TNFRSF21 Human

What is TNFRSF21 and what are its primary functions in human cells?

TNFRSF21, located on chromosome 6 in humans, functions as a receptor involved in various programmed cell death mechanisms including apoptosis, necroptosis, and pyroptosis . Its role appears to be highly context-dependent:

• In the nervous system: Induces apoptosis when binding to cleaved amino-terminal fragments of amyloid precursor protein (APP)

• In vascular endothelial cells: Causes necroptosis (not apoptosis) when bound to APP activated by tumor cells

• In osteosarcoma: Functions as a tumor suppressor by inhibiting cell proliferation and motility through activation of necroptotic pathways

Research methods to characterize TNFRSF21 function typically include:

• qPCR for mRNA expression analysis

• Western blotting for protein detection (recommended antibody dilution: 1:1000)

• Co-immunoprecipitation for interaction partner identification

• Functional assays (proliferation, migration, invasion) following manipulation of expression levels

How is TNFRSF21 gene expression regulated in different human tissues?

TNFRSF21 expression is regulated through multiple mechanisms:

Transcriptional regulation:

• Tissue-specific transcription factors (can be identified through ChIP assays)

• Epigenetic modifications (methylation analysis reveals tissue-specific patterns)

Post-transcriptional regulation:

• MicroRNAs, notably miR-20a-5p in head and neck squamous cell carcinoma (HNSCC), target TNFRSF21 mRNA

• Post-transcriptional regulation mechanisms are key for swift protein production in response to stimuli

Methodological approaches for expression analysis:

• RNA-seq or qPCR for tissue-specific expression profiling

• Single-cell RNA-seq for cellular heterogeneity analysis within tissues

• Luciferase reporter assays with the TNFRSF21 promoter or 3'UTR regions

• Western blotting with phospho-specific antibodies for protein level confirmation

What signaling pathways does TNFRSF21 activate in human cells?

TNFRSF21 participates in several critical signaling pathways related to cell death:

Necroptosis pathway:

• Upregulates phosphorylation of RIPK1 (at S166), RIPK3 (at S227), and MLKL (at S358)

• This phosphorylation cascade is essential for necroptotic cell death execution

Apoptosis pathway:

• In nervous system cells, triggers caspase-dependent apoptosis following binding to APP fragments

Pyroptosis pathway:

• When activated by α-ketoglutarate, induces caspase 8 to gasdermin C-mediated pyroptosis

• This pathway appears to be APP-independent

The specific pathway activated appears to depend on cell type, binding partners, and cellular context. Researchers typically employ Western blotting with phospho-specific antibodies to track pathway activation, combined with pathway inhibitors (e.g., Necrostatin-1 for RIPK1, Z-VAD-FMK for caspases) to confirm specificity.

What are the known binding partners of TNFRSF21 and how can these interactions be studied?

Confirmed binding partners:

• Amyloid Precursor Protein (APP): Interaction leads to different outcomes depending on cell type

• RIPK1, RIPK3, MLKL: While direct binding is not explicitly confirmed, TNFRSF21 regulates their phosphorylation status

Recommended methods for interaction studies:

A comprehensive approach would combine multiple methods, as each has distinct strengths and limitations for detecting different types of interactions.

How can TNFRSF21 be effectively knocked down or overexpressed in experimental models?

Knockdown approaches:

• siRNA transfection: Transient knockdown (3-7 days)

• shRNA via lentiviral vectors: Stable knockdown with antibiotic selection

• CRISPR-Cas9: Complete knockout using guide RNAs targeting exonic regions

Overexpression methods:

• Plasmid-based transient transfection: Maximum expression at 24-48h post-transfection

• Lentiviral/retroviral stable overexpression: For long-term studies

• Inducible expression systems (Tet-On/Off): For controlled, dose-dependent expression

Validation requirements:

• qPCR confirmation of mRNA changes

• Western blotting verification of protein levels (antibody dilution 1:1000)

• Functional assays relevant to TNFRSF21 (cell death assays, proliferation, migration)

Based on published osteosarcoma studies, overexpression of TNFRSF21 inhibits proliferation and motility of osteosarcoma cells, providing a functional readout for successful manipulation .

What cell and animal models are most appropriate for studying TNFRSF21 function?

Cell models with established TNFRSF21 research:

• Osteosarcoma cell lines: Show downregulated TNFRSF21 expression compared to normal controls

• Head and neck squamous cell carcinoma (HNSCC) lines: Targeted by miR-20a-5p

• Vascular endothelial cells: Exhibit TNFRSF21-mediated necroptosis when exposed to tumor-activated APP

• Neuronal cells: Show TNFRSF21-mediated apoptosis following APP binding

Animal models:

• Xenograft models using cells with modulated TNFRSF21 expression

• Orthotopic implantation for tissue-specific effects

• Conditional knockout models for tissue-specific TNFRSF21 deletion

Emerging models:

• Patient-derived organoids for personalized assessment

• Human tissue explants for ex vivo studies

• CRISPR-engineered mouse models with human TNFRSF21 substitutions

Cell type selection should be guided by the specific research question, as TNFRSF21 functions differently across tissue contexts.

What is the role of TNFRSF21 in cancer progression and metastasis?

TNFRSF21 exhibits context-dependent roles in cancer, primarily functioning as a tumor suppressor:

In osteosarcoma:

• Expression is downregulated in cancer cell lines

• Overexpression inhibits proliferation and motility

• Promotes necroptosis via RIPK1/RIPK3/MLKL phosphorylation

• Lower necroptosis score (indicating more active necroptosis) correlates with better prognosis

In head and neck squamous cell carcinoma (HNSCC):

• miR-20a-5p, which targets TNFRSF21, is upregulated in HNSCC

• This miRNA promotes proliferation and invasion through TNFRSF21 downregulation

Research approaches:

• Expression analysis in clinical samples using immunohistochemistry

• Correlation studies using TCGA and GEO datasets

• Single-cell RNA-seq for tumor microenvironment heterogeneity assessment

• Functional assays following TNFRSF21 manipulation (proliferation, migration, invasion)

• Xenograft models for in vivo validation

Understanding the molecular mechanisms underlying TNFRSF21's tumor suppressive functions could reveal new therapeutic strategies for cancers where this pathway is dysregulated.

How does TNFRSF21 contribute to programmed cell death mechanisms?

TNFRSF21 contributes to three major programmed cell death pathways in a context-dependent manner:

Necroptosis:

• In osteosarcoma, promotes necroptosis through phosphorylation of:

  • RIPK1 at S166

  • RIPK3 at S227

  • MLKL at S358

• This leads to membrane rupture and inflammatory cell death

Apoptosis:

• In neural cells, binding to APP fragments induces apoptosis

• Likely involves caspase cascade activation

Pyroptosis:

• When activated by α-ketoglutarate, induces caspase 8 to gasdermin C pathway

• Results in inflammatory cell death characteristic of pyroptosis

The detection and differentiation of these pathways requires specific methodological approaches:

Researchers should employ pathway-specific inhibitors (Necrostatin-1 for necroptosis, Z-VAD-FMK for apoptosis) to confirm the specific mechanism at work.

What are the contradictions in current research about TNFRSF21's role in cell death pathways?

Several apparent contradictions in TNFRSF21 function warrant further investigation:

Cell type-dependent outcomes:

• Neural cells: TNFRSF21-APP interaction induces apoptosis

• Vascular endothelial cells: TNFRSF21-APP interaction causes necroptosis

• Osteosarcoma cells: TNFRSF21 promotes necroptosis via RIPK1/RIPK3/MLKL

Stimulus-dependent pathways:

• α-ketoglutarate activation: Leads to pyroptosis via caspase-8/gasdermin C (APP-independent)

• APP fragment binding: Results in either apoptosis or necroptosis depending on cell type

Methodological approaches to resolve these contradictions:

• Direct comparative studies using identical stimulation conditions across cell types

• Receptor complex analysis using proximity labeling techniques

• Domain-specific functional studies using truncated or chimeric constructs

• Phosphoproteomics to identify differential signaling pathways

• In vivo validation with tissue-specific conditional models

Resolving these contradictions is essential for therapeutic development, as TNFRSF21-targeted interventions may have opposite effects in different tissues.

How can single-cell analysis be used to study TNFRSF21 heterogeneity in tumor microenvironments?

Single-cell technologies offer powerful approaches to characterize TNFRSF21 heterogeneity:

Single-cell RNA sequencing (scRNA-seq):

• Enables comprehensive profiling of TNFRSF21 expression across all cell types

• Osteosarcoma single-cell data (GSE162454) has been analyzed using:

  • 'Seurat' R package for quality control and clustering

  • 'MAESTRO' for cell-type annotation

• Allows calculation of necroptosis scores for individual cells

Analytical approaches:

• Principal Component Analysis (PCA) to distinguish cell states

• Cell-cell communication analysis using 'CellChat' R package

• Calculation of necroptosis score (NS) at single-cell level

Key findings from single-cell studies:

• Necroptosis shows significant heterogeneity in osteosarcoma at single-cell level

• Cell communication patterns of malignant cells with high NS correlate with tumor progression

• This suggests complex interactions between TNFRSF21-mediated necroptosis and the tumor microenvironment

Recommended workflow:

• Quality control and normalization of single-cell data

• Clustering and cell type annotation

• TNFRSF21 expression mapping across clusters

• Calculation of pathway scores (e.g., necroptosis score)

• Cell-cell communication network analysis

• Integration with clinical or experimental outcomes

This approach provides unprecedented resolution for understanding TNFRSF21 biology in complex tissue environments.

How might TNFRSF21 be targeted therapeutically in human diseases?

Based on current research, several therapeutic approaches targeting TNFRSF21 show promise:

For cancers with downregulated TNFRSF21 (e.g., osteosarcoma, HNSCC):

• miRNA inhibitors targeting miR-20a-5p to restore TNFRSF21 expression

• Small molecules to enhance TNFRSF21 expression or activity

• Gene therapy approaches to restore TNFRSF21 function

For necroptosis modulation:

• Agonists to enhance TNFRSF21-mediated necroptosis in cancer cells

• Combined TNFRSF21 activation with immune checkpoint inhibitors

For inflammatory conditions with excessive cell death:

• Antagonistic antibodies to block TNFRSF21 signaling

• Small molecule inhibitors of downstream effectors (RIPK1, RIPK3)

Considerations for therapeutic development:

• Cell type-specific effects require careful targeting strategies

• Potential for opposite effects in different tissues necessitates localized delivery

• Biomarkers to identify patients likely to respond (e.g., necroptosis score)

As research progresses, personalized approaches based on TNFRSF21 pathway status could emerge as effective strategies.

What cutting-edge technologies are advancing TNFRSF21 research?

Several emerging technologies are transforming TNFRSF21 research:

Spatial omics:

• Spatial transcriptomics to map TNFRSF21 expression within tissue architecture

• Multiplex imaging to visualize TNFRSF21 with multiple markers simultaneously

• Imaging mass cytometry for high-parameter spatial protein analysis

Advanced genetic engineering:

• CRISPR base editing for precise modification of TNFRSF21 regulatory elements

• CRISPR activation/inhibition systems to modulate expression without DNA modification

• Knock-in reporter systems for live monitoring of TNFRSF21 activity

Protein structure and interaction:

• AlphaFold2 and other AI approaches for structure prediction

• Hydrogen-deuterium exchange mass spectrometry for dynamic structural analysis

• Time-resolved crosslinking mass spectrometry for interaction kinetics

Functional genomics:

• Genome-wide CRISPR screens to identify TNFRSF21 pathway components

• Single-cell CRISPR perturbation with RNA-seq readout

• Pooled in vivo CRISPR screens for pathway modifiers

Data integration platforms:

• Multi-omics integration frameworks connecting genomics, transcriptomics, and proteomics

• Machine learning approaches to predict TNFRSF21 pathway activity from multiple data types

• Network medicine approaches to position TNFRSF21 within disease pathways

These technologies will enable more comprehensive understanding of TNFRSF21 biology and accelerate therapeutic development.