Recombinant Human Fibroblast growth factor 4 protein (FGF4), partial (Active)

What is the structure and basic characteristics of recombinant human FGF4?

Recombinant human FGF4 is a 25 kDa secreted, heparin-binding member of the fibroblast growth factor family. The human FGF4 cDNA encodes 206 amino acids with a 33 amino acid signal sequence and a 173 amino acid mature protein. The mature protein contains an FGF homology domain with a heparin binding region near the C-terminus. The commercially available recombinant form typically spans from Ser54 to Leu206 of the native sequence and is often produced in E. coli expression systems .

Recombinant human FGF4 shares high sequence homology with other mammalian species:

• 91% amino acid identity with mouse FGF4

• 82% amino acid identity with rat FGF4

• 94% amino acid identity with canine FGF4

• 91% amino acid identity with bovine FGF4

This high conservation enables cross-species activity in experimental systems .

What receptors does FGF4 interact with and how does this influence experimental design?

FGF4 primarily signals through FGF receptors FGF R1c, 2c, 3c, and 4. When designing experiments to study FGF4 function, it is essential to verify the expression of these receptors in your experimental system. If studying a specific receptor-mediated effect, consider using receptor-specific inhibitors or cell lines with defined receptor expression profiles to isolate signaling pathways .

For proper experimental design, verify receptor expression via RT-PCR or Western blotting before initiating FGF4 treatment studies. The spatiotemporal expression of these receptors is developmentally regulated, so developmental stage and tissue type should be carefully considered when designing experiments .

How should recombinant human FGF4 be reconstituted and stored for optimal activity?

Reconstitution Protocol:

• For carrier-containing preparations: Reconstitute lyophilized protein at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin.

• For carrier-free preparations: Reconstitute at 100 μg/mL in sterile PBS.

Storage Recommendations:

• Store reconstituted protein in working aliquots at -20°C or -80°C.

• Avoid repeated freeze-thaw cycles which can significantly reduce protein activity.

• Use a manual defrost freezer rather than automatic defrosting units.

When establishing dose-response relationships, note that the effective dose for stimulating cell proliferation in NR6R-3T3 mouse fibroblast cells (ED50) is typically 0.25-1.25 ng/mL. This information is essential for determining appropriate concentration ranges in your experiments .

How does FGF4 regulate macrophage polarization in inflammatory conditions?

Recent research demonstrates that FGF4 serves as a novel regulator of macrophage phenotype switching, particularly in autoimmune hepatitis (AIH). FGF4 reduces M1 macrophage polarization while potentially promoting M2 phenotypes, suggesting a therapeutic role in inflammatory conditions .

Mechanism of Action:

• FGF4 activates the phosphatidylinositol 3-kinase (PI3K)–protein kinase B (AKT) signaling pathway in macrophages.

• This activation leads to reduced M1 macrophage levels.

• Consequently, hepatic inflammation is mitigated through immunomodulation.

In experimental models using concanavalin A (ConA)-induced AIH, both genetic approaches (hepatocyte-specific Fgf4 knockout mice) and pharmacological administration of FGF4 have confirmed this regulatory role. Notably, macrophage depletion compromises the therapeutic efficacy of FGF4, confirming macrophages as the primary cellular mediators of FGF4's anti-inflammatory effects .

For researchers studying inflammatory conditions, these findings suggest FGF4 as a potential therapeutic target worthy of further investigation. When designing macrophage polarization experiments, consider including PI3K inhibitors (such as LY294002 at 25 μM) as controls to demonstrate pathway specificity .

What experimental approaches can detect and quantify FGF4 activity in biological samples?

Functional Assays:

• Proliferation Assays: The gold standard for measuring FGF4 bioactivity is the NR6R-3T3 mouse fibroblast proliferation assay. A typical ED50 for this effect is 0.25-1.25 ng/mL. Quantify cell numbers using MTT, crystal violet, or automated cell counting methods .

• Phosphorylation Analysis: Measure activation of downstream signaling molecules (particularly phospho-AKT) via Western blotting following FGF4 stimulation. This approach is particularly relevant when studying the PI3K-AKT pathway .

• Macrophage Polarization Assessment: Analyze M1/M2 marker expression by flow cytometry, qPCR, or immunofluorescence following FGF4 treatment. This is especially relevant for inflammatory disease models .

Controls to Include:

• Positive control: Commercial FGF4 with known activity

• Negative control: Heat-inactivated FGF4

• Signaling pathway controls: Include PI3K inhibitors (e.g., LY294002) to confirm mechanism specificity

How is FGF4 expression dysregulated in pathological conditions, and what are the implications for experimental model selection?

Research on gastrointestinal stromal tumors (GISTs) has revealed distinct mechanisms of FGF4 dysregulation in different tumor subtypes:

• In SDH-deficient GISTs: FGF4 overexpression results from methylation of an FGF insulator region, representing an epigenetic mechanism of dysregulation.

• In quadruple WT GISTs (KIT/PDGFRA/SDH/RAS-P WT): FGF4 overexpression is driven by genomic structural changes, specifically recurrent focal duplication of the FGF3/FGF4 locus. The copy of FGF4 positioned closer to the ANO1 super-enhancer is preferentially expressed .

These findings highlight the importance of characterizing the molecular mechanisms of FGF4 dysregulation in your experimental model. When studying FGF4 in disease contexts, researchers should:

• Assess both epigenetic and genomic structural alterations

• Consider gene copy number variations

• Examine potential enhancer interactions affecting gene expression

This research also suggests that therapeutic approaches targeting the FGFR pathway might be effective across different GIST subtypes despite distinct mechanisms of FGF4 dysregulation .

What are the critical variables to control when designing experiments involving FGF4?

When designing experiments to investigate FGF4 function, consider these key variables:

Independent Variables:

• FGF4 concentration (typically 0-10 μg/mL, with 0.25-1.25 ng/mL being effective for proliferation assays)

• Treatment duration

• Cell type/tissue context

• Co-treatment with pathway inhibitors

• Genetic background (wild-type vs. knockout)

Dependent Variables:

• Cell proliferation

• Signaling pathway activation

• Gene expression changes

• Phenotypic outcomes (e.g., macrophage polarization)

Control for Confounding Variables:

• Serum factors (use serum starvation before FGF4 treatment)

• Endogenous FGF4 expression

• Expression levels of FGF receptors

• Cell density and passage number

For optimal experimental rigor, include appropriate randomization procedures and ensure adequate sample sizes for statistical power. If using complex experimental designs with multiple factors, consider factorial designs to efficiently test interactions between variables .

How should researchers design knockout or knockdown experiments to study FGF4 function?

When investigating FGF4 function through gene manipulation approaches:

For Tissue-Specific Knockout:

• Use Cre-loxP systems targeting Fgf4 as demonstrated in hepatocyte-specific knockout models (Fgf4−/−).

• Include Fgf4-floxed mice (Fgf4 fl/fl) as essential controls.

• Confirm knockout efficiency through gene expression analysis (qPCR) and protein detection (Western blot, ELISA).

For In Vitro Knockdown:

• Design siRNA or shRNA targeting conserved regions of FGF4 transcript.

• Validate knockdown efficiency at both mRNA and protein levels.

• Include scrambled siRNA controls.

• Consider rescue experiments with recombinant FGF4 protein to confirm specificity.

For knockdown experiments in cell culture systems, studies have successfully used both transient transfection approaches and stable lentiviral delivery systems, with the latter offering advantages for long-term studies .

What cell culture systems are most appropriate for studying FGF4 functions?

The selection of appropriate cellular models is critical for FGF4 research:

Recommended Cell Lines:

• NR6R-3T3 mouse fibroblasts: Standard cell line for FGF4 bioactivity assays with well-characterized response profiles.

• Macrophage cell lines: For studying inflammatory responses and polarization (RAW264.7, J774A.1).

• Primary bone marrow-derived macrophages: Offer more physiologically relevant responses than immortalized cell lines.

• AML12 hepatocytes: Useful for studying liver-specific FGF4 functions.

• Embryonic stem cells: Ideal for studying FGF4's role in pluripotency and self-renewal.

Culture Conditions for FGF4 Studies:

• Serum starvation (0.1-0.5% serum) for 12-24 hours before FGF4 treatment to reduce background signaling.

• For macrophage polarization studies, pre-treatment with FGF4 (optimally 0-10 μg/mL) for 30 minutes before LPS stimulation.

• For co-culture experiments, ensure at least three independent replicates to account for variability.

When studying inflammatory conditions, LPS at concentrations of 0-10 μg/mL with 24-hour incubation is commonly used to induce M1 polarization before assessing FGF4 effects .

How can inconsistent results with recombinant FGF4 be addressed in experimental settings?

Common Issues and Solutions:

• Loss of FGF4 Activity:

  • Verify protein integrity via SDS-PAGE (should show a single band at approximately 16 kDa)

  • Avoid excessive freeze-thaw cycles

  • Use carrier protein (BSA) for dilute solutions

  • Prepare fresh working solutions from frozen stocks

• Variable Cell Responses:

  • Confirm receptor expression in your cell system

  • Standardize cell density and passage number

  • Use positive controls (known FGF4-responsive cells)

  • Ensure heparin sulfate availability (can add exogenously if needed)

• Signaling Pathway Interference:

  • Serum-starve cells before treatment

  • Check for cross-talk with other growth factor pathways

  • Include pathway-specific inhibitors as controls

If experiencing reproducibility issues, consider using carrier-containing preparations for general applications and carrier-free versions only when absolutely necessary for specialized applications where BSA might interfere .

How does truncated FGF4 differ from full-length protein, and what are the experimental implications?

Two main FGF4 variants have been characterized:

• Full-length FGF4: The complete mature protein (Ser54-Leu206) that exhibits both mitogenic activity and receptor activation.

• Truncated 15 kDa isoform: An endogenously expressed C-terminally truncated variant that opposes the effects of full-length FGF4 and promotes differentiation rather than self-renewal in embryonic stem cells.

Additionally, engineered FGF4 analogs with N-terminal truncations (specifically Ala67–Leu206) demonstrate reduced dimerization capacity and altered activation of heparan sulfate-assisted FGFRs. These modified variants maintain activity comparable to wild-type FGF4 but with diminished mitogenic properties .

Experimental Implications:

• When studying stem cell self-renewal versus differentiation, consider the balance between full-length and truncated isoforms

• For therapeutic applications, modified FGF4 analogs might offer reduced tumorigenic potential

• In expression studies, ensure primers and antibodies can distinguish between isoforms

• For recombinant protein studies, verify which variant is being used

What are the most promising future research directions for FGF4 in disease models?

Based on current research findings, these areas offer significant potential for future FGF4 research:

• Autoimmune Liver Diseases:
  The demonstrated ability of FGF4 to modulate macrophage polarization through the PI3K-AKT pathway suggests therapeutic potential for inflammatory liver conditions. Future research could explore modified delivery systems, optimal dosing regimens, and combination approaches with existing immunomodulatory agents .

• Gastrointestinal Stromal Tumors:
  Different mechanisms drive FGF4 overexpression in GIST subtypes (epigenetic vs. genomic structural changes), yet both lead to increased FGF4 expression. This convergence suggests FGFR pathway inhibition as a potential common therapeutic strategy worth further investigation .

• Stem Cell Technologies:
  FGF4's dual role in promoting proliferation while also potentially facilitating differentiation depending on context and concentration warrants further exploration for regenerative medicine applications. Particular focus should be given to understanding the balance between full-length and truncated isoforms .

• Cardiovascular Applications:
  FGF4's potent angiogenesis-promoting properties have led to investigations for coronary artery disease therapy. Future research might refine delivery methods and target specific patient populations .

When designing studies in these areas, robust experimental designs with appropriate controls and mechanistic validation will be essential for meaningful advances .

What standardized methods exist for measuring FGF4-induced signaling pathways?

Recommended Methodological Approaches:

• Western Blotting for Pathway Activation:

  • Primary targets: phospho-AKT, total AKT

  • Secondary targets: phospho-ERK1/2, phospho-PI3K, total pathway components

  • Time course: Assess at 5, 15, 30, 60 minutes post-FGF4 treatment

  • Include pathway inhibitors (LY294002 for PI3K) as controls

• Transcriptional Response Analysis:

  • qRT-PCR for pathway-specific target genes

  • RNA-seq for global transcriptional changes

  • ChIP-seq for identifying direct transcriptional targets

• Flow Cytometry for Cellular Responses:

  • Particularly useful for macrophage polarization studies

  • Measure M1 markers (CD80, CD86) and M2 markers (CD206, CD163)

  • Include appropriate isotype controls and FMO (fluorescence minus one) controls

When comparing results across different experimental systems, standardize to a common positive control condition and report fold changes rather than absolute values to facilitate cross-study comparisons .

How can researchers effectively model FGF4's role in developmental processes?

Recommended Model Systems:

• Embryonic Stem Cell Models:

  • Mouse or human ES cells with controlled FGF4 exposure

  • Embryoid body formation assays

  • Differentiation protocols with and without FGF4 supplementation

• Transgenic Mouse Models:

  • Tissue-specific Cre-loxP conditional knockouts

  • Inducible expression systems for temporal control

  • Reporter lines for lineage tracing

• Ex Vivo Developmental Assays:

  • Limb bud cultures for studying outgrowth and patterning

  • Trophoblast stem cell cultures

  • Embryonic tissue explants

Critical Measurements:

• Expression patterns of FGF4 and its receptors during development

• Temporal coordination with other developmental signals

• Specific developmental outcomes (e.g., limb formation, trophectoderm maintenance)

These models allow researchers to recapitulate the spatially and temporally regulated expression of FGF4 and its receptors that is critical during embryonic development, particularly in the trophoblast inner cell mass and later in limb development .