A Novel Lipopolysaccharide-response Element Contributes to Induction of Nitric Oxide Synthase*

The gene encoding the high output isoform of nitric oxide synthase represents a large class of alarm and defense genes transcriptionally induced in response to bacterial lipopolysaccharide (LPS). The promoters of most of these genes contain at least two LPS-response elements, one of which commonly binds transcription factors of the NF- (cid:107) B/Rel family. Here a novel LPS-re-sponse element is identified in the inducible nitric oxide synthase promoter, termed LRE AA , which contains critical adenosine residues lying 19–20 base pairs down- stream of the proximal NF- (cid:107) B binding element (NF (cid:107) Bd). Both NF (cid:107) Bd and LRE AA are required for LPS-induced promoter activity. A protein partially recognized by antibody against transcription factor Oct-1 binds to the LRE AA element constitutively in untreated macrophages while contributing to a DNA-protein complex that includes NF- (cid:107) B p50 in macrophages treated with LPS. NF- (cid:107) B p50 and the LRE AA -binding proteins may together recruit an LPS-triggered transactivator of transcription.

iNOS, it is not sufficient (12,17). Additional transcription factors are required (12), which is consistent with the precedent that at least two-thirds of LPS-induced genes studied have more than one LPS-response element (5). In the present work, a second LPS-response element (LRE AA ) is identified in the iNOS promoter that has not been previously recognized in any gene.

EXPERIMENTAL PROCEDURES
Cell Culture--The macrophage cell line RAW 264.7 (American Type Culture Collection) was cultured in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 200 g/ml penicillin and streptomycin) as described (13).
Plasmids-Plasmids p7iNOS-CAT and p8.11iNOS-CAT contain the iNOS promoter and a reporter gene for chloramphenicol acetyltransferase (CAT). p7iNOS-CAT (18) contains a full-length promoter, including a basal promoter and an upstream enhancer; p8.11iNOS-CAT (12) contains only the basal promoter. Polymerase chain reaction-engineered mutations of element NFBd (from GGG to CTC) and of element LRE AA (from AA to CG) were individually introduced into p8.11iNOS-CAT to form p8.11 Bm and p8.11 LREm , respectively. Similarly, mutant constructs p7 Bm and p7 LREm were formed from p7iNOS-CAT. DNA sequence analysis confirmed the mutation of these elements without unwanted mutations elsewhere.
Transient Transfection and CAT Assays-RAW 264.7 cells were transfected by a modification of the DEAE-dextran procedure (19). The DNA used for transfection was prepared by the EndoFree plasmid kit (QIAGEN Inc., Chatsworth, CA). Media and DNA at the concentrations used contained Ͻ50 pg/ml LPS (QCL-1000 kit, BioWhittaker, Inc., Walkersville, MD). Cells were washed twice with serum-free RPMI and suspended at 5-9 ϫ 10 6 /ml in RPMI prewarmed to 37°C containing DEAE-dextran (250 g/ml) and 50 mM Tris (pH 7.4). 10 g of DNA was added to 2 ml of cell suspension at 37°C for 45 min with occasional shaking. Cells were shocked with 10% Me 2 SO for 1 min at room temperature, washed, and distributed to two 100-mm plates with 10 ml of complete medium. After 24 h at 37°C, 5% CO 2 , LPS (100 ng/ml) was added to some of the plates. About 16 h later, the cells were washed with ice-cold phosphate-buffered saline, resuspended in 0.25 mM Tris (pH 8.0), and frozen and thawed 3 times. Lysates were centrifuged (11,700 ϫ g, 10 min, 4°C), and the supernatant was heated at 65°C for 10 min to inactivate CAT inhibitors and then centrifuged as above. The supernatant (1 or 10 g) was assayed for CAT by a TLC method (20), and protein content was determined (21).
Oligonucleotides and Probes-Single-stranded oligonucleotides (Oligos Etc., Inc., Guilford, CT) were annealed with the complementary strand by polymerase chain reaction to form double-stranded oligomers with 5Ј overhang. To prepare probes (see Table I), double-stranded oligomers were filled in by the Klenow fragment of DNA polymerase I with [␣-32 P]dCTP and the three other nonradiolabeled dNTPs. To prepare competitors, all four dNTPs were nonradiolabeled.
Electrophoretic Mobility Shift Assay-Binding was tested in 15 l of solution by incubating 5 g of nuclear extract (22) with reaction buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 60 mM KCl, 12% glycerol, 1 mM dithiothreitol, 2 g of poly(dI-dC)⅐poly(dI-dC)) in the presence or absence of competitor or antibody for 20 min, followed by a 20-min incubation at room temperature with probe (Ն20,000 cpm). Products were electrophoresed at 30 mA for at least 3 h on 4.8% polyacrylamide gels in high ionic strength buffer (50 mM Tris, 380 mM glycine, 2 mM EDTA, pH ϳ8.5) (23), and dried gels were analyzed by autoradiography.
UV Cross-linking Analysis-50 g of nuclear extract and 10 6 cpm of bromodeoxyuridine-containing probe D Bm were reacted in a total volume of 60 l. The electrophoretic mobility shift assay gel was exposed to x-ray film for 10 min to localize the complex of interest. Excised gel was UV-irradiated (366 nm) 5 cm from an inverted transilluminator at 4°C for 60 min, boiled with sample buffer for 5 min, and analyzed by 10% SDS-polyacrylamide gel electrophoresis and autoradiography.

An LPS-response Element (LRE AA ) Distinct from NFBd Confers Inducibility of the iNOS Promoter by LPS-Construct
p8.11iNOS-CAT contains a fragment of the 5Ј-flanking region of the mouse iNOS gene (Ϫ85 to ϩ161) that includes the NFBd element (Ϫ85 to Ϫ76) and confers LPS-inducible promoter activity on transfected RAW 264.7 mouse macrophages (12). LPS responsiveness was lost in p8.13iNOS-CAT, from which the the NFBd element was eliminated (12). In this work, two mutated constructs derived from p8.11iNOS-CAT were prepared by (Fig. 1A) and tested for their promoter activity as induced by LPS (Fig. 1B). In p8.11 Bm , three nucleotides of the NFBd element were mutated, from GGGACTCTCC to CTCACTCTCC. This reduced LPS-induced promoter activity to 3.2 Ϯ 1.3% of wild type (mean Ϯ S.D., three experiments), consistent with an earlier finding that nuclear protein failed to bind to the NFBd element when it carried the same mutation (12). Surprisingly, mutation of two nucleotides (from AA to CG) located 19 -20 base pairs downstream from the 3Ј end of the NFBd element, as seen in p8.11 LREm , also nearly abolished LPS-induced promoter activity leaving only 2.0 Ϯ 0.3% as much activity as in wild type (mean Ϯ S.D., three experiments). This new LPS-response element will be called LRE AA . When the same mutations of elements NFBd and LRE AA were individually introduced into the full-length promoter construct p7iNOS-CAT ( Fig. 1A) to form p7 kBm and p7 LREm , LPS-induced transcription was also substantially reduced ( Fig. 1C). Therefore, both elements play an important role in the full-length promoter as well as in small fragments.
Contribution of Both LRE AA and NFBd to the Binding of Nuclear Proteins on the iNOS Promoter Correlates with Induction of the Gene by LPS-Oligonucleotides derived from the iNOS promoter with or without mutation (Table I) were used as probes or competitors in electrophoretic mobility shift assays to analyze the DNA binding activity of nuclear extracts from cells cultured with or without LPS. As described earlier (12), a cycloheximide-sensitive DNA-protein complex termed "X" was formed upon LPS induction when probe B was used ( Fig. 2A, lane 2). Two other faster migrating complexes, designated "Z" and "Y" in Fig. 2A (not labeled on Fig. 2A in Ref. 12), were present whether or not the cells were exposed to LPS. Without LPS, complex Z was strong and complex Y faint; LPS increased the amount of complex Y without appreciably changing the amount of complex Z (see Fig. 2 in Ref. 12).
Probe D is 19 nucleotides shorter than probe B at the 3Ј end but still contains elements NFBd and LRE AA . Probe D had the same binding activity as probe B, forming complexes X, Y, and Z with nuclear extracts from LPS-induced cells (Fig. 2A, lane  4). Thus, the nucleotides between Ϫ85 and Ϫ50 of the iNOS promoter are sufficient to sustain the formation of all three complexes.
Competition assays established that LRE AA together with NFBd contributed to the formation of these complexes. Complexes X and Y but not Z were competed by excess nonlabeled oligomer A containing element NFBd but not LRE AA (Fig. 2B,  compare lanes 2 and 4). Such competition did not occur with oligomer A kBm , whose NFBd element was mutated (Fig. 2B,  compare lanes 2 and 6). On the other hand, oligomer C, which contained the LRE AA element, blocked the formation of complex Z (Fig. 2B, compare lanes 1 and 7 without LPS and lanes 2 and 8 with LPS) and most of complex X (compare lanes 2 and 8). No competition was seen using oligomer C LREm with a mutated LRE AA element (lanes 9 and 10).
Results of transcription (Fig. 1B) and competition assays (Fig. 2B) were mirrored by tests of direct binding to mutated probes (Fig. 2, C-E). Taking the formation of complexes X, Y,  (13,14) and element NFBu (12) and interferon regulatory factor binding element (IRF-E) (34) indicated by arrows. Construct p8.11iNOS-CAT and its mutants p8.11Bm and p8.11LREm contain a fragment of promoter (Ϫ85 to ϩ161). The DNA sequence of the region from Ϫ85 to Ϫ50 is shown with elements NFBd and LRE AA (underlined) and the mutated nucleotides (asterisks). B and C, mutations of NFBd or LRE AA elements reduced the LPS-induced promoter activity in p8.11iNOS-CAT and in p7iNOS-CAT. Wild type and mutant constructs were transfected into RAW 264.7 cells with or without addition of LPS. The CAT activity of each was compared with that of respective wild type in the presence of LPS, which is set as 100%. Numerical results are means Ϯ S.D. for three or more experiments with each construct and for one the TLC results are illustrated. CAT activity driven by the wild type in response to LPS was 27% acetylation/10 g of protein/2 h for p8.11iNOS-CAT and 24% acetylation/1 g of protein/2 h for p7iNOS-CAT. and Z upon LPS induction as wild type DNA binding activity (Fig. 2C, lane 2), the incomplete formation of these complexes resulted from mutation of either NFBd (lane 3) or LRE AA (lane 4) in the context of probe D. Probe D kBm containing a point mutation of element NFBd did not sustain formation of complex X or Y but still formed complex Z (Fig. 2C, lane 3; note its slightly slower migration). On the other hand, complex Z and most of complex X were absent using probe D LREm containing the mutated element LRE AA ; in the meantime, complex Y became stronger and was accompanied by a complex migrating more slowly than Y (lane 4). Probe D kBmLREm , in which both elements are mutated, formed none of the complexes (lane 5). Probe C, containing LRE AA but not NFBd, formed complex Z alone (Fig. 2F, lanes 1 and 2), whereas no complex formed on the mutated probe C LREm (lanes 7 and 8). Thus, LRE AA and NFBd were the only two elements required for the binding of nuclear factors on the iNOS promoter between nucleotides Ϫ85 and Ϫ50.
The complex Z that formed with probe C or the mutated probe D kBm shared the same characteristics as the complex Z that formed with wild type probe D. Its formation was independent of exposure to LPS (Fig. 2, D and F, lanes 1 and 2) but dependent on the presence of element LRE AA . Formation of complex Z on probe D was competed by excess unlabeled oligomer C containing the element LRE AA (Fig. 2, D and F, lanes  3 and 4) but not by the oligomer C LREm in which LRE AA is mutated (Fig. 2, D and F, lanes 5 and 6).
On the other hand, the complex Y that formed on probe D LREm was more abundant than that formed on wild type probe D (Fig. 2C, compare lanes 2 and 4). Formation of complex Y required only the element NFBd, because complex Y that was formed with probe D LREm disappeared in a competition assay with NFBd containing oligomer A (Fig. 2E, lane 4) but not with oligomer A kBm containing the mutated NFBd element (not shown).
With probe D LREm , a small amount of complex X was still seen after LPS induction (Fig. 2E, lane 2). This residual complex X was competed by oligomer C (Fig. 2E, lane 6).
p50 and Oct-1-like Proteins Contribute to the DNA-Protein Complexes-Supershift assays gave information about some of the proteins comprising complexes X, Y, and Z. No reaction was detected with antibodies against NF-B p52, p65, c-Rel, or RelB, or against IRF-1, STAT1 (p91), c-Jun, c-Fos, C/EBP, Ets-1/Ets-2, NF-AT, Oct-2, or Pit 1, except for a partial supershift of complex X with large amounts of anti-c-Rel (not shown). In contrast, anti-NF-B p50 (a reagent that immunoblotted and supershifted authentic p50 overexpressed upon cell transfection; not shown) completely supershifted complex X (Fig. 3A, lane 2) while leaving complex Z untouched (Fig. 3, A and B,  lanes 1 and 2). Moreover, anti-p50 completely supershifted complex Y (marked by an asterisk in lane 2 of Fig. 3A), and such supershift was even clearer when complex Y was formed with probe D LREm (Fig. 3C, lane 3). Thus, p50 is a component of complexes X and Y but is not present (or is not accessible to the antibody) in complex Z.
Anti-Oct-1 (a reagent that supershifted authentic Oct-1 overexpressed upon cell transfection; not shown) partly supershifted complexes X and Z (Fig. 3, A and B, lanes 3 and 4). In contrast, anti-Oct-1 was nonreactive with complex Y as formed with probe D LREm (containing NFBd but not LRE AA ) (Fig. 3C,  lane 4). As noted above, probe D kBm only supported the formation of complex Z and did so whether or not the cells had been exposed to LPS (Fig. 3B, lanes 5 and 6).
Complex Z was subjected to UV cross-linking followed by SDS-polyacrylamide gel electrophoresis, revealing that the region containing element LRE AA bound at least three nuclear proteins with apparent molecular masses of ϳ160, ϳ100, and ϳ60 kDa (Fig. 4). DISCUSSION The most frequently implicated LPS-response element in mammalian promoters is the 10-base pair B element (GGGRNNYYCC) that binds transcription factors of the NF-B/Rel family (5,24). NF-B frequently associates with other transcription factors to impart specific regulation (25). The present work identifies an LPS-response element termed LRE AA including the dinucleotide AA downstream of NFBd in the mouse iNOS promoter. An NF-B-like binding site followed closely by an LPS-response element containing an AA dinucleotide was reported in the regulatory region of the major histocompatibility complex class II A ␣ k gene (26) and later in the mouse granulocyte colony-stimulating factor promoter (27), but  LRE AA . Other transcription factors known to bind the octamer motif include Pit-1 and the B cell-specific Oct-2 (29). However, with oligonucleotide probes derived from iNOS promoter and nuclear extracts from RAW cells, antibodies against Pit-1 and Oct-2 had no effect, while the antibody against Oct-1 caused a partial supershift in the two complexes (Z and X) whose formation depended on LRE AA . Thus, an Oct-1 like protein (OLP) is a candidate for one of the transcription factors interacting with LRE AA . UV cross-linking analysis indicated that the LRE AAdependent complex Z included at least three nuclear proteins with molecular masses of ϳ160, ϳ100, and ϳ60 kDa. None of these correspond to the molecular mass of Oct-1 (ϳ70 kDa). It is not known which, if any, of these three proteins binds anti-Oct-1 antibody to cause a partial supershift of complex Z. The ϳ100-kDa species corresponds in size to a component previously detected in complex X (12). The ϳ100 kDa protein that binds the LRE AA may be the same as the ϳ100 kDa protein that binds NFBd.
Sequence context strongly influences the composition of promoter binding complexes. The practice of using minimal probes to sustain complex formation militates against detecting factors that impart specificity to the induction of genes regulated by widely shared transcription systems such as NF-B. The present study used relatively long probes. Results with such probes were consistent with findings from reporter constructs including those representing point mutants in the full-length promoter. In earlier work (12) probe A, containing only the NFBd element, supported the LPS-activated binding of p50/ p65 and p50/c-Rel. However, this did not fully explain LPSinduced promoter activity of iNOS. First, activation of NF-B/ Rel is not sensitive to cycloheximide (12), but synthesis of iNOS mRNA in the cells under study was cycloheximide-sensitive (30). Second, activation of p50/p65 and p50/c-Rel peaked at 0.5 h after LPS induction and then decreased (12,16), but synthesis of iNOS mRNA continued for more than 24 h (11,18). Finally, NF-B/Rel was activated by LPS in an LPS-hyporesponsive macrophage cell line from C3H/HeJ mice, but iNOS was not induced. Thus, NF-B/Rel was not sufficient for LPS induction of iNOS (17). In contrast, probes B and D (Ref. 12 and present study) included not only the NFBd element but also downstream sequences that appear to be relatively specific for the iNOS gene. The complexes formed with probes B and D after LPS induction were different from those formed with probe A. In particular, complex X required both NFBd and LRE AA , contained additional protein(s) besides those of the NF-B/Rel family, lacked p65 and c-Rel, and was sensitive to cycloheximide (12).
Sequence analyses suggested that elements for binding of NF-IL6 are present in the iNOS promoter at positions Ϫ74 to Ϫ66 and Ϫ150 to Ϫ142 (14); the latter was protected by in vivo footprinting during LPS induction (15). However, mice rendered genetically deficient in NF-IL6 produced iNOS normally in response to LPS and interferon-␥ (31). Footprinting also showed protection of nucleotide at Ϫ58 (within the octamerlike sequence ATGCAAAA) after LPS induction (15). The present report demonstrates that the dinucleotide AA at Ϫ56 and Ϫ55 is critical to the formation of complex Z, which is inde- pendent of LPS induction, and that the mutation of AA to CG eliminates both protein binding and promoter activity.
Based on reporter constructs, binding assays, competition experiments, and antibody supershifts, it is hypothesized that both constitutive complex Z and inducible complex X required LRE AA and contained OLP, whereas inducible complexes X and Y required NFBd and contained NF-B p50. Since no direct interaction between NF-B and an Oct-1-containing complex has been reported and since p50 lacks a transactivation domain (25), it is postulated that LPS causes a distinct protein to bridge p50 and OLP, contributes to the formation of complex X, and either furnishes or recruits the transactivation domain that stimulates transcription of iNOS. The bridging protein may be the ϳ100 kDa species that seems common to complex X and complex Z, or the transactivating protein may bind the ϳ100 kDa species when it is bound to NFBd (an LPS-induced event) as well as to LRE AA (a constitutive event).
Together, NF-B p50 and the activated OLP-containing complex are proposed to recruit a transactivator to complex X, much as Bcl-3 supplies transactivating capacity by binding p50 dimers on DNA (32,33). Cloning of the proteins in complexes X and Z may provide fresh approaches to the pharmacologic control of iNOS expression and other responses to LPS.