綠茶表沒食子酸酯型唲茶素酸酯和第一型內皮素對於脂肪細胞中細胞激素訊息抑制物基因的交互作用

Green tea epigallocatechin gallate and endothelin-1 interact on suppressors of cytokine signaling genes in 3T3-L1 adipocytes

張欣蕙(Hsin-huei Chang)

指導教授 ： 高永旭

國立中央大學/理學院/生命科學系/博士(2014年)

摘要

第一型內皮素(endothelin-1)、細胞激素訊息抑制物-3 (suppressor of cytokine signaling-3; SOCS-3)和表沒食子酸酯型唲茶素酸酯(epigallocatechin gallate; EGCG)皆為調節脂質代謝與葡萄糖抗性的重要物質。然而至今仍未有清晰的證據顯示ET-1、SOCS-3和EGCG間的調節關係。因此，本論文欲探討ET-1對於SOCS-3的調節作用，並且更深入的了解EGCG是否會影響ET-1對於SOCS-3的作用。
在第一章中發現，ET-1會增加SOCS-1, -2, -3, -4, -5和-6的mRNA，但此作用並不會在SOCS-7和CIS-1中觀察到。ET-1對於SOCS-3的促進作用需要新RNA的合成，並且具有細胞特異性(cell-type specific)。ET-1對於SOCS-3 mRNA和蛋白質的促進作用是透過ERK、JNK、PI3K和JAK路徑。
第二章顯示，在3T3-L1脂肪細胞中，EGCG抑制ET-1所促進的SOCS-3基因表現是透過67LR和AMPK路徑。EGCG亦會抑制被ET-1所促進的SOCS-1、SOCS-2、SOCS-3、SOCS-4、SOCS-5和SOCS-6的mRNA表現，但此現象並沒有在SOCS-7和CIS-1中觀察到。EGCG也會抑制ET-1所增加的不同訊息蛋白的磷酸化表現，例如ERKs、p38、JNKs、cJUN和JAK2，其中也包含EGCG作用較不明顯的STAT-3蛋白。
在第三章，確立了從老鼠初代脂肪細胞和繼代脂肪細胞分離出的67LR為885鹼基對 (bp)與295個胺基酸，且其與大鼠和人類的基因序列相似性分別為96%和89%。67LR會依據不同組織與不同生長狀態而有不同的表現情形。67LR免疫血清的處理，阻斷了EGCG對於3T3-L1前脂肪細的生長抑制作用，此結果證實了EGCG抑制細胞生長作用須透過67LR作用。
於第四章中，成功將人類與老鼠的全長(67LR1-295)及不同缺失片段 (67LR1-200, 67LR1-150, 67LR1-100 and 67LR1-55) 67LR建構成Flag融合蛋白表現質體，並且進一步篩選出KB和MCF-7穩定表現細胞株。生長實驗指出，67LR的增生作用會依據癌細胞種類的不同與67LR蛋白的不同區域而有所不同。在MCF-7和KB的不同型67LR表現細胞株中處理EGCG，結果顯示67LR的151-200胺基酸位置對於67LR調節EGCG的抑制增生作用是很重要的。
第五章中，成功得到一隻帶有Rpsa-異型合子的老鼠。未來須得到Rpsa-/-同型合子的老鼠供研究所用。
我們歸納出ET-1會透過ETAR、ERK、JNK、JAK和PI3K路徑作用於特定的SOCS家族成員，但不包含ETBR路徑。而EGCG的抗ET-1 作用則是透過67LR和AMPK路徑。如同67LR被發現是EGCG的接受器、SOCS蛋白為胰島素訊息的抑制物，本篇論文的結果或許能有助於解釋EGCG和ET-1對於脂肪細胞功能和胰島素訊息的交互作用機制。

關鍵字

表沒食子酸酯型唲茶素酸酯 ； 第一型內皮素 ； 細胞激素訊息抑制物 ； 脂肪細胞

並列摘要

Endothelin (ET)-1, suppressor of cytokine signaling (SOCS)-3 and epigallocatechin gallate (EGCG) are important to regulate the lipid metabolism and insulin resistance, respectively. However, no clear evidence is showed the relationship among ET-1, SOCS-3 and EGCG. This dissertation was designed to understand the effect of ET-1 on modulating the SOCS-3 gene and further investigate whether EGCG regulated the effect of ET-1 on SOCS-3 gene.
Chapter One indicated that ET-1 upregulated the expression of SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, and SOCS-6 mRNAs, but not SOCS-7 or cytokine-inducible SH2-containing protein (CIS)-1 mRNAs. The ET-1 stimulation of SOCS-3 mRNA expression required new RNA synthesis and was cell-type specific. The stimulatory effects of ET-1 on SOCS-3 mRNA and protein expression were mediated through the ERK, JNK, PI3K, and JAK2 pathways.
Chapter Two showed that EGCG suppressed the ET-1-induced expression of the SOCS-3 gene in 3T3-L1 adipocytes through the 67LR and AMPK pathways. EGCG also suppressed the ET-1-stimulated expression of SOCS-1, -2, -4, -5 and -6 mRNAs, but not SOCS-7 or CIS-1 mRNAs. EGCG inhibited the ET-1-increased phosphorylation of different ET-1 signaling proteins, such as ERKs, p38, JNK, cJUN, AKT, JAK, and, to a lesser extent, STAT-3 proteins.
Chapter Three indicated that 67LR gene was isolated and sequenced with 885 bp and 295 amino acid (aa) from murine primary and secondary adipocytes and it had the 96% and 89% homology of nucleotide sequence to rat and human. The 67LR expression depended on tissue types and growth status. Treatment with 67LR antiserum blocked the inhibitory effect of EGCG on cell number in 3T3-L1 preadipocytes, suggesting the 67LR-dependent effect.
Chapter Four showed that different plasmids for recombinant full-length (67LR1-295) and truncated forms (67LR1-200, 67LR1-150, 67LR1-100 and 67LR1-55) of human and mouse 67LR were constructed with a Flag tag and stably cloned in KB oral cancer cells and MCF-7 breast cancer cells were established. Growth experiments indicated mitogenic effect of 67LR on cancer cells varies with cell types and different domains of the 67LR protein. Different forms of 67LR stably cloned MCF-7 and KB cells treated with EGCG showed that amino acid residues of the 67LR from 151-200 are important for modulating the antimitogenic effects of EGCG on MCF-7 and KB cancer cells.
Chapter Five was successful to generate one heterozygous 67LR gene (also called Rpsa-/flox alleles), the mice with Rpsa-/- alleles would be needed for a further study.
We conclude that ET-1 acts particular types of SOCS family members through the ETAR, ERK, JNK, JAK and PI3K but not ETBR pathways. The anti-ET-1 signaling effect of EGCG is mediated by 67LR and AMPK pathways. As the 67LR was discovered as an EGCG receptor and as the SOCS proteins were reported as an insulin signaling inhibitor, results of this dissertation could help explain the mechanism of how EGCG and ET-1 interacts on adipocyte functions and insulin signaling.

並列關鍵字

Epigallocatechin gallate ； Endothelin-1 ； Suppressors of cytokine signaling ； Adipocyte

參考文獻

Chapter 1:

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2. Minamoto, S., et al., Cloning and functional analysis of new members of STAT induced STAT inhibitor (SSI) family: SSI-2 and SSI-3. Biochemical and Biophysical Research Communications, 1997. 237(1): p. 79-83.

3. Starr, R., et al., A family of cytokine-inducible inhibitors of signalling. Nature, 1997. 387(6636): p. 917-921.

4. Greenhalgh, C.J. and Hilton D.J., Negative regulation of cytokine signaling. Journal of leukocyte biology, 2001. 70(3): p. 348-356.