肝臟生長因子受體在細胞遭受壓力下之啟動子表現調節

Abstract

肝損害是國人常見的健康問題也是最普遍因服用藥物而引起的器官毒性。目前已經有很多藥物被認為具有潛在的肝毒性而退出臨床上的應用。因此在人體服用前先行評估化學藥品對肝臟造成的影響並且建立一套完整的藥物毒性檢測系統便是一個重要的過程。過去的研究指出肝細胞生長因子(hepatocyte growth factor)與肝細胞生長因子受體(c-Met)的訊息傳遞在肝臟的生長與修復過程中扮演關鍵性的角色。因此本實驗的目的便是發展一體外(in vitro)的實驗模式，探討肝臟損傷中c-Met的表現調節。 本研究中，我們先從NCBI資料庫中取得人類c-Met 啟動子(promoter)序列，並且在含有luciferase或是enhanced green fluorescent protein等報導基因(reporter gene)的質體(plasmid)中構築322、649和952 bp等片段長度的c-Met promoter。接下來便將建構好的plasmid利用微脂體(liposome)轉染到大鼠正常肝臟細胞株(clone 9)。當promoter經RNA聚合酶結合進行轉錄作用時reporter gene的轉錄作用亦會被啟動。藉此我們便藉由冷光或螢光的訊號瞭解化合物在肝細胞中對c-Met promoter的表現調節情形。 經過plasmid轉染後我們發現322、649和952 bp的c-Met promoter在大鼠細胞中會被辨識，因此我們認為在人類與大鼠的c-Met promoter序列中有保留序列參與了c-Met的表現調節。為了瞭解clone 9細胞株在肝毒性藥物的影響下對c-Met基因的轉錄調節影響。我們分別對細胞處理半致死濃度的acetaminophen、D-galactosamine及thioacetamide等肝毒性藥物。實驗結果顯示20 mM acetaminophen, 60 mM D-galactosamine及150 mM thioacetamine均會顯著的抑制細胞中0.9 met-Luc的表現。因此我們推測在肝毒性藥物的刺激下會使c-Met的表現量下降，而c-Met的下降調節(downregulation)會減少與其配體(ligand)HGF的結合，進而降低肝細胞修復與再生的訊息傳遞。而在RT-PCR的實驗中，不管是正常或是肝損傷的細胞我們並沒有觀察到HGF的表現，但過去的研究指出HGF會由其他器官分泌藉由血液傳至肝臟細胞中，這不但可以說明我們的實驗結果，也指出肝損傷細胞其修復與再生的能力下降不僅是缺乏HGF的表現還包括了c-Met的表現量下降。因此我們認為不管HGF的表現量如何c-Met的調節作用的確可以當作一個生物指標(biomarker)來探討肝臟細胞受損的情形。 此外過去的研究也顯示在消化道腫瘤、黑色素瘤及肉瘤等癌細胞組織中c-Met gene會過量表現(overexpressin)。實驗的證據也指出c-Met gene overexpression最主要是因為基因不正常的轉錄調節所引起。而在我們的實驗中，我們發現MNNG、EMS等致癌物質均會顯著的增加細胞中0.9 met-Luc的表現。這不但與過去的研究相符合的，也指出我們的系統在未來也可以應用在致癌物質的篩選。 本研究已成功的建立了一套以冷光和螢光訊號來分析肝毒性藥物的系統。這個以細胞調節功能為分析基礎的系統可以當作一個藥物篩選平台來檢測可能具有潛在的肝毒性與致癌性物質。未來我們希望這樣的體外模式實驗能在藥物毒性篩選的研究中提供有用的科學資訊。

Liver damage is the most common organ toxicity caused by drugs and is a major health problem. Many drugs have been withdrawn from clinical use because of being recognized as potential hepatotoxins. The evaluation of the hepatic effects of a chemical before human exposure consequently represents a critical step and in vitro hepatotoxicity test is an important procedure of the pilot and high-throughput assay system. It has been demonstrated that hepatocyte growth factor/c-Met (hepatocyte growth factor receptor) signaling pathway is required for efficient liver regeneration and repair. Therefore, the aim of the present study is to develop an experimental in vitro model to study c-Met expression for hepatocyte poisoning. In this study, human c-Met promoter-Luc (luciferase) and c-Met promoter-EGFP (enhanced green fluorescent protein) were constructed by cloning 322-, 649-, and 952-bp of the 5□-flanking sequence of human c-Met into the promoterless Luc and EGFP-containing plasmid vector. We made use of a Rattus norvegicus normal liver cell line (clone 9) that expresses a recombinant luciferase or EGFP gene fused to c-Met promoter gene. Consequently, turning on the promoter gene causes the reporter gene to be turned on. Activation or suppression of the promoter gene by chemicals lead to production of reporter signal that ultimately generate fluorescence or luminescence. By transfection of 0.9, 0.6, 0.3 met-Luc/EGFP, we have clearly suggested that potential regulatory elements conserved between human and rat are likely to be actively involved in transcriptional regulation of the c-Met gene. To understand the hepatotoxic effect on transcriptional regulation of c-Met expression in clone 9 cells, the clone 9 cells were treated with LC50 concentration of acetaminophen, D-galactosamine, and thioacetamide. The results have shown that 20 mM acetaminophen, 60 mM D-galactosamine and 150 mM thioacetamine significantly reduced the c-Met promoter activity in the clone 9 cells harboring 0.9 met-Luc. Our results suggest that after hepatotoxic drugs-treated cells, downregulation of c-Met gene is crucial to regenerate because it decreases the activity of this receptor/ligand system with a lower targeting HGF action. In addition, the HGP expression was not detectable in the clone 9 cells treated with acetaminophen or not by the RT-PCR in this study. However, it is known that other organs can supply this protein to the liver through blood, which may account for its high levels in plasma of hepatitis patients. Therefore, it is reasonable to suggest that the unresponsiveness of hepatotoxic drugs-treated cells to the regeneration-promoting action of HGF may be due not to the lack of HGF, but rather to the lack of expression in the liver of the c-Met protooncogene coding for the HGF receptor. Our study suggests that regardless of the level of liver HGF, c-Met protooncogene seems the most active modulator of liver cell proliferation, and its induction might be a critical factor in determining the site specificity of the receptor/ligand system and might be a useful prognostic marker for evaluating liver poisoning. Furthermore past studies have shown that c-Met gene is overexpressed in a variety of neoplastic tissues such as carcinomas of the gastrointestinal tract, melanomas, and sarcomas. Experimental evidence suggests that overexpression of the c-Met gene is mainly due to aberrant transcriptional regulation. In this study we found that carcinogenic compounds such as MNNG and EMS significantly increaseed the c-met promoter activity in the clone 9 cells harboring 0.9 met-Luc. Our observation is in complete agreement with a past study that c-Met oncogen was activated after mutagen treatment in vitro [Park et al., 1986]. Hence we believe that this system can also be used to screen carcinogens. In the present study, we have developed luciferase and EGFP-base assays for the rapid screening of compounds possessing hepatotoxic or carcinogenic properties. This cell-based functional assay could provide a platform for identifying agents with potential hepatotoxic and carcinogenic activity. We hope that the results presented here will serve to accelerate the use of living cells in screens for drug discovery. The ultimate goal of in vitro experiments is to generate the type of scientific information needed to identify compounds that are potentially hepatotoxic or carcinogenic properties. For this purpose, both the design of the experiments and the interpretation of the results are critical.