Tumor necrosis factor
Tumor necrosis factor, formerly known as TNF-α, is a chemical messenger produced by the immune system that induces inflammation. TNF is produced primarily by activated macrophages, and induces inflammation by binding to its receptors on other cells. It is a member of the tumor necrosis factor superfamily, a family of transmembrane proteins that are cytokines, chemical messengers of the immune system. Excessive production of TNF plays a critical role in several inflammatory diseases, and TNF-blocking drugs are often employed to treat these diseases.
TNF is produced primarily by macrophages but is also produced in several other cell types, such as T cells, B cells, dendritic cells, and mast cells. It is produced rapidly in response to pathogens, cytokines, and environmental stressors. TNF is initially produced as a type II transmembrane protein, which is then cleaved by TNF alpha converting enzyme into a soluble form and secreted from the cell. Three TNF molecules assemble together to form an active homotrimer, whereas individual TNF molecules are inert.
When TNF binds to its receptors, tumor necrosis factor receptor 1 and tumor necrosis factor receptor 2, a pathway of signals is triggered within the target cell, resulting in an inflammatory response. sTNF can only activate TNFR1, whereas tmTNF can activate both TNFR1 and TNFR2, as well as trigger inflammatory signaling pathways within its own cell. TNF's effects on the immune system include the activation of white blood cells, blood coagulation, secretion of cytokines, and fever. TNF also contributes to homeostasis in the central nervous system.
Inflammatory diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease can be effectively treated by drugs that inhibit TNF from binding to its receptors. TNF is also implicated in the pathology of other diseases including cancer, liver fibrosis, and Alzheimer's, although TNF inhibition has yet to show definitive benefits.
History
In the 1890s, William Coley observed that acute infections could cause tumor regression, leading to his usage of bacterial toxins as a cancer treatment. In 1944, endotoxin was isolated from Coley's bacterial toxins as the substance responsible for the anticancer effect. In particular, endotoxin could cause tumor regression when injected into mice with experimentally induced cancers. In 1975, Carswell et al. discovered that endotoxin did not directly cause tumor regression, but instead induced macrophages to secrete a substance that causes tumors to hemorrhage and necrotize, termed "tumor necrosis factor."In the 1980s, TNF was purified, sequenced, and cloned in bacteria. Studies on recombinant TNF confirmed the anticancer potential of TNF, but this optimism faded when TNF injections were found to induce endotoxin shock. TNF was also discovered to be the same protein as cachectin, known to cause muscle wasting in mice. These findings demonstrated that TNF could be detrimental in excessive quantities. In 1992, TNF antibodies were found to reduce joint inflammation in mice, revealing TNF's role in inflammatory diseases. This led to the approval of the first anti-TNF therapy for rheumatoid arthritis in 1998.
Nomenclature
In 1985, TNF was found to have significant sequential and functional similarity with lymphotoxin, a previously discovered cytokine. This led to the renaming of TNF to TNF-α and lymphotoxin to TNF-β. However, in 1993, a protein with close similarity to lymphotoxin was discovered, termed lymphotoxin-β. In 1998, at the Seventh International TNF Congress, TNF-β was officially renamed to lymphotoxin-α, while TNF-α was renamed back to TNF. Nevertheless, some papers continue to use the term TNF-α.Evolution
The TNF and lymphotoxin-α genes are believed to be descended from a common ancestor gene that developed early in vertebrate evolution, before the Agnatha and Gnathostomata split. This ancestor gene was dropped from the Agnatha ancestor but persisted in the Gnathostomata ancestor. During the evolution of gnathostomes, this ancestor gene was duplicated into the TNF and lymphotoxin-α genes. Thus, while the ancestor gene is found across a variety of gnathostome species, only a subset of gnathostome species contain a TNF gene. Some fish species, such as Danio, have been found to contain duplicates of the TNF gene.The TNF gene is very similar among mammals, ranging from 233 to 235 amino acids. The TNF proximal promoter region is also highly conserved among mammals, and nearly identical among higher primates. The similarity of the TNF gene among fish is lower, ranging from 226 to 256 amino acids. Like mammalian TNF, the fish TNF gene has been shown to be stimulated in macrophages by antigens. All TNF genes have a highly conserved C-terminal module known as the TNF homology domain, due to its important role in binding TNF to its receptors.
Gene
Location
The human TNF gene is mapped to chromosome 6p21.3, residing in the class III region of the major histocompatibility complex, where many immune system genes are contained. The class III region is sandwiched between the HLA-DR locus on the centromeric side, and the HLA-B locus on the telomeric side. The TNF gene is 250 kilobases away from the HLA-B locus, and 850 kilobases away from the HLA-DR locus. The TNF gene is located 1,100 kilobases downstream of the lymphotoxin-α gene.Expression
TNF is produced rapidly in response to many stimuli by multiple cell types. Cell types that express TNF include T cells, B cells, macrophages, mast cells, dendritic cells, and fibroblasts, and stimuli that activate the TNF gene include pathogenic substances, cytokines from other immune cells, and environment stressors. A few such cytokines include interleukin-1, interleukin-2, interferon-γ, and TNF itself. TNF transcription is activated by a variety of signaling pathways and transcription factors, depending on the cell type and stimulus. TNF transcription does not depend on the synthesis of new proteins, enabling rapid activation of the gene.TNF gene expression is regulated by a proximal promoter region consisting of approximately 200 base pairs. Most of the binding sites within the proximal promoter region can recognize multiple transcription factors, enabling TNF to be activated by a variety of signaling pathways. As transcription factors bind to the promoter region, they also bind to coactivators, assembling into a large structure known as an enhanceosome. The composition of the enhanceosome depends on ambient factors within the cell, particularly nuclear factor of activated T-cells.
TNF expression is also regulated by DNA structure. DNA is coiled around histones, which is loosened by acetylation and condensed by methylation. Proteins that acetylate histones at the TNF promoter, particularly CREB-binding protein in T cells, are often critical for TNF expression. In contrast, several cell types that do not express TNF are highly methylated at the histones of the TNF promoter. Long-range intrachromosomal interactions can also regulate TNF expression. In activated T-cells, the DNA surrounding the TNF promoter circularizes, bringing promoter complexes closer together and enhancing transcription efficiency.
Transcription
The transcribed region contains 4 exons separated by 3 introns, for a total of 2,762 base pairs in the primary transcript and 1,669 base pairs in the mRNA. The mRNA consists of four regions: the 5′ untranslated region, which is not included in the TNF protein; the transmembrane portion, which is present in transmembrane TNF but not in soluble TNF; the soluble portion; and the 3′ untranslated region. More than 80% of the soluble portion is contained in the last exon, while the transmembrane portion is contained in the first two exons. The 3' untranslated region contains an AU-rich element that regulates the translation of TNF. In unstimulated macrophages, various proteins bind to the ARE to destabilize TNF mRNA, suppressing the translation of TNF. Upon activation, TNF translation is unsuppressed.Protein
TNF is initially produced as a transmembrane protein consisting of 233 amino acids. tmTNF binds to both TNFR1 and TNFR2, but its activity is primarily mediated by TNFR2. Upon binding to a receptor, tmTNF also activates signaling pathways within its own cell. tmTNF is cleaved by TNF alpha converting enzyme, which causes the extracellular portion to be secreted. After cleavage, the remaining tmTNF is cleaved again by SPPL2B, causing the intracellular portion to translocate to the nucleus. There, it is believed to regulate cytokine production, such as triggering the expression of interleukin-12.The secreted extracellular portion, denoted sTNF, consists of 157 amino acids. Unlike tmTNF, sTNF can only bind to TNFR1. The secondary structure of sTNF consists primarily of alternating strands that join into two sheets, known as antiparallel β-sheets. The two sheets are layered on top of each other, forming a wedge shape known as an antiparallel β-sandwich. Remarkably, this structure is similar to those seen on the coats of viruses. The last 9 residues of the C-terminus are locked into the middle strand of the bottom sheet, and are necessary for bioactivity.
Both tmTNF and sTNF are only bioactive as homotrimers, whereas individual monomers are inactive. The rate at which TNF trimers disassemble is constant, whereas the rate at which TNF trimers assemble increases with TNF concentration. This causes TNF to be mostly trimers at high concentrations, whereas TNF is mostly monomers and dimers at low concentrations. The coexistence of TNF dimers and trimers in dynamic equilibrium suggests that TNF might be a morpheein. Small molecules that stabilize TNF dimers and prevent the assembly of TNF trimers present a potential mechanism for inhibiting TNF.