Argininosuccinate synthase 1 suppresses cancer cell invasion by inhibiting STAT3 pathway in hepatocellular carcinoma.

Metastasis is the main reason for high recurrence and poor survival of hepatocellular carcinoma (HCC). The molecular mechanism underlying HCC metastasis remains unclear. In this study, we found that argininosuccinate synthase 1 (ASS1) expression was significantly decreased and down-regulation of ASS1 was closely correlated with poor prognosis in HCC patients. DNA methylation led to the down-regulation of ASS1 in HCC. Stable silencing of ASS1 promoted migration and invasion of HCC cells, whereas overexpression of ASS1-inhibited metastasis of HCC cells in vivo and in vitro. We also revealed that ASS1-knockdown increased the phosphorylation level of S727STAT3, which contributed to HCC metastasis by up-regulation of inhibitor of differentiation 1 (ID1). These findings indicate that ASS1 inhibits HCC metastasis and may serve as a target for HCC diagnosis and treatment.

Molecular characterization of argininosuccinate synthase and argininosuccinate lyase from the liver of the African lungfish Protopterus annectens, and their mRNA expression levels in the liver, kidney, brain and skeletal muscle during aestivation.

Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398 bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6 months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia-reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy.

Two hypomorphic alleles of mouse Ass1 as a new animal model of citrullinemia type I and other hyperammonemic syndromes.

Citrullinemia type I (CTLN1, OMIM# 215700) is an inherited urea cycle disorder that is caused by an argininosuccinate synthetase (ASS) enzyme deficiency. In this report, we describe two spontaneous hypomorphic alleles of the mouse Ass1 gene that serve as an animal model of CTLN1. These two independent mouse mutant alleles, also described in patients affected with CTLN1, interact to produce a range of phenotypes. While some mutant mice died within the first week after birth, others survived but showed severe retardation during postnatal development as well as alopecia, lethargy, and ataxia. Notable pathological findings were similar to findings in human CTLN1 patients and included citrullinemia and hyperammonemia along with delayed cerebellar development, epidermal hyperkeratosis, and follicular dystrophy. Standard treatments for CTLN1 were effective in rescuing the phenotype of these mutant mice. Based on our studies, we propose that defective cerebellar granule cell migration secondary to disorganization of Bergmann glial cell fibers cause cerebellar developmental delay in the hyperammonemic and citrullinemic brain, pointing to a possible role for nitric oxide in these processes. These mouse mutations constitute a suitable model for both mechanistic and preclinical studies of CTLN1 and other hyperammonemic encephalopathies and, at the same time, underscore the importance of complementing knockout mutations with hypomorphic mutations for the generation of animal models of human genetic diseases.

Reduced argininosuccinate synthetase is a predictive biomarker for the development of pulmonary metastasis in patients with osteosarcoma.

Pulmonary metastasis is the most significant prognostic determinant for osteosarcoma, but methods for its prediction and treatment have not been established. Using oligonucleotide microarrays, we compared the global gene expression of biopsy samples between seven osteosarcoma patients who developed pulmonary metastasis within 4 years after neoadjuvant chemotherapy and curative resection, and 12 patients who did not relapse. We identified argininosuccinate synthetase (ASS) as a gene differentially expressed with the highest statistical significance (Welch's t test, P = 2.2 x 10(-5)). Immunohistochemical analysis of an independent cohort of 62 osteosarcoma cases confirmed that reduced expression of ASS protein was significantly correlated with the development of pulmonary metastasis after surgery (log-rank test, P < 0.05). Cox regression analysis revealed that ASS was the sole significant predictive factor (P = 0.039; hazard ratio, 0.319; 95% confidence interval, 0.108-0.945). ASS is one of the enzymes required for the production of a nonessential amino acid, arginine. We showed that osteosarcoma cells lacking ASS expression were auxotrophic for arginine and underwent G(0)-G(1) arrest in arginine-free medium, suggesting that an arginine deprivation therapy could be effective in patients with osteosarcoma. Recently, phase I and II clinical trials in patients with melanoma and hepatocellular carcinoma have shown the safety and efficacy of plasma arginine depletion by stabilized arginine deiminase. Our data indicate that in patients with osteosarcoma, reduced expression of ASS is not only a novel predictive biomarker for the development of metastasis, but also a potential target for pharmacologic intervention.

Argininosuccinate synthetase is reversibly inactivated by S-nitrosylation in vitro and in vivo.

Prior studies have demonstrated that the substrate for NO synthesis, l-arginine, can be regenerated from the NOS co-product l-citrulline. This requires the sequential action of two enzymes, argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). AS activity has been shown to be rate-limiting for high output NO synthesis by immunostimulant-activated cells and represents a potential site for metabolic control of NO synthesis. We now demonstrate that NO mediates reversible S-nitrosylation and inactivation of AS in vitro and in lipopolysaccharide-treated cells and mice. Using a novel mass spectrometry-based method, we show that Cys-132 in human AS is the sole target for S-nitrosylation among five Cys residues. Mutagenesis studies confirm that S-nitrosylation of Cys-132 is both necessary and sufficient for the inhibition of AS by NO donors. S-nitroso-AS content is regulated by cellular glutathione levels and selectively influences NO production when citrulline is provided to cells as a protosubstrate of NOS but not when l-arginine is provided. A phylogenetic comparison of AS sequences suggests that Cys-132 evolved as a site for post-translational regulation of activity in the AS in NOS-expressing species, endowing NO with the capacity to limit its own synthesis by restricting arginine availability.

Argininosuccinate synthase expression is required to maintain nitric oxide production and cell viability in aortic endothelial cells.

Although cellular levels of arginine greatly exceed the apparent K(m) for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide (NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase (AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of l-arginine from l-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability.

Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients.

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.

Argininosuccinate synthetase: localization in astrocytes and role in the production of glial nitric oxide.

An antiserum raised against the peptide representing the partial sequence 196-222 of mouse liver argininosuccinate synthetase (ASS) was used to detect and localize the enzyme in cells of neural primary cultures. No ASS immunoreactivity was detected by Western blotting in homogenates of mouse pure astroglial cultures and rat astroglia-rich cultures. However, when the cultures had been treated with bacterial lipopolysaccharide, interferon-gamma, or a combination of both, ASS immunoreactivity was disclosed. Immunocytochemical examination of rat astroglia-rich cultures revealed a colocalization of ASS with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were some GFAP-positive cells showing no specific staining for ASS, and vice versa. Colocalization of ASS with the inducible isoform of nitric oxide synthase in the same cell was shown only occasionally; nitric oxide synthase was predominantly expressed in microglial cells. In rat neuron-rich primary cultures astroglial cells as well as neurons expressed ASS. Cells of mouse pure astroglial cultures were able to synthesize arginine and, consequently, nitric oxide from citrulline, but not from ornithine. The findings demonstrate that ASS is expressed in astroglial cells under conditions that stimulate long-lasting production of nitric oxide; a functional role of this enzyme in the latter process is implicated.