RARRES2 Human, HEK

What is RARRES2 and what are its primary functions in human biology?

RARRES2, located on chromosome 7q36.1, encodes a 14 kDa protein also known as chemerin that functions as both an adipokine and tumor suppressor. The gene comprises six exons and serves as a ligand for chemokine-like receptor-1 (CMKLR1), a G protein-coupled serpentine receptor . RARRES2 plays critical roles in inflammation and adipocyte differentiation, while simultaneously functioning as a tumor suppressor by promoting β-catenin phosphorylation and degradation . It also inhibits p38 mitogen-activated protein kinase (MAPK) phosphorylation and regulates cell proliferation, invasion, and tumorigenicity . Interestingly, RARRES2 may influence bone metabolism, with evidence suggesting a negative correlation with bone mineral density (BMD) . Heritability estimates indicate approximately 16.2% of the variation in circulating chemerin levels is attributable to genetic factors .

What experimental models are most effective for studying RARRES2 expression and function?

Multiple experimental models have demonstrated utility in RARRES2 research:

Cell Line Models:

• HEK293 cells: Show strong and sustained RARRES2 expression after transfection

• H295R cells: Adrenocortical carcinoma cells effective for both transient and stable RARRES2 expression studies

• SW13 cells: Adrenal cortex cells with weaker RARRES2 expression after transfection

Animal Models:

• Athymic nude mice: Effective for xenograft studies assessing RARRES2's tumor-suppressive effects

• NOD Scid gamma (NSG) mice: Also used successfully for xenograft studies, demonstrating similar results to athymic nude models

Expression Systems:

• Transient transfection for short-term studies yields significant effects on cell proliferation and invasion in HEK293 cells

• Stable polyclonal cell lines expressing RARRES2 enable assessment of long-term effects and dose-dependent relationships

• Recombinant protein administration can be used to examine potential extracellular effects, though surprisingly these show limited functional impact

How is RARRES2 expression regulated across different human tissues?

RARRES2 exhibits distinct tissue-specific expression patterns:

RARRES2 transcripts have been detected in liver, lung, adipose tissue, ovary, pancreas, heart, hypothalamus, and pituitary tissues, while expression in kidney and leukocytes was not detectable in studied species . In adrenocortical tissues, normal samples show significantly higher RARRES2 expression compared to benign tumors, while expression is further reduced in adrenocortical carcinomas (ACCs) . This progressive decrease suggests RARRES2 downregulation may correlate with increasing malignancy.

A significant correlation exists between RARRES2 mRNA and protein expression in adrenocortical tissue samples (r=0.3194, P=0.0062), indicating consistent transcriptional and translational regulation . At the genetic level, single nucleotide polymorphisms (SNPs) at the RARRES2 locus influence circulating chemerin concentrations, with rs7806429 showing the strongest association (p=7.8×10^-14), explaining approximately 2.0% of the variance in circulating levels .

What methods provide optimal detection and quantification of RARRES2 in experimental systems?

Several methodologies demonstrate effectiveness for RARRES2 detection:

mRNA Detection:

• Quantitative PCR (qPCR): Preferred for measuring RARRES2 transcript levels in tissue samples

• Microarrays: Effective for assessing mRNA expression in peripheral mononuclear cells

Protein Detection:

• Immunohistochemical (IHC) staining: Optimal for visualizing and quantifying RARRES2 protein in tissue sections with total signal intensity scoring systems

• Western blotting: Essential for confirming protein expression in cell lines after transfection

Functional Assessments:

• Cell proliferation assays: Effective for measuring RARRES2's impact on cell growth

• Cell invasion assays: Provide reliable readouts of RARRES2's effects on cell motility

• Clonogenic and soft agar colony formation assays: Valuable for assessing tumorigenicity and anchorage-independent growth

Through what molecular mechanisms does RARRES2 exert its tumor suppressor functions?

RARRES2 operates through several distinct molecular pathways:

β-catenin Pathway Regulation:
RARRES2 overexpression promotes β-catenin phosphorylation and degradation, reducing Wnt/β-catenin pathway activity . This mechanism is crucial since dysregulation of this pathway frequently contributes to cancer development.

MAPK Pathway Inhibition:
RARRES2 inhibits p38 MAPK phosphorylation, interfering with cellular stress responses that can drive cancer progression .

Cell Adhesion Molecule Regulation:
RARRES2 overexpression significantly decreases N-cadherin levels, potentially contributing to reduced cellular invasion . Interestingly, other extracellular matrix proteins (fibronectin, collagen I, collagen IV, laminin I, fibrinogen) remained unaffected in H295R cells .

Immune-Independent Mechanisms:
Studies with immunodeficient mouse models demonstrated that RARRES2 expression alone sufficiently suppresses tumor growth, indicating tumor suppression occurs through immune-independent mechanisms . This represents the first evidence for such an immune-independent tumor suppressor role for RARRES2.

Dose-Dependent Effects:
The degree of growth suppression correlates directly with RARRES2 expression levels, with higher-expressing cell lines showing greater inhibition of proliferation and tumorigenicity .

How do genetic variations in the RARRES2 gene influence expression and function?

Genetic variants significantly impact RARRES2 biology:

A meta-analysis of genome-wide association studies identified 30 SNPs at chromosome 7 within the RARRES2/LRRC61 locus reaching genome-wide significance for association with serum chemerin levels . The SNP rs7806429 showed the strongest association (p=7.8×10^-14, beta=-0.067), explaining approximately 2.0% of the variance in circulating chemerin levels .

All associated SNPs demonstrated linkage disequilibrium with rs7806429 (minimum r²=0.43) , suggesting potential shared functional effects. No significant SNP-sex interaction was observed for the genetic variants associated with chemerin levels, indicating consistent genetic regulation between males and females .

Tissue-specific effects were observed, with rs7806429 associated with RARRES2 mRNA expression in visceral adipose tissue in women (p<0.05 after adjusting for age and BMI) . In postmenopausal females, significant differences in bone mineral density at the right femoral neck (p=0.033) were observed between rs7806429 genotypes, with BMD significantly lower in TC heterozygotes compared to CC homozygotes .

What distinguishes the intracellular versus extracellular functions of RARRES2?

Research reveals important distinctions between intracellular and extracellular RARRES2 effects:

Intracellular Effects:
Overexpression of RARRES2 within cells consistently reduces cell proliferation, invasion, and tumorigenicity across multiple cell lines . Mechanistically, intracellular RARRES2 promotes β-catenin phosphorylation and degradation while inhibiting p38 MAPK phosphorylation .

Extracellular Effects:
Despite RARRES2 being characterized as a secreted protein that functions by binding to CMKLR1, exogenous addition of recombinant RARRES2 protein does not exhibit significant functional impacts in vitro . Various physiological concentrations of recombinant human chemerin tested on H295R, SW13, and HEK293 cells produced no significant changes in cell proliferation or invasion/migration .

This unexpected finding challenges the conventional understanding of RARRES2 primarily as an extracellular signaling molecule and suggests critical intracellular functions that operate independently of receptor binding. The research presents the first evidence that RARRES2's tumor suppressor activity may function primarily through novel intracellular mechanisms rather than through secretion and autocrine/paracrine signaling .

How does RARRES2 expression differ between normal tissues and pathological conditions?

RARRES2 shows distinct expression patterns across normal and pathological states:

In Adrenocortical Tissues:
RARRES2 protein expression is significantly lower in benign tumors compared to normal adrenocortical tissues, and even lower in ACC samples . This pattern appears consistent at both mRNA and protein levels, suggesting transcriptional downregulation during tumorigenesis .

Cell Line Expression:
Endogenous expression of RARRES2 and its receptor CMKLR1 is barely detectable in all tested ACC cell lines, consistent with the observed downregulation in tumor samples .

In Bone-Related Conditions:
Serum RARRES2 levels are significantly elevated in osteoporotic postmenopausal females compared to non-osteoporotic and osteopenic individuals . Serum RARRES2 levels function as independent negative predictors of BMD at the right and left femoral neck .

Contradictory Findings:
Some studies report decreased RARRES2 levels in postmenopausal osteoporotic females , contradicting other findings. These inconsistencies highlight the complexity of RARRES2 regulation across different pathological contexts and emphasize the need for further research to reconcile these apparent contradictions.

What protocols optimize RARRES2 overexpression studies in HEK293 cells?

Based on published research, the following protocols yield optimal results:

Transient Transfection:
HEK293 cells demonstrate the strongest and most sustained RARRES2 expression after transient transfection compared to other cell lines (H295R, SW13) . Standard lipid-based transfection reagents provide effective delivery, with expression confirmation via Western blotting.

Functional Assessment Timeline:
Cell proliferation assays should be conducted during peak expression periods, as transient RARRES2 overexpression significantly reduces HEK293 cell proliferation . Cell invasion assays also show reliable results during this window, while migration assays appear less informative as RARRES2 does not significantly affect this parameter in HEK293 cells .

Experimental Controls:
Empty vector controls are essential to account for transfection effects. For dose-response studies, establishing multiple stable cell lines with varying RARRES2 expression levels provides valuable insights into concentration-dependent effects .

Extracellular Studies:
When investigating potential extracellular effects, recombinant human chemerin can be added to culture media at physiological concentrations, though previous research suggests limited functional impact through this mechanism .

What assays most effectively measure RARRES2-induced cellular changes?

Several assays provide reliable measurement of RARRES2's functional effects:

Cell Proliferation:
Standard proliferation assays effectively detect RARRES2-induced growth inhibition in both transient and stable expression systems . Effects appear cell-type dependent, with stronger inhibition observed in HEK293 cells compared to ACC cell lines .

Invasion Assessment:
Transwell invasion assays with matrigel coating reliably detect RARRES2-mediated reduction in invasive capacity . This effect is observable in both transiently transfected cells and stable lines, with both H295R and HEK293 cells showing reduced invasion upon RARRES2 overexpression .

Tumorigenicity Measurement:

• Clonogenic assays: RARRES2-overexpressing stable cell lines show dramatically inhibited colony formation

• Soft agar colony formation assays: Anchorage-independent growth is significantly reduced in RARRES2-expressing cells

• Both assays demonstrate correlation between RARRES2 expression levels and inhibition magnitude

Molecular Markers:

• N-cadherin levels: Decrease significantly with RARRES2 overexpression

• β-catenin phosphorylation: Increases with RARRES2 expression

• p38 MAPK phosphorylation: Decreases with RARRES2 expression
  These molecular changes provide effective biochemical markers for RARRES2 activity.

What considerations are critical when designing xenograft studies to investigate RARRES2 function?

Xenograft studies require careful planning across multiple parameters:

Mouse Model Selection:
Both athymic nude and NOD Scid gamma (NSG) mice have demonstrated utility, with RARRES2 overexpression significantly suppressing tumor growth in both models . This consistency across immunodeficient models indicates either is suitable for studying RARRES2's immune-independent effects.

Cell Line Preparation:
Stable cell lines expressing either empty vector or RARRES2 should be established and characterized before xenograft studies . Expression levels must be verified and maintained across passages, with ideally multiple lines expressing different RARRES2 levels to assess dose-dependent effects .

Injection Methodology:
Subcutaneous injection into mouse flanks provides optimal accessibility for tumor measurement and monitoring .

Outcome Measurements:
Regular tumor size measurements throughout the study period combined with terminal tumor weight assessment at study endpoint provide complementary data points . Both parameters have shown consistent results in published studies.

Immune Considerations:
Using immunodeficient mouse models isolates immune-independent mechanisms, which has been crucial in demonstrating that RARRES2 functions as a tumor suppressor through novel pathways not requiring immune system involvement .

What approaches best elucidate RARRES2's role in tissue inflammation and metabolism?

Multiple complementary approaches provide insights into RARRES2's metabolic functions:

Expression Analysis:
Quantitative RT-PCR to measure RARRES2 mRNA expression in different tissue depots provides critical baseline data . Comparison between visceral and subcutaneous adipose tissue can reveal depot-specific effects, while analysis across different metabolic states (normal, obese, diabetic) illuminates regulatory patterns.

Genetic Approaches:
Genotyping SNPs within the RARRES2 locus, particularly rs7806429, which shows strong association with circulating chemerin levels, establishes genetic foundations . Expression quantitative trait locus (eQTL) analysis correlating genetic variants with gene expression in target tissues provides functional insights .

Clinical Correlations:
Measuring serum RARRES2/chemerin levels in relation to inflammatory markers across different metabolic conditions (obesity, diabetes, metabolic syndrome) establishes clinical relevance . Correlation with bone mineral density assessments provides insights into RARRES2's role in bone metabolism .

Evolutionary Perspective:
Comparative studies across species provide insights into conserved functions, with expression profile analysis in different primates suggesting evolutionarily conserved RARRES2 functions .

How should researchers reconcile contradictory findings regarding RARRES2 in pathological conditions?

Research presents conflicting observations regarding RARRES2 in certain conditions:

Reconciliation Strategies:

Methodological Standardization:
Different detection methods for measuring RARRES2 may contribute to discrepancies . Standardized assays and measurement techniques enable more reliable cross-study comparisons.

Population Stratification:
Different patient populations may exhibit distinct RARRES2 regulation patterns . Factors including age, ethnicity, comorbidities, and medication use should be carefully controlled and reported.

Temporal Dynamics:
RARRES2 levels may change dynamically during disease progression . Longitudinal studies tracking changes over time provide more comprehensive understanding than single-timepoint measurements.

Tissue vs. Circulating Levels:
Discrepancies between tissue expression and circulating levels may exist . Simultaneous assessment of both tissue and circulating RARRES2 provides more complete biological context.

Genetic Influence Analysis:
Genetic variants such as rs7806429 may influence RARRES2 expression differently across various conditions . Genotyping study subjects can help explain apparently contradictory findings through genetic stratification.

What are the implications of RARRES2's dual roles in adipogenesis and osteogenesis?

RARRES2's seemingly opposing effects on adipose and bone tissues present intriguing implications:

Functional Duality:
RARRES2 appears to facilitate adipogenesis while inhibiting osteogenesis , suggesting a potential role in regulating mesenchymal stem cell fate decisions.

Clinical Implications:

Osteoporosis Mechanisms:
Elevated RARRES2 levels may contribute to bone loss by inhibiting osteoblast function and/or promoting adipocyte differentiation in bone marrow . This mechanism could explain the negative correlation between serum RARRES2 and BMD observed in some studies.

Obesity-Bone Relationship:
RARRES2's dual effects may help explain the complex relationship between obesity and bone health . While obesity was traditionally considered protective against osteoporosis, obesity-related inflammation may negatively impact bone through mediators like RARRES2.

Therapeutic Target Potential:
RARRES2 represents a potential therapeutic target for conditions involving both adipose and bone tissues . Selective modulation of RARRES2 signaling might enable tissue-specific interventions.

Mechanistic Integration:
The Wnt/β-catenin pathway, which RARRES2 affects through promoting β-catenin degradation , plays crucial roles in both adipogenesis and osteogenesis, potentially representing a common mechanism underlying RARRES2's effects on both tissues.

How should genetic associations between RARRES2 variants and phenotypes be interpreted?

Genetic association studies require careful interpretation:

Effect Magnitude:
The strongest SNP association (rs7806429) explains approximately 2.0% of the variance in circulating chemerin levels . While statistically significant, this indicates most variation stems from other factors, requiring comprehensive multi-factor models.

Functional Context:
rs7806429 associates with RARRES2 mRNA expression in visceral adipose tissue in women , indicating tissue-specific and potentially sex-specific effects. Functional studies investigating the molecular mechanisms through which variants affect gene expression provide crucial mechanistic context.

Phenotypic Relationships:
rs7806429 genotypes show significant association with BMD at the right femoral neck, with TC heterozygotes exhibiting lower BMD compared to CC homozygotes . This suggests pleiotropic effects of RARRES2 genetic variants across multiple tissues.

Linkage Disequilibrium Considerations:
All SNPs in the associated cluster demonstrate linkage disequilibrium with rs7806429 , challenging identification of the specific causal variant(s). Fine mapping and functional studies are necessary to pinpoint the specific variant(s) responsible for observed associations.

What statistical approaches best analyze RARRES2 expression and functional data?

Several statistical approaches enhance RARRES2 data analysis:

Data Distribution Assessment:
Shapiro-Wilk's statistics should verify data normality , determining appropriate parametric or non-parametric testing approaches.

Group Comparisons:
For non-normally distributed data, non-parametric tests such as Kruskal-Wallis are optimal for comparing multiple groups . Post-hoc pairwise comparisons should employ the Dunn-Bonferroni approach to correct for multiple testing .

Correlation Analysis:
Correlation analysis between mRNA and protein expression provides insights into transcriptional and translational regulation . Appropriate correlation coefficients (Pearson's r or Spearman's rho) should be reported with p-values.

Regression Modeling:
Multivariate linear stepwise regression effectively predicts continuous outcomes (e.g., BMD) from RARRES2 levels and other variables . Logistic regression suits binary outcomes (e.g., osteoporosis status). Models should adjust for relevant covariates including age and menopausal status .

Genetic Association Analysis:
Genotypic and allelic frequencies should be calculated and tested for Hardy-Weinberg equilibrium . Association between genotypes and phenotypes requires appropriate statistical tests with multiple testing correction when examining multiple SNPs or phenotypes .