Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line.

Macrophages activated by exposure to cytokines and/or to endotoxin produce nitric oxide (NO.), a free radical that is a mediator of the host response to infection. Activation induces the expression of nitric oxide synthase, the enzyme that catalyzes formation of NO. from L-arginine and molecular oxygen. We report the cloning of a cDNA encoding the inducible nitric oxide synthase from a murine macrophage cell line, RAW264.7, exposed to interferon-gamma and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO.. Nitric production is blocked by the enzyme inhibitor, NG-monomethylarginine, and is independent of calcium/calmodulin. RAW264.7 cells demonstrate rapid accumulation of the nitric oxide synthase-encoding mRNAs upon activation. Comparison of the deduced amino acid sequence to the calcium/calmodulin-dependent nitric oxide synthase previously purified (Bredt, D. S., and Synder, S.H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685) and cloned (Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Synder, S. H. (1991) nature 351, 714-718) from rat brain identifies shared binding sites for the cofactors NADPH and flavins in the C-terminal half of both proteins and an additional conserved region near the N terminus that may recognize L-arginine and/or contribute to the active site.

Diverse roles for nitric oxide (NO') in different tissues have been identified. In macrophages, nitric oxide is an important mediator of tumoricidal (10, 11) and microbicidal activity, particularly against Cvptococcus neoformans (4) and intracellular organisms such as Toxoplasmgondii (12) and Leischmunia major (13). Production of NO' by endothelial cells is an important regulator of vascular resistance and blood pressure (5,14). Indeed, excess production of NO ' by macrophages and other cells exposed to endotoxin and interleukin-1 may contribute to the hemodynamic changes observed in septic * This work was supported by the Howard Hughes Medical Institute. 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.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M84373.
$ Supported by a hematology training grant from the National Institutes of Health. I Supported by the Howard Hughes Medical lnstitute.

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To whom correspondence should be addressed Rm. 925, Thorn Bldg., Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-5852;Fax: 617-732-6088. shock (15,16). In particular, a sepsis-related decrease in blood pressure may result from cGMP-mediated loss of vascular tone that is secondary to activation of smooth muscle guanylyl cyclase by NO' (17). Nitric oxide is also an important messenger molecule in the brain (18,19) and neurotransmitter in the peripheral nervous system (9).
Regulation of NO' production in different tissues is mediated by control of nitric oxide synthase activity. In endothelial cells and neurons, NOS' may be activated by phosphorylation (9) and requires Ca2+ and calmodulin (8). Purification (8) and cDNA cloning (9) have demonstrated that brain NOS is a 150,000-kDa protein that is related to cytochrome P450 reductase.
A second nitric oxide synthase (Mac-NOS) that is distinct from the brain/endothelium enzyme has been identified in macrophages activated by cytokines (1-3). Unlike brain NOS, activation of Mac-NOS requires protein synthesis and is independent of calcium/calmodulin. Recently, Mac-NOS has been purified (20-22). Although it is smaller (125-130 kDa) than brain NOS, both enzymes require FAD, FMN, and NADPH and demonstrate a similar K, for arginine (2-5 p~) and sensitivity to inhibition by arginine analogues such as Nc-monomethylarginine (L-NMMA).
In this report, we describe the cloning and functional expression of a cDNA encoding an inducible nitric oxide synthase from a macrophage-derived mouse cell line, RAW264.7, that has been activated by exposure to interferon-7 and lipopolysaccharide (LPS). Analysis of the deduced amino acid sequence demonstrates that Mac-NOS is related to brain NOS.

EXPERIMENTAL PROCEDURES
Cells and Reagents-RAW264.7 were obtained from American Type Tissue Collection and grown at 37 "C, 5% C02 in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetal calf serum. In experiments which required nitrite measurements, phenol red was omitted from the Dulbecco's modified Eagle's medium. Interferon-7 was purchased from Genzyme (Cambridge, MA). All other reagents were obtained from Sigma. The serotype of the LPS was Escherichia coli 0128B12.
Nitric Oxide Synthase Assay-NOS activity was measured as nitrite production (6,7) in injected oocytes. Stage 5 and 6 oocytes were removed from female Xenopus laevis and separated by incubation with collagenase as described (23). Each oocyte was injected with 50 nl of RNA (0.5-1.0 ng/nl) and incubated at 19 "C for 72 h in 50% Leibovitz L-15 medium (GIBCO) without phenol red. Unless otherwise indicated, the incubation medium contained 2 mM arginine and 0.67 mM calcium. Each assay was performed in triplicate using 5 oocytes per well. Nitrite was measured by mixing 50 pl of the oocyte supernatant with an equal volume of Griess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid) (24). The absorbance at 550 nM was measured NMMA, NG-monomethylarginine; LPS, lipopolysaccharide.
The abbreviations used are: NOS, nitric oxide synthase; L-and nitrite concentration determined using a curve calibrated from sodium nitrite standards. In the depletion studies, the oocytes were incubated in arginine-or calcium-free medium for 4 days prior to injection.
cDNA Cloning-RNA was prepared from RAW264.7 cells 6 h after exposure to interferon-y (50 units/ml) and LPS (20 ng/ml) by a guanidine isothiocyanate/CsCl procedure (25). Polyadenylated RNA (poly(A) RNA) was isolated by oligo(dT)-cellulose column chromatography and size-fractionated by density gradient centrifugation on 15-30% (w/v) sucrose gradients containing methylmercury hydroxide (26). After centrifugation at 4 "C for 16 h at 76,800 X g, 0.3-ml fractions were collected and assayed for nitric oxide synthase activity by injection into Xenopus oocytes. RNA from the positive fractions was combined, concentrated by ethanol precipitation, and used as a template to synthesize a cDNA library that was ligated into the phage vector, lambda ZAP I1 (Stratagene). DNA was prepared from amplified pools of IO5 phage and used as template in a polymerase chain reaction (40 cycles, 48 "C annealing temperature) using primers from Poly(A) RNA from activated RAW264.7 cells was fractionated on 15-30% sucrose gradients, and aliquots were assayed for nitric oxide synthase-encoding activity by measuring nitrite production in injected oocytes. Fractions containing the greatest activity (10 and 11) were used as the template to construct the cDNA library. suspected cofactor binding sites. Deoxyinosine was inserted at ambiguous codon positions (27). The sense oligonucleotide, AAITAC-TACCTIGATATIACIACICC, was derived from KYYLDITTP, an amino acid sequence conserved in enzymes that bind NADPH and FAD (9, 28). The antisense oligonucleotide, AAIGGIGCIATICCIG-TICCIGGICC, encodes GPGTGIAPF, an amino acid sequence conserved in the NADPH-ribose binding site (9, 28). The 450-base pair product of the polymerase chain reaction was radiolabeled with ['"PI dCTP and used to screen the cDNA library by plaque hybridization (29). Phage identified by hybridization were isolated, and plasmids containing the inserts were rescued by superinfection with helper fl bacteriophage as described by the manufacturer (Stratagene). The nucleotide sequence of the inserts from these plasmids was determined by dideoxynucleotide chain termination using Sequenase 2.0 (U. S. Biochemical Corp.) (30). pMac-NOS, a plasmid containing the single long open reading frame identified by the analysis of the nucleotide sequence, was constructed by combining inserts from two overlapping clones through a shared AflIII site into the expression vector, pGEM (Promega).
Functional Expression of cDNA Clone-pMac-NOS was digested with KpnI, blunted with T4 polymerase, and used as a template to prepare RNA by in vitro transcription using SP6 RNA polymerase (Stratagene). Each oocyte was then injected with 50 nl (0.5 ng/nl).
Northern Blot Anulysis-Poly(A) RNA was prepared as above from activated and control RAW264.7 cells. Five pg was separated on an agarose-formaldehyde gel, transferred onto a nitrocellulose filter, and hybridized to a 3'P-labeled insert from pMac-NOS (29). The blot was washed (0.2 X SSC, 0.1% sodium dodecyl sulfate 65 "C) and autoradiographed for 1 h.
Sequence Analysis-All programs used for sequence analysis were from the University of Wisconsin Genetics Computer Group (31).

RESULTS AND DISCUSSION
Previous studies demonstrated that nitrite production by RAW264.7 cells activated by exposure to y I N F and LPS correlated with induction of NOS activity (1-3). We observed an arginine-dependent increase in nitrite production by frog oocytes injected with poly(A) RNA from activated, but not from resting, RAW264.7 cells. Incubation of injected oocytes in the presence of L-NMMA, an arginine analogue that inhibits NOS (14), blocked nitrite production (data not shown). In . r( 0 addition, sucrose gradient sedimentation of this RNA identified a 5-6-kilobase fraction that was enriched for nitriteproducing activity (Fig. 1). This RNA is large enough to encode the 130-kDa NOS previously purified from these cells (20-22).
A cDNA library was constructed from the RNA in the fractions enriched for this activity. Our screening strategy took advantage of previous reports of conserved amino acid residues in the cofactor binding regions of several NADPH/ flavin-dependent proteins (28), including brain-NOS (9). We synthesized oligonucleotides that encode these conserved residues and used them as primers in a polymerase chain reaction on phage DNA obtained from the library. A single 450-base pair product was generated and used as a hybridization probe to screen the library. Analysis of the nucleotide sequence obtained from several overlapping clones that hybridized to this probe revealed a single long open reading frame that encoded a protein of greater than 130 kDa.
To test if this open reading frame encoded NOS, a fulllength cDNA was constructed from two overlapping clones and inserted into the pGEM plasmid vector (Promega). RNA transcribed from this clone (pMac-NOS) was injected into oocytes and evaluated for NOS activity. These oocytes demonstrated arginine-dependent nitrite production that was inhibited by L-NMMA (75 p M ) , and this inhibition was reversed by excess arginine (Fig. 2). Nitrite production did not require the presence of Ca2+ in the incubation medium and was not inhibited by trifluoperazine (200 p~) , an inhibitor of brain NOS (9) and calmodulin-mediated reactions in frog oocytes (32). Hybridization of pMac-NOS insert to poly(A) RNA identified three transcripts in activated, but not in resting, RAW264.7 cells (Fig. 3). The most prominent transcript is 5.0 kilobases in length; two larger transcripts were also identified and may represent use of alternative polyadenylation sites. We conclude that pMac-NOS encodes an inducible nitric oxide synthase (Mac-NOS) that is expressed in activated macrophages. The nucleotide sequence of the pNOS insert is shown in Fig. 4a. Examination of the deduced amino acid sequence identifies two potential translation start sites (33) that encode proteins of 130,574 and 118,067 kDa. Translation from both potential initiator methionine residues could, in part, explain the reported migration of purified NOS as multiple bands between 125 and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (22).
Comparison of the deduced amino acid sequence of Mac-NOS to the brain NOS demonstrates that the two enzymes are related (Fig. 4b).
In addition to the conservation of cofactor binding sites in the C-terminal portion of both proteins, there is also a conserved region (residues 157-476 in Mac-NOS) near the N terminus (Fig. 4c). We speculate this region may comprise an arginine recognition domain and/or the active site and, therefore, may be conserved in other nitric oxide synthases. In addition to macrophages, inducible NOS activity has been identified in hepatocytes (34), neutrophils (35,36), and smooth muscle (37). These tissues could express additional members of a family of related nitric oxide synthases that contain this domain. Induction of NO' synthesis in macrophages by cytokines is likely to result from transcriptional activation of the NOSencoding gene. Expression of pMac-NOS will provide the opportunity to examine the contribution of nitric oxide to the tumoricidal and microbicidal activity displayed by macrophages. Furthermore, this clone will provide tools to examine the regulation, structure, and function of the inducible Mac-NOS.