Recombinant Human Fibroblast growth factor 7 (FGF7) (Active)

What is the structure and basic function of human FGF7?

Human FGF7 is a 194-amino acid protein with the active form spanning from Cys32 to Thr194 . It belongs to the fibroblast growth factor family and exhibits specificity for both heparin/heparan sulfate and receptor complexes on epithelial cells. FGF7 primarily functions as a potent epithelial cell-specific growth factor, stimulating cell proliferation and migration. It plays critical roles in wound healing, tissue regeneration, and embryonic development, particularly in epithelial tissues .

What are the optimal methods for producing recombinant human FGF7?

Recombinant human FGF7 can be produced using bacterial or mammalian expression systems, each with distinct advantages:

Bacterial Expression (E. coli):

• Fusion with glutathione-S-transferase (GST) at the N-terminus followed by proteolytic removal of GST while bound to heparin significantly improves yields

• Supplementation of culture medium with 10-100 mM MgCl₂ enhances GST-FGF7 yields to approximately 17 mg per liter per OD₆₀₀ in BL21(DE3)pLysS strain

• This modification improves yields of active 54ser-FGF7 by approximately five-fold after proteolytic excision of the GST portion

Mammalian Expression (CHO cells):

• Provides N-glycosylated FGF7, which may have enhanced stability and activity for certain applications

• While bacteria-produced FGF7 is not glycosylated, CHO cell-produced FGF7 contains post-translational modifications that can influence biological activity, particularly for wound healing applications

How can I verify the biological activity of recombinant FGF7?

The biological activity of recombinant FGF7 can be verified through several assays:

• Cell Proliferation Assay: Using 4MBr-5 rhesus monkey epithelial cells, where the ED₅₀ for FGF7-induced proliferation typically ranges from 6-60 ng/mL

• Wound Healing Assay: Measuring the migration rate of epithelial cells in scratch assays

• Receptor Binding Assay: Assessing binding to FGFR2 and FGFR1 receptors

• Downstream Signaling Activation: Monitoring MAPK pathway activation through phosphorylation of ERK1/2

• In vivo Assessment: Evaluating wound healing capability in animal models using parameters such as hydroxyproline content, expression of growth factors (FGF, VEGF, TGF), and histological assessment of epidermalization

Which receptors does FGF7 primarily interact with?

FGF7 primarily interacts with Fibroblast Growth Factor Receptor 2 (FGFR2), with secondary binding to FGFR1 . The receptor specificity of FGF7 is as follows:

• FGFR2 (Primary receptor): FGF7 shows highest affinity for the IIIb splice variant of FGFR2, which is predominantly expressed in epithelial cells

• FGFR1: FGF7 can also interact with FGFR1, though with lower affinity than FGFR2

• Heparan sulfate proteoglycans: Act as co-receptors that stabilize the FGF7-FGFR complex and enhance signaling efficiency

This receptor specificity explains the epithelial cell-targeted activity of FGF7 and its restricted biological effects compared to other FGF family members.

What signaling pathways are activated by FGF7?

FGF7 activates several intracellular signaling cascades through its interaction with FGF receptors:

• MAPK/ERK Pathway: Primary pathway activated by FGF7, leading to sustained intracellular mitogen-activated protein kinase (MAPK) activity

• PI3K/AKT Pathway: Contributes to cell survival signals

• β-catenin Signaling: FGF7 induces cytoplasmic accumulation and nuclear translocation of β-catenin, which is particularly important in osteocytes

• PLCγ/PKC Pathway: Activated downstream of FGFR phosphorylation

The activation of these pathways collectively mediates the biological effects of FGF7, including cell proliferation, migration, differentiation, and survival, depending on the cellular context.

How can FGF7 be effectively used in wound healing studies?

FGF7 has shown significant efficacy in wound healing applications with the following experimental approaches:

Delivery Methods:

• Direct application: Purified FGF7 applied directly to wounds

• Collagen patches (CP) combined with FGF7: This combination has shown superior wound healing compared to FGF7 alone or untreated controls

Assessment Parameters:

• Hydroxyproline content measurement: Indicator of collagen synthesis

• Growth factor expression analysis: FGF, VEGF, and TGF

• Histological evaluation: Epidermalization assessment using H&E staining

• Blood vessel formation: Angiogenesis evaluation on days 7 and 14 post-treatment

Research shows that wounds treated with collagen patches containing FGF7 (CP+FGF7) exhibited the most effective healing, with the highest expression of hydroxyproline and growth factors on day 7 post-exposure, compared to FGF7-only, CP-only, and untreated controls .

What is the role of FGF7 in stem cell differentiation studies?

FGF7 plays a critical role in directing stem cell differentiation, particularly in pancreatic lineage development:

• Islet Organoid Differentiation:

  • FGF7 is added at specific stages of differentiation: stage 2 (50 ng/ml), stage 3 (50 ng/ml), and stage 4 (2 ng/ml)

  • Precise modulation of FGF7 concentration and duration is essential for pancreatic lineage development

  • FGF7 treatment influences ACE2 expression during differentiation, with removal of FGF7 resulting in decreased ACE2 expression

• Epithelial Tissue Development:

  • FGF7 supports the differentiation of epithelial stem cells

  • Adult stem cells from human lung biopsy tissue can be cultured with FGF7 in lung organoid expansion medium to form functional organoids

This indicates that FGF7 is a key regulator in tissue-specific stem cell differentiation and can be manipulated to control developmental pathways in experimental models.

How does FGF7 influence ACE2 expression and what are the implications for SARS-CoV-2 research?

Recent research has uncovered a significant role for FGF7 in regulating ACE2 expression, particularly relevant to SARS-CoV-2 infection studies:

• Mechanism of ACE2 Upregulation:

  • FGF7 interacts with FGFR2 and FGFR1 to upregulate ACE2 expression predominantly in β cells

  • During islet organoid differentiation, ACE2 expression increases significantly during maturation stages, with FGF7 treatment promoting this increase

  • Removing FGF7 from differentiation medium silences the FGF7-FGFR pathway, eliminating ACE2 expression in stages 4 and 5

• Implications for SARS-CoV-2 Infection:

  • Enhanced ACE2 expression within islets facilitates SARS-CoV-2 infection

  • This leads to impaired insulin secretion in pancreatic β cells

  • The finding suggests that modulating FGF7 signaling could be a potential strategy to protect β cells from SARS-CoV-2 infection

This research provides important insights into the molecular mechanisms underlying COVID-19 complications related to glucose metabolism and identifies FGF7 as a potential therapeutic target.

What is the role of FGF7 in cancer research, particularly in rhabdomyosarcomas?

FGF7 has emerged as a significant factor in cancer biology, particularly in fusion-positive rhabdomyosarcomas:

• FGF7-FGFR2 Autocrine Signaling:

  • Patient samples show higher mRNA levels of FGFR2 and FGF7 in fusion-gene-positive (PAX3-FOXO1) versus fusion-gene-negative rhabdomyosarcomas

  • FGF7 secretion occurs during serum starvation of PAX3-FOXO1 rhabdomyosarcoma cells

  • Genetic silencing of FGFR2 or FGF7 decreases cell viability, supporting the existence of an FGF7-FGFR2 autocrine loop

• Therapeutic Targeting:

  • FGFR inhibitors (NVP-BGJ398, nintedanib, dovitinib, and ponatinib) show greater efficacy against fusion-gene-positive rhabdomyosarcoma cell lines

  • FGFR inhibition with NVP-BGJ398 reduces cell viability and shows synergistic effects with SN38 (active metabolite of irinotecan)

  • In vivo, NVP-BGJ398 inhibits xenograft growth, suggesting potential for therapeutic applications

These findings highlight FGF7 as both a biomarker and potential therapeutic target in certain cancers, particularly fusion-positive rhabdomyosarcomas.

How does FGF7 influence osteocyte function in bone biology?

FGF7 has been identified as a key regulator of osteocyte function through the following mechanisms:

• Gap Junction Communication:

  • FGF7 increases the expression of Connexin43 (Cx43) in osteocytes

  • This promotes cell processes in the form of gap junctions between osteocytes

  • FGF7 mRNA levels are higher relative to other FGF family members in both osteocyte cell lines (MLO-Y4) and bone tissue

• β-catenin Signaling Pathway:

  • FGF7's modulation of osteocyte cell processes occurs through FGF7-induced cytoplasmic accumulation and nuclear translocation of β-catenin

  • This signaling pathway is crucial for osteocyte communication and function

These findings expand our understanding of FGF7's role beyond epithelial tissues and highlight its importance in bone cell behavior, physiology, and potentially pathology.

What are the optimal storage and handling conditions for recombinant FGF7?

To maintain the biological activity of recombinant FGF7, the following storage and handling conditions are recommended:

• Storage Temperature:

  • Long-term storage: -80°C in small aliquots to avoid repeated freeze-thaw cycles

  • Working stocks: -20°C for up to 3 months

• Buffer Composition:

  • Recommended buffer: PBS with 0.1-1% BSA or human serum albumin as a carrier protein

  • Addition of 1-10 mM DTT may help maintain activity by preventing oxidation of cysteine residues

  • For E. coli-derived FGF7, heparin (1-10 μg/ml) in the storage buffer can enhance stability

• Reconstitution:

  • Use sterile, cold buffer for reconstitution

  • Gently mix by swirling or inverting the vial, avoiding vigorous shaking

  • Allow protein to sit for 30 minutes at 4°C after reconstitution

• Working Concentration:

  • For cell culture applications, effective concentrations typically range from 6-60 ng/mL

  • For wound healing applications, concentrations of 2-50 ng/mL have been reported as effective

How can I optimize FGF7 production in bacterial expression systems?

Based on research findings, the following strategies can optimize FGF7 production in bacterial systems:

• Expression Construct Design:

  • Use GST fusion at the N-terminus of FGF7

  • Include a specific protease cleavage site between GST and FGF7

  • The construct should encode the mature form of FGF7 (Cys32-Thr194)

• Host Strain Selection:

  • BL21(DE3)pLysS strain has been shown to be effective for FGF7 expression

  • Consider using strains with enhanced disulfide bond formation for improved folding

• Culture Conditions:

  • Supplement culture medium with 10-100 mM MgCl₂

  • This modification has been shown to improve yields to approximately 17 mg per liter per OD₆₀₀

  • Optimize induction conditions: temperature, IPTG concentration, and induction time

• Purification Strategy:

  • Immobilize GST-FGF7 on heparin-Sepharose before proteolytic cleavage

  • This approach improves yields of active 54ser-FGF7 by approximately five-fold after GST removal

These optimizations significantly enhance the cost-effectiveness of producing recombinant FGF7 in bacteria for research applications.

How does FGF7 differ functionally from other FGF family members?

FGF7 possesses unique characteristics that distinguish it from other members of the FGF family:

• Receptor Specificity:

  • FGF7 shows specificity for FGFR2-IIIb and, to a lesser extent, FGFR1, while other FGF family members may bind to multiple FGFR subtypes

  • This receptor specificity contributes to FGF7's epithelial cell-targeted activity

• Tissue Expression and Function:

  • FGF7 is predominantly expressed in mesenchymal cells but acts on neighboring epithelial cells

  • In contrast to the broader expression patterns of FGF1 and FGF2

  • FGF7 mRNA levels are particularly high in osteocytes compared to other FGF family members

• Production Challenges:

  • Recombinant FGF7 production from bacteria yields lower recoveries compared to FGF1 and FGF2, with GST-FGF7 fusion protein yields being approximately 10% of that for FGF1

• Biological Applications:

  • FGF7's specific role in wound healing and epithelial regeneration makes it particularly valuable for these applications

  • Its role in regulating ACE2 expression distinguishes it in the context of SARS-CoV-2 research

Understanding these differences is crucial for researchers selecting the appropriate FGF family member for their specific experimental applications.