Interleukin (IL)-8, a prototypic chemokine, is rapidly induced by the pro-inflammatory cytokine IL-1 but is barely detectable in noninduced cells. Although there is clear evidence that the transcription factor NF-κB plays a central role in inducible IL-8 transcription, very little is known about the cis-elements and trans-acting factors involved in silencing of the IL-8 promoter. By sequence comparison with the interferon-β promoter, we found a negative regulatory element (NRE) in the IL-8 promoter overlapping partially with the NF-κB response element. Here we show that an NF-κB-repressing factor (NRF) binds to the IL-8 promoter NF-κB-NRE. Reduction of cellular NRF by expressing NRF antisense RNA results in spontaneous IL-8 gene expression. In contrast, IL-1-induced IL-8 secretion is strongly impaired by expressing NRF antisense RNA. Mutation of the NRE site results in loss of NRF binding and increased basal IL-8 transcription. On the other hand IL-1-induced IL-8 transcription is decreased by mutating the NRE. These data provide evidence for a dual role of the NRF in IL-8 transcription. Although in the absence of stimulation it is involved in transcriptional silencing, in IL-1-induced cells it is required for full induction of the IL-8 promoter.

Although nuclear factor (NF)-κB plays a central role in mediating cytokine-stimulated human inducible nitric-oxide synthase (hiNOS) gene transcription, very little is known about the factors involved in silencing of the hiNOS promoter. NF-κB-repressing factor (NRF) interacts with a specific negative regulatory element (NRE) to mediate transcriptional repression of certain NF-κB responsive genes. By sequence comparison with the IFN-β and IL-8 promoters, we identified an NRE in the hiNOS promoter located at −6.7 kb upstream. In A549 and HeLa human cells, constitutive NRF mRNA expression is detected by RT-PCR. Gel shift assay showed constitutive NRF binding to the hiNOS NRE. Mutation of the −6.7-kb NRE site in the hiNOS promoter resulted in loss of NRF binding and increased basal but not cytokine-stimulated hiNOS transcription in promoter transfection experiments. Interestingly, overexpression of NRF suppressed both basal and cytokine-induced hiNOS promoter activity that depended on an intact cis-acting NRE motif. By using stably transformed HeLa cells with the tetracycline on/off expression system, reduction of cellular NRF by expressing antisense NRF increased basal iNOS promoter activity and resulted in constitutive iNOS mRNA expression. These data demonstrate that the transacting NRF protein is involved in constitutive silencing of the hiNOS gene by binding to a cis-acting NRE upstream in the hiNOS promoter.

Transcriptional regulation of the interferon‐β (IFN‐β) gene is characterized by strict constitutive repression and virus‐specific activation. Previous studies have shown that the IFN‐β promoter is constitutively repressed by a negative regulatory element (NRE). Isolated NRE acts as a constitutive and position‐independent silencer on the NF‐κB‐binding sites. Here, we describe the identification and functional characterization of the NRE‐binding protein, called NRF (NF‐κB‐repressing factor), which abolishes the transcriptional activity of the bordering NF‐κB‐ binding sites by a distance‐independent mechanism. Deletion studies show that a minimal repression domain of NRF is sufficient to exert its inhibitory effect. In vitro , NF‐κB proteins bind to purified NRF by a direct protein–protein interaction. We demonstrate that NRF is a ubiquitous and constitutive nuclear protein. In fibroblasts, the expression of the NRF antisense RNA releases the endogenous IFN‐β gene transcription. Our data strongly suggest that the NRF‐mediated inhibition of NF‐κB is a critical component of the IFN‐β gene silencing prior to viral infection.

The ubiquitous transcription factor NRF (NF-κB repressing factor) is a constitutive transcriptional silencer of the multifunctional cytokine interferon-β. NRF mRNA contains a long 5′ untranslated region (5′UTR) predicted to fold into a strong secondary structure. The presence of stable hairpins is known to be incompatible with efficient translation by ribosomal scanning. Using dicistronic reporter gene constructs, we show that the NRF 5′UTR acts as an internal ribosome entry site (IRES) which directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES in various cell lines is at least 30-fold higher than that of picornaviral IRESs. The NRF 5′UTR also functions as a translational enhancer in the context of monocistronic mRNAs. Our results indicate that the NRF 5′UTR contains a highly potent IRES, which may allow for an alternate mode of translation under physiological conditions in which cap-dependent translation is inhibited.

The human interferon‐beta (IFN‐beta) promoter contains several functional domains that contribute to its virus‐inducible regulation. One of them, PRDII, and NF‐kappa B‐binding sequence, can function as a constitutively activating element. Due to the presence of a negative regulatory domain that mediates a constitutive repression the natural IFN‐beta promoter is silent in the non‐induced state. Within this domain we have delimited an 11 bp element that acts as a negative regulatory element (NRE) of PRDII. Although the NRE is physically overlapping with PRDII in the IFN‐beta promoter, it acts as a position‐independent silencer of PRDII. Virus infection, which leads to the transcriptional activation of the IFN‐beta promoter, does not alter the negative activity of the NRE on an isolated PRDII. It is the cooperative effect of PRDI and PRDII that is able to overcome the NRE function after virus infection. By UV cross‐linking analysis using uninduced and virus‐induced nuclear extracts, we show that two factors with molecular masses of approximately 95 and 100 kDa bind to the NRE.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine and has been implicated in inflammatory diseases. However, little is known about the regulation of MIF in adipose tissue and its impact on wound healing. The aim of this study was to investigate MIF expression in inflamed adipose and determine its role in inflammatory cell recruitment and wound healing. Adipose tissue was harvested from subcutaneous adipose tissue layers of 24 healthy subjects and from adipose tissue adjacent to acutely inflamed wounds of 21 patients undergoing wound debridement. MIF protein and mRNA expression were measured by ELISA and RT-PCR. Cell-specific MIF expression was visualized by immunohistochemistry. The functional role of MIF in cell recruitment was investigated by a chemotaxis assay and by flow cytometry of labeled macrophages that were injected into Mif^–/–and wildtype mice. Wound healing was evaluated by an in vitro scratch assay on human fibroblast monolayers. MIF protein levels of native adipose tissue and supernatants from acutely inflamed wounds were significantly elevated when compared to healthy controls. MIF mRNA expression was increased in acutely inflamed adipose tissue indicating the activation of MIF gene transcription in response to adipose tissue inflammation. MIF is expressed in mature adipocytes and in infiltrated macrophages. Peripheral blood mononuclear cell migration was significantly increased towards supernatants derived from inflamed adipose tissue. This effect was partially abrogated by MIF-neutralizing antibodies. Moreover, when compared to wildtype mice, Mif^–/–mice showed reduced infiltration of labeled macrophages into LPS-stimulated epididymal fat pads in vivo. Finally, MIF antibodies partially neutralized the detrimental effect of MIF on fibroblast wound healing. Our results indicate that increased MIF expression and rapid activation of the MIF gene in fat tissue adjacent to acute wound healing disorders may play a role in cell recruitment to the site of inflammation and wound healing.