©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Diversity in the 5`-Untranslated Region of Cytokine-stimulated Human Inducible Nitric Oxide Synthase mRNA (*)

Shan C. Chu (§) , Hai-Ping Wu , Tyrone C. Banks , N. Tony Eissa , Joel Moss

From the (1) Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Inducible nitric oxide synthase, the critical enzyme responsible for the enhanced synthesis of nitric oxide in inflammatory states, is widely expressed in mammalian cells. To evaluate potential regulatory roles of the 5`-untranslated region (UTR) in the human inducible nitric oxide synthase gene, the transcription initiation sites and structure of the 5`-UTR of human inducible nitric oxide synthase were examined. Freshly isolated human alveolar macrophages, bronchial epithelial cells, and several types of cultured cells were evaluated following stimulation with cytokines ( i.e. interferon-, interleukin-1, tumor necrosis factor-, and interleukin-6). The mRNA was analyzed by reverse transcription-polymerase chain reaction, Northern analysis, and 5`-rapid amplification of cDNA ends. Despite the presence of a TATA box in the promoter region, multiple transcription initiation sites were observed, some extending several hundred base pairs upstream from the main TATA-directed initiation site. Alternative splicing in the 5`-UTR of human inducible nitric oxide synthase mRNA resulted in further diversity. The TATA-independent inducible nitric oxide synthase mRNA transcripts were up-regulated by cytokines. The long and complex 5`-UTRs contain eight partially overlapping open reading frames upstream of the putative inducible nitric oxide synthase ATG, which may have an important role in translational regulation of human inducible nitric oxide synthase mRNA.


INTRODUCTION

Nitric oxide (NO)() appears to be critical in the physiology or pathophysiology of every organ system (1, 2, 3, 4) . The enzyme responsible for NO synthesis, nitric oxide synthase (NOS), converts L-arginine to L-citrulline and NO. The three human NOS genes include an inducible type (iNOS), whose activity is Ca-independent, and two constitutive, Ca/calmodulin-dependent types termed neuronal and endothelial constitutive NOS (1, 2, 5) . The genes for these three isoforms are located on different chromosomes.

The human iNOS gene, located on chromosome 17 at position 17cen-q11.2, is a 37-kilobase pair, 26-exon gene with the translational start codon in exon 2 and stop codon in exon 26 (6) . In 425 bp of its 5`-flanking region are found a TATA box and consensus sequences for three interferon- response elements, an NF-B site, an A activator-binding site, and a degenerate (but palindromic) tumor necrosis factor response element. These findings are consistent with the observation that the iNOS gene is inducible by cytokines. Expression of the human iNOS gene has been documented in a variety of cell types ( i.e. hepatocytes, respiratory epithelial cells, and macrophages) (2, 3) , and the human iNOS cDNA has been cloned from hepatocytes, chondrocytes, and a human colorectal adenocarcinoma cell line, DLD-1 (7, 8, 9) . Sequence analysis reveals only a few nucleotide differences among these cDNAs, consistent with the existence of a single human iNOS gene.

There is increasing evidence that the 5`- and 3`-untranslated regions (UTRs) of many mRNAs play an important role in the regulation of gene expression by influencing mRNA stability and translational efficiency (10) . We hypothesized that, due to the diversity and multiplicity of transcription element consensus sequences in the promoter region of the iNOS gene, previously unidentified transcription initiation sites could exist in the 5`-UTR. To evaluate this hypothesis, the 5`-UTR of human iNOS mRNA was characterized by using reverse transcription-polymerase chain reaction (RT-PCR), Northern analysis, and 5`-rapid amplification of cDNA ends (RACE). The study demonstrates that, due to multiple transcription initiation sites and alternative splicing, there exist multiple forms of exon 1 in iNOS mRNA transcripts from human epithelial cells and alveolar macrophages, with production of these iNOS mRNAs enhanced by cytokines.


MATERIALS AND METHODS

Cell Culture

A549 cells (American Type Culture Collection (ATCC) CCL 185), a human alveolar type II epithelium-like lung carcinoma cell line, were maintained in Ham's F-12K medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (all from Biofluids). DLD-1 cells (ATCC, CCL 221), a human colorectal adenocarcinoma cell line, were maintained in RPMI 1640 medium (BioWhittaker) with the same supplements used for the A549 cells. T84 cells (ATCC, CCL 248), a human colon carcinoma cell line, were maintained in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with 5% heat-inactivated fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. Normal human bronchial epithelial primary culture (HBEC) cells (Clonetics), isolated from the large bronchi and trachea of normal humans, were cultured in serum-free, modified LHC-9 medium (Clonetics). To induce iNOS mRNA, A549, DLD-1, and HBEC cells were treated with a mixture of recombinant human cytokines containing 100 units/ml interferon-, 0.5 ng/ml interleukin (IL)-1, 10 ng/ml tumor necrosis factor-, and 200 units/ml IL-6 (9) . All of these were from Boehringer Mannheim except IL-1, which was from Genzyme. All experiments were carried out with confluent cells.

Isolation of Normal Human Bronchial Epithelial Cells and Alveolar Macrophages

Normal human bronchial epithelial (NHBE) cells were obtained as described previously from the large bronchi and trachea of six nonsmoking, normal volunteers (age range, 21-45 years) by fiberoptic bronchoscopy, using a standard cytology brush (Microvasive) (11, 12) . The cells were immediately suspended in RPMI 1640 medium at 4 °C, pelleted by centrifugation, and lysed in 4 M guanidinium thiocyanate, 25 mM sodium acetate, pH 5.2, 0.5% Sarkosyl, and 0.1 M -mercaptoethanol. Alveolar macrophages were obtained from one of the six volunteers by bronchoalveolar lavage (13) and purified by adherence on plastic for 1 h at 37 °C in serum-free RPMI 1640 medium supplemented with 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (14) . After washing three times with ice-cold Hanks' balanced salt solution (Mediatech), macrophages were lysed in 4 M guanidinium thiocyanate, 25 mM sodium acetate, pH 5.2, 0.5% Sarkosyl, and 0.1 M -mercaptoethanol.

Analysis by RT-PCR of the 5`-End of Human iNOS mRNA

Total RNA was extracted from cells by the guanidinium thiocyanate-CsCl gradient method (15) . The 5`-ends of iNOS mRNAs from all cell types were analyzed by RT-PCR. The cDNA was synthesized using Moloney murine leukemia virus RNase H reverse transcriptase (Superscript II RNase H RT; Life Technologies, Inc.), random hexanucleotide primer (Pharmacia Biotech Inc.), and oligo(dT) primer (Pharmacia Biotech Inc.) in 20 µl of reverse transcriptase buffer (50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl, 10 mM dithiothreitol, and 0.5 mM each of dATP, dGTP, dCTP, and dTTP).

Oligonucleotides were synthesized using an automated DNA synthesizer (Applied Biosystems) and purified on Sephadex G-25M (PD-10) (Pharmacia Biotech Inc.). All PCR amplifications were performed using the DNA thermal cycler (Perkin-Elmer). The 5`-ends of human iNOS mRNA transcripts (after conversion to cDNA) were amplified for 25 cycles (94 °C for 20 s, 60 °C for 20 s, and 72 °C for 40 s), except that 28 cycles were used for alveolar macrophages. Taq DNA polymerase (Stratagene) was used with sense primer NO7 (5`-AAACAACTCTCTGGATGGCATGG-3`, -59 to -37 in the 5`-flanking region) and antisense primer NO6 (5`-CTGGGTCCTCTGGTCAAACTTTTG-3`, 653 to 630 in exon 5) (see Fig. 1 ); the transcription initiation site described by Chartrain et al. (6) is defined as +1. The PCR products were evaluated by Southern analysis (16) . 20% of the PCR product of each sample was size-fractionated on 1.5% agarose gel in 1 TBE buffer (89 mM Tris, pH 8.3, 89 mM boric acid, 2 mM EDTA). DNA on duplicate gels was blotted onto Nytran membranes (Schleicher & Schuell) and fixed to the membrane by UV irradiation (UV Stratalinker; Stratagene). One membrane was hybridized with an [-P]dATP-labeled (at the 3`-end) oligonucleotide probe (probe A, 5`-GATCCTCACATGCCGTGGGGAGGACAATGGGGTTGCATCCAGCTTGACCAG-3`, 525 to 475 in exon 4), washed, evaluated by autoradiography (see Fig. 1A), and then scanned by a PhosphorImager (Molecular Dynamics); relative amounts of exon 1 and 1 transcripts were calculated using the total units of both transcripts as 100%. The other membrane was hybridized with a 191-bp, [-P]dATP-labeled iNOS cDNA probe (probe Exon 1), prepared by random priming, encompassing the entire sequence of exon 1 (see Fig. 1 B).


Figure 1: Alternative transcription initiation sites and deletion of previously reported exon 1 in iNOS mRNA in human cells. At the top is shown a portion of the 5`-flanking region and the structure of the mature 26-exon human iNOS mRNA. The TATA box in the 5`-flanking region, the start codon ( ATG) in exon 2, and stop codon ( TGA) in exon 26 are indicated. Shown also are the positions of primers ( NO7 in the 5`-flanking region and NO6 in exon 5) used in RT-PCR, the antisense oligonucleotide probe A ( A) in exon 4, and the PCR-generated Exon 1 probe ( Exon 1) used for hybridization with amplified mRNA transcripts. Total RNA was extracted from cells with or without prior stimulation by cytokine mixture ( CM) and converted to cDNA, which was amplified by PCR for 25 cycles (28 cycles for AMs) using NO7 and NO6 primers and then evaluated by Southern analysis. A, hybridization with [-P]dATP-labeled oligonucleotide probe A. PCR products of 712 and 486 bp, respectively, corresponding to iNOS mRNA with (exon 1) and without previously reported exon 1 plus the adjacent 35 bp at its 5`-flanking region (exon 1) were detected. B, hybridization with [-P]dATP-labeled iNOS cDNA Exon 1 probe encompassing only the sequence of exon 1. The 486-bp bands were not detected except for some nonspecific hybridization in lanes 2 and 4. C, -actin. -Actin-specific primers and probe were used for RT-PCR and Southern hybridization, respectively. Sources of RNA are indicated above the lanes. A549 alveolar type II epithelium-like lung carcinoma cells ( lanes 1 and 2), DLD-1 colorectal adenocarcinoma cells ( lanes 3 and 4), HBEC cells ( lanes 5 and 6), T84 colon adenocarcinoma cells ( lane 7), freshly isolated NHBE cells ( lane 8), and AMs ( lane 9) were evaluated. HO was used in the RT-PCR as a negative control ( lane 10).



As a control, -actin mRNA was evaluated by a similar technique, except that the primers were specific for -actin transcripts (HAG3, 5`-ATGAAGATCAAGATCATCGCACCC-3` and HAG4, 5`-CACCAAGCCACCGACTTGTCTTCC-3`); amplification was performed for only 20 cycles, and a nested PCR-generated cDNA probe was used (see Fig. 1C) (17, 18) .

Nucleotide Sequence of iNOS mRNA Transcripts with and without Previously Reported Exon 1

After preliminary observations of the RT-PCR products suggested the presence of two different iNOS transcripts (712-bp ``exon 1'' transcripts and 486-bp ``exon 1'' transcripts), the exon 1 and exon 1 transcripts of RT-PCR products from DLD-1 cells were excised from the agarose gel, purified (QIAEX gel extraction kit; QIAGEN Inc.), and sequenced by the dideoxy chain termination method (19) using a double-stranded DNA cycle sequencing system (Life Technologies, Inc.) with NO7 and NO26 (5`-TGATACTGAAGGTCATCCTGTGTC-3`, 404 to 381 in exon 3) as sequencing primers.

Northern Analysis of the 5`-End Structure of Human iNOS mRNA

The 5`-end of human iNOS mRNA transcripts was further evaluated by Northern analysis (20) . Total RNA (20 µg/lane) from A549 and DLD-1 cells, with and without stimulation by cytokines, was subjected to formaldehyde-agarose gel electrophoresis, transferred to Nytran membranes, and hybridized with various oligonucleotide probes (probe A, same as the probe A used for Southern analysis of RT-PCR products; probe B, 5`-CCAAAGGGAGTGTCCCCAGCTTGTGTACAGTTAGCTAATTTATGACTGTGA-3`, -100 to -150; probe C, 5`-TCACAGTCATA-AATTAGCTAACTGTACACAAGCTGGGGACACTCCCTTTGG-3`, -150 to -100; probe D, 5`-GGCCAAGCCACATGGCCTCACTTTCAGCATCTGGGAGATTTTTTCCTCAGC-3`, -306 to -356) labeled with [-P]dATP at the 3`-end (see Fig. 2, A-D). Probes A, B, and D were antisense; probe C corresponded to the sense strand of probe B. As a control, glyceraldehyde-3-phosphate dehydrogenase mRNA was evaluated with a 1.0-kilobase pair human glyceraldehyde-3-phosphate dehydrogenase cDNA probe (see Fig. 2 E) (21) .


Figure 2: Effect of cytokines on human iNOS mRNA with transcription initiation sites upstream of the TATA box. At the top is shown a portion of the 5`-flanking region and the structure of the mature 26-exon human iNOS mRNA, as well as positions of oligonucleotide probes used for Northern analysis: antisense probe A in exon 4, antisense probe B, sense probe C, and antisense probe D upstream of TATA box. A549 and DLD-1 cells were incubated with or without a cytokine mixture ( CM) for 8 h before extraction of RNA. Northern blots were hybridized with [-P]dATP-labeled iNOS oligonucleotide probes A, B, C, or D ( panels A-D, respectively) or with [-P]dATP-labeled 1.0-kilobase pair human glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) cDNA clone ( panel E).



Analysis by 5`-RACE of Human iNOS mRNA

The results of RT-PCR and Northern analysis indicated that only a small fraction of iNOS mRNA transcripts was transcribed from upstream of the reported transcription initiation site, but more than half of these lacked the sequence -35 to 191 (see ``Results''). 5`-RACE (instead of primer extension) using gene-specific primers and a 5`-AmpliFINDER RACE kit (Clontech) were used to further characterize the 5`-end of human iNOS mRNA.

RNA from cytokine-stimulated A549 cells (2 µg of poly(A) RNA, which was purified from total RNA on oligo(dT)-cellulose (5 Prime 3 Prime, Inc.)), and from freshly isolated NHBE cells (20 µg of total RNA) was reverse transcribed with a gene-specific antisense primer (NO32, 5`-CTGCCCCAGTTTTTGATCCTCAC-3`, 539 to 517 in exon 4) by avian myeloblastosis virus reverse transcriptase. The first strand of cDNA was ligated to an anchor (ANCH, 5`-P-CACGAATTCACTATCGATTCTGGAACCTTCAGAGG-NH-3`) containing a phosphate group at the 5`-end to facilitate ligation and an amine group at the 3`-end to prevent self-ligation and amplified by two rounds of PCR. For the first round PCR, the sense primer was ANCH-2 (5`-CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG-3`) and the antisense primer was NO60 (5`-GGGGGCTTTCTCCACATTGTTG-3`, 361 to 339 in exon 2). For the second round PCR, the sense primer was ANCH-2 and antisense primer was NO30AP (5`-CTCGCTCGCCCACTCTATGGCTTTACAAAGCAGGTC-3`). PCR products were subcloned into pDIRECT vector (Clontech) and subjected to sequence analysis using a double-stranded DNA cycle sequencing system (Life Technologies, Inc.).


RESULTS

Human iNOS mRNA Transcripts with Deletion of Previously Reported Exon 1

Evaluation of iNOS mRNA transcripts in the region encompassing the 5`-flanking region and exons 1-5 by RT-PCR (Fig. 1, top panel) demonstrated two different transcripts in cytokine-stimulated A549 cells, DLD-1 cells, and HBEC cells (Fig. 1 A, lanes 2, 4, and 6), resting DLD-1 and T84 cells (Fig. 1 A, lanes 3 and 7), and freshly isolated NHBE cells and alveolar macrophages (Fig. 1 A, lanes 8 and 9). iNOS transcripts were not observed in unstimulated A549 or HBEC cells (Fig. 1 A, lanes 1 and 5). Results from NHBE samples from five other normal volunteers were similar to lane 8 of Fig. 1(data not shown). The size difference (226 bp) between these two amplified fragments (712 and 486 bp) was a little larger than the size of exon 1 (191 bp). Southern analysis with an exon 4-specific probe (probe A) demonstrated that both fragments contained the exon 4 sequence. In contrast, Southern analysis with an exon 1-specific probe (probe Exon 1) demonstrated that the 486-bp fragment did not contain the exon 1 sequence (Fig. 1 B), although some hybridization with the PCR products from cytokine-stimulated A549 and DLD-1 cells was observed. Thus, some iNOS mRNA transcripts included previously reported exon 1 sequences whereas others did not. The exon 1 transcripts were more abundant than exon 1 transcripts in all samples with visible bands on autoradiography.

Sequence analysis of the 5`-end of the 712-bp fragment (derived from mRNA transcripts that included exon 1, as shown by Southern analysis of RT-PCR products) demonstrated that, as expected, mRNA transcripts corresponding to this amplification product contained sequences of the 5`-flanking region adjacent to exon 1, exons 1 and 2, and the 5`-end of exon 3. Consistent with the Southern analysis, sequence analysis of the 486-bp fragment (derived from mRNA transcripts that did not include exon 1) demonstrated that iNOS mRNA represented by this fragment indeed did have a deletion of the entire sequence of the previously reported exon 1 (1 to 191) plus the adjacent 35 bp of 5`-flanking region (-1 to -35). Altogether, these data clearly demonstrated that some iNOS mRNA transcripts were initiated upstream of the TATA box, and more than half of these transcripts lacked exon 1 plus 35 bp upstream (-35 to 191).

Regulation by Cytokines of Human iNOS mRNA Initiated Upstream of the TATA Box

Northern analysis with an exon 4-specific antisense probe (probe A) demonstrated that iNOS mRNA transcripts of A549 and DLD-1 cells were increased by a cytokine mixture of interferon-, IL-1, tumor necrosis factor-, and IL-6 (Fig. 2 A). Using antisense probe B in the 5`-flanking region (-100 to -150), Northern analysis demonstrated a similar pattern (Fig. 2 B) to that with probe A, although the exposure time during autoradiography required to obtain bands of similar density was much longer. The data suggested that only a small proportion of iNOS mRNA was transcribed from upstream of the TATA box. In contrast, with the more upstream antisense probe D (-306 to -356) or the sense probe C (-150 to -100, sense strand of probe B), no iNOS mRNA could be detected by Northern analysis except for some nonspecific hybridization on 28 S rRNA (Fig. 2, C and D). The data clearly demonstrated that cytokines enhanced the production of iNOS mRNA transcribed from upstream, as well as downstream, of the TATA box.

5`-RACE of Human iNOS mRNA

Shown in Fig. 3 are the results of 5`-RACE with mRNA from cytokine-stimulated A549 cells (Fig. 3 B) and freshly isolated NHBE cells (Fig. 3 C). A total of 128 independent A549 clones and 73 NHBE clones were sequenced to determine the 5`-end of the first strand cDNA. Consistent with a previous report (6) , a predominant transcription initiation site (55 of 128 A549 clones and 26 of 73 NHBE clones) was located 30 bp downstream of the first nucleotide of the TATA box. Consistent with the results of RT-PCR and Northern analysis, eight A549 clones and five NHBE clones had transcription initiation sites upstream of the major site. Of these, four A549 clones and four NHBE clones lacked exon 1 and 35 bp upstream (-35 to 191), three A549 clones and one NHBE clone did not have this deletion, and one A549 clone lacked the sequence -221 to 191. Many clones had apparent transcription initiation sites downstream of the major initiation site. Some of these likely resulted from incomplete reverse transcription by the avian myeloblastosis virus reverse transcriptase ( i.e. some transcription initiation sites might actually be located upstream of those defined by 5`-RACE). The structural diversity in the 5`-UTR of human iNOS mRNA, demonstrated by the 5`-RACE study, is summarized in Fig. 4 .


Figure 3: Analysis by 5`-RACE of human iNOS mRNA from A549 and freshly isolated NHBE cells. A, the 5`-flanking region, exon 1, and part of exon 2 of human iNOS mRNA are shown. Positions of consensus sequences for transcription factors, the TATA box ( TATA), the major transcription initiation site ( +1), and the start codon ( ATG) are indicated. Shown also are sequences of 5`-splice sites of exon 1B (5`-GTGGGT-3`; GTGGGT) and exon 1A (5`-GTGAGT-3`; GTGAGT; see Fig. 4 and ``Results'' for details). B, 5`-RACE of human iNOS mRNA from cytokine-stimulated A549 cells. 2 µg of poly(A) RNA was reverse transcribed with a gene-specific antisense primer (NO32 in exon 4). First strand cDNA was ligated to an anchor primer. First round PCR (sense primer ANCH-2; antisense primer NO60 in exon 2) and second round PCR (sense primer ANCH-2; antisense primer NO30AP) followed. PCR products were subcloned into pDIRECT vector and subjected to DNA sequence analysis. Each clone is represented by a separate symbol. , clone transcribed from 1 or downstream; , clone transcribed from upstream of 1 that includes previously reported exon 1; , clone transcribed from upstream of 1 with deletion of sequence -35 to 191; , clone transcribed from upstream of 1 with deletion of sequence -221 to 191. Of 128 clones sequenced, 55 had transcription initiation sites at 1. C, 5`-RACE of iNOS mRNA from NHBE cells. Experimental procedures were the same as those in B except that 20 µg of total RNA from freshly isolated NHBE were used for reverse transcription. Of the 73 clones sequenced, 26 had transcription initiation sites at 1.




Figure 4: Structural diversity in the 5`-untranslated region of human iNOS mRNA. Shown are 5`-end structures of human iNOS mRNA transcripts with exon numbers in boxes. The start codon ( ATG) in exon 2 is indicated. Exon 1a contains the sequence -35 to -1. Exon 1A contains a sequence with the 5`-end located between -221 and -37 and the 3`-end at -36. Exon 1B contains a sequence with 5`-ends either at or upstream of -223 and the 3`-end at -222. A, iNOS mRNA transcribed from or downstream of the major transcription initiation site ( in Fig. 3). Sequences of the 5` splice site and part of the 3` splice site of intron 1 are shown. B, iNOS mRNA transcribed from upstream of the major transcription initiation site containing exon 1 ( in Fig. 3). C, iNOS mRNA transcribed from upstream of the major transcription initiation site lacking sequence -35 to 191 ( in Fig. 3). The 5` splice site sequence of exon 1A (5`-GTGAGT-3`) is identical to that of exon 1. D, iNOS mRNA transcribed from upstream of the major transcription initiation site lacking sequence -221 to 191 ( in Fig. 3). The 5` splice site sequence of exon 1B (5`-GTGGGT-3`) differs from that of exons 1 and 1A by one nucleotide.



Five AUGs were noted in the 5`-UTR (-270 to 264), located at -256, -65, -45, -40, and 87, which possibly serve as start codons for upstream open reading frames (uORFs). Combined with deletion of the sequence -35 to 191 (Fig. 4 C) or -221 to 191 (Fig. 4 D), eight small uORFs were found: 1) -256 to -212; 2) (-256 to -222)-(192 to 210); 3) -65 to 1; 4) (-65 to -36)-(192 to 230); 5) -45 to 9; 6) (-45 to -36)-(192 to 199); 7) (-40 to -36)-(192 to 210); and 8) 87 to 149.


DISCUSSION

We describe here the complexity of the 5`-UTR of human iNOS mRNA. Multiple forms of exon 1 were observed in human alveolar macrophages and respiratory and intestinal epithelial cells, resulting from multiple transcription initiation sites and alternative splicing (Fig. 4). Of the 201 clones sequenced in the 5`-RACE study, only 12 (6%) had transcription initiation sites located upstream of the TATA box. In contrast, no structural diversity was found in the 3`-UTR by using RT-PCR (data not shown). The major transcription initiation site defined by 5`-RACE in our study is the same as the only transcription initiation site reported previously (6) . No other TATA-directed transcription initiation site was found in the upstream 395 bp. Although the majority of iNOS mRNA transcripts were generated as expected in a TATA-containing gene, a small proportion of iNOS mRNA transcripts were apparently TATA-independent. The TATA-independent mRNA transcripts, including the alternatively spliced forms, were also up-regulated by cytokines (Fig. 2), which is consistent with the findings in many TATA-less genes (22) and with the presence of multiple transcription element consensus sequences involved in cytokine-stimulated gene expression. The occurrence of alternative splicing in the 5`-UTR may be explained in part by the presence of a sequence in this region either identical (Fig. 4 C) or similar (Fig. 4 D) to the reported 5` splice site sequence for intron 1 (Fig. 4, A and B).

Similar to the human iNOS gene, the mouse iNOS gene contains a TATA box 30 bp upstream of the reported transcription initiation site and multiple consensus sequences for the binding of transcription factors in the 5`-flanking region (23) . Using RT-PCR, TATA-independent iNOS mRNA transcripts were found in RAW 264.7 mouse macrophages stimulated by interferon- and bacterial lipopolysaccharide, although no alternative splicing in the 5`-UTR was observed (data not shown).

It had been observed that a TATA-containing promoter can yield transcripts with multiple downstream initiation sites (24, 25, 26, 27) . For example, transcription initiation occurs at multiple sites between 10 and 111 bp downstream from the TATA box in the human L-plastin gene (24) . Although heterogeneity in the upstream initiation sites of TATA-containing promoters is not widely reported, the presence of multiple consensus sequences for transcription factors might account for the observed diversity.

There is increasing evidence that the 5`- and 3`-UTRs of many mRNAs play an important role in the regulation of gene expression (10) . To assure efficient translation, most eukaryotic mRNAs have a short 5`-UTR and no AUGs upstream of the translation initiation site. However, in 5-10% of vertebrate mRNAs, the first AUG is not the translational initiation site of the major open reading frame (ORF) (28, 29) . Some 5`-UTRs of these mRNAs dramatically inhibit translation at major ORFs (30, 31, 32, 33) . For example, the translational inhibition by uORFs of the GCN4 gene, which encodes a transcriptional activator of amino acid biosynthetic genes, has been well studied in Saccharomyces cerevisiae(34, 35, 36) . Similar to human iNOS mRNA, the retinoic acid receptor 2 mRNA has a long (461 nucleotides) 5`-UTR, which contains five partially overlapping uORFs (37) . Recently, Zimmer et al.(38) demonstrated that uORFs in the 5`-UTR of the retinoic acid receptor 2 mRNA inhibited its translation in a tissue-specific manner. In human iNOS mRNA, the long and complex 5`-UTR contains eight partially overlapping uORFs prior to the expected iNOS AUG, which could have an important role in the regulation of protein synthesis.

It appears that NHBE cells and AMs, as well as T84 and DLD-1 cells, without apparent cytokine stimulation, contained significant amounts of iNOS mRNA (Fig. 1). In DLD-1 cells, the amount of iNOS mRNA was substantially increased by cytokines. It would thus appear that a low level of iNOS mRNA is present in some cells. In agreement, expression of human iNOS at the protein level has been observed in the normal epithelium of large, cartilaginous airways (39) . It is possible that environmental stimuli result in the induction of iNOS mRNA in NHBE cells and AMs, secondary to an increase in cytokine levels above basal. In cells in culture, such as DLD-1 and T84 colon carcinoma cells, autocrine stimulation from endogenous production of cytokines or similar factors might conceivably result in the increase in iNOS mRNA. In contrast to these cells, no significant amount of iNOS mRNA was observed in unstimulated A549 or primary culture of normal human bronchial epithelial cells. These observations are consistent with the conclusion that iNOS mRNA might be induced in some cells without stimuli from other cells whereas other cells might be more dependent on outside stimuli. Thus, the autocrine loop may be cell- or tissue-specific.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Pulmonary-Critical Care Medicine Branch, Bldg. 10, Rm. 6D03, 10 Center Dr. MSC 1590, NIH, Bethesda, MD 20892-1590. Tel.: 301-496-9479; Fax: 301-496-2363.

The abbreviations used are: NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; IL, interleukin; HBEC, normal human bronchial epithelial primary culture; NHBE, normal human bronchial epithelial; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); RACE, rapid amplification of cDNA ends; UTR, untranslated region; ORF, open reading frame; uORF, upstream ORF; AM, alveolar macrophage.


ACKNOWLEDGEMENTS

We thank Dr. Martha Vaughan, Dr. Vincent C. Manganiello, and Dr. Bruce Trapnell for critical reviews of the manuscript and Carol Kosh for expert secretarial assistance.


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