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A critical role for C/EBPß binding to the AABS promoter response element in the human iNOS gene¹

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

²Correspondence: University of Pittsburgh, Starzl Transplant Institute, 3459 Fifth Ave., UPMC Montefiore, 7 South, Pittsburgh, PA 15213-2582, USA. E-mail: gellerda{at}msx.upmc.edu

SPECIFIC AIMS

The human inducible nitric oxide synthase (hiNOS) gene is pathophysiologically expressed in a tissue-specific manner. The molecular basis for this regulation has not yet been elucidated. The purpose of this study was to identify the molecular mechanisms accounting for liver-specific hiNOS transcription.

PRINCIPAL FINDINGS

1. Identification of a unique A-activator binding site (AABS) at -192 bp in the human iNOS promoter that specifically binds C/EBPß transcription factor
Our initial DNA sequence analysis of the human iNOS promoter indicated an AABS at -192 bp of hiNOS 5'-flanking region. This site is a 9/9 nucleotide (GTGATGTAA) match for AABS consensus motif (GTGNNGYAA), which was reported to mediate liver-specific gene expression. However, the mere presence of a DNA binding element does not imply a functional cis-acting motif. To identify whether nuclear proteins interact with the AABS in the hiNOS promoter, gel shift assays were performed on CM-stimulated rat hepatocytes and A549 human cells using wild-type or mutant oligos specifically designed from DNA sequence between -202 to -174 in the hiNOS promoter. A strong constitutive protein-DNA complex was observed in rat hepatocytes that was increased by CM treatment. Competition with 100-fold of excess cold wild-type AABS oligo, but not cold mutant AABS oligo, abrogated the gel shift complex, indicating specificity for AABS. This protein-DNA binding complex was also detectable in A549 human lung cells but not as strongly as in the rat hepatocytes. To identify the specific transcription factor binding to the AABS element at -192 bp, antibody supershift experiments were performed. A significant supershift was seen with the antibody against C/EBPß transcription factor. Antibodies against C/EBP, C/EBP transcription factors failed to elicit a supershift of the protein DNA complex. The data suggest that C/EBPß transcription factor plays a functional role in transcriptional regulation of hiNOS gene by interacting with the cis-acting AABS motif in the proximal hiNOS promoter.

2. Site mutation of AABS results in a loss of basal human iNOS promoter activity in human liver cells and significantly inhibits cytokine-induced promoter activity
To further determine a putative role for AABS element in hiNOS transcription, we used site-directed mutagenesis to generate a mutant AABS element in the wild-type -7.2 kb hiNOS luciferase promoter construct. Three liver cell types—primary rat hepatocytes, human AKN-1 and HepG2 cell lines—and three nonhepatic cell lines—Hela cervix, DLD-1 colon, and A549 lung cell lines—were transiently transfected with either wild-type or hiNOS-luc AABS mutant promoter constructs. Promoter activity was measured as relative luciferase activity in the lysed cells. In each cell line, the luciferase activity of mutant AABS was normalized as relative percentage of the luciferase activity of wild-type construct. As shown in Fig. 1 A, mutation of the AABS motif significantly reduced the basal promoter activity in the three hepatic cells, but did not significantly inhibit the basal activity in the three nonhepatic cells. Thus, the AABS element identified in the hiNOS promoter is a cis-acting DNA element that mediates liver-specific basal hiNOS transcriptional activity. Mutation of AABS significantly decreased the cytokine induced promoter activity in all cells tested (Fig. 1B ). This suggests that C/EBPß binding to AABS functions as a "switch point," which is necessary for cytokine-inducible hiNOS gene expression in a non-tissue-specific manner.

View larger version (56K): [in this window] [in a new window]   Figure 1. Effect of mutation of AABS on basal and cytokine-inducible human iNOS promoter activity in various cell types. Luciferase activity of the -7.2 kb wild-type human iNOS promoter was normalized to 100%. Luciferase activity of the transfected AABS mutant construct is shown relative to WT. A) Effect of AABS mutation on basal hiNOS promoter activity. B) Effect of AABS mutation on cytokine-inducible hiNOS promoter activity. *P < 0.05 vs. WT.

3. Overexpression of LAP trans-activates basal hiNOS gene transcription in a liver-selective manner by binding AABS
LAP overexpression and cotransfection with the hiNOS promoter were examined in multiple hepatic and nonhepatic cell lines. Overexpression of LAP produced an sixfold increase in hiNOS basal promoter activity in rat hepatocytes, human liver AKN-1 cells, and HepG2 cells but increased the basal activity in nonhepatic cells by only one- to twofold (Fig. 2 A). This suggests that LAP protein is more efficient at trans-activating basal hiNOS gene transcription through AABS in hepatic cells vs. nonhepatic cells. To reinforce the notion that LAP can boost basal hiNOS transcriptional activity, we examined the ability of LAP to modulate endogenous iNOS mRNA expression in cultured cells. Using RT-PCR, iNOS mRNA was not detected in resting (basal) cells transfected with empty vector (Fig. 2B ). Transfection of cells with a plasmid overexpressing LAP yielded detectable iNOS mRNA in resting rat hepatocytes as well as human A549 and AKN-1 cells. When endogenous iNOS mRNA was induced by standard cytokines stimulation, overexpression of LAP did not further increase the mRNA levels, consistent with the inability of LAP cotransfection to "superinduce" CM-stimulated hiNOS promoter activity. Overexpression of LIP partially inhibited CM-stimulated hiNOS mRNA in the A549 cells (Fig. 2B ), consistent with the ability of LIP to inhibit CM-induced hiNOS promoter induction.

View larger version (48K): [in this window] [in a new window]   Figure 2. Effect of overexpression of LAP on basal hiNOS promoter activity and mRNA level in different cell types. A) Percent increase in basal wild-type hiNOS promoter activity after LAP overexpression. B) RT-PCR analysis of the mRNA transcripts of hiNOS gene expression in A549, AKN-1, and rat hepatocytes after transfection with vector, LAP, or LIP expression plasmids. ß-Actin was used as internal control in the same RT-PCR reaction.

CONCLUSIONS

Promoter regions required for cytokine-induced hiNOS transcription have been mapped far upstream in the 5'-flanking region of the hiNOS gene from -4 to -16 kb. Functionally important NF-B, Stat-1, and AP-1 elements have been identified in the hiNOS promoter. However, no information exists regarding the function of AABS in governing hiNOS transcription. Further, the precise mechanisms regulating liver-specific basal expression of the hiNOS gene have not been previously reported. The major and novel findings of these experiments are the following: 1) AABS is a functional cis-acting DNA element that regulates basal hiNOS transcriptional activity in a relative liver-specific manner; 2) the C/EBPß isoforms LAP and LIP are positive and negative trans-acting nuclear factors, respectively, that bind to the cis-acting AABS motif in the proximal hiNOS promoter to regulate transcription; 3) AABS is a critical DNA response element that is necessary, but not sufficient for cytokine inducibility of hiNOS gene expression in a non-tissue-specific pattern.

Here, we proposed a model (Fig. 3 ) regarding the functional role AABS in transcriptional regulation of hiNOS gene transcription. Liver cells enriched in C/EBPß with strong AABS binding maintain high levels of basal transcription for hiNOS. In nonliver cells, AABS has weaker binding or no functional binding, lowering the basal transcription level of hiNOS. With cytokine stimulation, AABS binding is necessary but not sufficient for high levels of hiNOS transcription. AABS still needs to synergistically work with other transcription factors such as NF-B, AP-1, and Stat-1, which have been shown to bind upstream cis-acting response elements after cytokine stimulation.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic diagram.

In conclusion, functional binding between AABS and the active form of C/EBPß (LAP) is essential for liver-specific transcriptional regulation of the hiNOS gene. It is also necessary but not sufficient for cytokine-induced transcriptional regulation of the hiNOS gene in a non-tissue-specific manner.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1172fje; doi: 10.1096/fj.02-1172fje

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