SFRP4 Human, sf9

What is SFRP4 and what signaling pathways does it modulate?

SFRP4 belongs to the Secreted Frizzled-Related Protein family, containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It functions primarily as a soluble modulator of Wnt signaling pathways . SFRP4 serves multiple biological roles, including regulation of adult uterine morphology and function, increasing apoptosis during ovulation through modulation of FZ1/FZ4/WNT4 signaling, and exerting phosphaturic effects by specifically inhibiting sodium-dependent phosphate uptake . Expression profiles show SFRP4 in proliferative endometrium, several types of ovarian, endometrial and breast tumors, mesenchymal cells, and cardiomyocytes. Notably, SFRP4 expression in ventricular myocardium correlates with apoptosis-related gene expression and is up-regulated in failing myocardium .

Interestingly, recent research reveals that SFRP4's antagonistic effects on FSH (Follicle-Stimulating Hormone) action occur neither via canonical (CTNNB1-dependent) nor non-canonical WNT signaling mechanisms, but through a GSK3β-dependent pathway involving AMPK, which leads to antagonization of AKT activity .

Why are Sf9 insect cells used for SFRP4 production in research applications?

The Spodoptera frugiperda Sf9 insect cell line is preferentially used within the baculovirus expression vector system for producing complex mammalian proteins like SFRP4 for several methodological reasons . These cells efficiently process eukaryotic post-translational modifications, including complex glycosylation patterns essential for proper SFRP4 folding and function. The baculovirus expression system allows for high-yield production of proteins that retain their native biological activities more effectively than bacterial expression systems .

It's important to note that Sf9 cells have been found to produce endogenous retroviral-like particles containing reverse transcriptase activity, as detected using the PCR-enhanced reverse transcriptase (PERT) assay . While this characteristic is particularly relevant for viral vaccine and gene therapy product development, researchers working with SFRP4 should consider this when designing control experiments to distinguish observed effects between the protein itself and potential contaminants from the expression system .

What are the recommended storage and handling conditions for SFRP4 produced in Sf9 cells?

For optimal retention of SFRP4 activity and stability, researchers should follow these evidence-based storage protocols:

• Short-term storage (2-4 weeks): Store at 4°C if the entire vial will be used within this period .

• Long-term storage: Store frozen at -20°C, with the addition of a carrier protein (0.1% HSA or BSA) to enhance stability .

• Avoid multiple freeze-thaw cycles as these can significantly compromise protein integrity and activity .

When handling the protein, maintain sterile conditions and consider aliquoting the stock solution to minimize freeze-thaw cycles. For experimental work, SFRP4 is typically provided at 0.5 mg/ml concentration in a buffer containing phosphate-buffered saline (pH 7.4) and 10% glycerol . Before use in biological assays, researchers should verify protein purity (>90% by SDS-PAGE is standard for most research applications) .

What methodologies are most effective for studying SFRP4 methylation status in tumor samples?

When investigating SFRP4 methylation status in tumor samples such as high-grade astrocytomas, researchers typically employ methylation-specific PCR (MSP) following bisulfite conversion of DNA . The methodological workflow involves:

• DNA isolation from tumor samples and appropriate controls

• Bisulfite treatment to convert unmethylated cytosines to uracils while leaving methylated cytosines unchanged

• Methylation-specific PCR using primers designed to distinguish between methylated and unmethylated sequences

• Gel electrophoresis analysis of PCR products to determine methylation status

For comprehensive analysis, researchers should complement MSP with assessment of SFRP4 protein expression, commonly performed using immunohistochemistry and evaluated semi-quantitatively. The intensity of protein expression can be quantified using image analysis software such as ImageJ (National Institutes of Health, United States), examining protein expression in approximately 200 cells within tumor hot-spot areas. Immunopositivity is then typically assessed using the Immunoreactivity Score (IRS) .

To validate experimental findings, researchers frequently corroborate their results with public databases such as cBioPortal, COSMIC, and LOVD for genomic and prognostic correlation . This multi-faceted approach provides a more comprehensive understanding of the relationship between SFRP4 promoter methylation and protein expression in tumors.

How does SFRP4 influence autophagy in granulosa cells and what mechanisms are involved?

SFRP4 induces autophagy in granulosa cells through a novel signaling mechanism involving the AKT-mTORC1-ULK1 pathway . Methodological approaches for investigating this phenomenon typically involve:

• Primary cultures of granulosa cells (from wild-type mice) treated with recombinant SFRP4 and/or FSH

• Analysis of autophagy markers through western blotting, focusing on:

  • LC3-I to LC3-II conversion

  • p62/SQSTM1 levels

  • Phosphorylation status of mTOR and ULK1

The autophagy-promoting effect of SFRP4 occurs through GSK3β-dependent antagonism of AKT activity via a mechanism involving AMPK . This leads to decreased mTORC1 activity and subsequent activation of ULK1, which initiates the autophagy process. In comparative studies, granulosa cells from Sfrp4-null mice show increased FSH-stimulated AMPK, AKT, and FOXO1 phosphorylation levels compared to wild-type controls, confirming SFRP4's role in this pathway .

To comprehensively investigate these mechanisms, researchers can use pharmacologic inhibitors of specific signaling effectors (like AMPK inhibitors or mTOR inhibitors) in combination with SFRP4 and FSH treatments to dissect the pathway components. Autophagy can be further assessed using fluorescent markers (GFP-LC3) or transmission electron microscopy to visualize autophagic structures .

How does SFRP4 antagonize FSH action through the GSK3β-AMPK-AKT signaling pathway?

SFRP4 antagonizes FSH action through a previously uncharacterized GSK3β-AMPK-AKT signaling mechanism, distinct from its traditional role as a WNT signaling antagonist . Investigating this pathway requires a multi-modal experimental approach:

• Transcriptional analysis: Treat primary cultures of granulosa cells with FSH and/or SFRP4, then evaluate gene expression by RT-qPCR and RNA-seq. This reveals that SFRP4 decreases both basal and FSH-stimulated mRNA levels of FSH target genes .

• Signaling pathway dissection: Use pharmacological inhibitors to identify that SFRP4's effects are GSK3β-dependent but CTNNB1-independent. This involves treating granulosa cells with specific inhibitors before exposure to FSH and/or SFRP4 .

• Phosphorylation analysis: Assess AMPK, AKT, and FOXO1 phosphorylation states through western blotting. SFRP4 antagonizes AKT activity through AMPK, leading to hypophosphorylation of FOXO1 and decreased expression of FSH and FOXO1 transcriptomes .

• Comparative studies: Compare signaling responses in wild-type versus Sfrp4-null granulosa cells to validate pathway components. Sfrp4-null cells show increased FSH-stimulated AMPK, AKT, and FOXO1 phosphorylation compared to wild-type controls .

• Bioinformatic analyses: Analyze RNA-seq data (processed using pipelines like GenPipes v.3.1.2) to identify differentially expressed genes between control and SFRP4-treated conditions using tools such as DESeq2 .

This methodological framework establishes SFRP4 as a novel regulator that blunts FSH responsiveness in granulosa cells through manipulation of the GSK3β-AMPK-AKT pathway rather than through canonical WNT signaling mechanisms .

What experimental approaches can be used to investigate SFRP4's role in bone formation and resorption?

Although the search results don't directly address SFRP4's role in bone formation, we can draw methodological insights from related studies on transferrin receptor 2 (Tfr2), another protein involved in bone metabolism . Similar experimental approaches could be applied to investigate SFRP4:

• μCT analysis: Quantify bone parameters including:

  • Bone volume per total volume (BV/TV)

  • Cortical bone mineral density (BMD)

  • Trabecular number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp)

• Biomechanical testing: Assess bone strength using three-point-bending tests to measure maximum force parameters .

• Dynamic histomorphometry: Use calcein double staining to visualize and quantify bone formation rate per bone surface (BFR/BS) .

• Serum biomarker assessment: Measure bone formation markers (P1NP) and bone resorption markers (CTX) using ELISAs .

• Histological analysis: Perform tartrate-resistant acid phosphatase staining to identify and quantify osteoclasts .

• Cell-specific knockout models: Generate conditional knockout mice (using systems like Cre-loxP) to delete SFRP4 in specific cell types (osteoblasts or osteoclasts) to determine cell-specific functions .

• Gene expression profiling: Conduct RNA-seq analysis of osteoblasts with and without SFRP4 to identify differentially expressed genes involved in bone formation and resorption .

What techniques are most effective for evaluating SFRP4 protein expression in high-grade astrocytomas?

Research on SFRP4 expression in high-grade astrocytomas demonstrates that a multi-faceted approach yields the most comprehensive results . The recommended methodological workflow includes:

• Immunohistochemistry (IHC): This is the primary technique for detecting SFRP4 protein expression in tumor samples. The protocol involves:

  • Deparaffinization and antigen retrieval of tissue sections

  • Incubation with anti-SFRP4 antibodies

  • Detection using appropriate secondary antibodies and chromogenic substrates

  • Semi-quantitative evaluation based on staining intensity and percentage of positive cells

• Quantitative image analysis: Using software like ImageJ to determine the intensity of protein expression in approximately 200 cells within the tumor hot-spot area. This provides a more objective measurement than visual scoring alone .

• Immunoreactivity Score (IRS): Calculating an IRS based on both staining intensity and percentage of positive cells for standardized assessment across samples .

Findings from these techniques revealed that SFRP4 protein expression in glioblastomas was very weak or non-existent in 86.7% of samples, moderate in 13.3%, while strong expression was not observed . The data showed a significant inverse correlation between astrocytoma grade and SFRP4 protein expression, suggesting its potential role as a tumor suppressor in these malignancies .

How should RNA-seq data be processed when studying SFRP4-related gene expression?

Based on the methodology described in the research on SFRP4's effects on granulosa cells, the following RNA-seq data processing workflow is recommended :

• Quality control and preprocessing:

  • Use tools like Trimmomatic for quality and adaptor trimming of raw reads

  • Assess library technical quality using Picard-tools (v.1.92) and Samtools (v.1.1)

  • Filter out PCR duplicates and fragments with poor mapping quality (q < 255)

• Alignment and quantification:

  • Align preprocessed reads to the appropriate reference genome (e.g., mm10 for mouse) using the STAR aligner

  • Quantify gene transcription levels over annotated genes (e.g., Ensembl genes)

• Differential expression analysis:

  • Use DESeq2 to measure differential gene expression between control and SFRP4-treated conditions

  • Apply appropriate statistical corrections for multiple testing

• Bioinformatic interpretation:

  • Perform pathway enrichment analysis to identify biological processes affected by SFRP4

  • Compare SFRP4-regulated genes with other relevant transcriptomes (e.g., FSH-responsive genes)

  • Validate key findings using RT-qPCR for selected genes

This methodological approach provides a comprehensive view of SFRP4's effects on the transcriptome, allowing researchers to identify novel pathways and mechanisms through which SFRP4 exerts its biological functions.

What quality control measures should be implemented when working with SFRP4 produced in Sf9 cells?

When working with SFRP4 produced in Sf9 cells, researchers should implement several quality control measures to ensure experimental reliability:

• Purity assessment: Verify protein purity is greater than 90.0% using SDS-PAGE . This is critical as contaminants may influence experimental outcomes.

• Functional validation: Confirm biological activity through appropriate functional assays (e.g., Wnt signaling reporter assays or phosphate uptake inhibition assays) before using in experiments .

• Testing for endogenous retroviral-like particles: Given that Sf9 cells produce reverse transcriptase activity, consider testing for the presence of retroviral-like particles using the PCR-enhanced reverse transcriptase (PERT) assay, especially for sensitive applications .

• Physical characterization: Confirm molecular weight (approximately 40-57 kDa on SDS-PAGE due to glycosylation) and proper folding through circular dichroism or other structural analysis methods .

• Glycosylation analysis: As SFRP4 is a glycosylated protein, consider analyzing glycosylation patterns using techniques such as mass spectrometry to ensure consistency between batches .

• Batch consistency: Maintain strict quality control between batches by comparing protein activity, purity, and structural characteristics to establish reproducibility .

• Endotoxin testing: Perform endotoxin testing, especially for in vivo applications or cell-based assays sensitive to endotoxin contamination.

These measures help ensure that experimental outcomes reflect genuine SFRP4 activity rather than artifacts from the expression system or protein preparation.