1
FOXL2
β
2 ... 4 1 ... 5 2 ... 13 1. ... 13 2. ... 13 3. ... 14 4. ... 15 5. ... 15 6. GST-pull down ... 16 7. ... 18 8. ... 18
9. short-interference RNA FOXL2 ... 20
10. RNA PCR ... 21 11. ... 22 3 ... 23 1. FOXL2 ... 23 2. FOXL2 ERβ ... 23 3. FOXL2 ERβ ... 24 4. ERβ FOXL2 ... 26 5. ERα FOXL2 ... 26 6. ERβ FOXL2
3 ... 27 7. FOXL2 aromatase ... 27 4 ... 29 5 ... 33 ... 34 ... 35 ... 36 ... 47
4 40 FOXL2 FOXL2 FOXL2 ER β FOXL2 FOXL2 ERβ FOXL2 ERβ
5 1 22 26.1 22.9 24 30.8 29.2 4.7 6.3 1 Figure 1 World
Health Organization: WHO 1
1 10 5 1 2007 3 1.3% 26.4% 9% 700 30 4,5
6 6 Figure 2 Figure 3 6 primary ovarian insufficiency: POI
40 7,8 POI 3-6 FSH 40 mIU/mL POI 9 20 0.01% 30 0.1% 40 1% 10 POI 9 POI
7 10 15 10 POI 4 30 POI X FMR-1 Xq27.3 1 exon CGG repeat 60 200 POI 11 FMR-2 FMR-1 1Xq28 microdeletion 12 BMP15 Xp11.2 homodimer 13 FSH receptor 2p21-p16 7 exon 566 189 Ala Val 14 LHβ LHβ
mutation POI 15 NOGGIN NOGGIN
BMP4, 7 16 NOBOX 7q35
Homeobox 17 GALT 9q13
60 70 POI 18
POI
FOXL2 forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 POI
8
BPES type 1 type 2 20 BPES type 1
BPES type1
BPES type 2 19 Figure 4 BPES
3q23 FOXL2 2001 19 BPES type 1 FOXL2 Q129X, W204X type 2 FOXL2 A224_A234dup BPES type1 1 2 Figure 5 21 FOXL2 forkhead DNA forkhead 14 379
1 22 Figure 6 FOXL2 forkhead
44 110
DNA DNA forkhead 3 α-helix
9 23,24 FoxA DNA DNA 23 forkhead forkhead FoxC2, -D2, -D3, -G1 23 MAP 23 FOXL2 19 FOXL2
FOXL2 12.5 dpc day post coitum 25
FOXL2 26 FOXL2
FOXL2
FOXL2
SOX9
10
28 FOXL2 gonadotropin
releasing hormone receptor GnRHR steroidogenic acute regulatory protein StAR
aromatase 29-31 FOXL2 Q219X POI FOXL2
FOXL2 StAR aromatase
P450scc FOXL2 Q219X StAR aromatase
P450scc 26,31 FOXL2 aromatase
aromatase
29,32 FOXL2 aromatase
FOXL2 aromatase 29,33 FOXL2
aromatase FOXL2
aromatase 30,34 FOXL2 aromatase
FOXL2
FOXL2
95%
FOXL2 C134W 35 FOXL2
11 FOXL2 Estrogen receptor: ER 43 ER α β 2 ERα ERβ 37 ERα 6q25 ERβ 14q23 ER 17β-estradiol: E2 38 ERα 595 ERβ 530 A/B C D E F 5
Figure 7 DNA C ERα ERβ 95%
E 55%
A/B activation function-1: AF-1
AF-2 37
ER ER 2 DNA
12
ERE DNA ER
Figure 8 AP-1 activating protein-1 SP-1
stimulating protein-1 ERE
MAP ER 39 ER ERα ERβ 40 ER 41 ERα αERKO luteinizing hormone; LH ERβ βERKO 42,43 βERKO
13
ERα AP-1 ERα
44 Cyclooxygenase-2 COX-2 AP-1
ERα ERα COX-2 FOXL2
44
ERβ FOXL2
FOXL2
FOXL2
ERα ERβ FOXL2
ERβ FOXL2 ERβ
FOXL2 ERβ ERβ
ER ERβ aromatase
FOXL2 ERβ
14 2
1.
17β-estradiol (E2) Sigma Aldrich (St. Louis, MO, USA)
2.
KGN
293T BRC ( , )
COV434
Health Protection Agency Culture Collection (Salisbury, UK) 293T
COV434 10% fetal bovine serum FBS Dulbecco's
modified Eagle medium DMEM
45
3594 (2)
KGN 10% FBS
DMEM/F-12 FBS DMEM Sigma Aldrich E2
E2 10-9 M
E2 pH
phenol red 46 phenol
red-free
15 3.
KGN COV434 FOXL2 ERβ
KGN DMEM/F12 10% FBS 100 mm dish
TNE 10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, protease inhibitor cocktail
, , 10 20
10 anti-ERβ Santa
Cruz, Dallas, TX, USA 2 µg 4 1
Protein G Sepharose 4 Fast Flow 25 µg GE Healthcare UK Ltd, Little Chalfont,
UK 4 16
COV434 pcMV-myc-FOXL2 (
100 mm dish 1.5 106 cells / dish plating pCMV-myc-FOXL2 2 µg Effectene (Qiagen, Duesseldorf, Germany)
) 2 anti-c-Myc
agarose affinity gel Sigma Aldrich 25 µg
10% SDS-PAGE Immobilon-P Millipore, Billerica, MA, USA
10% TBS-T 10 mM
16
anti-FOXL2 MBL, , anti-ERβ Santa Cruz TBS-T
1 anti-rabbit IgG 1:3000 Cell Signaling Technology, Danvers,
MA, USA TBS-T 1 TBS-T 3
horseraddish peroxidase ECL advance
GE Healthcare UK Ltd ImageQuant LAS 4000
GE Healthcare UK Ltd 3
4.
pCMV-myc-FOXL2 Bae Department of Life
Science, College of Natural Science, Chung-Ang University,
pcDNA Flag ERβ pcDNA3 Invitrogen, Camarillo, CA, USA
pcDNA-Flag ERβ
48 GST ERβ AF-1/2 ERβ AF-1 1 149 AF-2
248 530 pGEX 4T-1 vector GE healthcare
49
ERE
GGTCAGAGTGACC 3 thymidine kinase
pGL3 basic
17
AdMLP Luc 48
5.
324
32 42
15 gonadotropin releasing hormone
(GnRH) 10% 4 µm organosilane 10 mM pH 6.0 microwave 0.3%
0.1% NP-40 5% Bovine serum Albumin PBS FOXL2
4oC
preimmune IgG PBS EnVision Kit DAKO, Glostrup,
Denmark horseradish peroxidase Diaminobenzidine
18 6. GST-pull down
GST-pull down ERβ FOXL2
GST GST ERβ AF-1
GST ERβ AF-2 BL 21 Codon Plus Stratagene, Santa Clara, CA,
USA 37 LB OD600 0.4~0.5 Isopropyl-β-D-thiogalactopyranoside 0.1 mM GST IPTG 30 2 PBS +10% Glycerol 10 mM EDTA Triton X-100 1% 4 Glutathione-Sepharose 4B GE Healthcare UK Ltd GST
4 SDS-PAGE Coomassie Brilliant
Blue
19
E2 10-9M vehicle NET-N+ 20 mM Tris-HCl, pH
7.5, 200 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, protease inhibitor cocktail
pCMV-myc-FOXL2 ( 100 mm
dish 1 106 cells / dish plating pCMV-myc-FOXL2 2 µg
Effectene (Qiagen) 2 ) 293T 4 60 NET-N+ buffer 6 10% SDS-PAGE Immobilon-P anti-FOXL2 3 7. DMEM/F12 10% FBS 6 well (35 mm ) TNE 10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, protease inhibitor cocktail
10 20 10
10%
SDS-PAGE Immobilon-P Millipore
20
mM NaCl, 0.05% Tween 20 1 anti-FOXL2 MBL
anti-β-Actin Santa Cruz TBS-T 1 anti-rabbit IgG
1:3000 anti-mouse IgG : 1:3000 Cell Signaling TBS-T 1
TBS-T 3
horseraddish peroxidase ECL advance GE Healthcare UK Ltd ImageQuant LAS 4000 GE Healthcare UK Ltd
3
8.
FOXL2 ERα ERβ
293T phenol red-free
DMEM 5% FBS
12 well dish 1 105 cells / well plating Effectene Qiagen
Lane 1~4: E2 1: pcDNA3 2: pcDNA3 Flag ERβ 3: pCMV-myc-FOXL2 4:
pcDNA3 Flag ERβ + pCMV-myc-FOXL2 Lane 5~8: 10-9 M E2
5: pcDNA3 6: pcDNA3 Flag ERβ 7: pCMV-myc-FOXL2 8: pcDNA3 Flag ERβ +pCMV-myc-FOXL2
21
Lane 1: E2 pcDNA3 Flag ERα Lane 2~4: 10-9 M E2 2:
pcDNA 3: pcDNA3 Flag ERβ 4: pcDNA3 Flag ERβ + pCMV-myc-FOXL2
Lane 1~3; E2 1: pM + pcDNA3 2: pM ERβ AF-2 + pcDNA3 3: pM ERβ AF-2 + pCMV--myc-FOXL2 Lane 4~6: 10-9 M E2 4: pM + pcDNA3
5: pM ERβ AF-2 + pcDNA3 6: pM ERβ AF-2 + pCMV-myc-FOXL2
well ERE tk Luc 250 ng
well 17m8 AdMLP Luc 250 ng
well internal control phRL CMV Luc 1 ng
pcDNA3 pcDNA3 Flag ERβ pCMV-myc- FOXL2 pM ERβ AF-1 pM ERβ AF-2 0.1 µg
24 Dual Luciferase Reporter System Promega firefly luciferase
Renilla luciferase 3
3
9. short-interference RNA siRNA FOXL2
6 FOXL2 siRNA
22
254-272 84-91 283-301 94-101 773-791
257-264 808-826 269-276 FOXL2-RNAi Thermo Scientific ,
Lifetechnologies , , Sense
5’-UCGCGAAGUUCCCGUUCUA-3’ 5’-AAGAAGGGCUGGCAAAAUA-3’ 5’-ACACACGCGUGCAGAGCAU-3’ 5’-GUAGUGAACUCGUACAAUG-3’
siRNA All Stars Negative Control siRNA 1027281: Qiagen
siRNA Lipofectamine RNAimax Lifetechnologies
, , siRNA 10 nM
FOXL2 PCR
10. RNA PCR
FOXL2 ERβ ERβ
aromatase mRNA
10% FBS DMEM/F12 negative control
23
5% FBS DMEM/F12
48 Isogen ,
, RNA RNA ReverTra Ace TOYOBO,
, cDNA PCR LightCycler
2.0 DX400 Roche Applied Science, Penzberg, Germany PCR 95oC 10
55 oC 10 72 oC 10 1 40
PCR PCR
glyceraldehyde-3-phosphate dehydrogenase
GAPDH mRNA mRNA
aromatase Forward 5'- GAGAATTCATGCGAGTCTGGA -3' Reverse 5'- CATTATGTGGAACATACTTGAGGACT -3' StAR Forward 5'- AAACTTACGTGGCTACTCAGCATC -3' Reverse 5'- GACCTGGTTGATGATGCTCTTG -3' GAPDH Forward 5'-GAAGGTGAAGGTCGGAGTC-3'
24
Reverse 5'-GAAGATGGTGATGGGATTTC-3'
LightCycler 2.0 DX400 LightCycler FastStart DNA Master SXBR Green I Roche Applied Science
11.
StatView Version 5
for Windows (SAS, Cary, NC, USA) One-factorial
25 3 1. FOXL2 FOXL2 FOXL2 FOXL2 FOXL2 FOXL2 Figure 9 2. FOXL2 ERβ KGN FOXL2 C134W KGN anti-ERβ anti-FOXL2 ERβ FOXL2 Figure 10 COV434 FOXL2 COV434 pCMV-myc-FOXL2
anti-c-Myc agarose affinity gel anti-ERβ
FOXL2 ERβ COV434
26 3. FOXL2 ERβ in vitro
Glutathione-Sepharose 4B GE Healthcare UK Ltd GST-ERβ AF-1
GST-ERβ AF-2 E2 10-9 M vehicle
pCMV-myc-FOXL2 293T
GST 10% SDS-PAGE
FOXL2 ERβ AF-1 FOXL2 ERβ AF-2
ERβ E2 10-9 M
Figure 12
4. ERβ FOXL2
293T ERE tk Luc phRL
CMV Luc FOXL2 ERβ E2
ERβ E2 10-9 M
FOXL2 ERβ
Figure 13
5. ERα FOXL2
27
AP-1 44 FOXL2 ERβ
ERβ ERα 293T
ERE tk Luc phRL CMV
Luc FOXL2 ERα E2
ERα E2 10-9 M
FOXL2
Figure 14 Figure 13 FOXL2 ERβ E2
6. ERβ FOXL2
FOXL2 ERβ E2 E2
ERβ
293T ERβ AF-2
17m8 AdMLP Luc phRL CMV Luc
ERβ AF-2 E2 Figure 15 FOXL2
28 FOXL2 ERβ ERβ aromatase 51,52 FOXL2 ERβ FOXL2 aromatase Lipofectamine
RNAimax Lifetechnologies All stars Negative Control siRNA FOXL2 siRNA
5% FBS 48 aromatase mRNA
PCR FOXL2 siRNA FOXL2
Figure 16 FOXL2 aromatase mRNA
Figure 17 Aromatase 51,52
29 4 FOXL2 POI FOXL2 POI 19 FOXL2 POI FOXL2 FOXL2 FOXL2 26,52 FOXL2
FOXL2 ERα19 ERβ
FOXL2 ERβ
FOXL2 ERβ
ERβ aromatase FOXL2
FOXL2 ERβ aromatase
30
FOXL2 aromatase
FOXL2 DNA
15-17 Forkhead 5’ [(G/A) (T/C) (C/A)
A A (C/T) A] 3’ 54 FOXL2
GTCAAGG (T/C) 55 aromatase
II39 FOXO aromatase
FOXO TGTTT FOXO
FOXL2 30
aromatase II ERE half site56 FOXL2
ERβ aromatase ERβ 37 ERβ ERα 2 ERβ ERα ERβ ERα 37 ERβ 43 aromatase
31 LH mRNA mRNA 42 ERβ 57 ERα 43 ERα ERβ Figure 8 FOXL2 ERβ ERβ FOXL2 FOXL2 ERβ
Figure 18 FOXL2 ERβ
FOXL2 FOXL2 FOXL2 StAR FOXL2 Q219X StAR 26 FOXL2 FOXL2 ERβ 26 FOXL2 ERβ
32 BPE type1 19,2158 BPE type1 FSH E2 BPE type1 E2 E2 ERβ FOXL2 FOXL2 FOXL2
33 5
FOXL2
ERβ FOXL2
FOXL2 ERβ AF-1 AF-2
FOXL2 ERβ
35 AF-1, AF-2; activation function-1, 2
AP-1; activating protein-1
BPE; blepharophimosis/ptosis/epicanthus inversus syndrome DMEM; Dulbecco's modified Eagle medium
E2; 17β-estradiol
ER; estrogen receptor
ERE; estrogen responsive element FBS; fetal bovine serum
FOXL2; forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 FSH; follicle stimulating hormone
GST; glutathione S-transferase
GnRHR; gonadotropin releasing hormone receptor NLS; nuclear localization signal
NR; nuclear receptor LH; luteinizing hormone
POI; primary ovarian insufficiency StAR; steroidogenic acute regulatory
36
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46 Res 157, 48-52 (2011).
47 Figure 1
48
Figure 2 ART
1978 1992
ART; assisted reproductive technology
49
Figure 3 2012 ART
ART; assisted reproductive technology
50 Figure 4 BPE
51 Figure 5 BPE
BPE
52
Figure 6 FOXL2 forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2
379 1 forkhead
53
Figure 7 ER α β
ER α β 2 ERα 595 ERβ 530
activation function -1 (AF-1)
activation function -2 (AF-2) AF-1 AF-2
DNA ERα ERβ 95%
54 Figure 8 ERβ genomic function
ER genomic function
ER ERβ
DNA ERα E2; 17β-estradiol
ER; estrogen receptor
55 Figure 9 FOXL2 (n=15) anti-FOXL2 (1:100) FOXL2 FOXL2 (FOXL2 / ) primordial follicle 43% (73/169)
intermediatry follicle 68%(96/141) primarymordial follicle 81% (52/64) secondary follicle 84% (38/45) antral follicle 68% (42/62) corpus luteum 40% (7/17)
56
Figure 10 FOXL2 ERβ
KGN FOXL2 C134W
KGN FOXL2 ERβ
KGN
anti-ERβ 2 µg Protein G Sepharose 4 Fast Flow anti-FOXL2 (1:500)
anti-rabbit IgG (1:3000) ECL advance anti-ERβ
3 FOXL2; forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 ERβ; estrogen receptor β
57
Figure 11 FOXL2 ERβ
COV434 FOXL2
COV434 FOXL2 ERβ
COV434 pCMV-myc-FOXL2
anti-c-Myc agarose affinity gel (beads 25µg) anti-ERβ (1:250)
anti-rabbit IgG (1:3000) ECL advance
anti-ERβ
3
FOXL2; forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 ERβ; estrogen receptor β
58 Figure 12 FOXL2 ERβ in vitro
GST FOXL2 ERβ in vitro
GST Glutathione-Sepharose 4B
GST ERβ AF-1 AF-2 GST GST
ERβ AF-2 E2 10-9 M vehicle
pCMV-myc-FOXL2 293T
GST 300µg 10% SDS-PAGE
(anti-FOXL2 : 1:500, anti-rabbit IgG : 1:3000)
GST ERβ AF-1 / AF-2 FOXL2
GST ERβ AF-2 FOXL2
3 GST; glutathione S-transferase
FOXL2; forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 ERβ; estrogen receptor β
59
Figure 13 ERβ E2 FOXL2
ERβ E2 FOXL2
293T 100 ng, ERE
tk Luc 250 ng internal control phRL CMV Luc 1 ng
Effectene vehicle E2 10-9 M
24 Dual Luciferase Reporter System Promega
firefly luciferase
Renilla luciferase 3
3
one-factorial ANOVA Bonferroni/Dunn
post hoc test FOXL2 ERβ E2
* p < 0.05
FOXL2; forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 ERβ; estrogen receptor β
60
Figure 14 ERα E2 FOXL2
ERα E2 FOXL2
293T 100 ng, ERE
tk Luc 250 ng internal control phRL CMV Luc 1 ng
Effectene vehicle E2 10-9 M
24 Dual Luciferase Reporter System Promega
firefly luciferase
Renilla luciferase 3
3
one-factorial ANOVA Bonferroni/Dunn
post hoc test FOXL2 ERα E2
(N.S. ; not significant)
FOXL2; forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 ERα; estrogen receptor α
61
Figure 15 ERβ AF-2 FOXL2
ERβ FOXL2
293T 100 ng,
17m8 AdMLP Luc 250 ng internal control phRL CMV Luc 1 ng
Effectene vehicle
E2 10-9 M 24 Dual Luciferase Reporter System
Promega firefly luciferase
Renilla luciferase
3 3
one-factorial ANOVA
Bonferroni/Dunn post hoc test FOXL2
ERβ AF-2 **p < 0.01
FOXL2; forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 ERβ; estrogen receptor β
62
Figure 16 siRNA FOXL2
FOXL2 siRNA FOXL2
FOXL2-RNAi
siRNA Lipofectamine RNAimax siRNA
10 nM 48
FOXL2 (anti-FOXL2 1:500, anti-rabbit IgG : 1:3000)
β-Actin (anti-β-Actin : 1:500, anti-mouse IgG : 1:3000)
15 µg SDS PAGE FOXL2-RNAi
FOXL2 3
63
Figure 17 FOXL2 aromatase
FOXL2 ERβ
aromatase mRNA
PCR aromatase
FOXL2-RNAi siRNA Lipofectamine
RNAimax 48 RNA
aromatase mRNA PCR
Mann- Whitney U test
FOXL2 aromatase
mRNA FOXL2 ERβ
(n=6, * p<0.05)
FOXL2; forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 ERβ; estrogen receptor β
64 Figure 18 FOXL2 ERβ
FOXL2 ERβ ER
ERβ
FOXL2; forkhead/hepatocyte nuclear factor 3 gene family of transcription factor L2 E2; 17β-estradiol
ER; estrogen receptor
