Recombinant Human Proepiregulin (EREG), partial  (Active)

What is recombinant human Epiregulin and what are its key structural features?

Recombinant human Epiregulin (EREG) is a 5.6 kDa monomeric protein belonging to the EGF family of growth factors. The mature form corresponds to amino acid residues Val63-Leu108 of the precursor protein, often with an N-terminal methionine when expressed in E. coli expression systems . Commercial preparations are typically derived from bacterial expression systems and are available in various formulations, including those with carrier proteins and carrier-free versions . The protein contains essential disulfide bonds that maintain its bioactive tertiary structure, which is crucial for receptor binding and downstream signaling activities .

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

Epiregulin functions as a ligand for the EGF receptor (EGFR/ErbB1) and ErbB4, but does not bind to ErbB2 or ErbB3 . It plays critical roles in multiple physiological processes including:

• Stimulation of cell proliferation in keratinocytes, hepatocytes, fibroblasts, and vascular smooth muscle cells

• Regulation of inflammation and tissue repair mechanisms

• Contribution to wound healing through promotion of angiogenesis and vascular remodeling

• Involvement in reproductive biology, particularly in oocyte maturation

• Modulation of epithelial cell function in various tissues

Unlike some growth factors, Epiregulin has also been observed to inhibit the growth of certain tumor-derived epithelial cell lines, suggesting a context-dependent role in cell proliferation regulation .

What are the optimal reconstitution and storage conditions for maintaining EREG activity?

For optimal reconstitution of lyophilized recombinant human Epiregulin:

The product is typically shipped at ambient temperature but should be stored immediately at recommended temperatures upon receipt . Working aliquots should be prepared to minimize freeze-thaw cycles, as repeated freezing and thawing can significantly reduce protein activity.

What concentration ranges of EREG are typically effective in cell-based assays?

The effective dose for Epiregulin varies by cell type and experimental endpoint. For proliferation assays using Balb/3T3 mouse embryonic fibroblast cell lines, the ED50 (effective dose for 50% maximal response) is typically in the range of 0.125-0.75 ng/mL . This concentration range provides a useful starting point for experimental design, though optimization may be necessary for specific cell types or experimental conditions.

When designing dose-response experiments, a recommended approach is to test a logarithmic series of concentrations (e.g., 0.01, 0.1, 1, 10, and 100 ng/mL) to establish the appropriate working range for your specific experimental system .

What is the difference between carrier-free and BSA-containing EREG preparations, and how does this impact experimental design?

The primary differences between carrier-free and BSA-containing EREG preparations are:

How can EREG activity be verified in experimental systems?

To verify EREG activity in your experimental system, consider the following methodological approaches:

• Proliferation assays: Measure cell proliferation in responsive cell lines like Balb/3T3 mouse embryonic fibroblasts, where the ED50 should be approximately 0.125-0.75 ng/mL .

• Receptor phosphorylation: Assess EGFR and ErbB4 tyrosine phosphorylation by Western blotting after EREG stimulation .

• Downstream signaling: Monitor activation of canonical pathways downstream of EGFR/ErbB4, including MAPK/ERK and PI3K/AKT pathways.

• Gene expression analysis: Measure changes in expression of known EREG-responsive genes.

• Biological response validation: For specific research contexts, verify the expected biological outcomes, such as cell migration, angiogenesis, or differentiation.

Comparison to a positive control (such as EGF) and inclusion of appropriate inhibitor controls (e.g., EGFR inhibitors) can provide additional validation of specificity .

How does EREG signaling differ from other EGF family ligands, and what implications does this have for experimental design?

Epiregulin demonstrates several unique signaling characteristics compared to other EGF family ligands:

• Receptor specificity: While Epiregulin binds specifically to EGFR (ErbB1) and ErbB4, it does not bind ErbB2 or ErbB3 . This differs from ligands like Neuregulin-1, which predominantly signals through ErbB3 and ErbB4.

• Receptor activation patterns: Epiregulin can activate homodimers of both ErbB1 and ErbB4, as well as all possible heteromeric combinations of the four ErbB family members .

• Bimodal activity: Unlike most EGF family members, Epiregulin has been observed to inhibit the growth of certain epithelial tumor cell lines while stimulating others, suggesting unique signaling outcomes .

• Binding affinity: Epiregulin typically demonstrates lower binding affinity to EGFR compared to EGF, potentially resulting in different signaling kinetics and dynamics.

For experimental design, these differences suggest that:

• When comparing EGF family ligands, equivalent molar concentrations rather than mass-based concentrations should be used

• Experiments should include appropriate receptor expression profiling in the cell systems used

• Time-course studies may reveal important differences in signaling kinetics between EREG and other family members

• Downstream pathway analyses should consider both canonical and non-canonical signaling outputs

What are the key methodological approaches for studying EREG's role in tumor microenvironments?

Investigating Epiregulin's role in tumor microenvironments requires multi-faceted experimental approaches:

• Expression analysis in clinical samples:

  • Immunohistochemistry to localize EREG protein in tumor tissue

  • RNA-seq or qPCR to quantify EREG mRNA levels

  • Single-cell analysis to identify specific cell populations expressing EREG

• Functional studies in co-culture systems:

  • Use of transwell or direct co-culture models combining tumor cells with stromal components

  • Selective inhibition of EREG signaling using neutralizing antibodies or genetic approaches

  • Assessment of paracrine effects on proliferation, migration, and invasion

• In vivo models:

  • Xenograft models with EREG-overexpressing or EREG-knockout cells

  • Orthotopic models that better recapitulate tissue-specific microenvironments

  • Use of antibody-drug conjugates targeting EREG for therapeutic assessment

• Mechanistic investigations:

  • Analysis of EREG-induced changes in tumor-associated macrophages or fibroblasts

  • Assessment of angiogenic responses through endothelial cell assays

  • Evaluation of immunomodulatory effects using immune cell functional assays

Several published studies have employed these approaches to demonstrate EREG's role in colorectal, bladder, lung, and kidney carcinomas .

What are common challenges in EREG-based experiments and how can they be addressed?

Additional quality control measures should include:

• SDS-PAGE analysis to confirm protein integrity (should show a single band at approximately 5-6 kDa)

• Endotoxin testing for sensitive applications

• Biological activity assessment in a well-characterized assay system

How can researchers distinguish between EREG-specific effects and other EGF family ligand effects in experimental systems?

To distinguish EREG-specific effects from those of other EGF family ligands:

• Receptor profiling and manipulation:

  • Characterize the expression profile of ErbB receptors in your experimental system

  • Use receptor-specific siRNAs or CRISPR-based approaches to selectively modulate individual receptors

  • Employ receptor-selective inhibitors when available

• Comparative studies:

  • Perform parallel experiments with multiple EGF family ligands at equivalent molar concentrations

  • Analyze temporal dynamics of signaling activation, as different ligands may induce different kinetic profiles

  • Examine differences in biological outcomes and dose-response relationships

• Molecular approaches:

  • Utilize EREG-specific neutralizing antibodies

  • Generate EREG knockout cell lines using CRISPR/Cas9 technologies

  • Employ receptor mutants with altered binding specificity

• Downstream signaling analysis:

  • Comprehensive phosphoproteomic analysis to identify EREG-specific signaling nodes

  • Transcriptomic profiling to identify EREG-specific gene expression signatures

  • Analysis of receptor trafficking and degradation patterns

Studies have demonstrated that combining these approaches can effectively separate EREG-specific signaling from that of other family members, revealing unique biological functions in contexts such as wound healing and tumor progression .

How can EREG be utilized in cancer research models and what methodological considerations are important?

Epiregulin has emerged as an important factor in various cancer types, with several methodological approaches proving valuable:

• Expression correlation studies:

  • Analysis of EREG expression in patient samples correlated with clinical outcomes

  • Investigation of EREG as a biomarker for therapy response, particularly for EGFR-targeted therapies

  • Meta-analysis of public cancer genomics datasets for EREG expression patterns

• Functional studies in cancer models:

  • EREG overexpression or knockdown in cancer cell lines to assess effects on proliferation, migration, and invasion

  • Use of recombinant EREG to stimulate cancer cells in combination with therapeutic agents to assess modulation of drug sensitivity

  • Investigation of EREG-blocking antibodies as potential therapeutic strategies

• Targeting approaches:

  • Development of antibody-drug conjugates targeting EREG for selective delivery of cytotoxic agents to cancer cells

  • Testing of combination approaches with established therapies

  • Evaluation of EREG as an immunotherapy target

Research has shown particularly promising applications in colorectal cancer, where antibody-drug conjugates targeting EREG have demonstrated robust anti-tumor activity . Additionally, EREG has been implicated in Head and Neck Squamous Cell Carcinoma, where its expression correlates with sensitivity to EGFR inhibitors like Erlotinib .

What are the methodological approaches for studying EREG's role in inflammatory and wound healing processes?

To investigate Epiregulin's functions in inflammation and wound healing:

• In vitro wound healing models:

  • Scratch assays with primary keratinocytes or fibroblasts with EREG treatment

  • Transwell migration assays to assess cellular motility responses

  • 3D organotypic culture systems to evaluate tissue architecture and repair mechanisms

• Inflammation models:

  • Macrophage polarization studies in response to EREG stimulation

  • Assessment of inflammatory cytokine production in relevant cell types

  • Neutrophil recruitment and activation analyses

• In vivo approaches:

  • Cutaneous wound healing models in wild-type and EREG-deficient animals

  • Inflammatory disease models (e.g., colitis, arthritis) with EREG neutralization or supplementation

  • Tissue-specific conditional knockout models to dissect cell type-specific contributions

• Mechanistic investigations:

  • Analysis of angiogenesis markers and vascular remodeling in response to EREG

  • Assessment of extracellular matrix production and remodeling

  • Investigation of epithelial-mesenchymal interactions during the repair process

These methodological approaches have revealed that EREG contributes significantly to wound healing by regulating angiogenesis and vascular remodeling while stimulating cell proliferation essential for tissue repair .