Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells

Molecular Brain Research 90 (2001) 165–173 www.elsevier.com / locate / bres

Research report

Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells Kohichi Kawahara a , Tomomi Gotoh b , Seiichi Oyadomari b , Makoto Kajizono a , Akihiko Kuniyasu a , Keiko Ohsawa c , Yoshinori Imai c , Shinichi Kohsaka c , Hitoshi Nakayama a , *, Masataka Mori b a

Department of Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5 -1 Ohe-Honmachi, Kumamoto 862 -0973, Japan b Department of Molecular Genetics, Kumamoto University School of Medicine, 2 -2 -1 Honjo, Kumamoto 860 -0811, Japan c Department of Neurochemistry, National Institute of Neuroscience, 4 -4 -1 Ogawahigashi, Kodaira, Tokyo 187 -8851, Japan Accepted 20 March 2001

Abstract Nitric oxide (NO) produced by activated microglia has been implicated in many pathophysiological events in the brain including neurodegenerative diseases. Cellular NO production depends absolutely on the availability of arginine, a substrate of NO synthase (NOS). Murine microglial MG5 cells were treated with bacterial lipopolysaccharide (LPS) and interferon-g (IFN-g), and expression of inducible NO synthase (iNOS) and arginine-supplying enzymes was investigated by RNA blot analysis. iNOS mRNA was strongly induced after treatment and reached a maximum at 6–12 h. mRNA for argininosuccinate synthetase (AS), a citrulline–arginine recycling enzyme, increased at 6 h and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and AS proteins were also induced. In addition, mRNA encoding the cationic amino acid transporter-2 (CAT-2) was strongly induced shortly after treatment. Induction of mRNAs for iNOS, AS, and CAT-2 by LPS / IFN-g was also observed following stimulation of rat primary microglial cells. These results strongly suggest that both arginine transport by CAT-2 and citrulline–arginine recycling are important for high-output production of NO in activated microglial cells.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorder of the nervous system Topic: Infectious disease Keywords: Nitric oxide; Nitric oxide synthase; Citrulline–NO cycle; Argininosuccinate synthetase; Cationic amino acid transporter; Microglia

1. Introduction Microglia, which are distinguished from other glial cells by their origin and function, play important roles in immune responses in the central nervous system (CNS) [16,23,31]. However, like immune cells in other organs, microglia may play a dual role, amplifying the effects of inflammation and mediating cellular degeneration as well as protecting the CNS. In brain injuries and neurodegenerative diseases, microglia are activated and accumu*Corresponding author. Tel.: 181-96-371-4357; fax: 181-96-3727182. E-mail address: [email protected] (H. Nakayama).

late in affected areas [12,21,27]. In addition, concentrations of several cytokines, including interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a), increase with mechanical lesions [22]. Activated microglia appear to be directly involved in propagation of neuropathological events: microglial activation leads to production of several cytotoxic factors, including nitric oxide (NO) [4,16,27]. Therefore, understanding how activation of microglia is regulated may lead to new drug discovery targets for treatment of neurodegenerative diseases. NO produced by activated microglia is thought to be an important contributor to neuronal damage seen in neurodegenerative diseases. Inducible NO synthase (iNOS) is up-regulated in activated microglia [6,13,14,40,44]. Cel-

0169-328X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00100-0

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lular NO production depends absolutely on availability of arginine, a NOS substrate. Microglia, like most cells, have no complete arginine biosynthetic pathway and arginine is supplied in part by the cationic amino acid transporter (CAT) [35]. Alternatively, arginine is synthesized in the ‘citrulline–NO cycle’, in which arginine is regenerated from citrulline, a coproduct of the NOS reaction, via successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) [41,45,47]. Arginine transport increases in activated macrophages [6,37] and CAT-2 mRNA as well as iNOS mRNA is induced in activated macrophages [9,36] and vascular smooth muscle cells [15]. Furthermore, iNOS and AS are co-induced in response to immunostimulants in endothelial cells [33], macrophages [19,29,36], and vascular smooth muscle cells [18]. Nevertheless, little is known about how arginine is supplied to activated microglia, where a large amount of NO is thought to be generated. To investigate this question, we mainly used murine microglial MG5 cells [34] and treated them with LPS and IFN-g and evaluated the expression of iNOS, arginine-recycling enzymes, and CAT isoforms in this study. The MG5 microglial cell line was established from a primary culture derived from the p53-deficient mouse [34]. The cell line can be maintained much longer than the primary-cultured microglia. The MG5 cell line also retains several properties characteristic of wild-type microglia: namely, expression of the marker proteins Mac-1, F4 / 80, LFA-1, MHC class I- and II-antigens, and Iba1; 59-nucleotidase activity, phagocytic activity, and proliferation in response to mitogenic factors. Furthermore, it can release NO at similar levels to wild-type microglia. Here we report the co-induction of iNOS, AS, and CAT-2 mRNAs and proteins in stimulated murine MG5 cells and rat primary microglial cells as well. These results strongly suggest that both arginine transport by CAT-2 and citrulline–arginine recycling are important for high-output production of NO in activated microglial cells.

into poly-L-lysine coated flasks (75 cm 2 ) at a density of 2310 7 cells / flask in DMEM containing 10% FCS, penicillin (100 U / ml), and streptomycin (100 mg / ml). The cells were maintained at 378C in a humidified air atmosphere containing 5% CO 2 . One week after seeding, floating and weakly attached cells were harvested after shaking the flask vigorously (150 rpm for 2 h). The resulting suspension was re-seeded into plastic dishes (Falcon 1001, Lincoln Park, NJ, USA) and allowed to adhere at 378C. After 30 min, unattached cells were removed and microglia were isolated as strongly adhering cells. Both MG5 and primary microglial cells were treated with a combination of Escherichia coli LPS (1 mg / ml; serotype 0127:B8, Sigma, St. Louis, MO, USA) and mouse IFN-g (100 U / ml; Life Technologies, Inc., Rockville, MD, USA) for indicated periods.

2.2. RNA blot analysis Total RNA from MG5 cells (2310 6 cells / 100-mm dish) or primary microglial cells (1310 6 cells / 100-mm dish) was isolated with the ISOGEN reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer’s recommendations. After electrophoresis through formaldehydecontaining agarose gels, RNAs were transferred to nylon membranes. Hybridization was done with the following digoxigenin-labeled antisense RNA probes derived from rat: iNOS [29], AS [46], AL [46], CAT-2 [36] and CAT-1. Chemiluminescence signals derived from hybridized probes were detected on X-ray films using the DIG luminescence detection kit (Boehringer Mannheim, Mannheim, Germany), and were quantified using a MacBas Bioimage Analyzer (Fuji Photo Film Co., Tokyo, Japan). The template plasmid for CAT-1 was prepared as follows: a partial cDNA for rat CAT-1 corresponding to nucleotides 202–807 (GenBank, accession no. L10152) was isolated by PCR of cDNA made from rat brain RNA, and inserted into the HincII site of pGEM–3Zf(1) (Promega, Madison, WI, USA), yielding pGEM–rCAT1-1.

2. Material and methods

2.3. Immunoblot analysis

2.1. Cell culture and treatment

MG5 cells (2310 6 cells / 100-mm dish) were homogenized in 25 mM Tris–HCl (pH 7.4) containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mg / ml pepstatin A, 1 mg / ml leupeptin, and 10 mg / ml soybean trypsin inhibitor. After centrifugation, the supernatants were subjected to sodium dodecylsulfate–polyacrylamide gel electrophoresis, and proteins were electrotransferred to polyvinylidene difluoride membranes. Immunodetection was performed using the ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, UK), according to the manufacturer’s protocol. A monoclonal antibody against mouse iNOS was obtained from Transduction Laboratories (Lexington, KY, USA). The antiserum used against rat AS has been described elsewhere [46]. Protein concentrations were

MG5 microglial and A1 astrocyte-like cell lines were established from p53-deficient mice [34]. MG5 cells were maintained in A1 conditioned medium. A1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 20 U / ml penicillin, and 20 mg / ml streptomycin. Rat primary microglia were isolated from primary cultures of rat brain as previously described [30]. Briefly, the cerebral cortex from 2-day-old Wistar rats was minced and treated with trypsin (2.5 mg / ml) and DNase (2000 U / ml) at 378C for 10 min. The dissociated cells were filtered through a 100-mm pore nylon mesh and seeded

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determined by the Coomassie brilliant blue method (BioRad Laboratories, Hercules, CA, USA). Bovine serum albumin was used as the standard.

3. Results

3.1. Induction of iNOS, AS, and CAT-2 mRNAs by LPS and IFN-g For the investigation, we first used murine MG5 microglial cell line [34]. Fig. 1 shows the effects of LPS (1 mg / ml) and IFN-g (100 U / ml) treatment on mRNAs encoding iNOS, AS, and CAT-2. iNOS mRNA was not

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evident in untreated cells but was markedly induced by LPS and less strongly so by IFN-g. In addition, iNOS mRNA was induced synergistically by a combination of the two treatments (Fig. 1B). A lower concentration of IFN-g (10 U / ml) in combination with LPS gave nearly maximal induction when measured at 12 h (data not shown). The level of iNOS mRNA induced by LPS / IFN-g was similar to that seen in LPS / IFN-g activated murine macrophage-like RAW 264.7 cells [17] that were used as a positive control (Fig. 1A). mRNA encoding AS, an enzyme required for citrulline– arginine recycling, is present in untreated cells. AS mRNA was induced 4- to 6-fold by LPS, IFN-g and their combination (Fig. 1B); however, the effects of the com-

Fig. 1. Effect of LPS and IFN-g on mRNAs encoding iNOS, AS and CAT-2 in murine MG5 cells. (A) Cells were treated with LPS (1 mg / ml), IFN-g (100 U / ml), or their combination for 12 h. Total RNAs (2.0 mg) were subjected to blot analysis. Positive controls (Con) are total RNA (2.0 mg) from LPS / IFN-g activated murine macrophage-like RAW 264.7 cells [17] for iNOS mRNA, and that from rat liver for AS and CAT-2 mRNAs. The positions for 28S and 18S rRNAs are shown on the right. The bottom panel shows ethidium bromide staining of 28S and 18S rRNAs as a control for RNA loading. (B) Results in A were quantified and are shown as means6S.D. (n53). The maximal values are set at 100%.

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bined agents were less than additive. The mRNA level induced by LPS / IFN-g was similar to that observed in liver where AS, a urea cycle enzyme, is highly expressed (Fig. 1A). CAT-2 mRNA was expressed at a low level in untreated cells, induced about 6-fold by LPS, induced 7-fold by IFN-g and induced 15-fold by a combination of the treatments. The level induced by LPS / IFN-g was higher than that seen in the liver. Thus, mRNAs encoding iNOS, AS and CAT-2 were co-induced by LPS and IFN-g separately and maximally by their combination, suggesting that these proteins are involved in related metabolic functions. However, the induction profiles differed among these proteins, suggesting that their expression may be differentially regulated.

3.2. Kinetics of induction of iNOS, AS and CAT-2 mRNAs in MG5 cells We next examined the time course of mRNA induction for iNOS, AS, AL, CAT-2, and CAT-1 after LPS / IFN-g treatment (Fig. 2). iNOS mRNA was induced 2 h after treatment, reached a maximum at 6–12 h, then decreased slowly but remained at high levels at 24 and 48 h. AS mRNA began to increase at 6 h with a time lag, reached a maximum at 12 h (an approximate 5-fold increase), decreased at 24 h, and returned to control levels at 48 h. In contrast, mRNA for AL, another enzyme required for citrulline–arginine recycling, remained unchanged up to 24 h after treatment but slightly decreased at 48 h. The expression level of AL mRNA was several-fold lower than that seen in the liver where AL is highly expressed as part of the urea cycle. CAT-2 mRNA increased 2 h after treatment, reached a maximum at 12 h, then decreased and returned to very low levels at 48 h. In contrast, CAT-1 mRNA decreased gradually after treatment and became undetectable within 24 h.

3.3. Induction of iNOS and AS proteins by LPS and IFN-g In order to determine whether induction of iNOS and AS mRNAs paralleled expression of their respective proteins, we did immunoblot analysis of these proteins. Fig. 3A shows such an analysis of iNOS and AS proteins in MG5 cells treated with LPS and IFN-g for 24 h. iNOS protein, which was undetectable in untreated cells, was induced by LPS, less strongly by IFN-g, and most strongly by their combination. The level induced by LPS / IFN-g was somewhat higher than that seen in LPS / IFN-g activated RAW 264.7 macrophages. These protein levels correspond approximately to the observed mRNA levels. AS protein was induced strongly and to a similar extent by both LPS and IFN-g, but a combination of both agents did not lead to further induction. The induced levels were

similar to those seen in the liver. Again, these results correspond to the observed mRNA levels. Fig. 3B and C show the time course of induction of iNOS and AS proteins in MG5 cells stimulated with LPS / IFN-g. iNOS protein began to increase 6 h after stimulation, increased up to 24 h and was somewhat decreased at 48 h. AS protein was present before treatment and increased gradually up to 48 h. Thus, iNOS and AS proteins were induced later than their respective mRNAs, as would be expected.

3.4. The effect of LPS /IFN-g on microglial primary cultures Finally, we examined the effect of LPS / IFN-g on primary cultures of microglia isolated from rat brain. As shown in Fig. 4, primary microglial cells stimulated by LPS / IFN-g induced expression of iNOS, AS, and CAT-2 mRNAs, an observation similar to that for MG5 cells. The level of iNOS mRNA induced by LPS / IFN-g was higher than that seen in LPS / IFN-g activated RAW 264.7 cells used as a control. The mRNA levels for AS and CAT-2 induced by LPS / IFN-g were also higher than those observed in liver. In untreated primary culture cells, expression of AS and CAT-2 mRNAs was not evident, although both were weakly expressed in MG5 cells. Astrocyte-conditioned medium or cell passage may induce the weak expression of these mRNAs in MG5 cells. In summary, cells derived from microglia primary culture as well as the MG5 cell line displayed apparent co-induction following LPS / IFN-g treatment of mRNAs encoding iNOS, AS, and CAT-2, indicating that upregulation of a program of proteins involved in arginine metabolism is inherent to microglial cells.

4. Discussion Chronic glial activation is generally considered a consequence of neuronal death seen in neurodegenerative diseases such as Alzheimer’s, Huntington’s and Parkinson’s disease. Recent evidence, however, suggests that toxic substances released from glial cells might promote the neurodegenerative processes that occur in these diseases. Liveratore et al. [25] provided strong evidence that NO produced by glial cells participates in the cascade of events leading to the degeneration of neurons in mice that are rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. In their study, they showed that iNOS is induced in microglial cells, which then produce NO. Dehmer et al. [11] reported similar results. Furthermore, NO produced by iNOS in activated glial cells promotes neurodegenerative processes in amyotrophic lateral sclerosis [2]. Various inflammatory mediators such as endotoxin, cytokines, and Alzheimer’s disease-associated peptides

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Fig. 2. Time course of expression of mRNAs encoding iNOS, AS, AL, CAT-2, and CAT-1 in murine MG5 cells after LPS / IFN-g treatment. (A) Cells were treated with LPS (1 mg / ml) plus IFN-g (100 U / ml) for the indicated periods. Total RNAs (2.0 mg) were subjected to blot analysis. Positive controls (Con) are total RNA (1.0 mg) from LPS / IFN-g activated RAW 264.7 cells for iNOS mRNA, RNA (2.0 mg) from rat liver for AS, AL, and CAT-2 mRNAs, and RNA (2.0 mg) from rat brain for CAT-1 mRNA. One lane for CAT-1 mRNA at 0 h is missing. The bottom panel shows ethidium bromide staining of 28S and 18S rRNAs. (B) Results in A were quantified and are shown as means6ranges (n52), except for CAT-1 mRNA at 0 time (n51). The maximal values are set at 100%.

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Fig. 3. Immunoblot analysis of iNOS and AS in murine MG5 cells stimulated by LPS and IFN-g. (A) The cells were treated with LPS (1 mg / ml), IFN-g (100 U / ml), or their combination for 24 h. (B) The cells were treated with LPS (1 mg / ml) plus IFN-g (100 U / ml) for the indicated periods. Cell extracts (10 and 20 mg of protein for iNOS and AS, respectively) were subjected to immunoblot analysis using the anti-mouse iNOS monoclonal antibody (1:500 dilution) or anti-AS antiserum (1:500 dilution). Positive controls (Con) are extracts (5 mg of protein) from LPS / IFN-g activated RAW 264.7 cells for iNOS or from rat liver for AS. (C) Results in B were quantified and are shown as means6ranges (n52). The maximal values are set at 100%.

activate microglia and induce iNOS expression in vitro [7,13,14,28,40,44]. Kitamura et al. [22] reported that intrahippocampal injection of LPS plus IFN-g induced iNOS protein in microglial cells in vivo. Moreover, this

Fig. 4. Effect of LPS / IFN-g on mRNAs encoding iNOS, AS and CAT-2 in rat primary microglial cells. The cells were treated with LPS (1 mg / ml) plus IFN-g (100 U / ml) for the indicated periods. Total RNAs (1.0 mg) were subjected to blot analysis. Positive controls (Con) are total RNA (1.0 mg) from LPS / IFN-g activated RAW 264.7 cells for iNOS mRNA, and that from rat liver for AS and CAT-2 mRNAs. The bottom panel shows ethidium bromide staining of 28S and 18S rRNAs as a control for RNA loading.

treatment induced delayed neuronal apoptosis in rat hippocampus [26]. The present study demonstrates that AS is co-induced with iNOS in murine microglial MG5 cells by treatment with LPS and IFN-g at both mRNA and protein levels. A combination of the two agents promoted maximal induc-

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tion of both iNOS and AS. Induction of AS mRNA and protein was somewhat delayed compared to that of iNOS, suggesting that AS, which is present prior to stimulation, is sufficient for citrulline–arginine recycling at the early stages of stimulation. In contrast, induction of AL did not occur in MG5 cells by treatment with LPS and IFN-g, similar to observations reported in rat aortic smooth muscle cells [18], RAW 264.7 macrophages [33] and rat C6 glioma cells [47]. Basal levels of AL may be sufficient for the citrulline recycling reaction. Schmidlin and Wiesinger [39] reported that AS was induced predominantly by LPS / IFN-g in astrocytes in mixed glial primary cultures, while iNOS expression was localized primarily to microglia. This suggests the presence of an ‘intercellular’ citrulline–NO cycle, which includes production of NO and citrulline from arginine in microglia, transfer of citrulline to astrocytes and conversion to arginine, and transfer of arginine from astrocytes back to microglia. More recently, however, the same group reported that AS was expressed almost exclusively in microglial cells and at very low levels in astrocytes after striatal microinjection of LPS / IFN-g, suggesting that the citrulline–NO cycle operates in microglial cells in vivo, although co-induction of iNOS was not shown [20]. Thus, with respect to AS induction in microglia, there remained a discrepancy between the in vivo and in vitro results [20]. The present study clearly demonstrates co-induction of AS and iNOS following exposure to LPS / IFN-g in an activated microglial cell line and in primary cultures of microglia. Although whether an intercellular citrulline–NO cycle exists in brain is an open question, it is certain that microglia can regenerate arginine from citrulline. In the present study, we found that CAT-2 mRNA was also co-induced with iNOS mRNA in stimulated cells. The time course of CAT-2 mRNA induction was very similar to that of iNOS mRNA. In contrast, CAT-1 mRNA, which encodes a constitutively expressed isoform of CAT, decreased with time. This finding suggests that CAT-2 is more critical than CAT-1 for arginine uptake required for NO production in activated microglia. In murine macrophages [9,36] and vascular smooth muscle cells [15], cytokine treatment leads to parallel increases in iNOS and CAT-2 mRNAs, with little effect on CAT-1 mRNA levels. By contrast, in activated C6 glioma cells [47] and PC12 neuronal cells [48], iNOS and AS are markedly co-induced, whereas CAT-2 is not. Furthermore, arginine transport is not increased in activated C6 cells [38]. Therefore, the route of arginine supply for NO production may differ between microglia and astrocytes. Recently, functional analyses of CAT-2 arginine transport and the citrulline–NO cycle on sustained NO production were performed. Nicholson et al. [32] reported that cytokine activated macrophages from Cat2 2 / 2 mice revealed a 92% reduction in NO production and a 95% reduction in arginine uptake. On the other hand, Zhang et al. reported that AS and iNOS are co-induced in activated

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C6 glioma cells [47] and PC12 neuronal cells [48] and that these cells are able to produce NO from citrulline as well as from arginine, indicating that citrulline–arginine recycling occurs in these cells. A number of studies on microglia NO generation were reported ([10,42,43] for example). They have been shown that microglia from mouse and rat effectively secrete large amounts of NO, while those from human and hamster generate very small even no amounts of NO. In addition, LPS, a potent NO inducer in rodent cells, does not induce NO in human glial cells when it was used alone or even in combination with cytokines [8,24]. Thus, the induction and regulation of iNOS in human glial cells seems substantially different from that in rodents. However, Ding et al. [13] reported that human microglia are capable of synthesizing iNOS and NO in response to cytokine stimulation. In addition, iNOS mRNA expression has been demonstrated in the brains of multiple sclerosis patients [3,5]. Furthermore, in AIDS patients, iNOS protein was detected and its expression level was thought to be correlated with clinical dementia [1]. Clearly, more research is needed to elucidate the NO production from human microglia as well as human monocytes. But, this demonstration may be rather difficult in vivo, because astrocytes and microglia cooperatively function in generating the innate immune response of brains [27]. Taken together, our results strongly suggest that both arginine transport by CAT-2 and citrulline–arginine recycling are important for high-output production of NO in activated microglial cells. Intervention in the supply of arginine might represent a new therapeutic approach to the treatment of NO-mediated neurodegenerative damage in the brain.

Acknowledgements We thank Dr. W.Y. Zhang for technical advice. This work was supported, in part, by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (to H.N. and M.M.) and the Fugaku Trust for Medicinal Research (to H.N.).

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