||The cellular and molecular mechanisms of FGF9 induced steroidogenesis, testis development and tumorigenesis.
||Institute of Basic Medical Sciences
Fibroblast growth factor 9
纖維母細胞生長因子可以透過調節細胞增生、遷移、分化與存活來幫助組織的發育與修復。在先前的研究當中就指出纖維母細胞生長因子九的缺失會使雄性胚胎產生性別倒轉的現象，並且纖維母細胞生長因子九也被發現會促進萊氏細胞的固醇類生合成。這些研究顯示了纖維母細胞生長因子九對於雄性個體的重要性。然而，在許多的癌症(像是肺癌、胃癌以及大腸癌)也發現了纖維母細胞生長因子九的大量表現。因此，研究纖維母細胞生長因子九在正常細胞與癌症細胞當中的差異對於雄性來說相當的重要。而在本篇當中，我們證實纖維母細胞生長因子九可以促使細胞的固醇類生成並且活化蛋白激酶B (Akt)、c-Jun氨基末端激酶 (JNK)、絲分裂原活化蛋白激酶 (p38)與細胞外調節蛋白激(ERK)。我們進一步去觀察睪丸發育過程當中，纖維母細胞生長因子九與纖維母細胞生長因子受器的表現情形。我們發現，在生長與成熟的過程當中，睪丸會持續表現纖維母細胞生長因子九與其受器。在小鼠妊娠第17-18天以及出生後35-65天的期間，纖維母細胞生長因子九會大量的表現在曲細精管之間的區域。關於纖維母細胞生長因子受器的表現，纖維母細胞生長因子受器1與受器4會在睪丸的各處表現。而纖維母細胞生長因子受器2與3在各處也都會表現之外，纖維母細胞生長因子受器2會在小鼠妊娠第16-18天時增加在曲細精管當中的表現，而纖維母細胞生長因子受器3會在小鼠妊娠第17-18天增加在曲細精管間的表現。在出生後35-65天，纖維母細胞生長因子受器2會在精細胞與萊氏細胞中大量表現，而纖維母細胞生長因子受器3則廣泛的表現在睪丸當中。根據纖維母細胞生長因子九與纖維母細胞生長因子受器2與3表現位置的相似性，我們推測纖維母細胞生長因子九會參與在睪丸的發育當中來調控睪丸的生長發育。而在睪丸發育的過程當中，前驅細胞的生長與成熟對於精細胞與固醇類的生成有著相當大的影響。然而，過量的纖維母細胞生長因子九表現卻會造成癌症。在本篇研究當中，我們發現纖維母細胞生長因子九會促進萊氏前驅細胞(TM3)與萊氏腫瘤細胞(MA-10)的生長複製。在訊息路徑當中我們發現，纖維母細胞生長因子九會個別活化前驅細胞中的Akt, JNK, p38, ERK與磷脂質脂解酶 (PLC)γ-1以及腫瘤細胞中的ERK。而在細胞週期方面，纖維母細胞生長因子九可以增加兩種細胞當中週期蛋白 D1, 週期蛋白 A, 週期蛋白依賴型激酶1 與週期蛋白依賴型激酶2的表現量。而在調控細胞週期的分子當中，我們發現纖維母細胞生長因子九可以增加視網膜母細胞瘤蛋白 (Rb)的磷酸化程度。我們也發現纖維母細胞生長因子九會個別增加腫瘤細胞中的纖維母細胞生長因子受器1-4與前驅細胞中的纖維母細胞生長因子受器1,3,4。除次之外， 在癌細胞當中，纖維母細胞生長因子九與不同抑制劑的共同處理會導致腫瘤細胞的磷酸化AKT的上升與磷酸化ERK的下降；纖維母細胞生長因子九與不同抑制劑的共同處理也會在正常細胞當中促進磷酸化AKT的上升。這個結果顯示MAPK的抑制可能會導致AKT的活化。總而言之，纖維母細胞生長因子九所刺激的Akt, JNK, p38與ERK活化、固醇類生合成的增加，還有與纖維母細胞生長因子受器2,3的共同表現顯示了纖維母細胞生長因子九的重要性，除此之外，纖維母細胞生長因子九可以各別活化前驅細胞中的Akt, JNK, p38, ERK與PLCγ-1以及腫瘤細胞當中的ERK，並且調節週期蛋白與週期蛋白依賴型激酶來促使細胞增生。總結以上結果，纖維母細胞生長因子九對於男性性腺的發育與固醇類生合成相當的必要，然而，纖維母細胞生長因子九的過度表現卻會促使萊氏腫瘤細胞的增生與癌症的進程。
Fibroblast growth factors can modulate the signal of cell proliferation, migration, differentiation and survival to regulate the tissue development and repair. In previous studies, the deficient of FGF9 caused male-to-female sex reversal in XY mouse embryo and FGF9 could promote steroidogenesis in primary Leydig cell. These evidences indicate the crucial role of FGF9 in male individual. However, over-expression of FGF9 was demonstrated to participate in different cancers, such as lung, gastric, prostate and colon cancer. According to these observations, it is important to investigate the detail role of FGF9 in male. In the present study, FGF9 could promote steroidogenesis and activate Akt, JNK, p38 and ERK signals in mouse Leydig cells at the same time. We further investigated the expressional profiles of FGF9 and FGF receptors in testes during development to verify the role of FGF9 in male gonad. FGF9 and FGFRs continuously expressed in the mouse testis from birth to adult. At 17-18 days post coitum (dpc) and postnatal day (pnd) 35-65, FGF9 was highly expressed in the interstitial region. Compared with the evenly and widely expressional patterns of FGFR1 and FGFR4, FGFR2 expression increased in seminiferous tubules at 16-18 dpc and FGFR3 expression increased in interstitial region at 17-18 dpc. In postnatal stage, FGFR2 extensively expressed with higher expression at spermatids and Leydig cells on 35-65 pnd and FGFR3 widely expressed in the whole testis. FGF9 is correlated with the temporal expression profiles of FGFR2 and FGFR3 and possibly associated with testis development. In gonad development, proliferation and differentiation of progenitor cells are important for spermatogenesis and steroidogenesis. However, over-expression of FGF9 participated in cancer initiation and progression. It is important to clarify the function of FGF9 in normal and tumor cells. In the present study, we observed that FGF9 could promote cell proliferation in progenitor (TM3) and tumor (MA-10) Leydig cell lines. Regarding the mechanism investigations, FGF9 activated Akt, ERK, JNK, p38 and PLCγ-1 pathways in TM3 cells and ERK pathway in MA-10 cells, respectively. Regarding cell cycle study, FGF9 increased cyclin D1, cyclin A, cyclin dependent kinase (CDK) 1 and CDK2 protein expressions in TM3 and MA-10 cells. In addition, FGF9 promoted the phosphorylation of retinoblastoma protein (Rb) in both cells. We also demonstrated that FGF9 stimulated FGFR1-4 expression in MA-10 cells and FGFR1, 2 and 4 in TM3 cells, respectively. In addition, p38, JNK or ERK inhibitors could promote Akt phosphorylation but reduce ERK phosphorylation with FGF9 treatment in MA-10 cells, and p38, JNK or ERK inhibitors also increased Akt phosphorylation with FGF9 treatment in TM3 cells. In summary, FGF9 activated Akt, JNK, p38 and ERK to promote steroidogenesis and in Leydig cells, and had expressional correlation with FGFR2 and FGFR3 during testes development. Furthermore, FGF9 activated Akt, ERK, JNK, p38 and PLCγ-1 signals in TM3 Leydig progenitor cells and ERK signal in MA-10 Leydig tumor cells to induce cell proliferation, respectively. FGF9 also promoted cell cycle progression with cyclin/CDK up-regulation in Leydig progenitor and tumor cells. By using serve combined immunodeficiency (SCID) mice, FGF9 significantly promoted MA-10 cell proliferation in vivo. In conclusion, FGF9 plays essential role in steroidogenesis and development in male gonads. However, abnormal expression of FGF9 could cause tumor Leydig cell proliferation inducing tumorigenesis.
Table of contents
Abstract in Chinese I
Table of contents VI
List of Tables IX
List of Figures X
Chapter 1: Introduction 1
1.1. FGF and FGFR 1
1.2. Phospholipase C gamma pathways 2
1.3. Mitogen activated protein kinases signaling pathways 2
1.4. Phosphatidylinositide 3-kinases/Akt signaling pathways 3
1.5. Fibroblast growth factor 9 4
1.6. Hormone production 4
1.7. Testes development 5
1.8. Leydig progenitor cells 6
1.9. Leydig tumor cells 6
1.10. Cyclin/CDK regulation pathways 7
1.11. Objectives 7
Chapter 2: Materials and Methods 10
2.1. Chemicals 10
2.2. Animals 11
2.3. Isolation of mouse primary Leydig cells 11
2.4. Cell culture 12
2.5. Radioimmunoassay 12
2.6. Immunoblotting 12
2.7. Immunohistochemistry 13
2.8. MTT assay 14
2.9. Flow cytometry 15
2.10. Allograft tumor analysis 15
2.11. Ethics statement. 15
2.12. Statistical analysis 16
Chapter 3: Results 17
3.1 FGF9 increased testosterone production on mouse Leydig cells. 17
3.2. FGF9 increased the phosphorylation level of Akt, JNK, p38 and ERK1/2 in primary Leydig cells. 17
3.3. FGF9 increased the phosphorylation level of Akt, JNK and ERK1/2 in MA-10 Leydig tumor cells. 18
3.4. Effects of selective inhibitors on FGF9 induced Akt, JNK, p38 and ERK1/2 activation in mouse Leydig cells. 18
3.5. FGF9 and its receptors were expressed in mouse testes. 19
3.6. FGF9 was expressed in embryonic and postnatal mouse testis. 19
3.7. FGFR1, 2, 3 and 4 were expressed in embryonic and postnatal mouse testis. 20
3.8. FGF9 increased the proliferation rate in mouse Leydig cell lines. 21
3.9. FGF9 activated PI3K signaling pathway in mouse Leydig cells. 22
3.10. FGF9 activated MAPK and PLCγ signaling pathways in mouse Leydig cells. 23
3.11. FGF9 regulated cell cycle distribution in Leydig cell lines. 23
3.12. FGF9 induced cell cycle protein expression in mouse Leydig cell lines. 24
3.13. FGF9 effect on p53 and Rb in mouse Leydig cell lines. 25
3.14. FGF9 regulated the expression of p21 and p27 in mouse Leydig cell lines. 25
3.15. FGF9 induced FGFR1-4 expression in mouse Leydig cells. 26
3.16. Inhibitor effect of FGF9-induced Akt, ERK1/2, JNK and p38 activation in mouse Leydig cell lines. 26
3.17. FGF9 promoted tumor growth in an allograft model of testicular cancer. 27
Chapter 4: Discussion 29
Chapter 5: Conclusion 38
Chapter 6: References 39
Chapter 7: Tables 50
Chapter 8: Figures 52
Chapter 9: Publications 89
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