FSTL1 Human, HEK

What is human FSTL1 and what are its primary biological functions?

FSTL1 (also known as Follistatin-related protein 1 or Follistatin-like protein 1, FRP) is a secreted glycoprotein involved in various physiological processes including angiogenesis, immune response regulation, cell proliferation, and differentiation. The protein plays critical roles in the development of multiple systems including the central nervous system, skeletal system, lungs, and ureter. FSTL1 initiates various signaling cascades by activating different cell surface receptors such as DIP2A, TLR4, or BMP receptors . In adipose tissue biology, FSTL1 is highly expressed in brown adipocyte progenitors, where it appears essential for maintaining brown adipose tissue (BAT) integrity and preventing its transformation into white adipose tissue with age .

What is the molecular structure and biochemical characteristics of human FSTL1?

Human FSTL1 is a full-length protein spanning amino acids 21 to 308. The protein sequence includes multiple functional domains and undergoes post-translational modifications, particularly glycosylation . Recombinant FSTL1 produced in HEK 293 cells contains the amino acid sequence: EELRSKSKICANVFCGAGRECAVTEKGEPTCLCIEQCKPHKRPVCGSNGKTYLNHCELHRDACLTGSKIQVDYDGHCKEKKSVS PSASPVVCYQSNRDELRRRIIQWLEAEIIPD... (continuing through position 308) . The protein functions as a secreted glycoprotein with significant extracellular matrix interactions, reflecting its role in tissue organization and cell-cell communication processes .

How does FSTL1 expression change during development and aging?

FSTL1 expression exhibits tissue-specific and age-dependent regulation. In brown adipose tissue, FSTL1 shows high expression in progenitor cells during early development but decreases significantly with age . Research using rabbit models demonstrates that during BAT involution (transformation to white adipose tissue), FSTL1 expression decreases in adipocyte progenitors alongside other marker genes including DLK1, CILP, GPC3, and COL12A1 . In human tissues, FSTL1 expression is dynamically regulated, with upregulation observed in epidermal keratinocytes following acute injury, where it promotes cell migration . These expression patterns suggest FSTL1 plays important roles in tissue development, maintenance, and repair processes that change throughout the lifespan .

What expression systems are optimal for producing research-grade human FSTL1?

HEK 293 cells represent a preferred expression system for recombinant human FSTL1 production, as they enable proper post-translational modifications, particularly glycosylation, which may be critical for FSTL1's biological activity. When expressed in HEK 293 cells, recombinant human FSTL1 can be produced with >95% purity and low endotoxin levels (<1 EU/μg), making it suitable for diverse research applications . The recombinant protein typically includes the functional domains spanning amino acids 21-308, often with affinity tags (such as histidine tags) to facilitate purification . Quality control methods for FSTL1 expressed in HEK systems typically include SDS-PAGE and HPLC analysis to confirm purity and integrity .

What purification strategies yield the highest quality FSTL1 for functional studies?

Although specific purification protocols aren't detailed in the available research, high-purity recombinant FSTL1 (>95%) with low endotoxin contamination (<1 EU/μg) has been successfully produced . Based on standard approaches for glycoproteins expressed in HEK 293 cells, optimal purification strategies likely involve multiple chromatography steps. The presence of histidine tags in recombinant FSTL1 suggests immobilized metal affinity chromatography (IMAC) as a primary purification method, potentially followed by ion exchange and/or size exclusion chromatography to achieve high purity. Endotoxin removal steps are critical, particularly for functional studies where inflammatory responses would confound results. The resulting purified protein should maintain appropriate glycosylation patterns and structural integrity for maximum biological activity in experimental systems .

How can researchers verify the functional activity of purified FSTL1?

Verification of FSTL1 functional activity should include multiple assays assessing its known biological functions. Based on FSTL1's reported activities, appropriate functional assays may include: (1) Cell migration assays using endothelial cells or keratinocytes, as FSTL1 promotes cell migration in wound healing contexts ; (2) Adipocyte differentiation assays, particularly examining its effects on brown adipocyte progenitor maintenance and differentiation ; (3) Signaling pathway activation assays, measuring WNT signaling activity, as genetic deletion of FSTL1 causes defective WNT signaling in mice ; (4) Receptor binding assays to confirm interaction with known receptors including DIP2A, TLR4, or BMP receptors ; and (5) Phosphorylation analysis, as site-specific phosphorylation appears important for FSTL1's promigratory function . These functional assays should be complemented by structural verification through methods like circular dichroism or mass spectrometry to confirm proper folding and post-translational modifications.

How does FSTL1 expression correlate with brown adipose tissue maintenance?

FSTL1 expression strongly correlates with brown adipose tissue maintenance across species. High-level expression of FSTL1 in adipocyte progenitors is associated with the constitutive presence of interscapular brown adipose tissue (iBAT) in mice . Conversely, the loss of FSTL1-expressing progenitors corresponds with BAT involution (transformation to white adipose tissue) in both rabbits and humans . Single-cell RNA sequencing reveals that FSTL1-high adipocyte progenitors constitute approximately 80.5% of stromal vascular fraction cells in neonatal rabbit iBAT but progressively decrease with age as the tissue transforms to white adipose tissue . Genetic deletion of FSTL1 in brown adipocyte progenitors causes iBAT atrophy in mice, further demonstrating its essential role in tissue maintenance . These findings collectively suggest that FSTL1 expression in progenitor cells is critical for maintaining the brown adipocyte phenotype and preventing transformation to white adipose tissue during aging .

What transcriptional changes occur in adipocyte progenitors during BAT involution?

Single-cell RNA sequencing of stromal vascular fraction cells from interscapular BAT reveals extensive transcriptional reprogramming during tissue involution. In neonatal rabbits, adipocyte progenitors (Group 1) highly express FSTL1 along with other markers including DLK1, CILP, GPC3, and COL12A1 . As BAT transforms to white adipose tissue at 3 and 12 weeks of age, these cells transition to Group 0 and Group 6 progenitors with decreased expression of BAT-associated markers . Concurrently, these cells begin expressing white adipocyte progenitor markers including DPP4 and PI16, indicating acquisition of white adipocyte progenitor characteristics .

Pathway analysis reveals that top activated pathways during BAT development include EIF2 and mTOR pathways, while LXR/RXR and PPAR signaling are inhibited during this transition . Particularly in adipocyte progenitors, genes activated by anti-adipogenic TGFB1 are highly expressed in early progenitors, while genes controlled by adipogenic factors PPARG, PPARA, and CEBPB increase in committed preadipocytes . This transcriptional shift appears to drive the tissue's transformation from energy-burning brown fat to energy-storing white fat during development .

How do rabbit and human FSTL1-expressing brown adipocyte progenitors compare?

Single-cell RNA sequencing has identified remarkably similar FSTL1-expressing brown adipocyte progenitor populations in both rabbit and human BAT. Comparison of upregulated marker genes between human Group 0 progenitors and rabbit Group 1 progenitors revealed 37 overlapping genes, with 9 genes showing particularly strong expression (log2-fold-change >0.5) in both species: DLK1, FSTL1, CILP, GPC3, COL12A1, MFAP4, FBN1, POSTN, and FBLN5 . Interestingly, 17 of these 37 shared marker genes encode extracellular proteins, and all 9 top marker genes encode either cytokines or extracellular matrix proteins, indicating high secretory activity as a common feature of brown adipocyte progenitors across species .

Both rabbit and human BAT undergo involution (transformation to white adipose tissue) at similar developmental stages, making rabbits a more appropriate model than mice for studying human BAT biology . These similarities suggest that molecular mechanisms governing BAT maintenance and involution may be conserved between rabbits and humans, with FSTL1-expressing progenitors playing a central role in both species .

What experimental approaches can effectively manipulate FSTL1 expression in adipocyte progenitors?

Based on research methodologies described in the literature, several approaches can effectively manipulate FSTL1 expression in adipocyte progenitors:

• Genetic deletion using tissue-specific Cre-loxP systems targeting brown adipocyte progenitors, which has successfully demonstrated FSTL1's role in iBAT development in mice

• Single-cell isolation and ex vivo culture of FSTL1-expressing progenitors, allowing detailed characterization of their differentiation potential

• Cell surface marker-based isolation using novel markers identified through scRNA-seq, such as ADGRD1 (GPR133) and SFRP4 for adipocyte progenitors

• Transcriptional profiling at single-cell resolution to track FSTL1 expression dynamics during differentiation and in response to environmental stimuli

• Phosphorylation site analysis and mutation studies to investigate how post-translational modifications affect FSTL1's function in progenitor cells

These approaches can be combined with functional assays measuring adipogenic differentiation, thermogenic capacity, and tissue maintenance to comprehensively assess FSTL1's role in adipose tissue biology .

How does FSTL1 phosphorylation affect its biological activities?

Research indicates that site-specific phosphorylation of FSTL1 significantly influences its biological activities, particularly its promigratory functions . While complete details of the phosphorylation mechanisms aren't fully described in the available literature, evidence suggests that phosphorylation regulates FSTL1's role in cellular migration, which is particularly important in contexts such as wound healing . FSTL1 is upregulated in epidermal keratinocytes following acute injury, where it promotes cell migration necessary for wound repair . The phosphorylation state likely affects FSTL1's interaction with cell surface receptors such as DIP2A, TLR4, or BMP receptors, thereby modulating downstream signaling cascades .

Understanding the specific phosphorylation sites, the kinases responsible, and the resulting conformational changes would provide valuable insights into how FSTL1's activity is regulated post-translationally and could reveal potential targets for therapeutic manipulation .

What signaling pathways interact with FSTL1 in tissue development and maintenance?

FSTL1 interacts with multiple signaling pathways that collectively regulate tissue development and maintenance:

• WNT signaling: Genetic deletion of FSTL1 causes defective WNT signaling and iBAT atrophy in mice, suggesting FSTL1 positively regulates this pathway critical for adipose tissue development

• TGF-β signaling: FSTL1-high adipocyte progenitors express genes activated by anti-adipogenic TGFB1, indicating potential crosstalk between FSTL1 and TGF-β signaling in maintaining progenitor identity

• BMP pathway: FSTL1 can activate BMP receptors, potentially modulating cell differentiation programs

• mTOR signaling: This pathway is activated during BAT development and thermogenesis, with potential connections to FSTL1 activity

• PPAR signaling: This adipogenic pathway is regulated during BAT development and may be influenced by FSTL1 expression

These interconnected pathways collectively regulate progenitor cell maintenance, differentiation, and tissue homeostasis, with FSTL1 appearing to play a central role in coordinating these processes, particularly in brown adipose tissue .

How might preserving FSTL1-expressing adipocyte progenitors impact metabolic health?

Maintaining or restoring FSTL1-expressing progenitors could potentially preserve BAT function throughout life, promoting energy expenditure and improving metabolic parameters . The authors propose that "preserving or regenerating FSTL1-high brown adipocyte progenitors may prevent BAT involution or rejuvenate aged BAT in elderly, thereby promoting metabolic fitness" . This approach represents a potential therapeutic strategy for addressing obesity and its comorbidities by targeting the fundamental cellular mechanisms of adipose tissue maintenance .

What experimental challenges exist in translating FSTL1 research to human applications?

Translating FSTL1 research from animal models to human applications faces several significant challenges:

• Model system limitations: Mice inadequately model human BAT involution, while rabbits provide a better but still imperfect model; these species differences complicate direct translation

• Developmental timing differences: Human BAT undergoes involution at different developmental stages compared to animal models, requiring careful consideration of timing in potential interventions

• Tissue accessibility: Human BAT is anatomically distributed differently than in animal models and accessing it for research or therapeutic targeting presents technical challenges

• Cellular heterogeneity: Single-cell analysis reveals complex cellular populations within adipose tissue that change with age, requiring sophisticated targeting approaches

• Delivery methods: Effectively delivering therapeutics specifically to FSTL1-expressing progenitors in humans would require advanced targeting systems not yet developed

• Post-translational regulation: The functional significance of FSTL1 phosphorylation and other modifications in human tissues requires further characterization

Addressing these challenges will require integrated approaches combining advanced single-cell technologies, improved human tissue models, and innovative delivery systems to effectively translate FSTL1 research into clinical applications .

What are the most promising methods for therapeutic modulation of FSTL1 function?

Based on current research, several approaches show promise for therapeutic modulation of FSTL1 function:

• Recombinant protein therapy: Administration of purified FSTL1 protein, similar to the HEK-produced version described in the literature, could potentially supplement endogenous FSTL1 activity in target tissues

• Progenitor cell preservation: Strategies to maintain or enhance FSTL1 expression in adipocyte progenitors could prevent BAT involution and promote metabolic health

• Signaling pathway modulation: Targeting the WNT signaling pathway or other pathways affected by FSTL1 could potentially mimic its effects on tissue maintenance

• Phosphorylation regulation: Modulating the phosphorylation state of FSTL1 could enhance its promigratory or other functions in specific contexts like wound healing

• Cell-based therapies: Transplantation of FSTL1-expressing progenitors or engineered cells could regenerate functional brown adipose tissue in elderly individuals

While significant research is still needed, these approaches offer potential avenues for therapeutic intervention based on FSTL1's established roles in tissue maintenance and cellular function .