RARRES2 Human

What is RARRES2 and what is its relationship with chemerin?

RARRES2 (Retinoic Acid Receptor Responder 2) is the gene that encodes chemerin, a chemoattractant protein with adipokine properties . Chemerin is also known as Tazarotene-induced gene 2 protein (TIG2) . The protein is initially secreted in an inactive form called prochemerin, which requires proteolytic cleavage at its C-terminus by inflammatory and coagulation serine proteases to become activated . This activation is crucial for chemerin to perform its biological functions, including acting as a ligand for the G protein-coupled receptor CMKLR1 .

In which human tissues is RARRES2 predominantly expressed?

RARRES2 exhibits tissue-specific expression patterns. According to the Human Protein Atlas data, the highest mRNA levels of human chemerin are found in liver, adrenal gland, pancreas, and white adipose tissue (WAT) . In contrast, RARRES2 transcripts are not detectable in B lymphocytes, monocytes, or granulocytes . Additional expression has been documented in skin (where it is highly expressed), spleen, prostate, ovary, small intestine, and colon . This differential expression pattern suggests tissue-specific roles of RARRES2 in various physiological contexts.

How is RARRES2 expression regulated at the genomic level?

RARRES2 expression is primarily regulated through DNA methylation of its promoter region. Research has demonstrated that the constitutive expression of chemerin is controlled by DNA methylation of the RARRES2 gene . The methylation status of the promoter region located from -735 to +258 bp relative to the transcription start site is particularly important, as this region contains all the binding sites for previously characterized transcription factors of RARRES2 .

The promoter can be divided into proximal (-252/+258 bp) and distal (-735/-253 bp) regions based on methylation levels . Different cell types show varying methylation patterns in these regions, which correlates with their RARRES2 expression levels. This epigenetic regulation contributes significantly to the tissue-specific expression patterns observed for RARRES2.

What are the primary biological functions of chemerin encoded by RARRES2?

Chemerin functions as:

• A chemoattractant that stimulates the chemotaxis of dendritic cells and macrophages to sites of inflammation .

• A ligand for the G protein-coupled receptor CMKLR1 .

• An adipokine involved in metabolic regulation, with associations to multiple metabolic phenotypes .

• A potential regulator of bone mineral density, acting as an independent negative predictor of BMD .

• A protein whose expression is inhibited in psoriatic lesions but can be activated by tazarotene in skin rafts and in the epidermis of psoriatic lesions .

These diverse functions position chemerin at the intersection of inflammatory and metabolic processes, making it a protein of significant interest in various physiological and pathological contexts.

What methodologies are most effective for studying RARRES2 gene methylation patterns?

For comprehensive analysis of RARRES2 methylation patterns, the following methodological approaches are recommended:

• Bisulfite Sequencing: This technique has been successfully employed to determine the methylation status of the murine RARRES2 promoter region . It involves treating DNA with bisulfite, which converts unmethylated cytosines to uracils while leaving methylated cytosines unchanged.

• Reporter Constructs: Establishing reporter constructs containing different portions of the chemerin promoter sequences provides valuable insights into the functional importance of specific regions. Key constructs include:

  • Chemerin_Full (position -735/+258 bp relative to the transcription start site)

  • Chemerin_Proximal (position -252/+258 bp)

  • Chemerin_Distal (position -735/-253 bp)

• DNA Fragment Analysis: Focus on the region ranging from -735 to +258 bp of the RARRES2 gene, as this contains all binding sites for previously characterized transcription factors .

• Correlation with Expression Data: Combine methylation analysis with expression studies using RT-QPCR to establish relationships between methylation status and transcriptional output .

These methodologies should be applied across different tissue types to capture the tissue-specific regulation of RARRES2, particularly comparing tissues with high expression (liver, WAT) versus those with low or undetectable expression (B-cells) .

How do genetic variants in the RARRES2 locus affect circulating chemerin levels?

Genetic variants in the RARRES2 locus significantly influence circulating chemerin levels. A genome-wide meta-analysis revealed that genetic variation in the RARRES2 locus plays a crucial role in regulating circulating chemerin concentrations . Specifically:

• Key SNP rs7806429: This SNP showed the strongest evidence for association with chemerin levels (p = 7.8×10^-14, beta = -0.067), explaining approximately 2.0% of the variance in circulating chemerin .

• Expression Quantitative Trait Loci (eQTL) Analysis: rs7806429 has been associated with mRNA expression of RARRES2 in visceral adipose tissue, particularly in women, suggesting sex-specific effects .

• Other Associated SNPs: Additional SNPs within the RARRES2/LRRC61 cluster were in linkage disequilibrium with rs7806429, with at least moderate correlation (minimum r² = 0.43) .

• Peripheral Blood Mononuclear Cells (PBMCs) Analysis: In PBMCs, rs7806429 showed strong effects on expressed transcripts for AL359058 (p = 5.1×10^-10, explained variance = 4.2%) and for LRRC61 (p = 7.8×10^-9, explained variance = 3.7%) .

Researchers investigating genetic influences on chemerin levels should incorporate these variants in their study designs and consider sex-specific effects when analyzing results.

What experimental approaches can effectively assess the role of RARRES2 in inflammatory processes?

To investigate RARRES2's role in inflammatory processes, researchers should consider these methodological approaches:

• Cytokine Stimulation Studies: Examine how acute-phase cytokines like interleukin 1β (IL-1β) and oncostatin M (OSM) regulate chemerin expression in different cell types. Previous research has shown these cytokines regulate chemerin expression in mouse primary adipocytes but not in hepatocytes, both in vitro and in vivo .

• Proteolytic Activation Analysis: Since chemerin is secreted in an inactive form (prochemerin) and requires proteolytic cleavage for activation , study the proteases involved in this process under various inflammatory conditions.

• Chemotaxis Assays: Quantify the ability of activated chemerin to stimulate chemotaxis of dendritic cells and macrophages to inflammation sites . These functional assays can reveal the potency of chemerin in recruiting immune cells.

• Receptor Binding Studies: Analyze the interaction between chemerin and its receptor CMKLR1 under different inflammatory states .

• Animal Models: Utilize both in vitro cell culture systems and in vivo mouse models to comprehensively understand RARRES2's inflammatory roles in different contexts .

• Psoriasis Research Models: Since RARRES2 is inhibited in psoriatic lesions but activated by tazarotene , psoriasis models can provide valuable insights into chemerin's inflammatory regulation.

An integrated approach combining these methods will provide a more complete picture of RARRES2's complex roles in inflammatory processes.

What is the association between RARRES2 and bone mineral density (BMD)?

Recent research has established significant associations between RARRES2 and bone mineral density:

• Negative Correlation: RARRES2 acts as an independent negative predictor of bone mineral density . This means increased levels of RARRES2 are associated with decreased BMD.

• Methodological Approach for BMD Studies:

  • Serum RARRES2 levels can be measured and correlated with BMD measurements using dual-energy X-ray absorptiometry (DXA).

  • Multivariate linear stepwise regression and logistic regression analysis should be used to predict BMD, with RARRES2 as an independent variable .

  • Models should be adjusted for age and menopausal age (in women) for accurate results .

• Genetic Analysis: The rs7806429 variant of RARRES2 has been studied in relation to BMD, with PCR amplifications followed by:

  • Restriction fragment length polymorphism (RFLP)

  • Amplification refractory mutation system (ARMS)

  • Allele-specific PCR

• Control Variables: When studying RARRES2-BMD associations, researchers should control for other factors that affect BMD, including serum calcium, phosphate, and alkaline phosphatase levels .

This association positions RARRES2 as a potential biomarker or therapeutic target in bone metabolism disorders, warranting further investigation into the underlying mechanisms.

How can researchers effectively study the methylation and transcriptional regulation of RARRES2?

For comprehensive analysis of RARRES2 methylation and transcriptional regulation, researchers should employ these integrated approaches:

• Promoter Region Analysis: Focus on the region from -735 to +258 bp relative to the transcription start site, which contains all binding sites for known transcription factors of RARRES2 .

• Promoter Constructs: Develop and utilize the following reporter constructs:

  • Chemerin_Full (-735/+258 bp)

  • Chemerin_Proximal (-252/+258 bp)

  • Chemerin_Distal (-735/-253 bp)

• Bisulfite Sequencing: Apply this technique to determine the methylation status of specific CpG sites within the promoter region .

• Cell Type Comparison: Compare methylation patterns and expression levels across multiple cell types with varying baseline RARRES2 expression (e.g., liver and white adipose tissue versus B cells) .

• Correlation Analysis: Perform correlation studies between RARRES2 mRNA levels and various phenotypic markers, including:

  • Anthropometric traits (% body fat)

  • Parameters of glucose homeostasis (fasting plasma glucose, glucose infusion rate)

  • Lipid metabolism markers (LDL-cholesterol)

• Sex-Specific Analysis: Given the observed sex differences in RARRES2 regulation, conduct separate analyses for males and females, particularly when studying adipose tissue expression .

This methodological framework allows for a comprehensive understanding of the epigenetic and transcriptional mechanisms governing RARRES2 expression in different physiological contexts.

What are the recommended approaches for producing and purifying recombinant RARRES2 protein?

For researchers working with RARRES2 protein, the following methodological guidelines are recommended:

• Expression Systems: Mammalian expression systems are preferred for producing recombinant human RARRES2 protein, as they ensure proper post-translational modifications .

• Protein Construction: Focus on expressing the region encoding Glu21-Ser157, which represents the mature protein sequence .

• Fusion Tags: A C-terminal 6His tag can be used for purification purposes without significantly affecting protein function .

• Purity Assessment: Ensure >95% purity as determined by reducing SDS-PAGE .

• Molecular Weight Verification: The expected molecular mass is approximately 16.9 kDa, with apparent molecular mass on SDS-PAGE of 14-16 kDa .

• Endotoxin Testing: Maintain endotoxin levels below 1.0 EU per μg as determined by the LAL method to ensure the protein preparation is suitable for biological assays .

These approaches ensure the production of high-quality RARRES2 protein suitable for various research applications, including functional studies, structural analyses, and antibody production.

What genomic techniques are most appropriate for studying RARRES2 gene variants?

For comprehensive analysis of RARRES2 genetic variations, researchers should consider these methodological approaches:

• PCR Techniques:

  • Use gene-specific primers for amplification of RARRES2 genetic regions

  • Employ restriction fragment length polymorphism (RFLP) for variant analysis

  • Utilize amplification refractory mutation system (ARMS) for specific allele detection

  • Apply allele-specific PCR for genotyping

• Sequencing Verification: Confirm PCR results through sequencing and analyze using specialized software like Chromas .

• Genome-Wide Association Studies (GWAS): For broader analysis, implement meta-GWAS approaches to identify additional variants associated with RARRES2 expression or function .

• eQTL Analysis: Combine genotype data with mRNA expression studies in relevant tissues (particularly adipose tissue) to identify expression quantitative trait loci .

• Hardy-Weinberg Equilibrium Testing: Ensure genotype distributions conform to Hardy-Weinberg equilibrium to validate genotyping quality .

These techniques provide a comprehensive toolset for investigating RARRES2 genetic variations and their functional consequences.

What are the most promising future research directions for RARRES2 in human health and disease?

Based on current understanding, several promising research directions for RARRES2 include:

• Metabolic Disease Biomarkers: Further exploration of RARRES2/chemerin as a biomarker for metabolic disorders, given its associations with multiple metabolic phenotypes and inflammatory markers .

• Bone Metabolism: Deeper investigation into the mechanisms by which RARRES2 negatively affects bone mineral density and potential therapeutic implications .

• Sex-Specific Regulation: More comprehensive studies on the sex-specific effects observed in RARRES2 expression and function, particularly in adipose tissue .

• Epigenetic Regulation: Expanded research on the DNA methylation patterns of RARRES2 across different tissues and disease states .

• Inflammatory Pathway Integration: Exploration of how RARRES2/chemerin integrates with broader inflammatory signaling networks, especially in conditions like psoriasis where its expression is altered .

• Therapeutic Targeting: Investigation of RARRES2 as a potential therapeutic target, particularly in inflammatory and metabolic disorders.

• Tissue-Specific Functions: Further elucidation of the tissue-specific roles of RARRES2, given its differential expression patterns across human tissues .