Expression profile and down-regulation of argininosuccinate synthetase in hepatocellular carcinoma in a transgenic mouse modelby Shih-Chang Shiue, Miao-Zeng Huang, Ting-Fen Tsai, Alice Chang, Kong Choo, Chiu-Jung Huang, Tsung-Sheng Su

J Biomed Sci

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RESEARCH Open Access

Expression profile and down-regulation of tase in hepatocellular

Moreover, this mouse model is a close reproduction of clinical behavior of ASS in HCC and is useful in testing

Shiue et al. Journal of Biomedical Science (2015) 22:10

DOI 10.1186/s12929-015-0114-6112, Taiwan

Full list of author information is available at the end of the articlearginine-depleting agents and for studies of the role of ASS in tumorigenesis.

Keywords: Argininosuccinate synthetase, Transgenic mouse model, Hepatocellular carcinoma, Embryo expression map, Brain expression map, Ventricular zone, Subventricular zone, Post-transcriptional regulation, Bacterial artificial chromosome, GFP reporter gene * Correspondence: su31659@gmail.com 1Institute of Microbiology & Immunology, National Yang-Ming University,

Taipei, Taiwan 2Department of Medical Research, Taipei Veterans General Hospital, Taipeicarcinoma in a transgenic mouse model

Shih-Chang Shiue1, Miao-Zeng Huang2, Ting-Fen Tsai3, Alice Chien Chang4, Kong Bung Choo5,6,

Chiu-Jung Huang7,8 and Tsung-Sheng Su1,2,3*

Abstract

Background: Argininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a ratelimiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express

ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter.

Results: We established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a posttranscriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx)-transgenic mice, the Tg (ASS-EGFP, HBx) double transgenic mice developed HCC in which ASS expression was down-regulated, as in clinical samples.

Conclusions: The BAC transgenic mouse model described is a valuable tool for studying ASS gene expression.argininosuccinate synthe© 2015 Shiue et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative

Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain

Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Shiue et al. Journal of Biomedical Science (2015) 22:10 Page 2 of 14Background

Argininosuccinate synthetase (ASS; EC 6.3.4.5) catalyzes the conversion of citrulline and aspartate to argininosuccinate, which is subsequently converted to arginine by argininosuccinate lyase. Arginine plays an important role in the synthesis of urea, nitric oxide (NO) and polyamines, among other metabolites [1]. In the process, ASS finetunes NO production to maintain cellular homeostasis in response to cellular and environmental stimuli. ASS is ubiquitously expressed but the highest enzyme activities are found in the urea cycle in the liver to eliminate ammonia [2,3].

Regulation of ASS expression is complex and dynamic.

Hormones, including glucocorticoid, glucagon and insulin, are major regulators of the expression of urea cycle enzymes, including ASS, in the liver [2]. We have reported an upstream cAMP response element (CRE) targeted by the CRE-binding protein (CREB) to mediate glucagon action [4]. On the other hand, ASS expression in non-hepatic cells has been shown to be induced by interleukin-1β through NF-κB activation via a putative

NF-κB binding site in the ASS promoter [5]. ASS gene expression also involves interactions between positive transcriptional factors c-Myc and Sp4 and negative factor HIF-1α in the proximal promoter [6]. Furthermore, we have described a novel post-transcriptional event in a canavanine-resistant variant of a human epithelial cell line that regulates the stability of ASS nuclear precursor

RNA, resulting in elevated ASS activities [7,8]. In addition, our recent study showed that the formation of the 3′-end of the human ASS mRNA is modulated by a highly polymorphic GT microsatellite located downstream of the poly (A) signal [9]. The identified post-transcriptional regulation events may have physiological relevance and is an added mechanism for tight regulation of ASS expression. Of clinical significance is the finding that ASS transcription is stimulated by glutamine and repressed by arginine in mammalian tissue cultures [10–12].

A number of tumor types, including hepatocellular carcinoma (HCC), melanoma, prostate, pancreatic and renal cancers, clearly show down-regulated ASS expression and are auxotrophic to arginine [13–15]. In HCC, down-regulated ASS expression has been linked to clinicopathological features and post-resectional patient survival [16,17]. Loss of ASS expression has been exploited as a predictive biomarker for cancers including malignant pleural mesothelioma and epithelial ovarian tumor, to name a few [18,19]. Clinical applications of an arginine-depleting enzyme, such as arginine deaminase (ADI), to treat ASS-deficient tumors have been tested, opening up new venues for further development of auxotrophic cancer therapy [15].