| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 84 === TGF-β可以抑制泌乳激素 (prolactin)mRNA之表現。本論文以GH3腦下垂體腫瘤細胞株為模型，尋找泌乳激素基因啟動子(promoter) 與 TGF-β抑制作用有關的 DNA序列。以不同區域的泌乳激素基因啟動子驅動 Luciferase gene 表現，發現 TGF-β可透過全長 2.5kb或近端 0.6kb與遠端 1.9kb啟動子抑制luciferase的表現。我們對近端啟動子作刪除 (deletion)分析發現 TGF-β無法透過只含泌乳激素基因的TATA box來抑制luciferase的表現。TGF-β對只含75bp prolactin promoter的抑制效果並不顯著。 但 TGF-β可經由含 -204bp，-l62bp及-130bp之 prolavtin 5' 端序列達到抑制的作用。我們將 -130PR-Luc上第一個或第二個 pit-1 binding site做突變，結果 TGF-β抑制作用仍存在。但是將兩個 pit-1 binding site同時突變 ，TGF-β只有 10% 的抑制效果。為了了解 TGF-β作用時是否透過啟動子上 CRE-或TRE-like序列，將5xTRE-CAT或5xCRE-Luc分別送入GH3細胞，做CAT或luciferase分析。發現TGF-β無法透過TRE或CRE來抑制CAT或 luciferase的表現。在遠端啟動子上有兩個TGF-β inhibitoryelement(TIE)，分別位於-1016bp與-1561bp上。以含 -1016bp之TIE或含兩個TIE(-1561bp，-1016bp)之PRL promoter接於luceferase gene之前，發現 TGF-β無法透過此TIE去抑制 luciferase之表現 。
將細胞先處理鈷離子，或鈣離子箝合劑(EGTA)，TGF-β抑制泌乳激素基因的現象就遭到阻斷。當細胞先處理cycloheximide後，也可阻斷TGF-β抑制泌乳激素基因的表現。顯示TGF-β的作用需要鈣離子和蛋白質的合成。以TPA(2μm，24hr)除去細胞內PKC，發現可降低TGF-β抑制泌乳激素的mRNA表現。但並不影響 TGF-β對泌乳激素基因的啟動子的抑制作用。綜合以上實驗，TGF-β 的抑制作用可經由泌乳激素啟動子近端 (-422/+33)與遠端(-2513/-620)區域抑制其表現。此外 pit-1 binding site對TGF-β的抑制可能是需要的，但可能不透過其上之CRE，TRE或TIE之序列。雖然TGF-β抑制 PRL mRNA 之表現需要PKC，但是它抑制啟動子的活性並不需要 PKC的參與。
Transforming growth factor-β (TGF-β) was previously shown to inhibit prolactin (PRL) gene transcription. In this study, we examined sequences on prolactm promoter that confer the TGF-β inhibition on PRL gene expression. Regions of DNA from PRL promoter were linked to the luciferase reporter gene, and the DNA constructs were analyzed for responsiveness to TGF-β inhibition in GH3 pituitary tumor cells. Luciferase assay revealed that sequence containing 2.5kb (-2567/+33), 0.6kb (-620/+33), or 1.9kb (-2567/-620) 5' flanking region of the promoter conferred TGF-β responsiveness. Analysis of deletion mutants generated from 0.6kb fragment indicated that the region of (-40/+33) containing TATA box, or (-75/+33) of the PRL promoter was not involved in TGF-β inhibition. (-204/+33), (-162/+33), (-130/+33) of the PRL promoter that contain from 4 to 2 pit-1 binding sites permitted response to TGF-β. Mutation on either the first or the second pit-1 binding site of (-130/+33) region still retained TGF-β inhibition. However, mutation of both pit-1 binding sites of the -130bp PRL promoter abolished the TGF-β inhibition. Transfection of reporter constructs linked to either 5 copies of TRE or CRE demonstrated that the TRE or CRE mediated gene expression was not affected by TGF-β. Promoter regions containing the consensus sequence for TGF-β inhibitory element (TIE) at -1016bp (-1016/+33) or two TIEs at -1561bp and -1016bp (-1585/+33) was not responsive to TGF-β. Inhibition by TGF-β was blocked by EGTA, cobalt, or cycloheximide, suggesting calcium or protein synthesis are required for TGF-β action. The effect of TGF-β on PRL mRNA expression could be significantly in inhibited in cells treated with 2μM PMA to deplete PKC. By contrast, the inhibition on PRL promoter was not affected by PKC down-regulation. All together our results indicate that proximal (-620/+33) and distal region (-2567/-620) of PRL promoter are sufficient to mediate TGF-β inhibition and pit-1 binding site may be involved. The inhibition of F-β on PRL promoter requires calcium and protein synthesis. PKC which plays a role in the TGF-β inhibition of PRL mRNA, is not involved in the hormone mediated inhibition on PRL promoter activity.
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