Tumor Necrosis Factor (TNF) Pathway

1. What is Tumor Necrosis Factor?

Tumor necrosis factor (TNF), a multifunctional pro-inflammatory cytokine within the TNF superfamily, is primarily secreted by macrophages. It plays a pivotal role in regulating numerous biological processes, including immune response, apoptosis, cell proliferation and differentiation, embryogenesis, and lipid metabolism. Historically, TNF was recognized for its anti-cancer properties. TNF ligands and their receptors are classified into two complementary superfamilies, referred to as the TNF superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF).

2. Ligands and Receptors of the TNF Superfamily

The TNFSF comprises 19 cytokine-like ligands, while the TNFRSF encompasses 29 receptors that bind to these ligands. Signaling mediated by interactions between TNFSF ligands and TNFRSF receptors regulates critical cellular processes, including survival, proliferation, differentiation, and effector activities in both immune and non-immune cells. This dynamic interplay underscores the central role of the TNF pathway in orchestrating physiological responses across diverse cellular contexts.

Fig.1 Interactions between multi-receptor TNF superfamily ligands (top) and their receptors (bottom).^1,4

Table 1. Cellular expression of ligands and receptors of the TNF/TNFR superfamilies

3. TNF Receptor Pathways

3.1 TNFR1 Pathway

In its unliganded state, TNFR1 remains inactive through the interaction of its intracellular death domain (DD) with the silencer of death domain (SODD), which prevents the recruitment of adaptor proteins such as TNFR-associated death domain protein (TRADD) and blocks apoptosis. Upon binding of trimeric TNF-α to the extracellular domain of TNFR1, receptor trimerization occurs, leading to the release of SODD and exposure of the intracellular DD.

3.2 TNFR2 Pathway

TNFR2, lacking a DD, instead contains motifs that enable interaction with TNF receptor-associated factors (TRAFs). Upon ligand binding, TNFR2 forms a complex with TRAFs, triggering downstream activation of NF-κB and JNK kinases. This signaling cascade promotes gene transcription programs that enhance cell survival, proliferation, and differentiation.

3.3 Relationship Between TNFR1 and TNFR2 Pathway

TNFR2 primarily functions to concentrate ligands near TNFR1, thereby amplifying TNFR1-mediated signaling. Unlike TNFR1, TNFR2 activation does not directly induce apoptosis but instead modulates apoptotic responses indirectly. Specifically, TNFR2 signaling enhances TNF secretion, which subsequently activates TNFR1 to induce apoptosis. Moreover, TNFR2 activation, through TRAF2, significantly potentiates TNFR1-driven apoptotic signaling.

Fig.2 TNFR1 (a) and TNFR2 (b) mediated signaling pathways.^2,4

4. Physiological Function

4.1 Immune Response

TNF functions as a master regulator of immune responses, orchestrating inflammatory processes. Upon encountering infection or tissue damage, TNF mobilizes the immune system by recruiting neutrophils and macrophages to the affected area. This action instigates a cascading release of additional inflammatory mediators, amplifying the immune response and coordinating pathogen clearance. Beyond its role in innate immunity, TNF also orchestrates adaptive immune responses by influencing the maturation and activity of T and B lymphocytes, thereby ensuring a robust and tailored defense against invading pathogens.

4.2 Apoptosis

TNF-α binding to TNFR1 triggers the assembly of a signaling complex that includes TRADD, TRAF2, receptor-interacting protein (RIP), and Fas-associated death domain protein (FADD). When TNFR1 transmits apoptotic signals, FADD is recruited to the complex, initiating a protease cascade that results in DNA fragmentation and apoptosis. In contrast, survival signaling through TNFR1 involves the recruitment of TRAF2. Subsequent protease activation leads to the engagement of cytoplasmic anti-apoptotic proteins, such as cellular inhibitor of apoptosis proteins (cIAPs), which inhibit apoptosis.

4.3 Cell Proliferation and Differentiation

TNF augments MHC class I antigen presentation in T cells and promotes IL-2-dependent proliferation of thymocytes and T cells. It elicits the production of lymphokines such as IL-2, CSF, and IFN-γ, and drives B cell proliferation and immunoglobulins secretion when stimulated by mitogens or exogenous antigens. TNF-α exerts growth factor-like effects on select tumor cells, synergizing with EGF, PDGF, and insulin to amplify proliferative signals and upregulate EGF receptor expression. Furthermore, TNF elevates the expression of proto-oncogenes like c-myc and c-fos, facilitating cell cycle transition from G0 to G1.

4.4 Embryogenesis

TNF-α, produced by mononuclear macrophages, is closely linked to embryonic development, with expression observed in both the endometrium and embryo, indicating its role in pregnancy maintenance. However, elevated TNF-α levels can disrupt endometrial decidualization and induce trophoblast apoptosis, leading to implantation failure or embryonic arrest. In contrast, reducing the cytotoxicity of decidual natural killer (dNK) cells can inhibit TNF-α expression, thereby maintaining a stable maternal-fetal interface and supporting ongoing pregnancy.

4.5 Lipid Metabolism

Lipid metabolism is essential for maintaining physiological equilibrium and is intricately linked to metabolic disorders, including obesity, insulin resistance, non-alcoholic fatty liver disease, and type 2 diabetes. TNF-α influences lipid metabolism through diverse mechanisms across various cellular and tissue contexts:

• Reduces cellular uptake of free fatty acids (FFAs) while stimulating lipogenesis.
• Promotes the breakdown of stored lipids through lipolysis activation.
• TNF represses the activity of key enzymes in lipid metabolism, thereby altering metabolic pathways.
• Regulates cholesterol homeostasis by influencing its synthesis and distribution.
• TNF regulates the synthesis and secretion of adipokines such as leptin and adiponectin, which are essential for metabolic homeostasis.

5. Regulation of Downstream Pathways

TNF exerts its wide-ranging effects by engaging and modulating several key downstream signaling pathways. The activation of these pathways is initiated by the binding of TNF to its receptors, primarily TNFR1 and TNFR2, setting off a complex cascade of intracellular events. Notably, TNFR1 and TNFR2 activate both common and distinct downstream signaling pathways, leading to both similar and distinct biological functions.

5.1 NF-κB Pathway

TNF signaling initiates a cascade that activates the IκB kinase (IKK) complex, which in turn phosphorylates IκB proteins. This phosphorylation event marks IκB for proteasomal degradation, releasing the NF-κB transcription factor from its inhibitory complex. Upon release, NF-κB translocates to the nucleus, where it induces the transcription of numerous genes associated with cell survival, pro-inflammatory responses, and proliferation.

Fig.3 TNF induced canonical NF-κB pathway.^3,4

5.2 MAPK Pathway

TNF signaling initiates the activation of mitogen-activated protein kinases (MAPKs), such as JNK, p38 MAPK, and ERK, which in turn regulate transcription factors controlling genes involved in apoptosis, inflammation, and cell growth. TNFR1 and TNFR2 both participate in MAPK activation but with distinct kinetic profiles and varying intensities. TNF typically induces the activation of JNK and ERKs through dual phosphorylation of tyrosine and threonine residues. Once activated, JNK kinase relocates to the nucleus, where it enhances transcriptional activity by phosphorylating the N-terminal activation domains of transcription factors.

5.3 Apoptosis Pathway

TRADD, upon recruitment to TNFR1 through its death domain (DD), interacts with the C-terminal DD of FADD, thereby incorporating FADD into the TNFR1 signaling complex. The N-terminal death effector domain (DED) of FADD subsequently initiates apoptosis by activating caspase family signaling pathways, such as MACH/RLICE (caspase-8), through dual phosphorylation of tyrosine and threonine residues. This activation cascade ultimately leads to the activation of caspases-3, -6, and -7, culminating in apoptosis.

6. Related Products

Creative Biolabs is a trusted provider of high-quality TNF-related antibodies for research applications. We offer custom antibodies targeting key components of the TNFSF and TNFRSF and NF-κB, MAPK, apoptosis signal pathways. Our products are optimized for various techniques, including Western blot, immunohistochemistry, and ELISA, supporting researchers in advancing their studies.