FSTL1 Human

What is the molecular structure of human FSTL1?

Human FSTL1 is a 308 amino acid secreted glycoprotein (45-55 kDa) belonging to the BM-40/SPARC/Osteonectin family. Its structure comprises:

• A 20 amino acid secretion signal peptide

• A cysteine-rich Follistatin (EGF- and kazal-like) domain

• Two EF-hand calcium-binding motifs (apparently non-functional)

• A von Willebrand Factor type C homology domain
  The human FSTL1 protein shares remarkable sequence conservation with other mammals: 94% amino acid identity with mouse, 95% with rat, 98% with bovine, and 99% with equine FSTL1. The secretion signal (amino acids 1-20) shows the highest species variability, while the remaining 272 amino acids demonstrate high conservation (94.4% similarity between human and mouse) .

How is FSTL1 post-translationally modified?

FSTL1 undergoes significant post-translational modifications, particularly glycosylation:

• Three potential sites for N-glycosylation and two for O-glycosylation have been identified in mouse Fstl1

• The glycosylation status significantly affects FSTL1's biological functions

• Non-glycosylated FSTL1 (bacterially produced) has been shown to increase cardiomyocyte proliferation

• Glycosylated FSTL1 (eukaryotically produced) protects cardiomyocytes from peroxidase-induced apoptosis and promotes fibroblast proliferation and migration via ERK1/2 phosphorylation
  This difference in glycosylation status explains some contradictory findings in FSTL1 research and highlights the importance of protein source when designing experiments.

What are the established methods for detecting FSTL1 in biological samples?

Multiple validated techniques exist for detecting FSTL1 in various biological contexts:

How should researchers optimize FSTL1 detection in different experimental settings?

For optimal FSTL1 detection:

• Tissue analysis: When performing immunohistochemistry, heat-induced epitope retrieval using basic antigen retrieval reagents significantly improves detection sensitivity. DAB staining with hematoxylin counterstaining provides excellent visualization of FSTL1 in tissues like placenta, where it localizes to endothelial cells in chorionic villi .

• Western blot: Use PVDF membranes and reducing conditions with appropriate immunoblot buffer systems. For human samples, validated antibodies detect FSTL1 at approximately 50 kDa .

• Cellular analysis: For immunofluorescence detection in cell lines, fix cells appropriately and use fluorophore-conjugated secondary antibodies. Counterstaining with DAPI helps visualize nuclear positioning in relation to FSTL1 expression .

• Expression analysis across multiple samples: Consider using methodological triangulation - combining qRT-PCR, western blot, and immunohistochemistry - to thoroughly characterize FSTL1 expression patterns, as demonstrated in studies of cervical carcinoma .

What is the significance of FSTL1 in cardiovascular diseases?

FSTL1 plays complex roles in cardiovascular health and disease:

• Biomarker potential: Circulating FSTL1 concentrations increase during various cardiac and vascular diseases:

  • Heart failure (HF)

  • Heart failure with preserved ejection fraction (HFpEF)

  • Acute coronary syndrome (ACS)

  • Chronic obstructive pulmonary disease

• Prognostic value: Elevated FSTL1 levels correlate with:

  • Mortality in ACS patients

  • Severity of chronic heart failure

  • Potential recovery prediction in end-stage heart failure patients receiving left ventricle assist device therapy

• Protective functions: FSTL1 appears cardioprotective by:

  • Promoting endothelial cell functions

  • Supporting revascularization after ischemia

  • Enhancing expression and activation of protein kinase AKT1
    These findings suggest FSTL1 could be both a therapeutic target for cardiovascular drug development and a prognostic biomarker for cardiovascular diseases.

How does FSTL1 function in cancer progression and what are the research contradictions?

FSTL1's role in cancer is complex and sometimes contradictory:

• Tumor suppression: Evidence suggests FSTL1 functions as a tumor suppressor in multiple cancers:

  • Expression is down-regulated in many human cancers

  • In cervical carcinoma, FSTL1 mRNA and protein levels are significantly reduced in tumor tissues compared to adjacent tissues

  • TCGA data shows a 3.2-fold reduction in FSTL1 mRNA in cervical tumors

  • High FSTL1 expression inhibits proliferation, motility, and invasion of cervical cancer cell lines (HeLa and C33A)

• Mechanistic actions:

  • Slows proliferation and matrix metalloproteinase (MMP)-dependent migration

  • Increases FAS-dependent apoptosis of tumor cell lines

  • Negatively regulates the BMP4/Smad1/5/9 signaling pathway in cervical carcinoma

• Research contradictions:

  • Some studies report pro-inflammatory effects while others show anti-inflammatory actions

  • Differential effects on cell proliferation depending on glycosylation status

  • Varied effects observed in different cancer types and cellular contexts
    These contradictions might be explained by:

• Extensive post-transcriptional regulation of FSTL1

• The existence of a microRNA (miR-198) encoded by the FSTL1 primary transcript in primates

• Multiple microRNA-binding sites in the 3′UTR affecting expression

• Differences in glycosylation patterns affecting function

How does FSTL1 interact with key signaling pathways?

FSTL1 participates in multiple signaling pathways with context-dependent effects:

• BMP/Smad signaling:

  • Functions as a BMP inhibitor in cervical carcinoma

  • Negatively regulates BMP4/Smad1/5/9 signaling

  • This inhibition correlates with decreased cell proliferation and invasion capacity

  • Treatment with BMP4 can partially rescue the inhibitory effects of FSTL1 overexpression

• AKT signaling:

  • Promotes expression and activation of protein kinase AKT1

  • This mechanism contributes to its cardioprotective effects

  • Enhances endothelial cell revascularization after ischemia

• TGF-β pathway:

  • First identified as a TGF-β-induced protein from a mouse osteoblast cell line

  • May modulate TGF-β signaling, affecting processes like endocardial to mesenchymal transition

• Inflammatory pathways:

  • Interacts with receptors including DIP2A, TLR4, and BMP receptors

  • Can both promote inflammatory cytokine secretion and inhibit inflammation in different contexts

What experimental approaches can elucidate FSTL1's contradictory roles in different tissues?

To address the complex and sometimes contradictory functions of FSTL1, researchers should consider:

• Tissue-specific conditional knockout models:

  • Studies using Tie2-Cre and S100A4-Cre mouse lines revealed distinct phenotypes

  • Endothelial/endocardial deletion resulted in dysfunctional mitral valves and HFpEF

  • Fibroblast-specific deletion affected cardiac repair after myocardial infarction

  • Consider using multiple tissue-specific Cre lines to compare effects across tissues

• Glycosylation status analysis:

  • Compare bacterially-produced (non-glycosylated) and eukaryotically-produced (glycosylated) FSTL1

  • Assess functional differences in the same experimental systems

  • Systematically study structure-function relationships

• Expression analysis in paired diseased/healthy tissues:

  • Collect matched tumor and adjacent tissues

  • Analyze both mRNA and protein expression using multiple methods

  • Compare with circulating levels as demonstrated in cervical carcinoma studies

• Signal pathway interrogation:

  • Use pathway inhibitors (e.g., dorsomorphin for BMP signaling)

  • Combine with FSTL1 overexpression or knockdown

  • Monitor downstream targets (e.g., p-Smad1/5/9, MMP2)

How can FSTL1 be leveraged as a biomarker in clinical research?

FSTL1 shows promise as a biomarker for several conditions:

• Cardiovascular diseases:

  • Increased circulating FSTL1 correlates with heart failure severity

  • High FSTL1 levels at LVAD implantation correlate with better recovery of ejection fraction

  • Consider measuring FSTL1 in longitudinal studies of cardiac patients

• Cancer progression:

  • FSTL1 mRNA reduction correlates with advanced cervical carcinoma FIGO stages

  • Monitor FSTL1 expression changes during cancer progression

  • Consider both tissue and circulating levels for comprehensive analysis

• Autoimmune conditions:

  • FSTL1 is a common rheumatoid arthritis auto-antigen

  • May promote inflammatory cytokine secretion or inhibit matrix metalloproteinase expression

  • Consider measuring anti-FSTL1 antibodies in autoimmune research
    When designing biomarker studies, researchers should note that tissue and circulating FSTL1 levels may not always correlate. For example, in cervical carcinoma, while tissue FSTL1 is significantly reduced, serum levels remain comparable between patients and healthy controls .

What are the key considerations for developing FSTL1-targeted therapeutic approaches?

When exploring FSTL1 as a therapeutic target, researchers should consider:

• Context-dependent effects:

  • Cardioprotective in heart and muscle tissues

  • Tumor suppressive in many cancers

  • Both pro- and anti-inflammatory depending on context

• Delivery considerations:

  • Recombinant protein therapy may require proper glycosylation

  • Gene therapy approaches must account for tissue-specific functions

  • Consider the impact of glycosylation on therapeutic efficacy

• Potential applications:

  • Cardiac regeneration after myocardial infarction

  • Cancer therapy, particularly for cervical carcinoma where FSTL1 suppresses tumor growth

  • Modulation of inflammatory responses in autoimmune conditions

• Experimental validation pipeline:

  • In vitro studies with multiple cell types

  • Ex vivo tissue models

  • In vivo conditional knockout and overexpression models

  • Dose-response studies with recombinant proteins of different glycosylation states

How can researchers address inconsistent FSTL1 detection in experimental samples?

When encountering detection issues:

• Protein source considerations:

  • Ensure antibodies recognize the appropriate epitopes based on species and post-translational modifications

  • Validated antibodies like Clone #229001R (for ELISA capture) and polyclonal antibodies (for detection) have proven efficacy

• Sample preparation optimization:

  • For tissue sections, heat-induced epitope retrieval significantly improves detection

  • For western blots, reducing conditions are essential

  • For cellular samples, proper fixation protocols enhance immunofluorescence results

• Detection system selection:

  • Use HRP-conjugated systems for high sensitivity in IHC and western blots

  • Consider fluorophore-conjugated antibodies for co-localization studies

  • For quantitative analysis, sandwich ELISA with validated antibody pairs offers reliable results

What strategies help resolve contradictory findings in FSTL1 research?

To address contradictory findings:

• Glycosylation status:

  • Always document the source of FSTL1 (bacterial vs. eukaryotic expression systems)

  • Consider testing both glycosylated and non-glycosylated forms in parallel

  • Report observed differences based on glycosylation state

• Experimental context:

  • Clearly define the cellular/tissue context in each experiment

  • Use multiple cell lines or primary cells to verify findings

  • Consider conducting experiments under both normal and stress conditions

• Signaling pathway analysis:

  • Monitor multiple pathways simultaneously (e.g., BMP/Smad, AKT, TGF-β)

  • Use specific pathway inhibitors to dissect mechanisms

  • Evaluate both immediate and delayed effects on signaling

• Comprehensive reporting:

  • Document experimental methods in detail, including antibody sources, clone numbers, and detection systems

  • Report negative findings alongside positive results

  • Consider methodological triangulation using multiple detection techniques By addressing these experimental considerations, researchers can better navigate the complexities of FSTL1 biology and contribute to a more coherent understanding of this multifunctional protein.