Recombinant Human Tumor necrosis factor receptor superfamily member 11B protein (TNFRSF11B), partial (Active)

What is TNFRSF11B and what are its primary biological functions?

TNFRSF11B, commonly known as Osteoprotegerin (OPG), functions primarily as a decoy receptor for TNFSF11/RANKL, effectively neutralizing its role in osteoclastogenesis. This protein inhibits osteoclast activation and promotes osteoclast apoptosis in vitro, playing a crucial role in bone homeostasis. The local ratio between TNFSF11 and TNFRSF11B appears to be a determining factor in maintaining bone health .

Additionally, TNFRSF11B may protect against arterial calcification and can act as a decoy receptor for TNFSF10/TRAIL, potentially providing protection against apoptosis. Research has demonstrated that TNFSF10/TRAIL binding to TNFRSF11B blocks the inhibition of osteoclastogenesis, suggesting a complex regulatory mechanism .

What are the common synonyms and nomenclature for TNFRSF11B in scientific literature?

In scientific publications, TNFRSF11B is referred to by several alternative names:

Researchers should be aware of these alternative designations when conducting literature searches to ensure comprehensive review of relevant studies .

What is the molecular structure and typical specifications of recombinant TNFRSF11B?

Recombinant Human TNFRSF11B typically consists of the fragment spanning amino acids 22-401 of the native protein. The molecular specifications are as follows:

The discrepancy between calculated and observed molecular weight is attributable to post-translational glycosylation, which significantly affects protein migration in SDS-PAGE under reducing conditions .

How is recombinant TNFRSF11B produced for research applications?

Recombinant Human TNFRSF11B for research purposes is predominantly produced using human embryonic kidney (HEK293) cells as an expression system. This mammalian expression system is preferred as it facilitates proper post-translational modifications, particularly glycosylation, which are essential for the protein's biological activity .

The production process typically involves:

• Transfection of HEK293 cells with expression vectors containing the TNFRSF11B gene (aa 22-401)

• Culture and expression of the protein with a C-terminal His-tag

• Purification via affinity chromatography

• Quality control through SDS-PAGE under reducing conditions

• Lyophilization with stabilizers such as mannitol (1%) and trehalose (5%) in PBS (pH 7.2)

For experimental use, the lyophilized protein is typically reconstituted at 0.5-1.0 mg/mL using sterile deionized water .

What methodologies are used to study TNFRSF11B overexpression in cartilage?

Studying TNFRSF11B overexpression in cartilage involves several specialized methodologies, as demonstrated in recent research on osteoarthritis:

• Lentiviral Transduction System: Human primary articular chondrocytes (hPACs) are isolated from articular cartilage and transduced with lentiviral particles carrying the TNFRSF11B gene to induce overexpression .

• 3D In Vitro Chondrogenic Models: Transduced cells are cultured in 3D spherical cartilage pellets to create tissue models that better recapitulate the in vivo environment compared to monolayer cultures .

• Expression Verification Methods:

  • RT-qPCR for mRNA expression quantification

  • Immunohistochemistry for protein localization

  • ELISA for protein quantification in culture medium

• Functional Assessments:

  • Alcian blue staining for glycosaminoglycan deposition

  • Immunohistochemistry for collagen types (COL1, COL2)

  • Analysis of matrix mineralization markers

These methodologies allow researchers to investigate the effects of TNFRSF11B overexpression on cartilage matrix composition, mineralization processes, and related gene expression changes .

How can researchers effectively analyze TNFRSF11B co-expression networks?

Analyzing TNFRSF11B co-expression networks requires sophisticated bioinformatic approaches as demonstrated in the RAAK study:

• RNA Sequencing Data Generation:

  • Collection of preserved and lesioned OA cartilage samples (e.g., n=57 preserved and n=44 lesioned)

  • RNA sequencing with appropriate quality control measures

• Correlation Analysis:

  • Spearman correlation to identify genes co-expressed with TNFRSF11B

  • Statistical significance threshold (p < 0.05)

  • Correlation strength threshold (typically |r| > 0.75)

• Network Construction:

  • Integration of differentially expressed genes between disease and control samples

  • Pathway analysis of correlated genes

  • Visualization of interaction networks

In the RAAK study, this approach identified 51 genes highly correlated with TNFRSF11B. The strongest positive correlations were found with CDH19 (r=0.88), ATP1A1 (r=0.87), and DIXDC1 (r=0.85), while the strongest negative correlations were with SLC15A3 (r=-0.81), MAPK11 (r=-0.81), and HLA-E .

What is the relationship between TNFRSF11B polymorphisms and fracture risk?

Meta-analyses of genetic and genome-wide association studies have revealed significant relationships between TNFRSF11B polymorphisms and fracture risk, particularly in older adults:

• Key Polymorphisms Studied:

  • rs2073618 (exonic)

  • rs3134069, rs3134070, rs3102735 (intronic)

• Statistical Findings:

  • Significant protective effects (13-37% risk reduction) in postmenopausal women (rs2073618)

  • Protective effects in subjects over 60 years and Western populations (rs3134069 and rs3134070)

  • Odds ratios ranging from 0.63 to 0.87 in protected populations

• Methodological Approach:

  • Allele-genotype model analysis (variant, wild-type, heterozygote)

  • Bonferroni correction for multiple comparisons

  • Meta-regression to examine heterogeneity sources

  • Strict criteria for evidence strength (I² = 0% and Pa < 0.00001)

• Sources of Heterogeneity:

  • Publication year

  • Study quality

  • Fracture type/site

  • Sample size

These findings suggest that TNFRSF11B polymorphisms may have protective effects against fractures, particularly in specific demographic groups, which has important implications for personalized medicine approaches to bone health .

How does TNFRSF11B contribute to osteoarthritis development?

TNFRSF11B has been identified as one of the highest upregulated genes in lesioned osteoarthritic cartilage. Research investigating its role in OA development has revealed:

• Expression Changes in OA:

  • Significant upregulation in lesioned versus preserved cartilage

  • Association with a gain-of-function mutation in families with early-onset OA with chondrocalcinosis

• Downstream Effects on Cartilage:

  • Impact on cartilage matrix deposition

  • Influence on matrix mineralization processes

  • Alterations in anabolic and catabolic markers involved in cartilage homeostasis

• Gene Expression Changes:

  • Significant upregulation of genes including FRZB (FD = 1.68, P = 2.3×10⁻²)

  • Upregulation of ASPN (FD = 2.61, P = 1.0×10⁻²)

  • Co-expression with multiple genes involved in cartilage metabolism

Understanding these mechanisms is crucial for developing therapeutic strategies targeting TNFRSF11B signaling in osteoarthritis treatment .

What is the emerging role of TNFRSF11B as an inflammatory biomarker?

Recent research has identified TNFRSF11B as a potential biomarker for inflammatory conditions, particularly in sepsis-acute respiratory distress syndrome (ARDS):

• Clinical Findings:

  • Significantly elevated levels in sepsis-ARDS patients compared to healthy controls

  • Highest plasma concentrations of 10-20 ng/mL in sepsis-ARDS patients

• Functional Significance:

  • Association with signal transduction, immune response, and inflammatory pathways

  • Correlation with vascular endothelial dysfunction

• Experimental Validation:

  • Increased levels in LPS-induced mouse models

  • Elevated expression in LPS-stimulated human umbilical vein endothelial cells (HUVECs)

  • Effects on endothelial junction proteins when HUVECs were stimulated with 10 ng/mL TNFRSF11B

• Protein Expression Changes:

  • Decreased levels of syndecan-1, claudin-5, VE-cadherin, occludin, aquaporin-1, and caveolin-1

  • Increased levels of connexin-43

These findings suggest that TNFRSF11B may serve as a novel predictive and diagnostic biomarker for vascular endothelium damage in inflammatory conditions such as sepsis-ARDS .

What are the critical quality control parameters for recombinant TNFRSF11B in experimental systems?

When utilizing recombinant TNFRSF11B in experimental systems, researchers should consider several critical quality control parameters:

Proper storage conditions (-20°C for lyophilized product) and reconstitution methods are also crucial for maintaining protein integrity. For experiments investigating TNFRSF11B effects on cellular systems, concentrations should be carefully determined based on physiological relevance (e.g., 10 ng/mL has been used to stimulate HUVECs based on levels found in inflammatory conditions) .

How can researchers distinguish between physiological and pathological effects of TNFRSF11B?

Distinguishing between physiological and pathological effects of TNFRSF11B requires careful experimental design:

• Concentration-Dependent Studies:

  • Titration experiments with recombinant protein (0.1-100 ng/mL range)

  • Comparison with physiological levels in healthy tissues (~1-5 ng/mL)

  • Comparison with pathological levels (e.g., 10-20 ng/mL in sepsis-ARDS)

• Temporal Analysis:

  • Short-term versus long-term exposure experiments

  • Pulse-chase studies to monitor adaptation and compensation

• Context-Specific Considerations:

  • Cell type-specific responses (e.g., chondrocytes vs. endothelial cells)

  • Tissue-specific microenvironments

  • Presence of interacting proteins (RANKL/TNFSF11, TRAIL/TNFSF10)

• Molecular Readouts:

  • Broad transcriptomic analysis (RNA-seq)

  • Proteomics to detect pathway activation

  • Specific functional assays (e.g., mineralization, apoptosis, barrier function)

What contradictions exist in current TNFRSF11B research findings?

Several contradictions and unresolved questions remain in TNFRSF11B research:

• Therapeutic Implications:

  • The drug strontium ranelate increases OPG expression to fight osteoporosis, but shows controversial results in OA treatment

  • This suggests context-dependent effects that require further clarification

• Tissue-Specific Functions:

  • Protective role in bone (preventing osteoporosis) versus potential pathological role in cartilage (OA development)

  • Beneficial versus detrimental effects in vascular tissues

• Genetic Associations:

  • While some polymorphisms show protective effects against fractures, other TNFRSF11B mutations are associated with early-onset OA

  • This suggests complex interactions with genetic and environmental factors

• Signaling Pathway Interactions:

  • The precise mechanisms by which TNFRSF11B interacts with various downstream pathways remain incompletely understood

  • Co-expression network analysis reveals associations with diverse gene sets, suggesting multiple functional roles

Resolving these contradictions requires integrated approaches combining genetic, molecular, cellular, and clinical studies with careful attention to experimental conditions and physiological context.

What are the latest methodological approaches for studying TNFRSF11B in disease models?

Cutting-edge methodological approaches for studying TNFRSF11B include:

• 3D Tissue Models:

  • Spherical cartilage pellets for OA research

  • Organ-on-chip systems for multi-tissue interactions

  • Patient-derived organoids for personalized disease modeling

• High-Throughput Screening:

  • CRISPR-Cas9 screens targeting TNFRSF11B pathway components

  • Small molecule libraries to identify modulators of TNFRSF11B signaling

• Advanced Imaging Techniques:

  • Live-cell imaging with fluorescently tagged TNFRSF11B

  • Super-resolution microscopy to visualize receptor-ligand interactions

  • In vivo imaging in animal models

• Integrative Omics Approaches:

  • Multi-omics integration (genomics, transcriptomics, proteomics)

  • Single-cell analysis to capture cellular heterogeneity

  • Network biology approaches to map TNFRSF11B interactions

These methodological advances are enabling more sophisticated investigations of TNFRSF11B's role in complex disease processes and may lead to novel therapeutic strategies targeting this pathway.

How can TNFRSF11B research findings translate into therapeutic applications?

Translating TNFRSF11B research into therapeutic applications involves several strategic considerations:

• Therapeutic Modalities:

  • Recombinant protein therapy (mimicking or antagonizing TNFRSF11B)

  • Monoclonal antibodies targeting TNFRSF11B or its ligands

  • Small molecule modulators of TNFRSF11B pathways

  • Gene therapy approaches to regulate TNFRSF11B expression

• Precision Medicine Approaches:

  • Genetic screening for TNFRSF11B polymorphisms to identify at-risk populations

  • Monitoring TNFRSF11B levels as a biomarker for disease progression

  • Patient stratification based on TNFRSF11B pathway activation

• Disease-Specific Considerations:

  • Osteoporosis: Increasing TNFRSF11B activity to inhibit bone resorption

  • Osteoarthritis: Modulating TNFRSF11B to prevent pathological mineralization

  • Inflammatory conditions: Targeting TNFRSF11B to preserve vascular integrity

• Delivery Challenges:

  • Tissue-specific targeting strategies

  • Maintaining protein stability and half-life

  • Controlling local versus systemic effects

Understanding the complex biology of TNFRSF11B in different disease contexts is essential for developing targeted therapeutic approaches that maximize efficacy while minimizing potential side effects.