ANXA8L1 Human

What is the basic structure of human Annexin A8/ANXA8L1?

Human Annexin A8 (ANXA8), also known as VAC-beta, is an approximately 37 kDa member of the Annexin superfamily of proteins. The protein consists of 327 amino acids (from Ala2-Pro327) and is encoded by the gene with accession number P13928 . ANXA8L1 (Annexin A8-like 1) is a variant that shares significant homology with ANXA8. Like other annexins, ANXA8 likely contains a core domain with four annexin repeats that mediate calcium-dependent binding to phospholipids. The protein's structure enables its function in membrane-related processes including endosome formation and membrane trafficking.

What are the primary cellular functions of ANXA8/ANXA8L1?

ANXA8 functions as a regulator of several cellular processes, including:

• Cell cycle regulation: ANXA8 expression is associated with quiescence in epithelial cells, with studies showing that ANXA8-positive cells in the mammary gland are mostly quiescent (Ki67-negative) .

• Membrane trafficking: ANXA8 has been linked to the formation of endosomes and epidermal growth factor receptor (EGFR) turnover in cellular models .

• Wnt signaling regulation: Research demonstrates that ANXA8 physically interacts with β-catenin and impacts the canonical Wnt signaling pathway, which is crucial for cell fate determination and differentiation .

• Cell surface protein presentation: Studies indicate ANXA8 is required for efficient cell surface presentation of certain proteins like CD63 and P-selectin in endothelial cells .

• Cell differentiation: ANXA8 appears to maintain certain cell phenotypes, as its downregulation can induce transdifferentiation in retinal pigment epithelium (RPE) cells .

What are the optimal methods for detecting ANXA8 protein expression in tissue samples?

For optimal detection of ANXA8 protein in tissue samples, researchers should consider the following methodologies:

• Immunohistochemistry (IHC): For paraffin-embedded tissue sections, use antigen retrieval with heat-induced epitope retrieval techniques. Based on the research protocol, anti-ANXA8 antibodies (such as Sheep Anti-Human ANXA8 Antigen Affinity-purified Polyclonal Antibody) can be used at concentrations of 1 μg/mL overnight at 4°C . Visualization can be achieved using appropriate secondary antibodies and detection systems such as HRP-DAB staining with hematoxylin counterstaining.

• Western blot: For protein lysates, use PVDF membranes probed with anti-ANXA8 antibodies (2 μg/mL concentration has shown good results with sheep anti-human ANXA8 antibody) . ANXA8 will be detected at approximately 36-37 kDa. Reducing conditions with appropriate immunoblot buffers are recommended for optimal results.

• Immunofluorescence: Double immunofluorescence staining is particularly useful for co-localization studies, such as examining ANXA8 expression in relation to other markers like ERα, Ki67, or c-kit .

When optimizing these protocols, it's essential to include appropriate positive controls (such as A549 human lung carcinoma cell line or human placenta samples) and negative controls to validate specificity.

How can researchers effectively manipulate ANXA8 expression in experimental models?

To effectively manipulate ANXA8 expression in experimental models, researchers can employ several approaches:

• RNA interference: siRNA targeting ANXA8 has been successfully used to suppress ANXA8 expression in cell lines such as ARPE19 cells. This approach allows for transient knockdown of ANXA8 to study resulting phenotypic changes .

• Overexpression systems: Transfection with ANXA8-GFP constructs has been used to study the effects of ANXA8 overexpression on Wnt signaling components and cell morphology . This approach is valuable for gain-of-function studies.

• CRISPR/Cas9 genome editing: For stable knockout models, CRISPR/Cas9 systems can be designed to target ANXA8 gene sequences, though specific protocols for ANXA8 editing were not detailed in the provided research material.

• Inducible expression systems: For temporal control of ANXA8 expression, inducible promoter systems could be employed, allowing researchers to study the effects of ANXA8 expression at specific time points.

When manipulating ANXA8 expression, it's crucial to validate the effectiveness of intervention using qRT-PCR for mRNA levels and Western blot or immunofluorescence for protein expression. The research shows that both siRNA knockdown and chemical treatments (such as FR) can effectively suppress ANXA8 expression, leading to observable phenotypic changes that can be analyzed .

How does ANXA8 interact with the Wnt/β-catenin signaling pathway?

ANXA8 plays a significant regulatory role in the Wnt/β-catenin signaling pathway through several mechanisms:

• Physical interaction with β-catenin: Co-immunoprecipitation experiments have demonstrated that ANXA8 physically associates with both total β-catenin and active β-catenin in epithelial cells . This interaction appears to be regulated by TGF-β1 signaling, as TGF-β1 treatment disrupts the ANXA8-β-catenin interaction, while blocking TGF-β1 signaling with the Alk5 inhibitor SB431542 restores it .

• Impact on β-catenin stability and localization: ANXA8 knockdown leads to decreased levels of both total and active β-catenin, with particularly marked reductions in nuclear β-catenin . This suggests that ANXA8 may protect β-catenin from degradation and/or promote its nuclear translocation.

• Regulation of GSK-3β: ANXA8 suppression increases GSK-3β expression, while overexpression of ANXA8 leads to increased GSK-3β phosphorylation . Since phosphorylated GSK-3β is less active in promoting β-catenin degradation, this suggests that ANXA8 may stabilize β-catenin partly through inhibiting GSK-3β activity.

• Effect on Wnt-related gene expression: Depletion of ANXA8 leads to decreased expression of Wnt ligands (Wnt2b, Wnt3a) and receptors (Frizzled-1, Frizzled-4), indicating that ANXA8 may also regulate the Wnt pathway at the transcriptional level .

These interactions position ANXA8 as an important modulator of Wnt signaling, with potential implications for cellular differentiation, proliferation, and tissue homeostasis.

What is the role of ANXA8 in mammary gland development and epithelial cell differentiation?

ANXA8 plays a specific role in mammary gland development and epithelial cell differentiation:

These findings suggest that ANXA8 functions as a mediator of quiescence in specific mammary epithelial progenitor populations, potentially maintaining a reserve of progenitor cells that can be activated during specific developmental stages.

How is ANXA8 implicated in breast cancer development and progression?

ANXA8 has significant implications in breast cancer, particularly in the basal-like subtype:

• Subtype-specific expression: ANXA8 is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers . It is not detected in the majority of breast cancers but in a distinct subset of CK5-positive, estrogen receptor (ER)α and progesterone receptor (PgR) negative breast cancers with poor prognosis .

• Prognostic significance: ANXA8 expression correlates with poor prognosis in breast cancer patients, as initially identified by microarray analysis by Perou et al. and confirmed at the protein level in subsequent studies .

• Link to cells of origin: ANXA8 is expressed in c-kit-positive/ERα-negative ductal luminal epithelial progenitor cells, which have been identified as the likely cells of origin for basal-like breast cancers . This suggests that ANXA8's expression in basal-like breast cancers may reflect the cellular origin of these tumors.

• Potential diagnostic utility: Given its association with a specific breast cancer subtype, ANXA8 may serve as a useful diagnostic marker for identifying basal-like breast cancers, particularly in combination with other markers .

Understanding the role of ANXA8 in breast cancer may provide insights into the biology of basal-like breast cancers and potentially identify new therapeutic targets or diagnostic approaches for this aggressive breast cancer subtype.

What other malignancies show altered ANXA8 expression, and what are the potential mechanisms?

ANXA8 expression alterations have been documented in multiple malignancies beyond breast cancer:

• Acute promyelocytic leukemia (APL): ANXA8 was first identified as being specifically overexpressed in APL, where it was found to be repressible by all-trans retinoic acid (ATRA) . This suggests a potential role in myeloid differentiation.

• Pancreatic cancer: Deregulation of ANXA8 has been observed in infiltrating adenocarcinomas of the pancreas .

• Cholangiocarcinoma: Altered ANXA8 expression has been documented in this biliary tract malignancy .

• Malignant pleural mesothelioma: ANXA8 expression changes have been noted in this aggressive thoracic malignancy .

• Melanoma: Studies have identified ANXA8 deregulation in melanoma .

• Squamous carcinoma of the uterine cervix: ANXA8 alterations have been observed in cervical cancer .

• Esophageal adenocarcinoma and Barrett's metaplasia: Both these conditions show changes in ANXA8 expression .

Potential mechanisms underlying ANXA8's role in cancer development include:

• Cell cycle regulation: ANXA8's role in maintaining cellular quiescence may be disrupted in cancer, potentially contributing to aberrant proliferation or, conversely, to cancer cell dormancy.

• Wnt signaling modulation: As ANXA8 interacts with β-catenin and influences Wnt signaling , disruption of this interaction could impact cell differentiation and proliferation.

• p53-mediated DNA damage response: ANXA8 has been identified as a target for p53-activated DNA damage response after treatment with adriamycin/doxorubicin or when p53 was overexpressed , suggesting a potential role in the cellular response to genotoxic stress.

• Membrane trafficking and receptor turnover: ANXA8's involvement in endosome formation and EGFR turnover might influence receptor tyrosine kinase signaling in cancer cells.

These findings highlight the diverse roles of ANXA8 across multiple cancer types and suggest that its functions may be context-dependent.

How can researchers effectively investigate the ANXA8-β-catenin interaction in different cellular contexts?

To effectively investigate the ANXA8-β-catenin interaction across different cellular contexts, researchers should consider the following approaches:

• Co-immunoprecipitation (Co-IP) with pathway modulation: As demonstrated in previous research, Co-IP can effectively detect the physical association between ANXA8 and β-catenin . Researchers should consider performing Co-IP under various conditions, including:

  • Treatment with Wnt pathway activators (e.g., Wnt3a)

  • Treatment with Wnt pathway inhibitors (e.g., DKK-1)

  • Modulation of TGF-β signaling (TGF-β1 treatment and/or Alk5 inhibitors like SB431542)

  • Different calcium concentrations, given ANXA8's calcium-binding properties

• Protein fractionation and localization studies: Since ANXA8 appears to influence β-catenin localization, researchers should examine cytosolic versus nuclear pools of both proteins using cellular fractionation followed by Western blotting . This should be complemented with immunofluorescence studies to visualize the co-localization patterns under different experimental conditions.

• Proximity ligation assays (PLA): This technique can detect protein-protein interactions in situ with high sensitivity and provide spatial information about where in the cell these interactions occur.

• FRET or BRET analysis: Fluorescence or bioluminescence resonance energy transfer techniques can provide information about the dynamics of ANXA8-β-catenin interactions in living cells.

• Domain mapping: Using truncation mutants of both ANXA8 and β-catenin can help identify the specific domains involved in their interaction.

• Functional readouts: Researchers should correlate physical interactions with functional outcomes by simultaneously monitoring:

  • β-catenin transcriptional activity using TOPFlash/FOPFlash reporter assays

  • Expression of canonical Wnt target genes (e.g., c-myc, Axin1, Lef1)

  • Cell phenotype changes (morphology, proliferation, differentiation)

• Cross-tissue comparison: Given that ANXA8 has been studied in mammary epithelial cells and retinal pigment epithelium , comparing the interaction characteristics across different tissue types could reveal context-dependent aspects of the interaction.

These approaches, used in combination, will provide a comprehensive understanding of how the ANXA8-β-catenin interaction is regulated and functions in different cellular contexts.

What methodological approaches are most effective for studying ANXA8's role in cell fate determination and transdifferentiation?

To effectively study ANXA8's role in cell fate determination and transdifferentiation, researchers should implement a multi-faceted approach:

• Temporal manipulation of ANXA8 expression:

  • Inducible knockdown or overexpression systems to control the timing of ANXA8 modulation

  • Time course analyses to track progressive changes in cell phenotype and molecular markers

  • Pulse-chase experiments to determine if transient versus sustained ANXA8 modulation has different effects on cell fate

• Single-cell analysis techniques:

  • Single-cell RNA sequencing to identify transcriptional trajectories during transdifferentiation

  • Mass cytometry (CyTOF) to simultaneously measure multiple protein markers at the single-cell level

  • Live-cell imaging of fluorescently tagged ANXA8 to track its dynamics during cell fate changes

• Lineage tracing methodologies:

  • In vivo lineage tracing of ANXA8-expressing cells using genetic reporters

  • Ex vivo organoid cultures derived from lineage-labeled cells

  • Cellular barcoding to track clonal populations during differentiation processes

• Pathway perturbation analysis:

  • Systematic modulation of signaling pathways (Wnt, TGF-β, etc.) in combination with ANXA8 manipulation

  • Chemical genetic screens to identify factors that enhance or suppress ANXA8-dependent phenotypes

  • Pathway-specific reporter systems to monitor signaling activities during transdifferentiation

• Epigenetic profiling:

  • ATAC-seq to assess chromatin accessibility changes associated with ANXA8 modulation

  • ChIP-seq to identify potential transcription factor binding sites affected by ANXA8

  • DNA methylation analysis to track epigenetic reprogramming during transdifferentiation

• Functional validation in relevant systems:

  • For retinal studies: 3D retinal organoids or explant cultures

  • For mammary gland studies: Mammary epithelial organoids or intraductal transplantation

  • Xenograft models to assess in vivo transdifferentiation potential

• Multi-omics integration:

  • Integrating transcriptomic, proteomic, and epigenomic data to build comprehensive models of ANXA8-mediated cell fate changes

  • Network analysis to identify key nodes and feedback loops in ANXA8-regulated processes

The research suggests specific cellular models where these approaches would be particularly relevant:

• ARPE19 cells for studying RPE-to-neuronal transdifferentiation

• Primary RPE cells as a more physiologically relevant system

• Kim-2 mammary epithelial cells for studying effects on mammary progenitor cell fate

• FACS-sorted mammary epithelial cell populations for targeted analysis of specific progenitor subtypes

By combining these approaches, researchers can develop a comprehensive understanding of how ANXA8 influences cell fate decisions and regulates transdifferentiation processes in different tissue contexts.

What are the key considerations for antibody selection and validation when studying ANXA8/ANXA8L1?

For effective research on ANXA8/ANXA8L1, careful antibody selection and validation are critical:

• Antibody specificity considerations:

  • Distinguish between ANXA8 and ANXA8L1: Researchers must verify whether antibodies can distinguish between ANXA8 and its closely related family member ANXA8L1, as these proteins share significant sequence homology .

  • Cross-reactivity with other annexins: Validate that antibodies do not cross-react with other members of the annexin family, which share structural similarities.

  • Species specificity: Confirm antibody reactivity across species if conducting comparative studies, as ANXA8L1 has been identified in multiple species including human, horse, cow, and dog .

• Validation techniques:

  • Western blot validation: Verify antibody specificity by Western blot, confirming a single band at the expected molecular weight (approximately 36-37 kDa for ANXA8) .

  • Positive controls: Include known ANXA8-expressing cells or tissues such as A549 human lung carcinoma cell line or placental tissue .

  • Knockout/knockdown controls: Use ANXA8 siRNA-treated samples as negative controls to confirm specificity .

  • Pre-absorption tests: Pre-incubate antibodies with purified ANXA8 protein to demonstrate binding specificity.

• Application-specific considerations:

  • For Western blot: Optimal concentrations around 2 μg/mL have been reported for sheep anti-human ANXA8 antibodies, with reducing conditions recommended .

  • For immunohistochemistry: Heat-induced epitope retrieval methods are necessary for paraffin sections, with antibody concentrations of approximately 1 μg/mL .

  • For immunofluorescence: Fixation methods can affect epitope accessibility; validate antibodies for specific fixation protocols.

  • For co-immunoprecipitation: Test antibodies specifically for their ability to immunoprecipitate native protein complexes without disrupting protein-protein interactions .

• Reproducibility factors:

  • Lot-to-lot variation: Monitor and document antibody lot numbers, as performance can vary between manufacturing batches.

  • Storage conditions: Follow manufacturer recommendations for antibody storage to maintain activity.

  • Documentation: Maintain detailed records of validation experiments to ensure reproducibility across studies.

Proper antibody selection and validation are essential for generating reliable, reproducible research findings on ANXA8/ANXA8L1 expression and function.

How can researchers distinguish between the functions of ANXA8 and other annexin family members in experimental systems?

Distinguishing between the functions of ANXA8 and other annexin family members requires specific experimental approaches:

• Gene-specific targeting strategies:

  • Design highly specific siRNAs or shRNAs that target unique regions of ANXA8 mRNA not shared with other annexin family members

  • Utilize CRISPR/Cas9 genome editing with guide RNAs targeting ANXA8-specific sequences

  • Verify knockdown/knockout specificity by measuring mRNA and protein levels of multiple annexin family members

• Rescue experiments:

  • After ANXA8 knockdown, perform rescue experiments with wild-type ANXA8 or mutant versions

  • Compare with rescue using other annexin family members to identify ANXA8-specific functions

  • Use expression constructs with mutations in key functional domains to identify critical regions

• Protein interaction profiling:

  • Perform immunoprecipitation followed by mass spectrometry to identify ANXA8-specific binding partners

  • Compare ANXA8 interactome with those of other annexin family members

  • Focus on unique interactions, such as ANXA8's specific association with β-catenin

• Domain swap experiments:

  • Create chimeric proteins containing domains from ANXA8 and other annexin family members

  • Test these chimeras in functional assays to map domain-specific functions

  • Identify which domains are responsible for ANXA8's unique effects on cellular processes

• Temporal and spatial expression analysis:

  • Map the expression patterns of multiple annexin family members under various conditions

  • Identify conditions where ANXA8 expression diverges from other family members

  • For example, focus on c-kit-positive/ERα-negative ductal luminal epithelial cells where ANXA8 has specific expression patterns

• Pathway-specific functional assays:

  • Develop assays specific to ANXA8's known functions, such as its effects on Wnt signaling

  • Compare effects of multiple annexin family members on these pathways

  • Identify annexin-specific effects on downstream targets and cellular phenotypes

• Context-dependent analysis:

  • Examine function across multiple cell types, as ANXA8 may have tissue-specific roles

  • Compare effects in transdifferentiation models, such as RPE cells and mammary epithelial cells

  • Identify cell types where ANXA8 functions are prominent compared to other annexins

By systematically applying these approaches, researchers can distinguish ANXA8-specific functions from roles shared with other annexin family members, providing a clearer understanding of ANXA8's unique contributions to cellular processes.

What are the most promising areas for future research on ANXA8's role in cellular plasticity and tissue homeostasis?

Several promising areas for future ANXA8 research emerge from current findings:

• ANXA8 in stem cell quiescence and activation:

  • Investigate whether ANXA8 functions as a general regulator of progenitor cell quiescence across multiple tissues

  • Study the molecular mechanisms by which ANXA8-positive cells exit quiescence during specific developmental or regenerative processes

  • Examine whether ANXA8 regulates the balance between symmetric and asymmetric cell divisions in progenitor populations

• ANXA8 in the integration of signaling networks:

  • Explore how ANXA8 coordinates Wnt and TGF-β signaling pathways

  • Investigate potential crosstalk with other key developmental pathways (Notch, Hedgehog, Hippo)

  • Develop systems biology approaches to model how ANXA8 functions as a node in cellular signaling networks

• ANXA8 in cellular reprogramming and regenerative medicine:

  • Determine whether modulation of ANXA8 can enhance cellular reprogramming efficiency

  • Explore ANXA8's potential as a target for promoting tissue regeneration

  • Investigate its role in maintaining epithelial identity and preventing inappropriate transdifferentiation

• ANXA8 in tissue-specific regulation:

  • Compare ANXA8 functions across additional epithelial tissues beyond mammary gland and retinal pigment epithelium

  • Identify tissue-specific binding partners that modify ANXA8 function

  • Develop tissue-specific ANXA8 knockout models to assess organ-specific phenotypes

• ANXA8 in disease contexts beyond cancer:

  • Investigate ANXA8's potential roles in fibrosis, which involves aberrant TGF-β signaling

  • Explore functions in degenerative conditions affecting epithelial tissues

  • Study potential roles in inflammatory processes, given annexins' membrane-binding properties

• Structural biology of ANXA8-protein interactions:

  • Determine the crystal structure of ANXA8 in complex with key binding partners like β-catenin

  • Identify critical interaction domains that could be targeted pharmacologically

  • Compare structural features with other annexin family members to understand functional specificity

• ANXA8 in aging and senescence:

  • Investigate whether ANXA8 expression changes during cellular and tissue aging

  • Explore potential roles in maintaining long-term quiescent stem cell populations throughout lifespan

  • Study relationships between ANXA8, cellular senescence pathways, and tissue homeostasis

These research directions build on current knowledge of ANXA8's roles in epithelial cell quiescence, Wnt signaling regulation, and cell fate determination while expanding into new territories with potential implications for regenerative medicine and disease treatment.

What technological advances would most benefit ANXA8 research in the coming years?

Several technological advances would significantly advance ANXA8 research:

• Advanced imaging technologies:

  • Super-resolution microscopy to visualize ANXA8 distribution at subcellular membrane domains

  • Live-cell imaging with ANXA8 biosensors to track real-time dynamics during signaling events

  • Correlative light and electron microscopy to determine precise membrane localization of ANXA8

  • Intravital imaging to observe ANXA8-expressing cells in their native tissue environment

• Single-cell multi-omics approaches:

  • Integrated single-cell RNA-seq and ATAC-seq to correlate ANXA8 expression with chromatin states

  • Spatial transcriptomics to map ANXA8 expression in relation to tissue architecture

  • Single-cell proteomics to detect low-abundance ANXA8 protein variants and post-translational modifications

  • Single-cell metabolomics to link ANXA8 expression with metabolic states

• Protein interaction and structural biology tools:

  • Proximity labeling methods (BioID, APEX) to map ANXA8's dynamic protein interaction network

  • Hydrogen-deuterium exchange mass spectrometry to identify conformational changes upon binding

  • Cryo-EM techniques to determine structures of ANXA8 in complex with membrane components

  • AlphaFold and other AI-based structure prediction tools to model ANXA8 interactions

• Genome editing and genetic screening technologies:

  • CRISPR base editing and prime editing for precise modification of ANXA8 coding sequences

  • CRISPR activation/inhibition screens to identify genes that modify ANXA8 function

  • CRISPR knockin of endogenous tags to study ANXA8 at physiological expression levels

  • Genetic screens in primary cell types relevant to ANXA8 function

• 3D tissue models and organoids:

  • Development of patient-derived organoids to study ANXA8 in human disease contexts

  • Bioprinted tissue models with controlled ANXA8 expression in specific cell populations

  • Organ-on-chip systems to study ANXA8 functions under physiological flow conditions

  • Co-culture systems to examine ANXA8's role in epithelial-stromal interactions

• In vivo monitoring and manipulation:

  • Optogenetic or chemogenetic tools to manipulate ANXA8 function with temporal precision

  • In vivo biosensors to monitor ANXA8-dependent signaling pathways in real-time

  • AAV-based approaches for tissue-specific ANXA8 modulation in adult animals

  • Non-invasive imaging techniques to track ANXA8-expressing cells longitudinally

• Computational and systems biology approaches:

  • Machine learning algorithms to identify patterns in ANXA8 expression across tissues and diseases

  • Network analysis tools to position ANXA8 within cellular signaling networks

  • Multi-scale modeling to link molecular interactions to tissue-level phenotypes

  • Predictive models for pharmacological targeting of ANXA8-dependent processes

These technological advances would enable researchers to address fundamental questions about ANXA8's dynamic behavior, context-specific functions, and roles in complex tissue environments, potentially leading to novel therapeutic approaches targeting ANXA8-dependent processes.