CYP7A1 Antibody

What is CYP7A1 and what is its primary biological function?

CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1) is a cholesterol 7α-hydroxylase that catalyzes the rate-limiting step in bile acid biosynthesis from cholesterol—representing the main pathway for cholesterol removal from the body . This 58 kDa enzyme contains 504 amino acids and plays a crucial role in lipid homeostasis and metabolism . As the initial and rate-determining enzyme in the classical pathway of bile acid synthesis, CYP7A1 converts cholesterol to 7α-hydroxycholesterol, initiating a cascade that ultimately produces primary bile acids. This conversion is essential for maintaining cholesterol balance and preventing conditions associated with cholesterol dysregulation .

What types of CYP7A1 antibodies are available for research applications?

Research-grade CYP7A1 antibodies are predominantly available as polyclonal antibodies developed in rabbits, such as the 18054-1-AP and CAB10615 products identified in the literature . These antibodies are typically generated using recombinant fusion proteins or peptide sequences corresponding to specific regions of human CYP7A1. For instance, CAB10615 uses an immunogen containing amino acids 390-504 of human CYP7A1 (NP_000771.2) . These antibodies demonstrate reactivity primarily with human samples, though some cross-reactivity with mouse and rat CYP7A1 has been documented . They are supplied in liquid form, generally with PBS buffer containing sodium azide and glycerol for stability, and are optimized for various applications including Western blotting, immunohistochemistry, immunofluorescence, and ELISA techniques .

What are the optimal conditions for using CYP7A1 antibodies in Western blot applications?

For Western blot applications with CYP7A1 antibodies, researchers should implement the following protocol for optimal results:

The specificity of antibody binding should be validated using known positive controls, and results should be interpreted in the context of appropriate experimental controls to ensure accuracy and reproducibility.

How should immunohistochemistry protocols be optimized when using CYP7A1 antibodies?

For immunohistochemistry applications with CYP7A1 antibodies, the following protocol optimizations are recommended:

• Tissue Preparation: CYP7A1 antibodies have been validated for use with human liver tissue and liver cancer samples. Formalin-fixed paraffin-embedded sections should be prepared at 4-6 μm thickness .

• Antigen Retrieval: Critical for successful staining, antigen retrieval should be performed using TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 can be used, though this may result in different staining intensity .

• Antibody Dilution: The optimal dilution range for IHC applications is 1:50 to 1:500, with specific dilution requiring optimization for individual experimental systems .

• Detection Systems: Polymer-based detection systems generally provide superior results compared to avidin-biotin systems for CYP7A1 detection.

• Controls: Positive controls should include normal human liver tissue, which expresses high levels of CYP7A1. Negative controls should include tissues known not to express CYP7A1 or primary antibody omission controls.

The staining pattern should be primarily cytoplasmic, reflecting the subcellular localization of this enzyme in the endoplasmic reticulum of hepatocytes. Interpretation of staining should consider that CYP7A1 expression can vary significantly based on nutritional status, time of day (due to circadian regulation), and pathological conditions affecting liver function.

How do genetic variants in CYP7A1 impact cholesterol metabolism and disease risk?

Genetic variation in CYP7A1 has significant implications for cholesterol homeostasis and disease susceptibility through multiple mechanisms:

• Promoter Variants: The promoter SNP rs3808607 has been extensively studied and shows associations with altered cholesterol levels. This regulatory variant affects transcription factor binding and consequently CYP7A1 expression levels .

• Enhancer Variants: Research has identified a functional SNP (rs9297994) located in a downstream enhancer region that interacts with the CYP7A1 promoter. Intriguingly, this variant has opposite effects on CYP7A1 mRNA expression compared to the promoter SNP rs3808607 .

• Combined SNP Effects: The interaction between these two SNPs (rs3808607 and rs9297994) creates a 2-SNP model that robustly predicts hepatic CYP7A1 mRNA expression, with expression levels varying by more than two orders of magnitude between different genotype combinations .

• Disease Associations: This 2-SNP model shows significant associations with:

  • LDL cholesterol levels

  • Risk of coronary artery disease

  • Response to statin therapy

  • Diabetes mellitus risk

These findings demonstrate that CYP7A1 genetic variants contribute to inter-individual differences in cholesterol metabolism and susceptibility to metabolic and cardiovascular disorders. The research highlights the importance of considering interactions between multiple regulatory variants rather than studying individual SNPs in isolation when investigating genotype-phenotype correlations .

What methodologies are used to identify and characterize regulatory regions affecting CYP7A1 expression?

Researchers employ several complementary approaches to identify and characterize regulatory regions that control CYP7A1 expression:

• Chromatin Conformation Capture (4C Assay): This technique identifies genomic regions that physically interact with the CYP7A1 promoter through three-dimensional chromatin looping. Studies have revealed several distal regions that interact with the CYP7A1 promoter, indicating long-range regulatory mechanisms .

• Chromatin Immunoprecipitation (ChIP-qPCR): This method identifies transcription factor binding sites within regulatory regions of CYP7A1 in hepatocytes, helping to elucidate the molecular mechanisms of its regulation .

• CRISPR-Mediated Genome Editing: Using CRISPR technology in hepatocellular carcinoma cell lines, researchers have identified novel CYP7A1 enhancer and repressor regions located more than 10 kb downstream of the CYP7A1 promoter .

• Allelic mRNA Expression Imbalance: This approach measures differences in expression between the two alleles of CYP7A1 in human liver samples to identify functional SNPs that affect gene expression in vivo .

• Reporter Gene Assays: These assays test the effects of specific SNPs on gene expression by inserting wild-type or variant sequences into reporter constructs and measuring differences in expression .

Through the integration of these complementary approaches, researchers have developed a more comprehensive understanding of the complex regulatory mechanisms controlling CYP7A1 expression, including the identification of previously unrecognized enhancer and repressor elements and the functional characterization of disease-associated genetic variants.

How is CYP7A1 expression regulated during liver regeneration, and what are the physiological implications?

CYP7A1 expression undergoes precise temporal regulation during liver regeneration, with significant physiological consequences:

• Biphasic Regulation: CYP7A1 expression is regulated in two distinct phases after 70% partial hepatectomy (PH):

  • Acute phase: Initial suppression occurs independent of farnesoid X receptor (FXR) and small heterodimer partner (SHP) signaling

  • Later phase: Both FXR and SHP become essential for regulating CYP7A1 expression

• Signaling Pathways: Multiple signaling cascades are involved in CYP7A1 suppression:

  • Hepatocyte growth factor (HGF) pathway

  • c-Jun N-terminal kinase (JNK) pathway
    These pathways are activated during the acute phase of liver regeneration and contribute to CYP7A1 suppression

• Physiological Significance: Experimental evidence demonstrates that suppression of CYP7A1 during liver regeneration is essential for:

  • Preventing hepatocyte death and liver damage

  • Enabling normal proliferation (shown by reduced BrdU-positive nuclei when CYP7A1 is overexpressed)

  • Preventing apoptosis (demonstrated by TUNEL staining)

  • Avoiding hepatocyte ballooning degeneration and fat accumulation

  • Reducing liver cytotoxicity (evidenced by lower ALT levels)

• Bile Acid Levels: Failure to suppress CYP7A1 after partial hepatectomy results in significantly higher levels of hepatic bile acids, which can be cytotoxic to regenerating liver tissue

This research reveals a critical protective mechanism whereby CYP7A1 suppression during liver regeneration prevents the accumulation of potentially toxic bile acids in the regenerating liver, highlighting the importance of coordinated regulation of metabolic processes during tissue repair and regeneration.

What are the interactions between CYP7A1 expression and inflammatory or stress response pathways?

CYP7A1 expression is intricately linked with inflammatory and stress response pathways through multifaceted interactions:

• JNK Signaling Pathway: The c-Jun N-terminal kinase (JNK) pathway plays a critical role in CYP7A1 suppression during liver regeneration and various stress conditions:

  • JNK1 is involved in CYP7A1 suppression independent of FXR and SHP during the acute phase of liver regeneration

  • This represents a rapid response mechanism that precedes the classical bile acid feedback regulation

• Inflammatory Cytokines: Though not explicitly detailed in the provided search results, research has established that inflammatory cytokines like IL-1β, TNF-α, and IL-6 can suppress CYP7A1 expression, representing a potential mechanism linking inflammation to altered cholesterol metabolism.

• Cellular Stress Responses: The suppression of CYP7A1 during liver regeneration appears to be part of a coordinated stress response that protects hepatocytes from bile acid-induced toxicity:

  • When this suppression is prevented (through adenoviral expression of CYP7A1), significant cellular damage occurs, including:

    • Ballooning degeneration of hepatocytes

    • Fat accumulation in the liver

    • Elevated serum ALT levels (a marker of liver damage)

    • Increased hepatic bile acid levels

• Regulatory Interactions: The temporal regulation of CYP7A1 involves complex interactions between multiple signaling pathways:

  • Early phase: Growth factor and stress-activated pathways (HGF, JNK)

  • Later phase: Nuclear receptor-mediated pathways (FXR, SHP)

These findings highlight CYP7A1 as a critical metabolic checkpoint that integrates stress signals, growth factor signaling, and bile acid feedback mechanisms to maintain homeostasis during physiological challenges. The coordinated suppression of CYP7A1 during stress appears to be an important adaptive mechanism to prevent bile acid toxicity when the liver's functional capacity is compromised.

What are common challenges in CYP7A1 antibody applications and how can they be addressed?

Researchers frequently encounter several challenges when working with CYP7A1 antibodies that require specific troubleshooting approaches:

• Variable Expression Levels: CYP7A1 expression shows significant diurnal variation and is affected by nutritional status:

  • Solution: Standardize sample collection times and feeding conditions when comparing CYP7A1 expression between experimental groups

  • Approach: Document time of day for sample collection and consider using pooled samples from multiple time points for certain studies

• Specificity Concerns: Cross-reactivity with other cytochrome P450 family members can occur:

  • Solution: Validate antibody specificity using known positive controls (HepG2, HuH-7, L02, and SMMC-7721 cells)

  • Approach: Include appropriate negative controls such as CYP7A1-knockout or knockdown samples when available

• Optimal Antigen Retrieval for IHC:

  • Solution: Test both recommended methods - TE buffer (pH 9.0) and citrate buffer (pH 6.0)

  • Approach: Perform side-by-side comparisons to determine which method yields optimal signal-to-noise ratio for specific tissue types

• Antibody Dilution Optimization:

  • Solution: Test a range of dilutions, starting with the manufacturer's recommended range (WB: 1:1000-1:6000; IHC: 1:50-1:500; IF/ICC: 1:50-1:500)

  • Approach: Perform titration experiments with serial dilutions to identify optimal concentration for each specific application

• Signal Detection Challenges in samples with low CYP7A1 expression:

  • Solution: Use signal amplification methods such as biotin-streptavidin systems for IHC or highly sensitive chemiluminescence substrates for Western blot

  • Approach: Increase exposure time for Western blots or primary antibody incubation time while monitoring background levels

Implementing these targeted approaches can significantly improve the reliability and reproducibility of experiments involving CYP7A1 antibodies across various research applications.

How can researchers accurately interpret CYP7A1 expression data in the context of genetic variation?

Accurate interpretation of CYP7A1 expression data requires consideration of genetic variation and several methodological approaches:

• Consideration of Multiple Regulatory SNPs: Research has demonstrated that interpretation of CYP7A1 expression requires analysis of multiple interacting SNPs:

  • The 2-SNP model (rs3808607 and rs9297994) explains hepatic CYP7A1 expression levels more robustly than either SNP alone

  • Expression levels can vary by more than two orders of magnitude based on the combined genotype

• Methodological Approaches:

  • Allelic Expression Imbalance: Measure allele-specific expression in heterozygous samples to detect cis-acting genetic effects on CYP7A1 expression

  • Haplotype Analysis: Consider linked SNPs as haplotypes rather than individual variants to better capture genetic effects on expression

  • Functional Validation: Use reporter gene assays to confirm the effects of specific variants on gene expression

• Contextual Factors to Consider:

  • Tissue-Specific Effects: CYP7A1 is predominantly expressed in the liver, so expression data from other tissues may not be representative

  • Environmental Interactions: Diet, circadian rhythm, and medication use can all influence CYP7A1 expression

  • Disease State: Conditions like diabetes, metabolic syndrome, and non-alcoholic fatty liver disease can alter CYP7A1 regulation

• Data Analysis Guidelines:

  • Standardize expression data to account for technical variability

  • Include genetic analysis of key regulatory variants when comparing expression between individuals

  • Consider regulatory pathway activity (FXR, LXR, SHP) when interpreting changes in CYP7A1 expression

  • Integrate genomic, transcriptomic, and functional data for comprehensive interpretation

By implementing these strategies, researchers can more accurately interpret CYP7A1 expression data, accounting for the complex interplay between genetic variation, environmental factors, and regulatory pathways that collectively determine enzyme levels and activity.

What cell and tissue models are most appropriate for studying CYP7A1 function and regulation?

When designing experiments to study CYP7A1, researchers should select appropriate models based on their specific research questions:

• Cell Lines:

  • Recommended Hepatocyte Cell Lines: HepG2, HuH-7, L02, and SMMC-7721 cells have been validated for CYP7A1 expression studies

  • Advantages: Easier to manipulate genetically, suitable for high-throughput screening

  • Limitations: Often have altered expression levels compared to primary hepatocytes

  • Application: Ideal for mechanistic studies of transcriptional regulation and protein-protein interactions

• Primary Hepatocytes:

  • Sources: Human, mouse, rat, or hamster hepatocytes have been documented to express CYP7A1

  • Advantages: More physiologically relevant expression patterns and regulation

  • Limitations: Limited lifespan, donor variability, challenging isolation procedures

  • Application: Better representation of in vivo regulation, suitable for acute responses to treatments

• Tissue Samples:

  • Validated Tissues: Human liver tissue and human liver cancer tissue have been confirmed for CYP7A1 expression studies

  • Advantages: Preserves tissue architecture and cell-cell interactions

  • Limitations: Complex cellular composition, potential confounding variables

  • Application: Studies of expression in different pathological states, regional distribution analysis

• Animal Models:

  • Considerations: Species differences exist in CYP7A1 regulation (particularly between rodents and humans)

  • Special Models: Liver regeneration models (70% partial hepatectomy) provide insights into stress-mediated regulation

  • Genetic Models: FXR−/− and SHP−/− mice have been used to elucidate regulatory pathways

  • Application: In vivo studies of physiological regulation and systemic effects

• Organoid Cultures:

  • Emerging Approach: Liver organoids represent a promising intermediate between cell lines and in vivo systems

  • Advantages: Retain 3D architecture while allowing experimental manipulation

  • Application: Long-term studies of regulatory mechanisms with more physiological cell organization

Selection of the appropriate model system should be guided by the specific research question, technical considerations, and the need to balance physiological relevance with experimental control and reproducibility.

What are the critical considerations for genetic association studies involving CYP7A1 variants?

When designing and interpreting genetic association studies involving CYP7A1 variants, researchers should address several critical considerations:

• Multi-SNP Analysis Approach:

  • Evidence Base: Research has demonstrated that individual SNPs may not capture the full genetic effect on CYP7A1 expression

  • Recommendation: Implement a multi-SNP model that accounts for interactions between regulatory variants

  • Example: The 2-SNP model (rs3808607 and rs9297994) shows robust association with clinical outcomes, while individual SNPs may not

• Functional Characterization:

  • Methodology: Use functional assays to validate the effects of identified variants:

    • Reporter gene assays to assess promoter/enhancer activity

    • CRISPR-mediated genome editing to confirm regulatory regions

    • Chromatin conformation capture to identify long-range interactions

  • Rationale: Statistical associations should be supported by functional evidence of biological impact

• Sample Size and Population Considerations:

  • Recommendation: Ensure adequate sample size for detecting modest genetic effects

  • Strategy: Consider population stratification and conduct analyses in different ethnic groups to validate findings

  • Application: Studies like CATHGEN (Catheterization Genetics) and Framingham have been used to validate CYP7A1 genetic associations

• Phenotype Definition:

  • Approach: Include both intermediate phenotypes (CYP7A1 expression, bile acid levels, cholesterol levels) and clinical endpoints (CAD, diabetes)

  • Rationale: Different variants may influence different aspects of CYP7A1 function and downstream effects

• Environmental Interactions:

  • Consideration: CYP7A1 is regulated by diet, medications, and circadian rhythms

  • Strategy: Document and account for these factors in study design and analysis

  • Implementation: Consider stratified analyses based on relevant environmental exposures

• Linkage Disequilibrium Structure:

  • Approach: Characterize the LD structure around CYP7A1 in the study population

  • Implementation: Selected tag SNPs should capture the major haplotype blocks

  • Finding: The enhancer SNP rs9297994 is in high linkage disequilibrium with promoter SNP rs3808607 but has opposite effects on expression