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Originally published In Press as doi:10.1074/jbc.M708697200 on February 8, 2008

J. Biol. Chem., Vol. 283, Issue 16, 11004-11013, April 18, 2008
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An NADPH Sensor Protein (HSCARG) Down-regulates Nitric Oxide Synthesis by Association with Argininosuccinate Synthetase and Is Essential for Epithelial Cell Viability*Formula

Yanmei Zhao{ddagger}§, Jinfang Zhang{ddagger}§, Huiying Li{ddagger}§, Yiyu Li§, Jie Ren§, Ming Luo{ddagger}, and Xiaofeng Zheng{ddagger}§1

From the {ddagger}National Laboratory of Protein Engineering and Plant Genetic Engineering and §Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing 100871, China and the Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received for publication, October 22, 2007 , and in revised form, January 24, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
NADPH is an important cofactor in many biosynthesis pathways that control fundamental cellular processes. We recently determined the crystal structure of HSCARG, with functions previously unknown, and demonstrated it is an NADPH sensor, which undergoes restructuring and redistribution in response to changes of intracellular NADPH/NADP levels. In this study, we identified argininosuccinate synthetase (AS), a rate-limiting enzyme in nitric oxide synthesis, as capable of associating with HSCARG and demonstrated further that HSCARG decreased nitric oxide synthesis by down-regulating AS activity, whereas AS overexpression up-regulated hscarg mRNA transcription, suggesting a negative feedback mechanism. A decrease in the NADPH/NADP+ ratio, induced by dehydroepiandrosterone treatment, enhanced the interaction between HSCARG and AS, which resulted in stronger inhibition of AS activity and nitric oxide production. The dimerization region of HSCARG, amino acids 153-189, was identified to undergo critical interactions with AS. Furthermore, the viability of HSCARG RNA interference-treated epithelial cells decreased significantly, accompanied by an increase of the activity of caspase-3, which suggested that the loss of viability was because of apoptosis. These results indicate that HSCARG regulation of AS activity is crucial for maintaining the intracellular balance between redox state and nitric oxide levels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO),2 a cellular signaling molecule, has been shown to be involved in vascular regulation, autoimmunity, and neurotransmission and impacts diverse biological processes, including cell survival (1-7). The impaired production of NO can result in the vascular dysfunction, whereas overproduction of NO will induce some diseases such as the cerebral infarction, diabetes mellitus, and neurodegenerative disorders (7-11). Arginine is the sole amino acid substrate that is required for the production of NO (12, 13), and its regeneration from citrulline, the co-product of NO synthesis, is rate-limited by argininosuccinate synthetase (AS) (13-16). In addition, the reducing reagent donor, NADPH, and oxygen are necessary for NO production (13, 17). For this reason, NO production is not only limited by the regeneration of arginine but is also affected by the intracellular NADPH concentration, which requires cross-talk between the signaling pathways of NADPH and NO.

In addition to its well known function in energy metabolism, NADH, along with its phosphorylated relative NADPH, has been recognized as an important regulatory molecule. Together, their roles are crucial in signaling pathways that control fundamental cellular processes, such as transcription, regulation of calcium homeostasis, and apoptosis (18-20). NAD mainly exists in its oxidized state (NAD+), whereas NADP is largely found in its reduced form, NADPH (21, 22). The predominant function of NADP is to maintain a pool in its reduced form to ensure a rapid regeneration of the defense systems to protect cells from oxidative damage. NADPH holds a key position in the cellular oxidative defense systems and is essential to NO synthesis, NADPH oxidase, and detoxifying pathways (23, 24). Because change of the intracellular ratio of NADPH/NADP+ affects many critical cellular metabolic processes, understanding the mechanisms and subcellular compartmentation of NADP+ and NADPH generation and their roles in cell physiology has attracted more attention recently (24, 25).

NADP exerts its function by associating with and regulating NADP-dependent proteins. The crystal structure of HSCARG, which we determined, revealed that an asymmetrical dimer forms with one protein molecule bound with an NADP(H) molecule and the other unoccupied, and the two protein molecules have dramatically different conformation (26). Changes in intracellular NADPH/NADP+ levels induced the redistribution of HSCARG, which associates with intermediate filaments, to spread to the cytoplasm and nucleus, suggesting that HSCARG is an NADPH sensor (26). The structure switch of HSCARG upon NADP(H) binding suggested a new regulatory mechanism in response to changes of intracellular NADPH or NADP levels. However, many issues regarding the regulating mechanisms of HSCARG still remain unclear, e.g. how does HSCARG regulate intracellular redox balance, whether HSCARG modulates the other biosynthesis processes, and so on, and requires further investigation.

In this study, we identified AS, one of the rate-limiting enzymes in NO synthesis, as a partner that could associate with HSCARG. The further assays indicated that HSCARG inhibited the production of NO by down-regulating AS activity. Decreased NADPH/NADP+ levels, which were induced by DHEA treatment, enhanced the interaction between HSCARG and AS and led to stronger inhibition of AS activity and NO production. The amino acids 153-189 of HSCARG, which are part of the NADPH binding sites and the dimerization interface, were found to be critical for AS interaction, which is consistent with the observation that NADPH concentration changes affected AS association with HSCARG. Furthermore, HSCARG was found to be essential for cell viability. Overexpression of HSCARG promoted cell proliferation, whereas the knockdown of HSCARG by RNAi treatment induced cell apoptosis. Thus, HSCARG is a regulatory protein that can sense intracellular levels of NADPH and balance NO production through the regulation of AS activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Plasmid Construction—HeLa and human embryonic kidney HEK293T cells were maintained in Iscove's modified Dulbecco's medium (Invitrogen), supplemented with 10% fetal calf serum (Hyclone), at 37 °C and 5% CO2.

AS and HSCARG cDNAs were inserted into prokaryotic expression vectors, pGEX-4T-1 (BamHI and NotI) and pET21DEST (Gateway cloning system), respectively, as well as into eukaryotic expression vectors, pRK-HA and pRK-FLAG, at the SalI and NotI enzyme restriction sites, respectively.

To ascertain the critical region of HSCARG that binds to AS protein, we constructed a series of HSCARG truncates (Fig. 5A). HSCARG amino acid truncates 1-219, 1-189, 153-299, 190-299, and 220-299 were amplified from full-length HSCARG by PCR using corresponding primers. Amplified products were then inserted into the eukaryotic expression vector pRK-FLAG at the SalI and NotI enzyme restriction sites. The resulting plasmids were verified by DNA sequencing.

Immunofluorescence Assay—The general determination for the subcellular localization of proteins was performed as described previously (26). Co-localization of HSCARG with AS was performed in HeLa cells that were co-transfected with FLAG-fused HSCARG and HA-tagged AS expression vectors. The cells were incubated with rabbit anti-HSCARG and monoclonal anti-HA antibody (Sigma), respectively, followed by fluorescent-labeled secondary antibodies (1:100 in phosphate-buffered saline), goat anti-rabbit IgG-TRITC, or goat antimouse IgG-fluorescein isothiocyanate and visualized by a laser scanning confocal microscope (Zeiss LSM 510). Nuclear DNA was stained with 1 µg/ml DAPI.

To examine HSCARG redistribution induced by AS overexpression or SNAP treatment, HeLa cells were either transfected with AS expression vector alone for 48 h or cultured with medium containing 100, 200, or 500 µM SNAP for 24 h. Cells transfected with empty vector or cultured without SNAP were used as controls, respectively. Cells were then incubated with rabbit polyclonal antibody, specific to HSCARG, followed by fluorescein isothiocyanate-labeled goat anti-rabbit IgG (Zhongshan Biotech). Cells were counterstained with DAPI and visualized using confocal microscope (Zeiss LSM 510).

To investigate whether the effect of SNAP on HSCARG distribution is cGMP-dependent, cells were treated with 500 µM SNAP in the presence or absence of 5 µM LY83583, a guanylyl cyclase inhibitor, for about 20 h. Then the subcellular localization of HSCARG was detected following the method described above.

Nuclear and Cytoplasmic Extracts Preparation and Detection—Highly purified nuclear extractions of the cells that were treated with or without SNAP were obtained by using the nuclear-cytosol extraction kit (Applygen Technologies, Inc.), following the protocol of the manufacturer. Briefly, cells were collected by centrifugation and resuspended in Cytosol Extraction Buffer A. After incubation on ice for 10 min, Cytosol Extraction Buffer B was added. Cells were then left on ice for 1 min and centrifuged at 1,000 rpm for 5 min at 4 °C to separate cytosol from nuclei. The supernatant (cytosol extract) was transferred to a new tube and stored at -70 °C in 20% glycerol. The pellet containing crude nuclei was washed with Cytosol Extraction Buffer A, centrifuged, and resuspended in pre-cold Nuclear Extract Buffer. After incubation on ice for 30 min, the samples were centrifuged at 13,000 rpm for 5 min at 4 °C. The supernatant containing nuclear extracts was transferred to a new tube. The nuclear and cytoplasmic extracts were subjected to Western blotting analyses using the rabbit polyclonal anti-HSCARG. The Cdc2 and actin proteins were used as loading controls.

In Vitro Pulldown Assay—Recombinant GST-fused AS and His-tagged HSCARG proteins were purified as described previously (27). The His pulldown assay was carried out by incubating nickel resin-associated recombinant His-tagged HSCARG with GST-fused AS protein at 4 °C for 3 h. The unbound proteins were removed using Tris-HCl buffer (pH 7.5). The remaining protein complex was eluted with 200 mM imidazole and separated using SDS-PAGE, followed by Western blot with anti-GST antibody. Control experiments were performed using only nickel resin-His-HSCARG-GST or nickel resin-GST-AS. GST pulldown assays were performed similarly by using GSH-Sepharose resin (BD Biosciences)-associated GST-fused AS protein and polyclonal anti-HSCARG antibody for Western blot detection.

To study the effect of NADPH on the interaction between AS and HSCARG, a GST pulldown assay was performed. GSH-Sepharose resin, which was bound to 10 µg of GST or GST-AS protein, was incubated with 10 µg of HSCARG proteins that contained various concentrations of NADPH for 3 h at 4 °C. The bound protein complex was subsequently eluted with 10 mM GSH buffer and detected by Western blot analysis using anti-GST and anti-HSCARG antibodies, respectively.

Co-immunoprecipitation and Western Blot Analysis—To examine HSCARG-AS interactions, co-immunoprecipitation and Western blot analyses were carried out according to procedures described previously (26).

RNAi Treatment and Verification—Four separate RNAi duplexes of HSCARG, hscarg1-4, were designed and inserted into the pGCsi vector. The target sequences for HSCARG cDNA are as follows: 1) CCTCATGGTGGACAAGAAA; 2) GCCTCCACTATGTGGTCTA; 3) CCAAGATGACCAGGTCATC; and 4) CCTTCATCGTGACCAATTA. The plasmids were subsequently transfected into HEK293T cells using Lipofectamine 2000, according to the manufacturer's protocol (Invitrogen). pGCsi-nonsilence was used as a negative control. Cells were collected 48 h post-transfection; the total RNAs and proteins were extracted, followed by RT-PCR and Western blot analyses to examine the effects of RNAi at both the mRNA and protein levels (28).


Figure 1
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  		FIGURE 1.
Identification of AS as an HSCARG-associated protein. A, lysates of HeLa cells were precipitated by anti-HSCARG sera and analyzed on SDS-PAGE. B, co-immunoprecipitation assays were performed to examine the interaction of HSCARG with AS. HEK293T cells were co-transfected with expression plasmids of FLAG-tagged HSCARG and HA-tagged AS, and cell lysates were immunoprecipitated (IP) with anti-FLAG, anti-HA, or control IgG, respectively, and then analyzed by Western blot with anti-HA plus anti-FLAG antibodies. C, in vitro pulldown assays confirmed direct interaction between HSCARG and AS proteins. His pulldown (left) and GST pulldown (right) were performed. Purified His-HSCARG bound to nickel resin (left) or GST-AS bound to GSH-Sepharose 4B (right) was incubated with purified GST-AS or His-HSCARG protein. The bound complex was then eluted with 200 mM imidazole or 10 mM GSH, respectively, and analyzed by Western blot with anti-GST (left) or anti-HSCARG (right) antibody. D, co-localization of HSCARG with AS in HeLa cells. The HeLa cells were co-transfected with expression plasmids of FLAG-tagged HSCARG and HA-tagged AS and double-stained with anti-HA (green) and anti-HSCARG (red) antibodies. DNA was stained by DAPI (blue). Scale bars equal 10 µm.

 
In Vitro AS Activity Assay—To examine the effect of HSCARG protein on AS activity, we assessed the accumulation of inorganic phosphate, which was produced as a reaction to AS catalyzing argininosuccinate synthesis, in the absence or presence of HSCARG. Equal amounts of AS and HSCARG proteins were added to each reaction, which contained 50 mM Tris-HCl (pH 7.8), 0.5 mM asparagine, 0.4 mM citrulline, 3 mM MgCl2, 1 mM ATP, and 2 units/ml pyrophosphatase in a final volume of 30 µl. Reactions were incubated for 20 min at 37 °C in 96-well microtiter plates. Subsequently, 120 µl of fluorescence reaction mixture (0.5% sulfuric acid, 0.25 mM ammonium molybdate, 0.05 mM hydrochloric thiamine) was added and incubated for 5 min at 37 °C, followed by termination with 120 µl of borax (100 mM). The accumulation of inorganic phosphate in the samples was measured on Synergy HT (BioTek Instrument) with excitation at 360 nm and emission at 460 nm. Results were compared with an inorganic phosphate standard curve that was made with KH2PO4.

Detection of NO Production—A fluorometric method was used to determine cellular NO production released into the medium (29). HSCARG was either overexpressed or knocked down by transfection of the corresponding plasmids into HEK293T cells in 6-well plates, respectively. Thirty six hours after transfection, the medium containing 10% fetal calf serum was removed, and 1 ml of Hanks' balanced salt solution buffer (1.26 mM CaCl2·2H2O, 5 mM KCl, 0.4 mM KH2PO4, 0.8 mM MgSO4·7H2O, 137 mM NaCl, 4 mM NaHCO3, 0.3 mM Na2HPO4, 5.5 mM glucose) was added, in the presence or absence of 100 µM DHEA. Media were then collected after 12 h, and the living cells were counted after trypan blue staining.

10 µl of freshly made 0.05 mg/ml 2,3-diaminonaphthablene (dissolved in 0.62 M HCl) was added to 100 µl of medium, followed by incubation at 20 °C for 10 min. The reaction was stopped by adding 5 µl of 2.8 M NaOH. The intensity of the fluorescent signal produced by the product 2,3-diaminonaphthotriazole was measured by excitation at 365 nm and emission at 450 nm on the Synergy HT (BioTek Instrument). To calculate the production of NO, NaNO2 was used to make the standard curve. The NO released by per 106 living cells was calculated. The effects of HSCARG truncations on NO production were examined followed the same procedure.

To check the influence of HSCARG on NO production in macrophage RAW 264.7 cells, cells were infected with or without the HSCARG recombinant adenoviruses. 48 h after infection, the NO released was determined as described above.

Cell Viability Assay—HEK293T cells were transfected with pRK-FLAG-HSCARG, HSCARG RNAi plasmids, and corresponding control vectors, respectively. Meanwhile, part of the HSCARG-depleted cells were either treated with 500 µM L-N(G)-nitroarginine methyl ester (L-NAME), a nonselective nitric-oxide synthase inhibitor, or 1 or 3 mM glutathione monoethyl ester (GSE), a well established membrane-permeable GSH donor. Forty hours later, cell viability was determined by the CellTiter 96® AQueous nonradioactive cell proliferation assay, according to the manufacturer's instruction (Promega). Cells grown in a 96-well plate were incubated with tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and an electron coupling reagent (phenazine methosulfate) for 1-4 h at 37 °C. The absorbance of the converted formazan by viable cells was measured at 490 nm. A standard curve was made by using a concentration series of cells, and cell viability was calculated subsequent to the different treatments.

Apoptosis Detection by Measurement of the Caspase 3 Activity—To measure the activity of caspase-3 (DEVDase), HEK293T cells were transfected with effective HSCARG RNAi plasmid and its control nonsilence vector, respectively. Forty eight hours after transfection, cells were collected, and the activity of caspase-3 was measured by using the CaspACETM assay system from Promega, according to the manufacturer's instruction (Promega). The p-nitroanilide released from the substrate, upon cleavage with DEVDase, was monitored by a spectrophotometer at 405 nm (DNM-9602, Prolong). The concentration of total proteins of each extract was also determined by the bicinchoninic acid (BCA) method (Applygen Technologies, Inc.), using a bovine serum albumin standard curve. The relative level of p-nitroanilide, which indicated caspase-3 activity, was normalized against the protein concentration.


Figure 2
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  		FIGURE 2.
High concentration of NO induces redistribution and up-regulates expression of HSCARG. A, overexpression of AS changed the localization of HSCARG in HeLa cells. 48 h after transfection with AS expression plasmid or empty vector (control), immunofluorescence was performed using anti-HSCARG antibody and then visualized by confocal microscope. B, SNAP treatment resulted in a change in HSCARG subcellular localization. HeLa cells were treated with 500 µM SNAP for 24 h, and the localization of HSCARG was detected by anti-HSCARG antibody. Scale bars equal 10 µm (A and B). C, Western blot analysis of the SNAP-induced HSCARG nuclear translocation. Cells were treated with or without 500 µM SNAP for 24 h, and then the nuclear and cytoplasmic extracts were subjected to Western blot analyses using the rabbit polyclonal anti-HSCARG. The Cdc2 and actin proteins were used as loading controls. The blot shown represents three independent experiments, and quantification of HSCARG protein amounts was performed using AlphaEase software (Alpha Innotech Corp.). D, both the overexpression of AS and SNAP treatment increased mRNA transcription of hscarg in HEK293T cells. E, HSCARG mRNA levels were up-regulated by proinflammatory cytokines, IL-1β and TNF{alpha}. RT-PCR was performed to examine the influence of the various treatments on hscarg expression (D and E). HEK293T cells were transfected either with AS expression plasmid to overexpress AS protein or treated with 500 µM SNAP for 24 h or 25 ng/ml cytokine (IL-1β or TNF{alpha}) for 48 h. The total RNAs were extracted, and RT-PCR was performed. Actin was used to correct for intersample variations. Images shown represent three independent experiments. Quantification of hscarg levels was performed using AlphaEase software (Alpha Innotech Corp.).

 
GSH Analysis—Cells that transfected with HSCARG RNAi plasmid were collected, and the GSH content of the cell lysates was determined by using a GSH assay kit (Beyond) followed the manufacturer's protocol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
AS, One of the Rate-limiting Enzymes of NO Production, Was Identified as the Partner That Interacts Directly with HSCARG—To identify the target proteins with which HSCARG may associate, immunoprecipitation experiments using anti-HSCARG sera were performed. Several specific bands were observed in SDS-PAGE (Fig. 1A), and mass spectrometry analysis revealed that AS, one of the rate-limiting enzymes in NO production, may be one of the target proteins that associate with HSCARG. The interaction between HSCARG and AS was confirmed by co-immunoprecipitation (Fig. 1B) (26). To further verify the physical interaction of these two proteins, an in vitro pulldown assay was performed using purified HSCARG and AS proteins (Fig. 1C). In the His pulldown assay, GST-tagged AS indicated an apparent affinity for HSCARG (Fig. 1C, left). In GSH-Sepharose resin, which was loaded with GST-AS, His-tagged HSCARG was found to bind at high levels (Fig. 1C, right).

Immunofluorescence analysis, using antibodies specific to HSCARG and HA, illustrated that HSCARG co-localized with AS protein. As shown in Fig. 1D, there was an obvious overlap between HSCARG (red) and AS (green) in the cytoplasm, mainly surrounding the nucleus. This result further validated the intracellular interaction of HSCARG with AS. Unexpectedly, we found that a fraction of HSCARG entered the nucleus when it was co-overexpressed with AS (Fig. 1D), whereas our previous studies showed that under normal culture conditions HSCARG was located primarily in the cytoplasm surrounding the nucleus (26). This new observation prompted us to examine the subcellular localization of HSCARG in AS-overexpressed cells.

Increase of NO Affects the Subcellular Localization and Expression of HSCARG—To assess whether overexpression of AS could alter the subcellular localization of HSCARG, cells were transfected with an AS expression plasmid, and the localization of HSCARG was mapped using anti-HSCARG polyclonal antibody. Cells transfected with an empty vector were used as controls. Compared with control cells, a fraction of HSCARG was clearly present in the nucleus of AS-transfected cells (Fig. 2A), indicating that overexpression of AS induced redistribution of HSCARG to the nucleus, where HSCARG might exert a potential regulatory role in gene activation by analogy to C-terminal binding protein (30, 31).

Recent studies demonstrated that AS is essential for endothelial NO production, and overexpression of AS increases the production of NO (13). To test whether HSCARG redistribution induced by AS overexpression is because of an increase in NO production modulated by AS, cells were treated with different concentrations (100, 200, and 500 µM) of SNAP, which is a NO donor, followed by an examination of the subcellular localization of HSCARG. In cells treated with 200 and 500 µM SNAP, NO released by SNAP indeed resulted in the re-localization of HSCARG; a visible amount of HSCARG, especially in the cells treated with 500 µM SNAP, shifted to the nucleus, compared with control cells devoid of the SNAP treatment (Fig. 2B and supplemental Fig. 1). In addition, HSCARG fluorescence intensity increased as well (Fig. 2B). Western blot analysis of the nuclear and cytoplasmic extracts following SNAP treatment revealed that in the nuclei of unstimulated cells, HSCARG can only be detected at a very low level, whereas in the SNAP-stimulated cells, the concentration of HSCARG is much higher (Fig. 2C), which further supports our immunofluorescence observations. These results demonstrated that an intracellular NO increase could induce redistribution of HSCARG, similar to the effects observed with DHEA (26).


Figure 3
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  		FIGURE 3.
HSCARG negatively regulates AS activity and NO production in epithelial cells. A, HSCARG overexpression inhibited production of NO. HEK293T cells were transfected with empty pRK vector (control), HSCARG or AS expression plasmids, or co-transfected with HSCARG and AS expression plasmids. The NO production in medium was examined. The experiments were repeated five times independently, and values were reported as mean ± S.D.; p value was determined by Student's t test. B, AS activity was inhibited by HSCARG directly. AS protein (1, 2, and 4 µg) was used to detect activity in the presence or absence of equal amounts of HSCARG protein. The values represent the means of five independent experiments, and bars denote the standard deviation. C, screening of effective HSCARG RNAi expression plasmids. HEK293T cells were transfected with four hscarg RNAi plasmids (hscarg 1-4, lanes 1-4) and nonsilence negative control (lane 5), respectively. 48 h later, the mRNA and protein expressions of HSCARG were examined by RT-PCR and Western blot analyses (upper panels). Actin was used to correct for intersample variations of RNA or protein. Images shown are representative from three independent experiments. Quantification of hscarg levels was performed using AlphaEase software (Alpha Innotech Corp.) (lower panel). D, a decrease in HSCARG expression facilitated NO production in HEK293T cells. Cells were transfected with HSCARG RNAi plasmids 3, 4, 1, and control vector, and the synthesis of NO was measured. The values represent the means of five independent experiments, and bars denote the standard deviation, and p value was ascertained by the Student's t test.

 
Because production of NO induces cGMP elevation (32, 33), to examine whether the redistribution of HSCARG induced by SNAP treatment is because of the increase of cGMP, cells were treated with SNAP in the presence or absence of LY83583, a cGMP inhibitor, and then the subcellular localization of HSCARG was determined. Our results showed that the addition of LY83583 inhibited the accumulation of HSCARG in the nucleus induced by SNAP treatment, and less HSCARG was shown in nuclear cells compared with the cells treated with SNAP alone (supplemental Fig. 2), suggesting that the effect of SNAP on the HSCARG redistribution is cGMP-dependent.

To investigate the effects of AS overexpression and SNAP treatment on gene expression of HSCARG, the total RNAs of the corresponding cells were extracted, and RT-PCR analyses were performed. Results demonstrated that not only the overexpression of AS but also the SNAP treatment could up-regulate hscarg expression (Fig. 2D), which was consistent with the immunofluorescence analysis (Fig. 2, A and B). Because NO production has been shown to be closely related to inflammation, and the proinflammatory cytokine IL-1β has previously been shown to up-regulate expression and activity of AS (34), we performed additional experiments to determine whether proinflammatory cytokines, such as IL-1β and TNF{alpha}, could also affect the expression of hscarg. RT-PCR analyses revealed that both IL-1β and TNF{alpha} induced an increase in hscarg gene expression at the transcriptional level (Fig. 2E), indicating that HSCARG might also be involved in the regulation of inflammation. This is associated with the vital roles that NADPH plays in the immune defense system (23, 24), and further work is required to delineate its mechanism.

HSCARG Is a Negative Regulator of NO Production and AS Activity—Through the experiments presented above, we found that HSCARG directly interacts with AS (Fig. 1), and its functions are closely related to NO production, as shown by NO-induced redistribution of HSCARG (Fig. 2). Subsequently, to test whether HSCARG expression can modulate NO production through the regulation of AS activity, we examined the effect of HSCARG overexpression on NO production in cells that were transfected with or without an AS expression vector. Trypan blue exclusion analysis was performed to estimate the cell number, and NO production was measured by nitrite production per 1 x 106 cells. Compared with the control cells, HSCARG overexpression resulted in a 63% decrease in NO production (p < 0.01) (Fig. 3A). NO production was indeed up-regulated in AS-overexpressing cells; however, HSCARG and AS co-overexpression dampened the NO increase resulting from AS overexpression (p < 0.05) (Fig. 3A). These data demonstrated that HSCARG down-regulated the production of NO, most likely through inhibition of AS activity. Moreover, in vitro AS activity assays indicated similar results. HSCARG strongly inhibited the activity of AS, as shown in Fig. 3B, confirming that HSCARG can reduce NO production by down-regulating AS activity.

Similar to what we showed in the epithelial cell above, the inhibition of HSCARG on NO production was also observed in a macrophage cell line, RAW 264.7 cells. Compared with the control, infection of RAW 264.7 cells with HSCARG recombinant adenoviruses resulted in a decrease of NO production (supplemental Fig. 3), suggesting that the regulation of HSCARG on NO production is similar in HEK293T cells and RAW264.7 cells.


Figure 4
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  		FIGURE 4.
Changes in NADPH concentration influence the interaction between HSCARG and AS and the regulation of HSCARG on AS activity. A, effects of DHEA and SNAP treatments on the association between HSCARG and AS were examined by co-immunoprecipitation and Western blot analyses. HEK293T cells were co-transfected with expression plasmids for FLAG-tagged HSCARG and HA-tagged AS for 24 h, and then treated with 100 µM DHEA or 500 µM SNAP for 24 h. Cell lysates were prepared and immunoprecipitated (IP) with anti-FLAG monoclonal antibody, and then analyzed by Western blot with anti-HA (left) and anti-FLAG (right) monoclonal antibodies. The expression of HSCARG and AS was confirmed by Western blot analysis with anti-FLAG (right) and anti-HA (left) antibodies, respectively. Cells transfected with HA-tagged AS alone served as negative controls in the anti-FLAG antibody immunoprecipitation assays, whereas cells co-transfected with FLAG-tagged HSCARG and HA-tagged AS were used as controls in the absence of DHEA or SNAP treatment. B, the addition of NADPH reduces the interaction of HSCARG with AS in vitro. GST pulldown assays were performed in the presence or absence of NADPH. Varying NADPH concentrations were added to 10 µg of purified HSCARG (0.3 µM) and incubated with 10 µg of GST or GST-AS-bound GSH resin. The bound proteins were eluted with 10 mM GSH and subjected to Western blot analysis using anti-GST monoclonal antibody and anti-HSCARG polyclonal antibody, respectively. Identical GST-AS, which bound to GSH resin, was used as a quantity control. C, effect of decreased NADPH, induced by DHEA treatment, on the ability of HSCARG to inhibit NO production. HEK293T cells were transfected with empty vector (control), HSCARG expression vector, AS expression vector, or co-transfected with HSCARG and AS expression vectors for 24 h, followed by treatment for 24 h with (+) or without (-) 100 µM DHEA, and NO production was assessed. The experiments were repeated five times independently, and the average is presented. The bars denote the standard deviation.

 
To further elucidate the role of HSCARG in the regulation of NO production, RNA interference experiments were performed to selectively knockdown HSCARG expression. Cells were transfected with four different HSCARG RNAi plasmids, pGCsi-hscarg1-4, and pGCsi-nonsilence, respectively. RT-PCR analysis showed that HSCARG RNAi treatment, especially hscarg3 and -4, specifically reduced HSCARG mRNA levels (Fig. 3C, upper left). HSCARG protein levels were examined by immunoblot analysis, and results indicated that HSCARG RNAi treatment with hscarg3 and -4 decreased cellular HSCARG levels obviously (Fig. 3C, upper right), which is consistent with RT-PCR analysis. The photodensitometric analysis of these bands was shown in the lower panel of Fig. 3C.

NO production in HSCARG-depleted cells that were transfected with pGCsi-hscarg3 or -4 was analyzed. Results showed that the knockdown of HSCARG resulted in a significant increase of NO production (p < 0.01) (Fig. 3D), whereas pGCsi-hscarg1 transfection, which showed no obvious reduction of hscarg mRNA levels (Fig. 3C), did not result in an enhancement of NO production when compared with control cells transfected with pGCsi-nonsilence (Fig. 3D). These data suggest that HSCARG is a negative modulator of NO production in epithelial cells.

An Altered NADPH/NADP+ Ratio Affects the Interaction between HSCARG and AS and Further Modulates AS Activity—DHEA is an uncompetitive inhibitor of glucose-6-phosphate dehydrogenase, which can result in a 30-40% decrease of intracellular NADPH/NADP+ ratio (35). Because DHEA treatment induces redistribution of HSCARG within cells (26), we investigated whether the altered NADPH/NADP+ ratio could affect the interaction between HSCARG and AS. If so, the question of whether a change in the association between HSCARG and AS would further influence inhibition of AS activity by HSCARG should be addressed. To answer these questions, cells were co-transfected with FLAG-tagged HSCARG and HA-tagged AS plasmids and cultured for 24 h, followed by further 24 h incubation in the absence (control) or presence of 100 µM DHEA. Co-immunoprecipitation experiments were performed to detect changes between HSCARG and AS protein interactions. As shown in Fig. 4A, compared with the control, more AS was co-precipitated in cells treated with DHEA (Fig. 4A, left), whereas similar amounts of HSCARG were precipitated by anti-FLAG antibody (Fig. 4A, right). These data indicated that a decrease of the intracellular NADPH/NADP+ ratio induced by DHEA enhanced the interaction between HSCARG and AS. In addition, we treated the cells with SNAP, and the results showed that, compared with control cells, less AS was co-precipitated by anti-FLAG antibodies in SNAP-treated cells (Fig. 4A). A possible explanation is that the exogenous NO released by SNAP inhibited the AS activity by S-nitrosylation modification and further attenuated the endogenous NO production and increased the intracellular NADPH level (7, 34, 36), and the increase of NADPH level may result in the impairment of the interaction between HSCARG and AS.

To further confirm that NADPH concentration changes directly affect the association of AS with HSCARG, different concentrations of NADPH were added to the mixture of purified HSCARG and AS proteins in vitro, followed by GST pulldown and Western blot assays. As shown in Fig. 4B, 0.3 µM NADPH apparently inhibited the interaction between HSCARG and AS. With the increase of the NADPH concentration, the interaction between HSCARG and AS decreased even further and was completely abolished when NADPH concentration reached 30 µM. These results suggest that the addition of NADPH indeed impaired the association of HSCARG with AS, and a possible reason is that NADPH competes with the binding site of AS to HSCARG.


Figure 5
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  		FIGURE 5.
Amino acids (aa) 153-189 of HSCARG serve as the critical region that interacts with AS. A, schematic representation of HSCARG-truncated mutations. B, co-immunoprecipitation assays were performed to examine the interaction between various HSCARG truncates and AS proteins. HEK293T cells were co-transfected with expression plasmids for HA-tagged AS and FLAG-tagged HSCARG truncates for 48 h. Cell extracts were prepared and immunoprecipitated (IP) with anti-FLAG monoclonal antibody. The precipitated complex (lower) and the expression of AS in cell lysates (upper) were detected by Western blot analysis using an anti-HA monoclonal antibody. C, expression of the HSCARG truncates was confirmed by Western blot assay using monoclonal anti-FLAG. D, effects of HSCARG truncations on NO production. HEK293T cells were transfected with empty pRK vector (control), HSCARG, or HSCARG truncates, including 1-189, 153-299, and 190-299 expression plasmids. The NO production in medium was examined as described under "Experimental Procedures." The experiments were performed three times independently, and values were reported as mean ± S.D. **, p < 0.01, cells transfected with HSCARG truncations were compared with cells transfected with control.

 
Furthermore, we examined whether enhanced association between HSACRG and AS, which was triggered by a reduced NADPH/NADP+ ratio, could further reduce AS activity that was down-regulated by HSCARG. NO production was compared with cells that overexpressed HSCARG, AS, or co-overexpressed HSCARG and AS, in the absence or presence of DHEA. In the absence of DHEA treatment, HSCARG overexpression led to a 60% decrease in NO production, whereas in the presence of DHEA, a 90% decrease in NO production was observed when compared with the control (Fig. 4C). Similarly, when cells that co-overexpressed HSCARG and AS were compared with cells that overexpressed AS, inhibition of AS by HSCARG resulted in a 37% decrease of NO production in the absence of DHEA, whereas this reduction increased to 52% with DHEA treatment (Fig. 4C).

HSCARG Interacts with AS, Mainly through Amino Acids 153-189—To elucidate the critical region in HSCARG that interacts with AS protein, a series of truncates, amino acids 1-219, 1-189, 153-299, 190-299, and 220-299, were designed to avoid disruption of the secondary structure and were inserted into the eukaryotic expression vector, pRK-FLAG (Fig. 5A). Cell lysates from cells, which were co-transfected with expression plasmids for each FLAG-tagged HSCARG truncate and HA-tagged AS, were subjected to co-immunoprecipitation. As shown in Fig. 5B, the N-terminal domain that contained truncates 1-219 and 1-189 was able to interact with AS, whereas truncates 190-299 and 220-299 did not co-precipitate with AS. Truncate 153-299, which contained part of the N-terminal domain and C-terminal domain, interacted considerably with the AS protein, indicating that the region of 153-189 of HSCARG is the crucial region responsible for association with AS. The expression of full-length HSCARG and truncates was verified by Western blot (Fig. 5C).

To further study the effects of these HSCARG truncations on NO production, we examined the NO production in cells that overexpressed HSCARG truncates 1-189, 153-299, or 190-299. As shown in Fig. 5D, truncates 1-189 and 153-299 that contain region 153-189 inhibited NO production, similar to full-length HSCARG, whereas truncate 190-299 that does not contain the 153-189 region had no obvious effect on the NO production. These data further support that region 153-189 is required for the interaction between HSCARG and AS. Only those truncations that contain region 153-189 can interact with AS stably and inhibit NO production.

Expression of HSCARG Is Essential for Cell Viability—Intracellular NO levels are closely related to cell apoptosis, and previous studies have shown that NO serves a dual function in cell viability, depending on the concentration and tissue type. NO can induce apoptosis at high concentrations yet inhibit apoptosis at low concentrations (32, 37-41). To determine the physiological role of HSCARG, we detected the cell viability of HEK293T cells that were either transfected with the HSCARG expression vector or treated with HSCARG RNAi. Interestingly, HSCARG overexpression led to an increase in cell viability when compared with the control (p < 0.05), whereas the knockdown of HSCARG through RNAi treatment resulted in a noticeable decrease in cell viability (p < 0.01) compared with the control (Fig. 6A), suggesting that HSCARG is essential for cell survival.

To further elucidate whether apoptosis was responsible for the observed loss of cell viability, we examined caspase-3 activity subsequent to HSCARG RNAi treatment. Compared with the control, caspase-3 activity increased by 3-fold within cells treated with HSCARG RNAi, and this activity could be suppressed by the addition of Z-VAD-FMK, a caspase-3 inhibitor (Fig. 6B), which demonstrates that a reduction in HSCARG expression induced caspase-3-dependent apoptosis.


Figure 6
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  		FIGURE 6.
Effect of HSCARG on cell viability. A, HEK293T cells were transfected with pRK-HSCARG, HSCARG RNAi plasmids, and corresponding control vectors, respectively. Forty eight hours after transfection, cell viability was measured. The values represent the means of four independent experiments, and the bars denote the standard deviation. The p value was ascertained by the Student's t test. B, caspase-3 activity in partially HSCARG-depleted HEK293T cells, in the absence or presence of caspase-3 inhibitor, Z-VAD-FMK. Following hscarg RNAi treatment, the caspase-3 activity of cell extracts was determined. The experiments were independently repeated three times, and values represent the means ± S.D. **, p < 0.01 versus samples that were transfected with pGCsi-nonsilence plasmid; #, p < 0.01 versus samples without Z-VAD-FMK. C, effect of L-NAME on cell viability of HEK293T cells. Cells were treated with HSCARG siRNA and 500 µML-NAME for 48 h, and then the cell viabilities were measured. Results shown are the means of four independent experiments. The p value was ascertained by the Student's t test. *, p < 0.05. RNAi-treated cells were compared with control cells. #, p < 0.05. Cells treated with RNAi and L-NAME were compared with cells treated with RNAi. D, GSH level in HSCARG-depleted HEK293T cells. Cells were transfected with HSCARG RNAi plasmid for 48 h, and then the GSH level was determined. Cells transfected with nonsilencing plasmid were used as control. *, p < 0.001. siRNA-treated cells were compared with control. E, the addition of GSE restored the loss of cell viability upon RNAi-treated HEK293T cells. Following HSCARG RNAi transfection, 1 mM (+) or 3 mM (++) GSE was added to increase the cellular GSH level. After 40 h of incubation, cell viabilities were measured. The values represent the means of four independent experiments, and the bars denote the standard deviation. The p value (*, p < 0.01) was ascertained by the Student's t test.

 
To examine whether the increase in NO seen upon HSCARG siRNA treatment is sufficient to induce apoptosis, cell viability was determined in HSCARG-depleted cells that were exposed to 500 µML-NAME, a nonselective nitric-oxide synthase inhibitor. As shown in Fig. 6C, the addition of L-NAME restored the cell viability lost upon HSCARG siRNA treatment, suggesting that the increase in NO seen upon HSCARG siRNA treatment was sufficient to induce apoptosis.

GSH is a major thiol-disulfide redox buffer and can protect cells against apoptosis (42, 43). Cellular GSH depletion has been found to be associated with decreased cell proliferation (42-45). To investigate whether the loss of cell viability upon HSCARG depletion is associated with a decrease of GSH, we determined the GSH levels of cells after HSCARG RNAi treatment, and the result showed that the GSH level in RNAi-treated cells was lower than that of the control cells (Fig. 6D). Further experiments were performed to examine whether the addition of GSE, a membrane-permeable GSH donor, can restore the loss of the cell viability induced by HSCARG RNAi treatment. Indeed, for the cells transfected with HSCARG RNAi plasmids, the addition of GSE visibly increased cell viability (Fig. 6E), suggesting that the decrease of cell proliferation caused by HSCARG depletion is associated with GSH depletion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
NO signaling results in pro-apoptotic or anti-apoptotic effects on cells, depending on a number of factors (32, 37, 40). One of these closely related conditions is the cellular redox status (46). Signaling pathways that regulate the redox status require cross-talk with NO signaling pathways to maintain cellular homeostasis. NO signaling is not only regulated by the supply of reductive/oxidative molecules that are required for enzymatic reactions, but also by regulatory protein molecules that act on different levels of NO signaling pathways, including enzyme activities related to NO production and subcellular localization of the NO production enzymes, as well as gene transcription and translation. In this study, HSCARG, a redox sensor protein (26), was found to associate and co-localize with AS, one of the rate-limiting enzymes in NO production. Co-localization took place in the cytoplasm, which suggests that HSCARG may exert its regulatory role on AS through direct interactions. A decrease in NADPH concentration, induced by DHEA treatment, enhanced the interaction between HSCARG and AS and strengthened the HSCARG-dependent down-regulation of AS activity, which further inhibited NO production (Fig. 4). These data provide evidence supporting our previously described hypothesis (26). Acting as a sensor of NADPH, the preexisting reservoir of HSCARG senses the subtle changes in intracellular NADPH/NADP+ concentration, triggers itself to restructure, and rapidly down-regulates AS activity, which leads to a decrease in NO production.

Determination of the critical region (amino acids 153-189) of HSCARG that interacts with AS further supported the effect of NADPH levels on the interaction between HSCARG and AS. Analysis of the HSCARG structure revealed that within the asymmetrical dimer, amino acids 153-189 of the NADP-bound molecule comprise the region at the dimer interface (26). In addition, Tyr-55 and Asn-158 form three hydrogen bonds between NADP+ and HSCARG (Fig. 7A) (26), which overlapped, at least partially, with the interaction region between HSCARG and AS. Furthermore, the docking of HSCARG with AS demonstrated that the interaction interface between these two proteins covered the region 153-189 (Fig. 7B). Alignment of the region 153-189 of the NADP+-free molecule II with that of the NADP+-bound molecule I revealed a conformational change occurred when HSCARG bound to NADP+. The NADP+-bound form became more ordered and lost its flexibility that facilitated its binding to the AS protein (Fig. 7A) (26). The structural information, combined with data that decreased NADPH induced by DHEA treatment, enhanced binding between HSCARG and AS, and adding NADPH in vitro impaired this interaction (Fig. 4, A and B), suggesting that NADPH is an allosteric regulator of HSCARG association with AS. When the intracellular NADPH level decreases, the NADPH molecule is released. The HSCARG asymmetrical dimer then changes to a co-factor-free monomeric form, binds to AS, and down-regulates AS activity, which leads to a reduction in NO production.


Figure 7
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  		FIGURE 7.
Structural analysis of the HSCARG region that is critical for AS binding. A, alignment of amino acids (aa) 153-189 of the HSCARG molecule II (yellow) with that of NADP+-bound molecule I. The protein is shown as ribbons, and the NADP+ molecule is shown as a stick model. B, docking of HSCARG (Protein Data Bank code 2EXX, copper-colored) with AS (Protein Data Bank code 2NZ2, red) showed that the interaction interface between these two proteins covers the region of amino acids 153-189 (yellow) of HSCARG.

 
The observation, however, that AS overexpression induced a relocalization of HSCARG was unexpected, because sequence analysis of HSCARG and AS indicated that both proteins appear to lack a nuclear localization sequence. Because AS expression has been shown to be essential for NO production (13, 14), we speculated that the redistribution of HSCARG, induced by AS overexpression (Fig. 2A), was because of an increase in NO levels. This evidence was supported by the fact that SNAP treatment also induced the same relocalization of HSCARG (Fig. 2, B and C, and supplemental Fig. 1). This new discovery suggests that in addition to the inhibition of AS activities in the cytoplasm, HSCARG has a second regulatory role, which may influence gene transcription of proteins related to NO production.

Our data as shown in Fig. 3 and Fig. 6 suggest that HSCARG is required to maintain a reduced level of NO in cells and therefore inhibit apoptosis, whereas an increase of NO levels upon HSCARG siRNA treatment inhibits cell viability because exposure to L-NAME can reduce the increase of NO production and subsequently restore the cell viability (Fig. 6C).

In conclusion, under normal NADPH concentrations, HSCARG forms an asymmetric dimer, resulting in shielding of the binding site for AS. Following a significant decrease of intracellular NADPH concentration, such as induced by DHEA treatment, the dimer of HSCARG dissociates and rapidly releases monomeric HSCARG, which binds AS. Association of HSCARG with AS impairs AS activity and immediately reduces the production of NO, which subsequently prevents apoptosis. The regulation of HSCARG on the cell viability is related to GSH levels in cells. Unlike other NAD/NADP regulatory proteins, which regulate gene expression through an indirect route (47, 48), the regulatory mechanism of HSCARG is unique. HSCARG is quick to respond to changes in the intracellular redox state. Regulation of AS activity by HSCARG is a critical cross-talk point to maintain a balance between the intracellular redox state and NO levels.

In addition, a group of NAD/NADP-dependent proteins, including NmrA, CC3, and C-terminal binding protein, has been shown to regulate gene transcription factors by sensing the cellular level of NAD/NADP (30, 47-50). Our observation that HSCARG enters the nucleus subsequent to a change in the intracellular redox state, together with our data that overexpression of HSCARG leads to NF{kappa}B inhibition,3 suggested that HSCARG may also target nuclear proteins, such as transcription factors, to further counter-balance the effects of the decreasing NADPH concentration. The precise mechanism of second level regulation of HSCARG on gene transcription requires further studies.


   FOOTNOTES
 
* This work was supported by National Science Foundation of China Grant 30670416, International Centre for Genetic Engineering and Biotechnology Project CRP/CHN05-01, and National High Technology and Development Program of China 863 Program 2006AA02A314. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. Back

1 To whom correspondence should be addressed. Tel.: 86-10-6275-5712; Fax: 86-10-6276-5913; E-mail: xiaofengz{at}pku.edu.cn.

2 The abbreviations used are: NO, nitric oxide; AS, argininosuccinate synthetase; DHEA, dehydroepiandrosterone; GSE, glutathione monoethyl ester; GST, glutathione S-transferase; L-NAME, L-N(G)-nitroarginine methyl ester; RT, reverse transcription; SNAP, S-nitroso-N-acetylpenicillamine; RNAi, RNA interference; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole; TNF, tumor necrosis factor; IL, interleukin; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; siRNA, short interfering RNA. Back

3 Q. Gan and X. Zheng, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We sincerely thank Drs. Zicai Liang, Quan Du, and Heping Cheng for providing the equipment used in this study and Geng Meng for helping with the structure analysis. We gratefully acknowledge the excellent technical assistance of Xinping Liu.



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