Recombinant Human Fibroblast growth factor 9 protein (FGF9), partial (Active)

What is FGF9 and what are its primary biological roles?

FGF9, also known as Glia-activating factor, is a member of the fibroblast growth factor family that possesses broad mitogenic and cell survival activities. It is involved in numerous biological processes including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion. The protein was initially isolated as a secreted factor that stimulates growth in cultured glial cells and is produced mainly by neurons in the nervous system, potentially playing a critical role in glial cell development. It forms part of a subfamily with FGF-16 and FGF-20, sharing 65-71% amino acid sequence identity .

How does recombinant FGF9 differ from native human FGF9?

Recombinant Human FGF9 typically contains the amino acid sequence Ala2-Ser208 or Leu4-Ser208 of the native protein. When expressed in systems like HEK293 cells, recombinant FGF9 maintains bioactivity comparable to native protein but offers greater consistency and purity for research applications. The recombinant protein exhibits the characteristic beta-trefoil structure of the FGF family and maintains similar receptor binding properties. In activity assays, recombinant FGF9 demonstrates an ED50 of 1.00-5.00 ng/mL for stimulating cell proliferation in Balb/3T3 Mouse Embryonic Fibroblast cells .

What receptors does FGF9 primarily interact with?

FGF9 primarily binds to FGFR3(IIIb) but also shows affinity for the IIIc splice forms of FGFR-1, FGFR-2, and FGFR-3. This receptor binding profile determines its tissue-specific activities and distinguishes it from other FGF family members. The interaction with these receptors initiates downstream signaling cascades that regulate various cellular processes including proliferation, differentiation, and migration, thereby influencing developmental processes and tissue homeostasis .

How does FGF9 contribute to sex determination during development?

FGF9 plays a vital role in male sex development, beginning with its expression in the bi-potent gonads of both females and males. Upon activation by SOX9, FGF9 forms a feedforward loop that increases the levels of both genes. This creates a positive feedback mechanism that upregulates SOX9 while simultaneously inactivating the female Wnt4 signaling pathway. In mouse models, deletion of the FGF9 gene results in a male-to-female sex reversal phenotype, highlighting its essential role in testicular embryogenesis. The timing and location of FGF9 expression are critical factors that influence proper male gonadal development .

What role does FGF9 play in lung morphogenesis?

In lung development, FGF9 is expressed in the mesothelium and pulmonary epithelium, where it maintains lung mesenchymal proliferation. The protein is critical for proper epithelial branching during lung formation. Studies have shown that inactivation of FGF9 results in diminished epithelial branching, and by the end of gestation, the underdeveloped lungs cannot sustain life, resulting in prenatal death. This demonstrates the essential nature of FGF9 in proper lung morphogenesis and respiratory system development .

How does FGF9 influence skeletal development and repair?

FGF9 stimulates chondrocyte proliferation, playing a significant role in skeletal development and repair. It works in conjunction with FGF18 to promote cartilage formation and bone growth. In mouse models, heterozygous FGF9 mutations result in compromised bone repair following injury. Additionally, mutations in the FGF9 gene are responsible for Elbow knee synostosis (Eks) in mice, causing joint fusions in the elbow and knee. In humans, FGF9 mutations that reduce receptor binding can result in multiple synostoses syndrome (SYNS), further demonstrating its crucial role in proper joint and skeletal development .

What are the optimal conditions for using recombinant FGF9 in cell culture experiments?

For optimal results in cell culture experiments, reconstitute lyophilized recombinant FGF9 at 250 μg/mL in sterile water. The protein should be stored at -20°C to -80°C and freeze-thaw cycles should be minimized. When designing experiments, an effective concentration range is typically 1-30 ng/mL, with the ED50 for cell proliferation being approximately 1.00-5.00 ng/mL in most responsive cell types. For studies examining cell proliferation or differentiation, a treatment duration of 24-48 hours is typically sufficient, though this may vary based on the specific cell type and experimental endpoint. Carrier-free formulations are recommended for applications where the presence of bovine serum albumin (BSA) might interfere with experimental outcomes .

How can I design experiments to study FGF9 interactions with other growth factors?

To study FGF9 interactions with other growth factors like IGF1, follow these methodological steps:

• Culture the cells of interest (e.g., granulosa cells) for 48 hours in medium containing 10% fetal calf serum (FCS).

• Wash cells with serum-free medium to remove residual growth factors.

• For synchronized experiments, serum-starve cells for 24 hours to bring them to G1 phase of the cell cycle.

• Treat cells with FGF9 (30 ng/mL) alone or in combination with other factors such as IGF1 (30 ng/mL) and FSH (30 ng/mL).

• Include appropriate controls (no treatment, single factor treatments).

• Assess outcomes at appropriate time points:

  • For proliferation: 48 hours post-treatment

  • For gene expression: 12-24 hours post-treatment

  • For protein accumulation: multiple time points (0h, 6h, 12h)

What molecular readouts are most informative when studying FGF9 effects on cell cycle regulation?

When studying FGF9 effects on cell cycle regulation, the following molecular readouts provide valuable information:

For optimal results, measure multiple parameters at different time points, as FGF9 effects may vary by cell type. For instance, in small-follicle granulosa cells, FGF9 increases CCND1 mRNA by 1.85-fold, while in large-follicle granulosa cells, it causes a 6-fold increase under similar conditions .

How do FGF9 and IGF1 signaling pathways interact to regulate cell proliferation?

At the molecular level, IGF1 significantly amplifies FGF9-stimulated CCND1 mRNA expression. In small-follicle granulosa cells, FGF9 alone increases CCND1 mRNA by 1.7-fold, but in the presence of IGF1, this increase jumps to 3.3-fold. This indicates that while both pathways independently promote proliferation, their interaction at the level of cell cycle regulatory genes creates a potentiated response, suggesting convergence of signaling pathways at critical regulatory nodes .

What are the mechanisms by which FGF9 inhibits myelination in multiple sclerosis models?

FGF9 inhibits myelination and remyelination through an indirect mechanism that surprisingly does not directly affect oligodendrocytes. Instead, FGF9 acts on astrocytes, causing them to secrete factors that inhibit proper myelin formation. This leads to the development of multi-branched "pre-myelinating" MBP+/PLP+ oligodendrocytes that interact with axons but fail to assemble functional myelin sheaths—a phenotype observed in chronically demyelinated multiple sclerosis lesions.

Transcriptional profiling reveals that FGF9 induces expression of tissue inhibitor of metalloproteinase-sensitive proteases in astrocytes, enzymes typically associated with extracellular matrix remodeling. Additionally, FGF9 upregulates the expression of pro-inflammatory chemokines Ccl2 and Ccl7, which contribute to the recruitment of microglia and macrophages into multiple sclerosis lesions.

This dual action creates a pathogenic environment that both prevents remyelination and promotes inflammation, potentially exacerbating axonal injury and loss in multiple sclerosis patients. This mechanism elucidates why increased glial expression of FGF9 in actively demyelinating lesions correlates with poor disease outcomes .

How does FGF9 signaling interact with the MAPK/ERK pathway in different cell types?

FGF9 activates the MAPK/ERK pathway in various cell types, with experimental evidence supporting this connection in theca cells (TC). When TC are treated with U0126, a specific inhibitor of MEK1/2 (the kinases that activate ERK1/2), the FGF9-induced increase in CCND1 mRNA is significantly attenuated. This suggests that FGF9 regulates gene expression, at least partially, through MAPK/ERK-dependent mechanisms.

The temporal dynamics of ERK activation following FGF9 treatment are rapid, with phosphorylation detectable within minutes and sustained for several hours. This activation pattern differs from other growth factors, which may exhibit more transient ERK activation profiles. The duration and intensity of ERK activation may contribute to the specific gene expression patterns induced by FGF9.

Furthermore, the interaction between FGF9 and other signaling pathways, such as those activated by LH, can modulate ERK activation. For example, in TC treated with IGF1, LH suppresses the FGF9-induced increase in CCND1 mRNA, reducing the enhancement from 16.2-fold to 6.5-fold, potentially through modulation of MAPK/ERK signaling .

What is the evidence linking FGF9 dysregulation to cancer progression?

Altered FGF9 expression or function has been implicated in several human cancers, including colon, endometrial, and ovarian cancers. These alterations correlate with disease progression, invasiveness, and patient survival. The mitogenic and cell survival activities of FGF9, combined with its roles in tissue repair and tumor growth, make it a significant factor in cancer biology.

In experimental models, FGF9 promotes cell proliferation through upregulation of cyclin D1 (CCND1) and activation of cyclin-dependent kinase 4 (CDK4), driving cells through the G1/S checkpoint of the cell cycle. This proliferative effect, combined with FGF9's ability to stimulate angiogenesis and interact with other growth factors, creates a microenvironment conducive to tumor growth and metastasis .

How might FGF9 modulation be used therapeutically in multiple sclerosis?

Given FGF9's role in inhibiting myelination and promoting inflammation in multiple sclerosis (MS), therapeutic strategies targeting FGF9 signaling present a promising avenue for MS treatment. Inhibiting FGF9 or blocking its downstream effects could potentially address two critical pathogenic pathways simultaneously: promoting remyelination and reducing inflammation.

Specifically, neutralizing antibodies against FGF9 or small molecule inhibitors of its receptor interactions could prevent the astrocyte-mediated inhibition of myelination. Alternatively, strategies that block the astrocyte-derived factors induced by FGF9, particularly tissue inhibitor of metalloproteinase-sensitive proteases, might allow oligodendrocytes to complete the myelination process despite elevated FGF9 levels.

Additionally, inhibiting the FGF9-induced expression of chemokines Ccl2 and Ccl7 could reduce inflammatory cell recruitment to lesion sites, potentially limiting demyelination and creating a more favorable environment for repair. This dual approach of promoting remyelination while limiting inflammation represents a comprehensive therapeutic strategy aligned with the pathophysiological understanding of MS .

What are the developmental consequences of FGF9 mutations in skeletal disorders?

FGF9 mutations have significant developmental consequences for skeletal formation and joint development. In mice, a specific mutation in FGF9 causes Elbow knee synostosis (Eks), resulting in joint fusions in the elbow and knee. These manifestations arise from aberrant chondrocyte proliferation and differentiation during critical developmental windows.

In humans, mutations that reduce FGF9's receptor binding capability can result in multiple synostoses syndrome (SYNS), characterized by joint fusions and skeletal malformations. The spectrum of skeletal abnormalities depends on the specific mutation and its effect on FGF9 function. Some mutations may completely abolish FGF9 activity, while others might result in altered receptor specificity or reduced binding affinity.

Heterozygous FGF9 mutant mice also demonstrate compromised bone repair following injury, suggesting that proper FGF9 signaling is essential not only for development but also for maintaining skeletal integrity throughout life. These findings highlight the critical role of precisely regulated FGF9 signaling in proper skeletal development and homeostasis .