Soluble klotho가 KLF4 pathway 활성을 통한 타액선 기능 조절 연구
- Issued Date
- 2020
- Abstract
- 타액선 기능 장애는 일반적으로 구강내 타액 부족으로 인한 구강 건조, 감연 및 충치를 유발합니다. 이 연구는 항 노화 유전자인 Klotho가 결핍된 마우스 노화 모델을 사용하여 노화에 따른 타액선의 유전적 및 기능적 변화를 연구하기 위해 수행하였다. 이 연구에서 klotho (-/-) 쥐의 타액선에서 KLF4의 감소를 확인하였고 soluble klotho과 발현은 KLF4 관련 신호전달 기전에 연관된 mTOR, AMPK, SOD1/2 활성화 및 발현을 유도했다. siRNA로 Klotho 발현억제는 KLF4의 발현은 억제하였다. klotho (-/-) 마우스 타액선에서 분과한 primary cultured salivary gland cells (PSGCs)에서 soluble klotho 과발현에 따를 KLF4의 증가는 타액선 기능유도와 관련된 (α-amylase, ZO-1 및 Aqua5)의 발현을 유도하였다. 3D 배양 시스템에서, 세포구체 응집체가 (cell sphere)가 soluble klotho와 KLF4 과발현 세포에서 관찰 되었으며, 또한 타액선기능 마커 단백질의 발현도 증가 하였다. |The dysfunction of salivary glands commonly induces dry mouth, infections, and dental caries caused by a lack of saliva. This study was performed to determine the genetic and functional changes in salivary glands using a klotho (-/-) mouse model. Here, we confirmed the attenuation of KLF4 expression in the salivary glands of klotho (-/-) mice. Soluble klotho overexpression induced KLF4 transcription and KLF4-mediated signaling pathways, including mTOR, AMPK, and SOD1/2. Silencing klotho via siRNA significantly down-regulated KLF4 expression. Additionally, we monitored the function of salivary glands and soluble klotho and/or KLF4 responses and demonstrated that soluble klotho increased the expression of KLF4 and markers of salivary gland function (α-amylase, ZO-1, and Aqua5) in primary cultured salivary gland cells from wild type and klotho (-/-) mice. In a 3D culture system, cell sphere aggregates were observed in soluble klotho- or KLF4-expressing cells and exhibited higher expression levels of salivary gland function‐related proteins than those in nontransfected cells. These results suggest that activation of the klotho-mediated KLF4 signaling pathway contributes to potentiating the function of salivary glands.
- Alternative Title
- Soluble klotho regulates the function of salivary glands by activating KLF4 pathways
- Alternative Author(s)
- NGUYEN CHI TAI
- Affiliation
- 치의생명공학과
- Department
- 일반대학원 치의생명공학과
- Advisor
- 안상건
- Awarded Date
- 2020-02
- Table Of Contents
- TABLE OF CONTENTS
TABLE OF CONTENTS..................................i
LIST OF FIGURES..................................iii
ABBREVIATIONS.....................................iv
ABSTRACT..........................................vi
I. INTRODUCTION....................................1
II. MATERIAL AND METHODS...........................4
2.1. Cell culture and reagents.......................4
2.2. Preparation of primary submandibular salivary gland cells from mice and in vitro culture...........4
2.3. Real-time quantitative PCR......................5
2.4. siRNA interference..............................6
2.5. Western blot analysis...........................6
2.6. Luciferase activity assay.......................7
2.7. Three-demensional matrigel spheroid culture.....8
2.8. Immunofluorescence analysis.....................8
2.9. Statistical analysis............................9
III. RESULTS......................................10
3.1. KLF4 expression in klotho (-/-) mouse organs...10
3.2. Klotho induces KLF4 expression.................12
3.3. Soluble klotho induces the KLF4-related pathway.............................................17
3.4. Soluble klotho and KLF4 regulate the p53/p21 and SOD1/2 pathways.....................................20
3.5. Soluble klotho induces the function of primary salivary gland cells (PSGCs)........................23
3.6. Soluble klotho-induced KLF4 induces differentiation into acinar-like cells..............27
IV. DISCUSSION AND CONCLUSION.....................33
V. REFERENCES ....................................38
VI.국문초록.......................................43
LIST OF FIGURES
Figure 1. Kruppel-like factor (KLF) 4 is downregulated in various tissues harvested from klotho (-/-) mice............................................11
Figure 2. The expression of KLF4 in wild-type and klotho (-/-) MEFs. qRT-PCR analysis of KLF4 and FOXO1 in wild-type and klotho (-/-) MEFs.......................................................................14
Figure3. Effects of soluble klotho on the expression of proteins belonging to the KLF4 pathway in wild-type and klotho (-/-) MEFs..........................16
Figure 4. Effects of si-klotho and siKLF4 on KLF4-related protein expression..................................................................19
Figure 5. The expression of salivary gland functional proteins in primary salivary gland cells (PSGCs)...............................................22
Figure 6. Functional restoration of PSGCs by soluble klotho and KLF4 expression..................................................................26
Figure 7. Effects of soluble klotho protein on the expression of proteins belonging to the KLF4 pathway in wild-type and klotho (-/-) MEFs.......................................................................28
Figure 8. The expression of KLF4 in HEK293 cells. (A) qRT-PCR analysis of KLF4 in soluble klotho-expressing HEK293 cells..................................30
Figure 9. The expression of salivary gland functional proteins induced by KLF4 in primary salivary gland cells (PSGCs)..................................31
Figure 10. The expression of salivary gland functional proteins in acinar cells (ACs)..........................................................32
ABBREVIATIONS
AMPK 5' adenosine monophosphate-activated protein kinase
ANOVA Analysis of variance
Aqua5 Aquaporin 5
Atp1a2 alpha2-Na+/K+-ATPase
Atp2a1 Ca2+-ATPase
EDTA Ethylenediaminetetraacetic acid
EGF Epidermal growth factor
EGFBP2 Epidermal growth factor binding type B
FBS Fetal bovine serum
FGF23 Fibroblast growth factor 23
FOXO1 Fork head box O1
HEK293 Human embryonic kidney 293 cells
IGF Insulin-like factor
INFγ Interferon gamma
KLF4 Kruppel-like factor 4
MEFs Mouse embryonic fibroblasts
mTOR Mammalian target of rapamycin
NGF Nerve growth factor
NFκB Nuclear factor kappa B
PARP Poly (ADP-ribose) polymerase
PBS Phosphate-buffered saline
PPARγ Proliferator-activated receptor-γ
PSGCs Primary salivary gland cells
P70S6K P70-S6 kinase 1
RT Room temperature
SD Standard deviation
sKL Soluble Klotho
SOD Superoxide dismutase
TGF Tumor growth factor
TNF Tumor necrosis factor
ZO-1 Tight junction protein 1
- Degree
- Master
- Publisher
- 치의생명공학과
- Citation
- 응웬 치타이. (2020). Soluble klotho가 KLF4 pathway 활성을 통한 타액선 기능 조절 연구.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/14067
http://chosun.dcollection.net/common/orgView/200000278386
- Appears in Collections:
- General Graduate School > 3. Theses(Master)
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