- 學位論文
助孕酮抑制人類臍靜脈內皮細胞增生之分子機轉
Molecular mechanisms underlying progesterone-induced proliferation inhibition in human umbilical vein endothelial cells
摘要
血管增生在女性的生殖週期中是一種重要的生理現象；其發生是經由原先已存在血管中之內皮細胞再新生而形成血管分支的過程。過去大量的實驗結果指出，性荷爾蒙是會調控生理性之血管增生現象。李文森 博士在1997年曾觀測到，在人類血管內皮細胞內是存在有助孕酮接受器的，而推測助孕酮可能對血管增生會有某些方面的影響。Vázquez 等人也於1999年提出透過助孕酮接受器的活化會阻礙血管的修復過程。這些發現意味著助孕酮可能是具有抑制血管內皮細胞增生的能力，但其分子機制卻尚未清楚。因此，本論文研究是想探討助孕酮是經由何種分子機制來調控血管內皮細胞增生現象以及其對血管增生的效應為何。 在論文第一部份，是針對助孕酮對血管增生所造成的效應與調控此效應的主要分子，予與探討。 在給予生理濃度內之助孕酮 (5-500 nmol/L) 於人類臍靜脈內皮細胞 (HUVEC) 會抑制去氧核糖核酸 (DNA) 的合成，且此抑制是隨著給予助孕酮濃度增加而更增強；以及隨引起效應時間增長而持續抑制。助孕酮的給予會增加p21與p27之訊息核醣核酸 (mRNA) 和蛋白質量的表現，並會增加p21與p27和細胞週期介素之蛋白激酶2 (cyclin-dependent kinase 2; CDK2)的複合體生成， 而非增加和細胞週期介素之蛋白激酶4 (CDK4) 的複合體生成。CDK2 的活性會隨著所給予助孕酮的濃度增加而更抑制。 藉由同時給予p21與p27反義寡核苷酸 (antisense oligonucleotide) 造成p21與p27的表現被抑制，會回復助孕酮所產生之胸腺嘧啶核甘標記嵌合 ([3H]thymidine incorporation) 的抑制。轉染 (transfection) 並表現 顯性抑制表現型p53 (dominant negative p53; DN-p53)會阻斷助孕酮所造成之DNA合成抑制，防止助孕酮所誘導增加之p21與p27的表現以及阻止具助孕酮濃度依賴性抑制之類微血管管腔形成 (capillary-like tube formation) 。給予p53小干擾RNA (p53 SiRNA) 會防止助孕酮所誘導增加p53，p21與p27 之蛋白質表現以及thymidine incorporation 的抑制。於鼠體內之細胞基質-血管增生測定 (matrigel angiogenesis assay) 中顯示出助孕酮具有抗血管增生的效應。 此部分的實驗結果指出，於生理濃度下之助孕酮是藉由p53依賴性的路徑致使產生具有抗細胞增生的能力。 於論文第二部份，是更近一步去探討助孕酮所誘導調控p53依賴性路徑之上游訊息途徑為何？給予助孕酮後會促使助孕酮接受器與Src 結合複合體的量和Src的活性增加；而此增加效應會被預處理助孕酮拮抗劑：RU 486所妨礙。在預先施予Src 抑制劑：PP2，Src Kinase Inhibitor I 與Ras 抑制劑：Ras inhibitory peptide，farnesyltransferase inhibitor 會防止助孕酮所增加之p53誘導下游分子活化的能力 (trans-activity)。助孕酮所誘導增加之p53蛋白質表現與Kras轉移至細胞膜的量會被預先給予PP2與Src Kinase Inhibitor I 所抑止。Transfection 並表現DN-Erk2會防止助孕酮所誘導之thymidine incorporation 的抑制以及助孕酮所誘導p53，p21和p27 之蛋白質表現量上升。 Raf-1 激酶 (Raf-1 kinase) 與Erk1/2 會被助孕酮誘導磷酸化，而此誘導之磷酸化現象會被預先施予Ras inhibitory peptide和sulindic sulfide 所抑制。藉由預先施予Src antisense oligonucleotide會防止助孕酮所誘導之p53 mRNA的表現量增加。此部分的實驗結果指出，助孕酮所誘導的Src/Kras/Raf-1/Erk訊息傳導途徑可能對調節p53的表現上升，進而促使p21與p27的蛋白質表現隨之增加，最後導致抑制靜脈內皮細胞之細胞增生，有所貢獻。 總括來說，由本論文的實驗發現，提供了一個證據來建議助孕酮本身在血管增生中也許是扮演了一個調節性的角色；而此調節可能是經由助孕酮所誘導之非基因型之訊息傳遞途徑來造成調節血管增生的作用。
並列摘要
Angiogenesis is a process of forming new blood vessels which are generated by endothelial cells of pre-existing blood vessels; it plays an important role in female reproductive cycle. A large body of experimental results indicates that physiological angiogenesis is under the control of sex hormones. Dr. Wen-Sen Lee observes the presence of progesterone receptor in human vascular endothelial cells in 1997 and speculated that progesterone might play some roles in the regulation of angiogenesis. In 1999, Vázquez et al. demonstated that activation of progesterone receptor could impair vascular repair process. These findings indicate that progesterone might produce anti-proliferative activity in vascular endothelial cells but the underlying mechanism is not clear. The aim of this thesis study is to investigate the molecular mechanisms underlying progesterone on proliferation of vascular endothelial and the effect of progesterone on angiogenesis. In the first part of this study, the research was aimed to investigate the effects of progesterone in angiogenesis and the associated key molecules involved in this regulation. The results showed that progesterone at physiologic levels (5-500 nmol/L) concentration- and time-dependently inhibited DNA synthesis of cultured human umbilical vein endothelial cells (HUVEC). The mRNA and protein levels of p21 and p27 were increased by progesterone treatment. Progesterone increased the formation of the CDK2-p21 and CDK2-p27 complexes, but not the CDK4-p21 and CDK4-p27 complexes. Moreover, the assayable CDK2, but not CDK4, activity was concentration-dependently decreased by progesterone treatment. The progesterone-induced inhibition in [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were suppressed by combined treatment with antisense oligonucleotide of p21 and p27. Transfection of HUVEC with dominant negative p53 blocked the progesterone-induced suppression of DNA synthesis, increases of p21 and p27 expression, and inhibition of capillary-like tube formation. Moreover, blockade of p53 induction with p53 siRNA prevented the progesterone-induced increases of p53, p21 and p27, and decrease of thymidine incorporation. Matrigel angiogenesis assay in mice demonstrated the antiangiogenic effect of progesterone in vivo. These results indicated that progesterone at physiologic levels exerts an anti-proliferation activity in HUVEC through a p53-dependent pathway. In the second part of this research, the signaling cascade of the progesterone-induced upregulation of p53 expression was further studied. Treatment of HUVEC with progesterone increased the formation of progesterone receptor-Src complex and the Src activity. These effects were prevented by pretreatment with a progesterone receptor antagonist, RU486. Luciferase reporting assay indicated that progesterone-induced increase of p53 trans-activity was abolished by treatment of HUVEC with PP2 (a Src inhibitor), Src Kinase Inhibitor I, Ras inhibitory peptide, or a farnesyltransferase inhibitor. Western blot analysis showed that pretreatment of HUVEC with PP2 or Src Kinase Inhibitor I prevented the progesterone-induced increase of p53 protein level and membrane translocation of Kras. Transfection of HUVEC with dominant negative Erk2 prevented the progesterone-induced decrease of thymidine incorporation and increases of the p53, p21 and p27 protein level. Moreover, progesterone-induced phosphoylation of Raf-1 and Erk1/2 was abolished by pretreatment with Ras inhibitory peptide or sulindac sulfide (a Raf-1 inhibitor). Semiquantitative RT-PCR revealed that administration of Src antisense oligonucleotide prevented progesterone-induced increase of p53 mRNA level. These results suggest that the Src/Kras/Raf-1/Erk signaling pathway might contribute to the progesterone-induced up-regulation of p53, which in turn increased the levels of p21 and p27 protein, and finally inhibited proliferation in HUVEC. Taken together, the findings of this thesis study suggest that progesterone alone might inhibit angiogenesis through a non-genomic signaling cascade.