Recombinant Human Tumor necrosis factor receptor superfamily member 12A protein (TNFRSF12A), partial (Active)

What is TNFRSF12A and what is its basic molecular structure?

TNFRSF12A, also known as TWEAK receptor (TWEAKR), fibroblast growth factor-inducible immediate-early response protein 14 (FN14), or CD266, is the smallest member of the Tumor Necrosis Factor Receptor superfamily. The gene encoding this protein is located on chromosome 16 in humans and chromosome 17 in mice, with approximately 93% sequence similarity between the two species . The protein initially consists of 128 amino acids with a single cysteine-rich domain (CRD), ultimately processed to a mature form of 102 amino acids .

Structurally, TNFRSF12A is a type I transmembrane protein characterized by disulfide bonds that form the cysteine-rich domains. The tertiary structure of the CRD features a beta-sheet with two strands, followed by a 3(10) helix and a C-terminal alpha-helix. This structure is stabilized by three critical disulfide bonds connecting Cys36-Cys49, Cys52-Cys67, and Cys55-Cys64 . Despite lacking a death domain typically found in other TNFR superfamily members, TNFRSF12A can still produce weak death signals in certain cellular contexts.

How is TNFRSF12A expressed in normal and pathological tissues?

TNFRSF12A exhibits a distinct expression pattern across human tissues. Under normal physiological conditions, TNFRSF12A is expressed at high levels in the heart, placenta, and kidney; at intermediate levels in lung, skeletal muscle, and pancreas; and at low levels in brain and liver . This expression pattern suggests tissue-specific functions across different organ systems.

In pathological contexts, TNFRSF12A shows significant upregulation. For instance, elevated TNFRSF12A expression has been documented in human liver cancer cell lines and hepatocellular carcinoma specimens . During acute liver failure (ALF), both TNFRSF12A and its ligand TWEAK show marked increases in expression within hepatic tissue . Bioinformatic analyses across multiple cancer types using data from TCGA, GEO, and Human Protein Atlas (HPA) have revealed that TNFRSF12A is upregulated in the majority of cancers and correlates with poor prognosis .

What signaling pathways does TNFRSF12A activate?

TNFRSF12A primarily signals through the NF-κB pathway, particularly activating the canonical NF-κB signaling cascade. When the TWEAK ligand binds to TNFRSF12A, it triggers intracellular signaling that leads to activation of various downstream molecules. RNA-sequencing analysis of TNFRSF12A-overexpressing colorectal cancer cells has revealed significant upregulation of several NF-κB pathway components .

The signaling cascade involves:

• Activation of the NF-κB pathway upon TWEAK/TNFRSF12A interaction

• Upregulation of NF-κB target genes, particularly those regulated by RELA (p65)

• Significant increases in expression of genes like TRAF1, NFKB2, and BIRC3

• Enhanced TNF signaling, cytokine-receptor binding, and inflammation-related processes

Additionally, in the context of acute liver failure, TNFRSF12A signaling activates receptor-interacting protein kinase 1 (RIPK1), which leads to RIPK1-dependent apoptosis of hepatocytes rather than necroptosis or pyroptosis . This mechanistic distinction is crucial for understanding the tissue-specific effects of TNFRSF12A signaling.

How can recombinant TNFRSF12A protein be effectively produced and purified?

Production of high-quality recombinant TNFRSF12A protein requires careful consideration of expression systems and purification strategies. Based on current methodologies, the HEK293 expression system has proven particularly effective for producing functional recombinant human TNFRSF12A . This approach allows for proper post-translational modifications essential for protein activity.

The recommended production methodology involves:

• Expression system selection: HEK293 cells are preferred for mammalian protein expression with appropriate post-translational modifications.

• Construct design: Optimal constructs include the extracellular domain (Glu28-Trp79) of human TNFRSF12A fused with an Fc tag and/or 6×His tag at the C-terminus for detection and purification .

• Purification process: Sequential purification using affinity chromatography (typically based on the His tag) followed by size exclusion chromatography achieves >90% purity as confirmed by SDS-PAGE .

• Endotoxin removal: Ensuring endotoxin levels below 0.1 EU/μg of protein using the LAL method is critical for downstream applications .

• Formulation: The purified protein is typically formulated in PBS (pH 7.4) and lyophilized for stability.

For reconstitution, researchers should centrifuge the vial before opening and reconstitute to a concentration of 0.1-0.5 mg/mL in sterile distilled water. To enhance stability during storage, addition of a carrier protein or stabilizer (e.g., 0.1% BSA, 5% HSA, 10% FBS, or 5% Trehalose) is recommended, and the solution should be aliquoted to minimize freeze-thaw cycles .

What are the most reliable methods for assessing TNFRSF12A activity in experimental settings?

Assessment of TNFRSF12A activity can be performed through several complementary assays that evaluate different aspects of its biological function:

• TWEAK-induced apoptosis inhibition assay: Functional TNFRSF12A can inhibit TWEAK-induced apoptosis in HT-29 human colon adenocarcinoma cells. The ED₅₀ for this effect typically ranges from 2-12 μg/mL in the presence of 1 μg/mL recombinant human TWEAK .

• Binding assays using functional ELISA: Immobilized Human TNFSF12 (TWEAK) at 2 μg/mL can bind Human TNFRSF12A with a linear detection range of 0.1-2.3 ng/mL, providing a quantitative measure of binding capacity .

• HUVEC proliferation inhibition assay: TNFRSF12A can inhibit TWEAK-dependent proliferation of HUVEC (human umbilical vein endothelial cells). The ED₅₀ for this effect is typically 20-80 ng/mL in the presence of 15 ng/mL recombinant human TWEAK .

• NF-κB pathway activation assessment: Measuring the activation of NF-κB signaling components, particularly RELA targets, can provide indirect evidence of TNFRSF12A activity. This can be assessed through reporter assays, western blotting for phosphorylated components, or quantification of downstream gene expression .

• RIPK1-dependent apoptosis measurement: In hepatocyte models, TNFRSF12A activity can be assessed by measuring RIPK1-dependent apoptosis using flow cytometry with annexin V/PI staining, caspase activity assays, or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) .

What experimental models are most appropriate for studying TNFRSF12A functions?

Selection of appropriate experimental models is crucial for studying the diverse functions of TNFRSF12A. Based on the current literature, the following models have proven particularly valuable:

• In vitro cellular models:

  • Human colon adenocarcinoma cell lines (e.g., HT-29, DLD-1) for studying TNFRSF12A's role in colorectal cancer progression

  • Human umbilical vein endothelial cells (HUVECs) for angiogenesis studies

  • Hepatocyte cell lines for studying acute liver failure mechanisms

  • Stable knockdown or overexpression cell lines to evaluate TNFRSF12A's function in various cellular processes

• In vivo models:

  • Thioacetamide (TAA) or acetaminophen (APAP)-induced acute liver failure mouse models for studying TNFRSF12A's role in hepatocyte death and liver injury

  • Subcutaneous xenograft models in BALB/c mice using TNFRSF12A-manipulated cancer cell lines to assess tumor growth and progression

  • Genetic knockout or transgenic mouse models for tissue-specific studies of TNFRSF12A function

• Human tissue samples:

  • Liver biopsies from ALF patients to correlate TNFRSF12A expression with disease severity

  • Cancer tissue microarrays for pan-cancer expression analysis

  • Paired tumor/normal tissue samples for comparative analysis of TNFRSF12A expression and pathway activation

Each model system offers unique advantages for investigating specific aspects of TNFRSF12A biology, from molecular signaling to physiological outcomes in disease contexts.

What is the role of TNFRSF12A in acute liver failure?

TNFRSF12A plays a critical role in the pathogenesis of acute liver failure (ALF), a severe clinical syndrome characterized by massive hepatocyte death leading to coagulopathy and hepatic encephalopathy with high mortality rates. Research has established that both TWEAK and TNFRSF12A expression are significantly upregulated during ALF induced by either thioacetamide (TAA) or acetaminophen (APAP) in experimental models, as well as in liver biopsies from ALF patients .

The mechanistic pathway by which TNFRSF12A contributes to ALF has been elucidated:

• During ALF, hepatic expression of both TWEAK and TNFRSF12A increases substantially

• The TWEAK/TNFRSF12A axis activates receptor-interacting protein kinase 1 (RIPK1) in hepatocytes

• This activation leads specifically to RIPK1-dependent apoptosis, rather than other cell death modalities such as necroptosis or pyroptosis

• The resulting massive hepatocyte apoptosis contributes to the severe liver injury characteristic of ALF

Importantly, inhibition of either the TWEAK/TNFRSF12A axis or RIPK1-dependent apoptosis significantly attenuates liver injury in experimental models of ALF . This finding identifies TNFRSF12A as a potential therapeutic target for ALF, which currently has limited treatment options beyond liver transplantation.

How does TNFRSF12A contribute to cancer progression?

TNFRSF12A has emerged as a significant factor in cancer biology, with elevated expression observed across multiple cancer types and correlation with poor prognosis. Comprehensive pan-cancer bioinformatics analysis using data from TCGA, GEO, and Human Protein Atlas has confirmed TNFRSF12A upregulation in most cancer types .

In colorectal cancer specifically, TNFRSF12A has been shown to promote tumor progression through several mechanisms:

• Enhanced cellular proliferation: Knockdown of TNFRSF12A inhibits colorectal cancer cell growth, while overexpression enhances proliferation both in vitro and in vivo .

• Increased clonogenic capacity: TNFRSF12A overexpression significantly enhances the colony-forming ability of colorectal cancer cells .

• Promotion of cell migration: Experimental evidence indicates that TNFRSF12A plays a role in enhancing the migratory capacity of cancer cells, potentially contributing to metastatic potential .

• Activation of NF-κB signaling: RNA-seq analysis of TNFRSF12A-overexpressing colorectal cancer cells revealed significant upregulation of NF-κB pathway components and target genes . The table below summarizes key differentially expressed genes in this pathway:

• TNFRSF12A/RELA/BIRC3 regulatory axis: Research has identified a regulatory axis whereby TNFRSF12A activates RELA (p65), leading to upregulation of BIRC3, which serves as a key downstream effector promoting cancer cell survival and proliferation .

Rescue experiments have confirmed the critical role of BIRC3 in mediating TNFRSF12A's effects on colorectal cancer cell growth, colony formation, and migration, indicating that BIRC3 is a key downstream factor in the oncogenic function of TNFRSF12A .

What immune and inflammatory pathways are modulated by TNFRSF12A?

TNFRSF12A significantly influences immune and inflammatory pathways across various physiological and pathological contexts. Gene Ontology (GO) and KEGG pathway analyses of TNFRSF12A-overexpressing cells have revealed several key inflammatory and immune-related processes modulated by this receptor :

• Enhanced TNF signaling pathway: TNFRSF12A overexpression leads to significant upregulation of TNF signaling components, creating potential feedback loops that may amplify inflammatory responses .

• Activation of NF-κB signaling: As a central regulator of inflammation, NF-κB pathway activation by TNFRSF12A drives expression of numerous pro-inflammatory genes .

• Altered cytokine-receptor interactions: TNFRSF12A modulates the expression of various cytokines and their receptors, potentially reshaping immune cell recruitment and activation .

• Promotion of chronic inflammation: Transcriptomic analysis indicates that TNFRSF12A overexpression creates a milieu favoring chronic inflammatory processes, which may contribute to disease progression in various contexts .

• Regulation of immune cell function: Although not directly addressed in the provided search results, TNFRSF12A's role in modulating inflammatory signaling suggests potential impacts on immune cell recruitment, activation, and function.

In the context of acute liver failure, TNFRSF12A-mediated activation of inflammatory pathways contributes to hepatocyte injury and death . In cancer, TNFRSF12A-driven inflammation may create a tumor-promoting microenvironment that facilitates cancer cell survival and progression . These findings highlight the importance of TNFRSF12A as a potential target for modulating inflammatory responses in various disease settings.

How might TNFRSF12A be targeted for therapeutic development?

Given its involvement in various pathological processes, TNFRSF12A represents a promising therapeutic target. Several approaches for therapeutic targeting of TNFRSF12A have been investigated or proposed:

• Direct inhibition of TWEAK/TNFRSF12A interaction: Blocking the binding of TWEAK to TNFRSF12A using neutralizing antibodies or decoy receptors has shown efficacy in experimental models of acute liver failure . This approach could potentially be extended to other inflammatory conditions where this axis plays a pathogenic role.

• Targeting RIPK1-dependent apoptosis: In acute liver failure, inhibiting RIPK1-dependent apoptosis downstream of TNFRSF12A activation provides an alternative therapeutic strategy . RIPK1 inhibitors are already in development for various inflammatory conditions and could be repurposed for TNFRSF12A-mediated pathologies.

• Development of TNFRSF12A-directed immunotherapeutics: Research has explored the development of TNFRSF12A× CD3 Bispecific T-cell Engagers (BiTEs) and TNFRSF12A-specific CAR-T cells, which have demonstrated promising anti-tumor efficacy both in vitro and in vivo . These approaches leverage the elevated expression of TNFRSF12A on cancer cells to direct immune responses against tumors.

• Targeting the TNFRSF12A/RELA/BIRC3 axis: Inhibition of downstream components of the TNFRSF12A signaling pathway, particularly BIRC3, represents another potential approach for blocking the oncogenic effects of TNFRSF12A . BIRC3 inhibitors could specifically counteract the pro-survival effects of TNFRSF12A overexpression in cancer cells.

• NF-κB pathway modulation: Given the central role of NF-κB signaling in mediating TNFRSF12A's effects, targeted inhibition of specific NF-κB components could potentially block pathological TNFRSF12A signaling while preserving other essential NF-κB functions .

Each of these approaches offers unique advantages and challenges, and the optimal therapeutic strategy likely depends on the specific disease context and desired outcomes.

What cellular processes are affected by TNFRSF12A signaling in different tissue contexts?

TNFRSF12A signaling affects a diverse array of cellular processes in a tissue-specific manner. Understanding these tissue-specific effects is crucial for predicting both the therapeutic potential and possible side effects of TNFRSF12A-targeted interventions:

• Hepatocytes:

  • Activation of RIPK1-dependent apoptosis during acute liver failure

  • Potential contribution to liver regeneration during injury repair

  • Low baseline expression in normal liver, with significant upregulation during pathological states

• Endothelial cells:

  • Promotion of angiogenesis and endothelial cell proliferation

  • Modulation of vascular integrity and permeability

  • Potential role in tumor vascularization

• Cancer cells:

  • Enhancement of proliferation, particularly in colorectal cancer cells

  • Promotion of clonogenic capacity and survival

  • Facilitation of migration and potentially invasion

  • Activation of pro-survival NF-κB signaling and upregulation of anti-apoptotic factors like BIRC3

• Immune cells:

  • Modulation of inflammatory responses

  • Potential influence on immune cell recruitment and activation

  • Contribution to the tumor microenvironment in cancer contexts

This tissue-specific diversity of TNFRSF12A functions underscores the importance of context-dependent research approaches when investigating its role in different physiological and pathological settings.

What are the emerging contradictions and controversies in TNFRSF12A research?

Current research on TNFRSF12A presents several interesting contradictions and unresolved questions that warrant further investigation:

• Pro-tumor vs. anti-tumor effects: While most evidence points to TNFRSF12A as a promoter of cancer progression, some studies have suggested potential cancer-suppressive functions in certain contexts . This contradiction highlights the complex, context-dependent role of TNFRSF12A in cancer biology and necessitates careful evaluation of its effects across different cancer types and stages.

• Cell death modalities: Despite lacking a canonical death domain, TNFRSF12A can induce cell death through RIPK1-dependent apoptosis in hepatocytes during acute liver failure . This contradicts the typical structure-function relationship observed in other TNFR family members and raises questions about the precise molecular mechanisms by which TNFRSF12A activates death signaling.

• Dual roles in inflammation: TNFRSF12A can promote inflammatory responses that contribute to pathology in conditions like acute liver failure , yet inflammation is also essential for tissue repair and regeneration. The balance between these potentially opposing functions remains poorly understood.

• Therapeutic targeting challenges: While TNFRSF12A represents a promising therapeutic target, its expression across multiple tissues raises concerns about potential off-target effects of systemic inhibition. Developing tissue-specific or context-selective approaches to TNFRSF12A targeting remains an important challenge.

• Interaction with other signaling pathways: The crosstalk between TNFRSF12A signaling and other pathways, including non-canonical NF-κB signaling and other TNFR family members, requires further clarification to fully understand the integrated signaling network in which TNFRSF12A operates.

Addressing these contradictions and controversies will require sophisticated experimental approaches, including tissue-specific conditional knockout models, single-cell analyses to capture cellular heterogeneity, and systems biology approaches to map the complex signaling networks influenced by TNFRSF12A.

What are the critical quality control parameters for recombinant TNFRSF12A protein production?

Ensuring the quality and functionality of recombinant TNFRSF12A protein is essential for reliable experimental outcomes. Researchers should consider the following critical quality control parameters:

• Purity assessment: Recombinant TNFRSF12A should achieve >90% purity as assessed by SDS-PAGE to minimize the influence of contaminants on experimental results .

• Endotoxin levels: Endotoxin contamination can significantly confound results, especially in inflammatory pathway studies. Endotoxin levels should be maintained below 0.1 EU/μg of protein, as measured by the LAL method .

• Proper folding and disulfide bond formation: Given the importance of the three disulfide bonds (Cys36-Cys49, Cys52-Cys67, and Cys55-Cys64) for the structural integrity of TNFRSF12A's cysteine-rich domain, verification of proper protein folding is essential . This can be assessed through circular dichroism spectroscopy or limited proteolysis.

• Functional activity validation: Multiple complementary assays should be employed to confirm biological activity:

  • Binding capacity to TWEAK ligand using ELISA

  • Inhibition of TWEAK-induced apoptosis in appropriate cell lines

  • Effect on HUVEC proliferation

  • Activation of downstream signaling pathways

• Stability assessment: Evaluation of protein stability under various storage conditions and after freeze-thaw cycles is crucial for maintaining consistent experimental results.

Implementing these quality control measures ensures that experimental outcomes reflect genuine biological effects rather than artifacts from suboptimal protein quality.

How can researchers effectively distinguish between different modes of cell death in TNFRSF12A studies?

Distinguishing between different modes of cell death is particularly important in TNFRSF12A research, as this receptor has been shown to induce RIPK1-dependent apoptosis rather than necroptosis or pyroptosis in certain contexts . Researchers can employ the following methodological approaches to accurately differentiate between cell death modalities:

• Apoptosis detection:

  • Annexin V/PI staining followed by flow cytometry: Early apoptotic cells are Annexin V-positive and PI-negative

  • Caspase activation assays: Measurement of caspase-3/7, -8, or -9 activity using fluorogenic substrates

  • PARP cleavage detection by western blot: Cleaved PARP is a hallmark of apoptosis

  • TUNEL assay: Detects DNA fragmentation characteristic of apoptosis

• Necroptosis detection:

  • Phosphorylation of MLKL: A key marker of necroptosis

  • Use of specific inhibitors: Necrostatin-1 (RIPK1 inhibitor) should block necroptosis

  • Membrane permeability assays: Propidium iodide uptake without annexin V positivity

  • Electron microscopy: Reveals necroptotic morphological features

• Pyroptosis detection:

  • Caspase-1 activation assays

  • IL-1β and IL-18 release measurements

  • Gasdermin D cleavage detection by western blot

  • LDH release assays

• Differential inhibitor studies:

  • z-VAD-fmk (pan-caspase inhibitor) to block apoptosis

  • Necrostatin-1 to inhibit RIPK1-dependent processes

  • GSK'872 to inhibit RIPK3 (necroptosis)

  • VX-765 to inhibit caspase-1 (pyroptosis)

In TNFRSF12A research specifically, the research has demonstrated that TWEAK/TNFRSF12A axis induces RIPK1-dependent apoptosis in hepatocytes during acute liver failure, as evidenced by the protective effects of RIPK1 inhibition and the lack of protection from necroptosis or pyroptosis inhibitors . This methodological approach of using specific inhibitors of different cell death pathways provides a robust framework for distinguishing between modes of cell death in various experimental contexts.

What are the best practices for analyzing TNFRSF12A expression and signaling in tissue samples?

Analysis of TNFRSF12A expression and downstream signaling in tissue samples requires careful consideration of methodological approaches to ensure accurate and reproducible results. Based on current research practices, the following best practices are recommended:

• Tissue collection and processing:

  • Rapid fixation in 10% neutral buffered formalin for IHC or flash-freezing for RNA/protein extraction

  • Careful orientation and sectioning of tissues to ensure representativeness

  • Inclusion of both tumor and adjacent normal tissue in cancer studies

  • Documentation of clinical parameters and patient demographics for correlation analyses

• Expression analysis methods:

  • Immunohistochemistry (IHC): Provides spatial information about TNFRSF12A expression

    • Use of validated antibodies with appropriate positive and negative controls

    • Quantitative scoring systems (e.g., H-score, Allred score) for objective assessment

    • Digital pathology tools for automated quantification when possible

  • RNA expression analysis:

    • qRT-PCR for targeted analysis of TNFRSF12A and related genes

    • RNA-seq for comprehensive transcriptomic profiling and pathway analysis

    • Single-cell RNA-seq to address cellular heterogeneity within tissues

  • Protein expression analysis:

    • Western blotting for semi-quantitative assessment of TNFRSF12A and signaling components

    • Proteomics approaches for unbiased profiling of pathway changes

    • Phospho-specific antibodies to assess activation status of signaling components

• Signaling pathway assessment:

  • Evaluation of NF-κB pathway activation:

    • Phosphorylation status of IκB and p65/RELA

    • Nuclear translocation of RELA using immunofluorescence or nuclear/cytoplasmic fractionation

    • Expression of NF-κB target genes, particularly BIRC3, TRAF1, and NFKB2

  • Analysis of RIPK1 activation and downstream apoptotic markers in liver samples :

    • RIPK1 phosphorylation status

    • Cleaved caspase-3 and PARP detection

    • TUNEL staining for apoptotic cells

• Data integration and analysis:

  • Correlation of TNFRSF12A expression with clinical parameters and outcomes

  • Pathway enrichment analysis of transcriptomic data to identify activated processes

  • Integration of multiple data types (genomic, transcriptomic, proteomic) for comprehensive understanding

  • Statistical approaches appropriate for the data type and experimental design

• Validation strategies:

  • Use of multiple independent cohorts for validation of findings

  • Complementary in vitro experiments using relevant cell types

  • Animal models to confirm mechanistic findings from human samples