Recombinant Human Tumor necrosis factor ligand superfamily member 11 (TNFSF11), partial (Active)

What is the molecular structure and genetic characterization of TNFSF11?

TNFSF11 is encoded by the TNFSF11 gene located at chromosome 13q14.11. The human TNFSF11 cDNA encodes a type II transmembrane protein of 317 amino acids with a predicted cytoplasmic domain of 47 amino acids, a 21 amino acid transmembrane region, and an extracellular domain of 249 amino acids. The extracellular domain contains two potential N-linked glycosylation sites. Mouse and human TNFSF11 share 85% amino acid identity, indicating strong evolutionary conservation .

The gene has several aliases including ODF, OPGL, sOdf, CD254, OPTB2, RANKL, TNLG6B, TRANCE, and hRANKL2. The protein exists in both membrane-bound and soluble forms, with the soluble form being particularly important for experimental applications .

What are the primary biological functions of TNFSF11 in normal physiology?

TNFSF11 serves multiple critical functions:

• Acts as a key factor for osteoclast differentiation and activation

• Functions as a ligand for osteoprotegerin (OPG)

• Serves as a dendritic cell survival factor

• Plays a role in T cell-dependent immune response

• Activates antiapoptotic kinase AKT/PKB through a signaling complex involving SRC kinase and TRAF6

• Regulates cell apoptosis

Targeted disruption of TNFSF11 in mice leads to severe osteopetrosis and lack of osteoclasts. Additionally, TNFSF11-deficient mice exhibit defects in early differentiation of T and B lymphocytes and fail to form lobulo-alveolar mammary structures during pregnancy .

How is TNFSF11 expression regulated in different tissue types?

TNFSF11 is primarily expressed in:

• T cells and T cell-rich organs (thymus and lymph nodes)

• Osteocytes, which comprise 90-95% of all bone cells and are the longest-living cell type in bone tissue

• Osteoblasts and stromal cells

Expression is regulated through multiple mechanisms:

• T cell receptor stimulation can rapidly upregulate TNFSF11 expression

• Epigenetic regulation through DNA methylation of the TNFSF11 promoter region

• Enhancer elements, including a recently discovered osteocyte-specific intronic enhancer

• Hormonal regulation, including correlations with progesterone levels

Cell death signaling has been shown to increase RANKL expression specifically in osteocytic cells, suggesting a link between cell senescence/death and TNFSF11 expression .

What are the optimal conditions for using recombinant human TNFSF11 in osteoclast differentiation assays?

Based on experimental data, the following protocol is recommended:

• Use RAW264.7 mouse monocyte/macrophage cell line as a model system

• Add recombinant human TRANCE/RANK L/TNFSF11 at concentrations between 1.5-7.5 ng/mL

• Include 2.5 μg/mL of a cross-linking antibody (Mouse Anti-polyHistidine)

• Monitor osteoclast differentiation through appropriate markers

The effective dose (ED50) for inducing osteoclast differentiation under these conditions is 1.5-7.5 ng/mL in the presence of the cross-linking antibody . This standardized approach allows for reproducible studies of TNFSF11-induced osteoclastogenesis in vitro.

How can researchers effectively detect and quantify TNFSF11 expression in experimental systems?

Multiple complementary approaches can be used:

• mRNA expression analysis:

  • RT-PCR or qPCR for TNFSF11 gene expression

  • RNA-seq for genome-wide expression profiling

  • Single-cell transcriptomic analysis to identify cell-specific expression patterns

• Protein expression analysis:

  • Western blotting for protein level quantification

  • ELISA for soluble RANKL in serum or culture supernatants

  • Immunohistochemistry for tissue localization

• Epigenetic analysis:

  • DNA methylation analysis of the TNFSF11 promoter region

  • Chromatin accessibility assays (DNase sensitivity)

When analyzing TNFSF11 expression, it's important to consider cell-type specificity, as expression patterns and regulatory mechanisms can vary significantly between osteocytes, osteoblasts, and immune cells .

What considerations are critical when designing genetic association studies involving TNFSF11 polymorphisms?

When designing genetic studies involving TNFSF11 polymorphisms, researchers should consider:

• Population stratification:

  • Ensure ethnically homogeneous study populations to avoid false-positive associations

  • Match cases and controls from the same geographical region

• Gender-stratified analysis:

  • The rs1021188 polymorphism has shown significant association with otosclerosis in men (p = 0.023) but not in women (p = 0.458)

  • This suggests the importance of sex-specific analytical approaches

• Linkage disequilibrium patterns:

  • 26 loci in the TNFSF11 gene have been found in linkage disequilibrium with rs1021188

  • Different populations exhibit unique allele enrichment/depletion patterns

• Epigenetic interactions:

  • Consider how polymorphisms might interact with epigenetic modifications

  • The rs1021188 polymorphism and DNA methylation changes in the promoter CpG sites within TNFSF11 may both play important roles in transcription regulation

What are the critical signaling pathways activated by TNFSF11 binding to its receptor?

TNFSF11 binding to RANK activates several signaling pathways:

• NF-κB pathway:

  • RANK undergoes receptor clustering during signal transduction

  • Recruitment of TNF receptor-associated factors (TRAFs), particularly TRAF6

  • Activation of the IKK complex and nuclear translocation of NF-κB

• MAPK pathways:

  • Activation of c-jun N-terminal kinase (JNK)

  • ERK and p38 signaling cascades

• AKT/PKB pathway:

  • RANKL activates antiapoptotic kinase AKT/PKB through a signaling complex involving SRC kinase and TRAF6

  • This pathway is involved in the regulation of cell apoptosis

Understanding these signaling mechanisms is crucial for developing targeted therapies that modulate specific aspects of TNFSF11 function.

How does the recently discovered intronic enhancer regulate TNFSF11 expression in osteocytes?

Research has identified a previously unknown osteocytic cell-specific intronic enhancer in the TNFSF11 gene locus that plays a crucial role in regulating RANKL expression . Key findings include:

• Cell-type specificity:

  • The enhancer is active specifically in osteocytic cells

  • Genetic deletion affects RANKL expression in osteocytes but not in osteoblasts or lymphocytes

• Functional significance:

  • Deletion of this intronic enhancer leads to a high-bone-mass phenotype

  • Results in decreased levels of RANKL in osteocytic cells and reduced osteoclastogenesis in adult stage

• Molecular regulation:

  • Bioinformatics analyses revealed that transcription factors involved in cell death and senescence act on this intronic enhancer region

  • Single-cell transcriptomic data demonstrated that cell death signaling increases RANKL expression in osteocytic cells

This osteocyte-specific enhancer provides a specialized regulatory element that links cellular senescence/death signals to RANKL expression, potentially facilitating targeted osteoclast formation at specific bone surfaces .

What epigenetic mechanisms regulate TNFSF11 expression and how can they be experimentally investigated?

Epigenetic mechanisms play crucial roles in regulating TNFSF11 expression:

• DNA methylation:

  • Significant differences in DNA methylation status have been observed in the TNFSF11 promoter region

  • Global DNA methylation levels show 4.53-fold decreases in females and 4.83-fold decreases in males with otosclerosis

  • Hypomethylation may contribute to increased disease risk by affecting gene expression

• Chromatin structure:

  • The TNFSF11 CpG-rich region is DNase-sensitive

  • Non-condensed chromatin permits transcriptional activation through greater accessibility of transcriptional machinery

• Transcription factor binding:

  • DNA methylation status affects transcription factor binding to the TNFSF11 promoter

  • In hypomethylated states, transcription factors can more readily bind DNA, allowing RNA polymerase to initiate transcription

Experimental investigation methods include:

• Bisulfite sequencing for DNA methylation analysis

• Chromatin immunoprecipitation (ChIP) for transcription factor binding

• DNase sensitivity assays for chromatin accessibility

• CRISPR-based epigenetic editing to manipulate methylation status

What is the role of TNFSF11 in pathological bone conditions?

TNFSF11 has been implicated in several bone pathologies:

• Osteoporosis:

  • Increased RANKL activity leads to excessive bone resorption

  • The balance between RANKL and osteoprotegerin (OPG) is disrupted

• Osteopetrosis:

  • RANKL deficiency results in reduced osteoclast formation

  • Leads to increased bone density and impaired bone remodeling

• Otosclerosis (OTSC):

  • The rs1021188 polymorphism in TNFSF11 is associated with OTSC in men

  • DNA hypomethylation of the TNFSF11 promoter is observed in OTSC patients

• Cancer-related bone disease:

  • TNFSF11 is implicated in bone metastases in breast cancer, prostate cancer, and multiple myeloma

  • Contributes to osteolytic lesions in multiple myeloma

Understanding the molecular mechanisms of TNFSF11 in these conditions provides opportunities for targeted therapeutic interventions.

How do genetic variations in TNFSF11 contribute to disease susceptibility across different populations?

Genetic variations in TNFSF11 show population-specific patterns in disease association:

• Population-specific risk alleles:

  • The rs1021188 CC genotype is associated with increased otosclerosis risk in Tunisian North-African populations

  • This SNP falls within a cluster grouping the subpopulations with African ethnicity

• Linkage disequilibrium patterns:

  • 26 loci in the TNFSF11 gene are in linkage disequilibrium with rs1021188

  • Different populations exhibit unique allele enrichment/depletion patterns

  • European populations are highly distinguished from American and African but closer to South Asian populations

• Sex-specific effects:

  • The rs1021188 C/T polymorphism shows significant association with OTSC in men (p = 0.023) but not in women (p = 0.458)

  • This suggests sex-specific genetic effects that may contribute to differences in disease prevalence

• Haplotype effects:

  • The haplotype of rs1021188 (C>T) and rs2324851 (A>G) may present a combined genetic risk factor for disease

  • These SNPs show similar allele enrichment/depletion patterns across populations

What novel therapeutic strategies targeting TNFSF11 are emerging for bone disorders?

Several innovative therapeutic approaches targeting TNFSF11 are being developed:

• Cell-specific targeting:

  • The discovery of the osteocyte-specific intronic enhancer provides an opportunity for developing osteocyte-targeted therapies

  • This could allow modulation of RANKL expression specifically in osteocytes without affecting other RANKL-producing cells

• Epigenetic modulation:

  • Targeting DNA methylation in the TNFSF11 promoter region may offer new therapeutic avenues

  • Both genetic polymorphisms and epigenetic modifications could be targeted simultaneously

• Pathway-specific inhibitors:

  • Development of inhibitors targeting specific downstream signaling pathways activated by RANKL

  • This approach could provide more selective intervention than global RANKL inhibition

• Sex-specific therapeutic strategies:

  • Given the observed sex differences in TNFSF11 regulation and genetic associations

  • Tailored approaches based on sex-specific mechanisms may improve therapeutic outcomes

How can single-cell technologies advance our understanding of TNFSF11 biology?

Single-cell technologies offer unique insights into TNFSF11 biology:

• Cell heterogeneity analysis:

  • Single-cell transcriptomics can reveal heterogeneity within populations of osteocytes, osteoblasts, and immune cells

  • Identifies specific cell subpopulations responsible for RANKL production in different contexts

• Regulatory network mapping:

  • Single-cell transcriptomic data analysis has demonstrated that cell death signaling increases RANKL expression in osteocytic cells

  • Enables identification of cell type-specific regulatory networks controlling TNFSF11 expression

• Temporal dynamics:

  • Single-cell trajectory analysis can map the temporal dynamics of RANKL expression during cellular differentiation and activation

  • Provides insights into how RANKL expression changes during bone remodeling processes

• Spatial context:

  • Spatial transcriptomics can reveal how RANKL-expressing cells are distributed within bone tissue

  • Helps understand the local microenvironment influencing RANKL production

These approaches provide unprecedented resolution for understanding the complex regulation and function of TNFSF11 in both physiological and pathological contexts.

What experimental models are most appropriate for studying the role of TNFSF11 in different disease contexts?

Different experimental models offer distinct advantages for studying TNFSF11 biology:

• In vitro models:

  • RAW264.7 mouse monocyte/macrophage cell line for osteoclast differentiation assays

  • Primary osteocyte cultures for studying intronic enhancer function

  • Co-culture systems of osteoblasts and osteoclast precursors

• Genetic mouse models:

  • Targeted disruption of TNFSF11 leads to severe osteopetrosis and lack of osteoclasts

  • Conditional knockout models targeting specific cell types (e.g., osteocyte-specific deletion)

  • Enhancer deletion models to study regulatory element function

• Disease-specific models:

  • Ovariectomized mice for studying postmenopausal osteoporosis

  • Tumor xenograft models for cancer-related bone disease

  • Models of inflammatory conditions affecting bone

• Human-derived systems:

  • Patient-derived cells for studying genetic variations

  • Induced pluripotent stem cells (iPSCs) differentiated into relevant cell types

  • Organoid systems modeling bone-related tissues

Selection of appropriate models should be guided by the specific research question, considering aspects such as species differences, cell type-specific mechanisms, and disease relevance.

What are the conflicting data in the literature regarding TNFSF11 function, and how might these be resolved?

Several areas of conflicting or incomplete understanding exist in TNFSF11 research:

• Cell source controversies:

  • While osteocytes are recognized as major sources of RANKL in adult bone , the relative contributions of different cell types (osteoblasts, T cells, etc.) in various physiological and pathological contexts remain debated

  • Single-cell approaches may help resolve these questions by quantifying cell type-specific contributions

• Sex-specific effects:

  • The gender-stratified analysis showed that TNFSF11 rs1021188 C/T was associated with otosclerosis in men (p = 0.023) but not in women (p = 0.458)

  • The molecular basis for these sex differences requires further investigation

• Enhancer function:

  • The newly discovered intronic enhancer regulates RANKL expression specifically in osteocytes

  • How this enhancer interacts with other regulatory elements and how it responds to various physiological stimuli requires further characterization

• Epigenetic regulation:

  • While DNA hypomethylation of the TNFSF11 promoter is associated with disease , the causality and mechanisms linking methylation changes to expression alterations remain incompletely understood

Resolution approaches:

• Integrative multi-omics approaches combining genomic, epigenomic, and transcriptomic data

• More comprehensive population studies with diverse ethnic backgrounds

• Advanced genetic engineering techniques to dissect regulatory elements

• Standardized methodologies to improve comparability between studies