Regulation of basal and induced expression of C-reactive protein through an overlapping element for OCT-1 and NF-kappaB on the proximal promoter - PubMed (original) (raw)

Regulation of basal and induced expression of C-reactive protein through an overlapping element for OCT-1 and NF-kappaB on the proximal promoter

Bhavya Voleti et al. J Immunol. 2005.

Abstract

C-reactive protein (CRP) is an acute phase protein produced by hepatocytes. A minor elevation in the baseline levels of serum CRP is considered an indicator of chronic inflammation. In hepatoma Hep3B cells, IL-6 induces CRP expression by activating transcription factors STAT3 and C/EBPbeta. IL-1 synergistically enhances the effects of IL-6. The first 157 bp of the CRP promoter are sufficient for IL-1 synergy. Previously, NF-kappaB, a transcription factor activated by IL-1beta in Hep3B cells, has been shown to increase endogenous CRP expression. The purpose of this study was to investigate the possible action of NF-kappaB on the 157 bp of the proximal promoter. In this study we show that NF-kappaB requires and acts synergistically with C/EBPbeta on the CRP-proximal promoter to regulate CRP expression. We located the regulatory element that consisted of overlapping binding sites for NF-kappaB (p50-p50 and p50-p65) and OCT-1. The kappaB site was responsible for the synergy between NF-kappaB and C/EBPbeta and was also necessary for the CRP transactivation by C/EBPbeta through the C/EBP site. Mutation of the kappaB site decreased the synergistic effect of IL-1beta on IL-6-induced CRP expression. Basal CRP expression increased dramatically when binding of both OCT-1 and NF-kappaB was abolished. Combined data from luciferase transactivation assays and EMSA lead us to conclude that the binding of OCT-1 to the promoter, facilitated by p50-p50 in a novel way, represses, whereas replacement of OCT-1 by p50-p65 induces CRP transcription in cooperation with C/EBPbeta. This model for CRP expression favors the variation seen in baseline serum CRP levels in a normal healthy population.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1

FIGURE 1

NF-κ_B acts on first 157 bp of the CRP promoter and synergizes with C/EBP_β to induce CRP promoter (−157/+3 or −300/−1)-driven luciferase expression. A representative experiment is shown; three independent experiments exhibited similar patterns. Cells were transfected with CRP promoter-Luc constructs (Luc-300 WT and Luc-157 WT) and plasmids encoding C/EBP_β_ (increasing doses) and NF-_κ_B (p50 and p65, 200 ng each). CRP transactivation was represented as the relative luciferase activity.

FIGURE 2

FIGURE 2

Localization of the _κ_B site on the CRP-proximal promoter. The nucleotide sequence of the CRP promoter between positions −74 and −43, relative to the transcription start site, is shown. The sequences of the putative _κ_B site centered at −69 and known binding sites for other transcription factors are boxed.

FIGURE 3

FIGURE 3

Binding of OCT-1 and NF-_κ_B to the composite OCT-1/_κ_B site. A, CRP promoter’s _κ_B site competes with the consensus _κ_B site for binding to NF-_κ_B. EMSA using radiolabeled consensus _κ_B site probe and nuclear extract from IL-_β_-treated cells as the source of NF-_κ_B; Competitor oligos 1–3, containing the putative _κ_B site, were derived from CRP promoter. Oligo 1, 19 bp containing the _κ_B site; oligo 2, 25 bp containing the _κ_B site; oligo 3, oligo 2 with mutated _κ_B site. B, Direct binding of NF-_κ_B to CRP promoter’s _κ_B site. EMSA used oligo 2 as a probe and nuclear extract from IL-_β_-treated cells. The competitors (200-fold excess of unlabeled oligos) and the Ab were added to the reaction mixtures before the addition of probe. Results were analyzed by a phosphor imager. The mobility of the free probe is not shown. A representative of three EMSAs is shown.

FIGURE 4

FIGURE 4

Binding of p50 and HNF-1 to oligo 2 and oligo 3, respectively. A, EMSA using oligo 2 as a probe and recombinant p50 (4 gel shift units/lane). B, EMSA using oligo 3 as a probe and nuclear extract from IL-_β_-treated cells. The competitors (200-fold excess of unlabeled oligos) and the Ab were added to the reaction mixtures before the addition of probe. Results were analyzed by a phosphor imager. The mobility of the free probe is not shown. A representative of three EMSAs is shown.

FIGURE 5

FIGURE 5

The κ_B site and the C/EBP site act together to regulate CRP expression. The basal Luc activity for each construct is considered 1, and Luc activities in response to C/EBP_β (80 ng) and NF-_κ_B (p50 and p65, 200 ng each) are plotted as fold induction over basal expression. The average ± SEM of three experiments are shown.

FIGURE 6

FIGURE 6

Synergistic effect of IL-1_β_ on IL-6-induced CRP expression is only partially mediated by NF-κ_B. The basal Luc activity for each construct is considered 1, and Luc activity in response to IL-6 and IL-1_β is plotted as the fold induction over basal expression. A representative experiment is shown.

FIGURE 7

FIGURE 7

The basal level of CRP expression was elevated from that of the promoter with the mutated _κ_B site. The basal Luc activities of mutated-STAT3 (m-ST) and m-_κ_B constructs are plotted as the fold induction over that of the WT construct whose basal activity was taken as 1. The average ± SEM of five experiments are shown.

FIGURE 8

FIGURE 8

A model showing the role of the overlapping _κ_B/OCT-1 sites functioning with the C/EBP site in regulating basal and induced CRP expression. A, Vacant OCT-1, κ_B, and C/EBP sites increase basal CRP expression. B, Binding of OCT-1 to its site requires previous transient binding of p50-p50 to the κ_B site. Once OCT-1 is bound, p50-p50 leaves its site. C, OCT-1-binding represses basal CRP expression. D, Cytokines such as IL-6 and IL-1_β activate C/EBP_β and NF-_κ_B p50-p65, respectively. A switch occurs between the repressor OCT-1 and p50-p65. Because the _κ_B site is only 16 bp away from the C/EBP site, a physical interaction between NF-κ_B and C/EBP_β is possible, resulting in induced CRP expression.

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References

    1. Agrawal A. CRP after 2004. Mol Immunol. 2005;42:927–930. - PMC - PubMed
    1. Kushner I. The phenomenon of the acute phase response. Ann NY Acad Sci. 1982;389:39–48. - PubMed
    1. Agrawal A, Simpson MJ, Black S, Carey MP, Samols D. A C-reactive protein mutant that does not bind to phosphocholine and pneumococcal C-polysaccharide. J Immunol. 2002;169:3217–3222. - PubMed
    1. Suresh MV, Singh SK, Agrawal A. Interaction of calcium-bound C-reactive protein with fibronectin is controlled by pH: in vivo implications. J Biol Chem. 2004;279:52552–52557. - PMC - PubMed
    1. Hurlimann J, Thorbecke GJ, Hochwald GM. The liver as the site of C-reactive protein formation. J Exp Med. 1966;123:365–378. - PMC - PubMed

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