Regulation of TNF-alpha
The proinflammatory cytokine TNF is produced by macrophages and cells of other lineages in response to a variety of extracellular signals. This response is very rapid and transient and includes a transcriptional component as well as posttranscriptional events. The transcriptional control occurs predominantly at the level of transcriptional initiation.
The approximately 1000 base pairs of the TNF gene's 5' flanking region contains a number of important regulatory elements that affect TNFs transcription in response to various stimuli. The basic promoter region is defined by the Goldberg-Hogness (TATA box) sequence, located about 20 bp upstream from the transcription site and about 200 bp form the translation start codon. The GC box/SP-1 binding site is about 20 bp farther upstream and inverted in orientation. Elements that may provide more specific control of TNF transcription are found farther upstream.
The 5' flanking region of the TNF gene contains multiple potential regulatory sites, including consensus sequences for the AP-1 and AP-2 sites, the cAMP-responsive element, and sequences similar to the kB sequences found in immunoglobulin and cytokine regulatory elements. This sequence has been demonstrated to be responsive to LPS and TNF stimulation. The 3' untranslated region contains a sequence element affecting posttranslational control of TNF through mRNA stability and translation efficiency. The functional importance and interactions of these regulatory elements remain undefined.
The TNF-promoter
Alignment of human and murine TNF promoter sequences reveals several regions of extended homology. The most noticeable conservation occurs within the first 200 nt of the promoter. This region contains several previously characterized regulatory elements, Egr-1, Ets, AP-1/ATF/CRE and site "kappa3", which is distantly homologus to kB consensus. The second region of extended homology spans about 50 nt in the promoter between -600 and -650 nt. It contains at least two kB-like binding motifes (somteimes caleld kB#2 and KB#2a).
No signifcant homology can be observed farther upstream of this region, although one kB-like motfiy (soemteimes caleld kB#1) is present in each ofthe genes in nonconserved positions.
An additional consesnsus kB site (kB#3) is located at -510 nt in the murine TNF promoter and has no homologous match in the human sequence.
Regulation by NF-kB
Four NF-kB sites (kB1, kB2, kB2a and kB3) have been idnetified in the murine TNFalpha promoter.
TNF-alpha expression is tightly regulated at the transcriptional level and is under the repressive influence of two NF-kB proteins p50 and RelB. A complex of homodimeric p50 and HDAC1 binds to kB sites and functions as a general repressor of NF-kB-dependent transcription in resting cells. On cell stimulation, the phosphorylated p65 subunit of newly formed nuclear p50/p65 heterodimers associates with CBP/p300 and this complex then replaces the repressive p50/p50-HDAC1 complex at kB sites to activate transcription.
NF-kB binding by the sites kB#2 and kB32a and KB#3 from the murine TNF gene promoter each binds a unique repertoire of NF-kB/Rel complexes. Site kB#3 predominantly binds NFKB1 homodimer and a constitutive non-NNF-kB complex. The binding by site KB#2a resembles that of site #3, with the exception of RelA-NFKB1 heterodimer, which binds to site kB#2a with much lower affinity.
Although mutation data shows that kB site #3 in the murine promoter, which has high affinity to both NF-kB1 (p50) homodimer and RelA-NF-kB1 (p65-050) heterodimer makes a contribution to maximal LPS inducibility, it may play a less important role than much weaker binding sites kB#2 and kB#2a.
Regulation of LPS Induction of the TNF promoter
LPS enhances TNF-alpha secretion partially through increasing nuclear availability of NF-kB dimers and c-Jun/ATF-2 dimers to bind their respective response elements in the TNFalpha promoter. Glucocorticoid exposure reduces the nuclear abundance of binding competent transcription factor complexes at both of these sites and correspondingly suppreses the activity of promoter reporter vectors incorporating these sites, when they are transfected into THP-1 cells.
LPS responsiveness of the murine TNF promoter is goverened primarily by two regions: 1) the proximal promoter region and 2) the distal regions between -665 and -510. This result suggests that the effects of NF-kB in LPS induced transcription of the TNF promtoer are mediated primarily by kB sites #2 and/or #2a.
NF-kB binding by sites kB#2, kB#2a and kB#3 from the murine TNF gene promoter each binds a unique repertoire of NF-kB/Rel complexes. Site kB#3 predominantly binds NFKBI homodimer and RelA-NFKB1 heterodimer, while site kB#2 binds several more slowly migrating RelA/cRel-continaing compelxes, but virtually no NFKB1 homodimer and a constitutive non-NF-kB complex.
NF-kB is not the only nuclear factor involved in activation of TNF transcription by LPS. LPS activaiton of TNF promoter is mediated by a concerted action of transcription factor binding sites and each of the kB sites with proven NF-kB binding is important for maximal activation.
A macrophage tumor cell line that negatively regulates LPS induced TNFalpha expression reportedly produced a factor which suppressed the induction of TNFalpha in macrophages through induction of the NF-kB p50 subunit.
Aspirin reportedly supressed LPS inducible NF-kB binding to an NF-kB binding site in the TNFalpha promoter, TNFalpha mRNA accumulation and protein secretion throughy stabilization of inhibitor kB (IkB)
Both MyD88 and tumor necrosis factor receptor associated factor 6 are commonly required for activation of the TNFalpha promoter by various Cdp-ODNs with different potencies. A dominant negative version of MyD88 cotransfected into RAW 264.7 cells also inhbited LPS mediated TNFalpha promoter activation.
Regulation by NF-kB independent components
It has been suggested that there is an NF-kB-independent component to LPS activation that can be mediated through known or perhaps not yet characterized binding sties in the TNF promoter.
Role of Introns
There is accumulating evidence that introns play a role in the regulation of transcriptional events. A comparison of intron 3 of the TNFα between human, porcine, and bovine genes demonstrates that it is the most conserved intron of the TNFα and the TNFβ gene. Compared with human intron 3, the similarity is 85% for the porcine and 81% for the bovine intron. Because there are some conserved transcription factor sequence elements located in this thrid intron, it may play a role in the modulation of TNFα expresion.
Chromatin requlation
It is widely accepted that chromatin structure can influence the expression of a gene. One approach to assess the functionality of a gene is to search for DNaseI hypersensitive sites (DH-sties), which indicate if a gene is actively transcribed. Such an analysis has been performed in human erythroleukemia (HEL) and myelomonocytic cells. No hypersensitive bands are detected in HEL cells. However, all myelomonocytic cells show a major DH site which is located within -100 to -200 of the TNFα promoter. This region includes sites for kappaB, CRE, AP-1, AP-2, and SP-1. This DH site is also seen in cells that are not activated. Interestingly, in activated cells there are no additional DH sites found. Band shift assays with this region show a specific binding for AP-1, AP-2 and NFkB sequences. AP-1 displays similar complexes before and after stimulation, but one complex is more intense when extracts from stimulated cells are used. AP-2 is shown to be involved in the specific binding of only one complex and only being present in extracts of stimulated cells. For NFkB nine specific complexes are detected. No difference is seen between extracts from unstimulated or stimulated cells.
Regulation by other cytokines
It is well established that expression of TNFalpha is inhibited by IL-10, an antiinflammatory cytokine produced by LPS activated that macrophages that suppresses LPS induced expression of several proinflammatory cytokines.
IL-4, transforming growth factor beta, prostaglandin E2 and glucocortoids also possess anti-inflammatory activities and inhibit production of TNF alpha and other cytokines.
