a critical role in the generation the endocochlear potential, which is required for normal hearing. The aim of this study was to determine the role of gap junctions and their constituents, hemichannels and Cxs in the cisplatin-induced ototoxic mechanism. As a result, I found that inhibition of gap junctional activity with either Cx43 siRNA transfection or a pharmacological drug, 18α-glycyrrhetinic acid in HEI-OC1 auditory cells caused a marked increase in cell viability with a decrease of apoptosis based on cleaved Poly (ADP-ribose) polymerase (PARP) and cleaved caspase 3 levels. Furthermore, I demonstrated that extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) pathways had a significant role in preventing cell death by inhibition of gap junctions. Next, I found that cisplatin did not affect the opening and closure of Cx43-hemichannels in HEI-OC1 cells and Cx43-transfected Cx-deficient HeLa cells. Accumulation of Cx43 was observed around the nuclei of cisplatin-treated cells, whereas Cx43 was scattered in the punctuated form in the cytoplasms as well as on the membranes in normal cells, suggesting that cisplatin may interrupt the trafficking of Cx43s to cell membranes in HEI-OC1 cells. In addition, I investigated whether Cx43 protein itself affected cell viability in response to cisplatin. I found that the knockdown of Cx43 in HEI-OC1 cells resulted in a significant increase in cell viability following cisplatin treatment independent of gap junctions. This response did not change following inhibition of Cx43 transport to the plasma membrane by Brefeldin A. HeLa cells expressing either full lengthen Cx43 and its N-terminal domain or its carboxyl tail, showed higher sensitivity to cisplatin than mock-treated cells. The mitochondrial Cx43 protein level was significantly increased in HEI-OC1 cells after cisplatin treatment in a time-dependent manner, which coincided with the release of mitochondrial cytochrome C into the cytoplasm. These findings suggest that Cx43 plays important roles on cisplatin-induced ototoxic damage through gap junction-dependent or independent processes in auditory cells. The control of the Cx43-mediated signaling may be a potential target for therapeutic strategies for drug-induced ototoxicity.
more목차
ABTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES v
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 8
1. Cell culture 8
2. Plasmid constructs and transfection 8
3. Fluorescence Recovery After Photobleaching (FRAP) 9
4. Scrape Load Dye Transfer (SLDT) Technique 10
5. Lucifer yellow dye uptake 11
6. Live/Dead assay 11
7. MTS assay 11
8. Western blot 11
9. Immunocytochemistry 12
10. Immunohistochemistry 13
11. Animals 13
12. Animal study 14
13. Scanning Electron Microscopy (SEM) 14
14. Auditory brainstem response (ABR) test 16
15. Statistical analysis 16
Ⅲ. RESULTS 17
1. Auditory cells expressed functional gap junctions 17
2. Gap junction activity was interrupted by cisplatin 22
3. Inhibition of gap junctions reduced cisplatin-induced apoptosis 27
4. Activation of ERK and Akt contributed to inhibition of gap junction-mediated
beneficial effects in response to cisplatin 33
5. Design of gap junctional and non-junctional condition in auditory cell culture 38
6. Effects of gap junction inhibition on cisplatin toxicity in the junctional and
non-junctional condition 41
7. Cx43 hemichannels was not changed by cisplatin treatment 43
8. HeLa cells transfected with cytoplasmic CT domain of Cx43 become sensitive
to the cisplatin 48
9. Cisplatin-induced toxicity decreases following Cx43 knockdown by inhibition
of Cx trafficking to the plasma membrane 51
10. Mitochondrial Cx43 has an impact on the cisplatin-induced damage in
HEI-OC1 cells 55
11. Phosphorylation status of Cx43 in primary organ of Corti cells treated with
gap junction blocker 57
12. Effects of CBX and 18α-GA on the cisplatin-induced hearing loss 59
Ⅳ. DISCUSSION 62
Ⅴ. CONCLUSION 70
REFERENCES 71
국문요약 82
