CYTL1 Human

What is CYTL1 and what are its basic characteristics?

CYTL1 is a secreted protein containing 136 amino acids that was first identified in CD34+ hematopoietic cells derived from bone marrow and cord blood . It encodes a polypeptide of 126 amino acid residues that displays characteristics of a secretory protein . The CYTL1 gene has been mapped to human chromosome 4p15-p16 . CYTL1 demonstrates substantial sequence similarities among human, mouse, rat, bovine, and dog homologs, with human CYTL1 being most closely related to bovine and dog orthologs, followed by mouse and rat .

Where is CYTL1 primarily expressed in human tissues?

CYTL1 is abundantly expressed in cartilaginous tissues, including the human articular cartilage and mouse inner ear . Gene profiling analysis reveals that human CYTL1 is most abundantly and specifically expressed in trachea (a cartilage-rich tissue) and CD34-positive cells . This tissue-specific expression pattern has led researchers to investigate its role in cartilage homeostasis and development. CYTL1 expression patterns vary in different cancer types, with some showing upregulation and others showing downregulation of CYTL1 .

What are the primary biological functions of CYTL1?

CYTL1 exhibits multiple biological functions that have been characterized through various experimental approaches:

• Chemotactic activity: CYTL1 can chemoattract human monocytes through the CCR2B-ERK pathway .

• Cartilage homeostasis: CYTL1 contributes to cartilage homeostasis and inhibits osteoarthritic cartilage destruction .

• Anti-inflammatory properties: CYTL1 can prevent inflammatory arthritis and downregulate the expression of inflammatory cytokines .

• Proangiogenic function: CYTL1 acts as a novel proangiogenic factor that can induce endothelial sprouting and vessel formation .

• Tumor suppression: In some cancers like breast cancer, CYTL1 functions as a tumor suppressor by inhibiting tumor metastasis and decreasing STAT3 phosphorylation .

• Bone homeostasis regulation: CYTL1 negatively regulates osteogenesis of mesenchymal stem cells and positively regulates osteoclastogenesis to modulate bone mass .

How does CYTL1 interact with cellular receptors and what signaling pathways are involved?

CYTL1 has been shown to interact primarily with the CCR2B receptor, which was identified through a series of experiments including chemotaxis, receptor internalization, and radioactive binding assays . The CYTL1-CCR2B interaction activates the ERK signaling pathway in human monocytes, leading to chemotactic responses .
The chemotactic activity of CYTL1 is sensitive to pertussis toxin, indicating the involvement of G-protein coupled receptor signaling . In tumor biology, CYTL1 has been observed to inhibit STAT3 phosphorylation, which may contribute to its tumor-suppressive effects in certain cancer types . The signaling mechanisms may differ depending on the cellular context and tissue type, which explains the varied biological effects of CYTL1 in different systems.

What is the relationship between CYTL1 and the CCR2 receptor?

Research has confirmed that CCR2B is a functional receptor of CYTL1 . This was demonstrated through multiple experimental approaches:

• Both human and mouse recombinant CYTL1 proteins showed chemotactic effects on macrophages from wild-type mice but not from Ccr2-/- mice .

• CYTL1-mediated chemotaxis in human monocytes is dependent on the CCR2B-ERK pathway .

• HEK293 cells expressing CCR2B or CCR2B-EGFP responded to CYTL1 in chemotaxis, receptor internalization, and radioactive binding assays .
  The structural similarity between CYTL1 and CCL2 (a known ligand for CCR2) likely contributes to their shared receptor specificity, though CYTL1 has distinct biological activities compared to CCL2 .

How does CYTL1 affect STAT3 signaling in tumor cells?

CYTL1 has been observed to inhibit tumor metastasis by decreasing STAT3 phosphorylation in multiple tumor types . In experimental settings using recombinant CYTL1 and CYTL1-overexpressing tumor cell lines, researchers found that while CYTL1 exerted no obvious effect on tumor cell proliferation, it significantly inhibited tumor cell migration and invasion, coinciding with decreased STAT3 phosphorylation .
This mechanism appears to be important for CYTL1's tumor-suppressive role in certain cancers such as breast cancer. The inhibition of STAT3 phosphorylation by CYTL1 may disrupt multiple downstream oncogenic pathways, as STAT3 is a known regulator of genes involved in cell proliferation, survival, and invasion .

How does CYTL1 expression vary across different cancer types?

CYTL1 expression shows remarkable variability across different cancer types, revealing its context-dependent roles in oncogenesis:

• Downregulation in multiple cancers: Bioinformatics analyses have revealed that CYTL1 expression is notably decreased and hypermethylated in various tumors, including breast and lung cancer .

• Upregulation in neuroblastoma: High levels of CYTL1 expression have been found in tumor tissues and cell lines of human neuroblastoma .

• Progressive upregulation in melanoma: Analysis of the GSE46517 and GSE114445 datasets showed that CYTL1 expression progressively increases from normal skin to nevi or malignant nevi to melanoma, suggesting its potential role in melanoma progression .

• Hypermethylation in lung squamous cell carcinoma: CYTL1 is overexpressed and heavily methylated in human lung squamous cell carcinoma, with hypermethylation consistent with its downregulation in SCC .
  These differential expression patterns suggest that CYTL1 may have tumor-type specific functions, acting as a tumor suppressor in some contexts and as an oncogenic factor in others.

What is the prognostic significance of CYTL1 expression in various cancers?

Analysis of CYTL1's prognostic impact across different tumors using the TIMER2.0 database revealed:

What mechanisms underlie CYTL1's tumor-suppressive effects in breast cancer?

In breast cancer, CYTL1 functions as a tumor suppressor through several mechanisms:

• Inhibition of metastasis: CYTL1 significantly inhibits tumor metastasis in experimental and spontaneous metastasis models of breast cancer in BALB/c mice .

• Reduction of cell migration and invasion: While showing no obvious effect on tumor cell proliferation, CYTL1 inhibits the migration and invasion of breast cancer cells .

• Suppression of STAT3 signaling: The anti-metastatic effects of CYTL1 are accompanied by decreasing STAT3 phosphorylation, which is a known driver of oncogenic pathways .

• Metabolic reprogramming prevention: Recent research has shown that in breast cancer cells, CYTL1 acts as a tumor suppressor by keeping NDUFV1 stable and preventing metabolic reprogramming .
  These findings suggest that CYTL1 exerts its tumor-suppressive effects in breast cancer primarily through inhibition of metastasis and invasion, rather than through direct effects on cell proliferation.

How does CYTL1 contribute to melanoma progression and immune evasion?

Recent research indicates that CYTL1 may have pro-oncogenic functions in melanoma through several mechanisms:

• Correlation with immune checkpoint genes: Analysis showed significant correlations between CYTL1 expression and immune checkpoint-related genes in skin cutaneous melanoma (SKCM), including PDCD1, CD274, HAVCR2, TIGIT, SIGLEC15, CTLA4, LAG3, and PDCD1LG2 .

• Impact on immune cell infiltration: The different copy statuses of CYTL1 affected immune cell infiltration compared to normal tissues, suggesting that CYTL1-mediated melanoma oncogenesis may involve tumor immune escape and interference with anti-tumor immunity .

• Promotion of invasion and migration: Knockdown of CYTL1 in A2058 melanoma cells significantly inhibited cell migration and invasion capabilities, consistent with the positive correlation between CYTL1 and epithelial-mesenchymal transition (EMT) .

• Limited effect on proliferation: CYTL1 knockdown had minimal impact on the proliferation of A2058 cells, suggesting its role in melanoma is primarily related to invasion and metastasis rather than tumor growth .
  These findings indicate that CYTL1 may be a valuable prognostic biomarker and a potentially effective therapeutic target in melanoma, especially BRAF-mutated melanoma .

What techniques are commonly used to study CYTL1 expression and function?

Researchers employ various techniques to investigate CYTL1 expression and functional properties:

• Gene expression analysis:

  • RT-qPCR for mRNA quantification in cells and tissues

  • RNA sequencing for transcriptomic analysis

  • Bioinformatics analysis of public datasets (TCGA, GTEx, etc.)

• Protein detection methods:

  • Western blot for protein expression analysis

  • Immunohistochemistry for tissue localization

  • ELISA for quantifying secreted CYTL1 in biological fluids

• Functional assays:

  • Chemotaxis assays to assess monocyte/macrophage attraction

  • Receptor internalization assays to study receptor-ligand interactions

  • Radioactive binding assays to confirm receptor specificity

  • Cell migration and invasion assays (wound healing, transwell) to assess metastatic potential

  • Angiogenesis assays (endothelial sprouting, vessel formation)

• Genetic manipulation:

  • Gene knockdown using siRNA or shRNA

  • Gene overexpression systems

  • Knockout mouse models (Cytl1−/−)

• Pathway analysis:

  • Phosphorylation status assessment (e.g., STAT3)

  • Signaling pathway inhibitors (e.g., pertussis toxin for G-protein coupled signaling)
    These methodologies provide complementary approaches to understand CYTL1's expression patterns, interaction partners, and functional effects in various biological contexts.

How can recombinant CYTL1 be produced for experimental studies?

The production of high-quality recombinant CYTL1 protein is crucial for functional studies and has been achieved through several approaches:

• Stable expression systems: Researchers have established stable expression systems to obtain high-quality CYTL1 protein . This typically involves:

  • Cloning the CYTL1 coding sequence into a suitable expression vector

  • Transfecting mammalian cell lines (like HEK293) with the expression construct

  • Selecting stable transfectants

  • Harvesting and purifying the secreted protein from cell culture supernatants

• Signal peptide optimization: Studies have demonstrated that replacement of the native signal peptide can improve expression and secretion efficiency . This approach may be particularly useful when high yields of recombinant protein are required.

• Post-translational modifications: It's important to consider that CYTL1 undergoes O-glycosylation, and the specific carbohydrate structure is needed for biological activity . Therefore, expression systems that support appropriate post-translational modifications (typically mammalian cell lines) are preferred over bacterial expression systems.

• Purification strategies: Common purification approaches include:

  • Affinity chromatography (using tagged versions of the protein)

  • Size exclusion chromatography

  • Ion exchange chromatography

  • Validation of protein purity by SDS-PAGE and Western blotting

• Activity validation: Following purification, the biological activity of recombinant CYTL1 should be confirmed using functional assays such as chemotaxis assays with monocytes or macrophages .

What animal models are available for studying CYTL1 function in vivo?

Several animal models have been developed to investigate CYTL1 function in different biological contexts:

• Cytl1 knockout mice: Cytl1−/− mice have been generated and characterized . These mice exhibit:

  • Higher bone mass than wild-type littermates

  • Resistance to ovariectomy-induced bone resorption

  • Inhibition of osteoclast activity and osteoclastogenesis of bone marrow-derived macrophages

  • Normal endochondral ossification and long-bone development

• Cancer metastasis models:

  • Experimental and spontaneous metastasis models of breast cancer in BALB/c mice have been used to demonstrate CYTL1's inhibitory effect on tumor metastasis .

  • These models can involve the injection of CYTL1-overexpressing tumor cell lines to assess effects on tumor growth and metastasis.

• Angiogenesis models:

  • In vivo models assessing vessel formation have shown that CYTL1 can trigger endothelial sprouting and vessel formation similar to VEGF-A .

  • These models can help understand CYTL1's role in vascularization and its potential therapeutic applications in vascular disorders.

• Inflammatory and cartilage-related models:

  • Models of inflammatory arthritis have shown that CYTL1 prevents inflammation and downregulates inflammatory cytokines .

  • Cartilage-specific models can help elucidate CYTL1's role in cartilage homeostasis and osteoarthritis progression.
    These animal models provide valuable insights into CYTL1's diverse functions in different physiological and pathological contexts, enabling researchers to investigate its potential as a therapeutic target.

How might CYTL1's dual roles in different cancer types be explained?

The contradictory roles of CYTL1 across different cancer types present an intriguing research question. Several hypotheses may explain these observations:

• Tissue-specific signaling networks: The downstream effects of CYTL1 likely depend on the specific signaling networks active in different tissue types or cancer contexts. In breast cancer, CYTL1 inhibits STAT3 phosphorylation and suppresses metastasis , while in melanoma, it appears to promote invasion and migration .

• Differential receptor expression: The expression levels of CCR2B and potentially other unidentified CYTL1 receptors may vary across cancer types, leading to different functional outcomes.

• Epigenetic regulation: CYTL1 is hypermethylated in various tumors , suggesting that epigenetic mechanisms play a role in regulating its expression and potentially its function in different cancer contexts.

• Microenvironmental influences: The tumor microenvironment, including immune cell populations and stromal components, varies considerably between cancer types and may modulate CYTL1's effects.

• Co-expression with regulatory partners: CYTL1's function may be influenced by co-expressed regulatory proteins that differ between cancer types.
  Future research using comparative multi-omics approaches across different cancer types could help elucidate the molecular basis for CYTL1's context-dependent functions.

What is the potential of CYTL1 as a therapeutic target or biomarker?

Based on current research, CYTL1 shows promise as both a biomarker and therapeutic target in several contexts:

How does CYTL1 interact with the immune system, and what are the implications for immunotherapy?

CYTL1's interaction with the immune system is complex and potentially significant for immunotherapeutic approaches:

• Chemotactic effects on immune cells:

  • CYTL1 chemoattracts human monocytes and macrophages via the CCR2B-ERK pathway .

  • This chemotactic activity suggests CYTL1 may influence immune cell recruitment to specific tissues, including tumors.

• Correlation with immune checkpoint molecules:

  • In melanoma, CYTL1 expression correlates significantly with immune checkpoint genes such as PDCD1, CD274, HAVCR2, TIGIT, SIGLEC15, CTLA4, LAG3, and PDCD1LG2 .

  • These correlations suggest that CYTL1 may be involved in mechanisms of immune evasion in cancer.

• Impact on immune cell infiltration:

  • The copy number status of CYTL1 affects immune cell infiltration in melanoma compared to normal tissues .

  • This finding indicates that CYTL1 may modulate the tumor immune microenvironment, potentially affecting responses to immunotherapy.

• Anti-inflammatory properties:

  • CYTL1 has been shown to prevent inflammatory arthritis and downregulate inflammatory cytokines .

  • In endothelial cells, CYTL1 induces anti-inflammatory and antiapoptotic genes rather than inflammatory genes .
    These observations suggest that CYTL1 may have significant implications for immunotherapeutic approaches, particularly in cancer types where it correlates with immune checkpoint molecules. Further research investigating how CYTL1 modulates the immune microenvironment and influences responses to immune checkpoint inhibitors would be valuable for developing combination therapeutic strategies.

How should contradictory findings on CYTL1 function be reconciled in research?

Researchers face several challenges when reconciling contradictory findings on CYTL1 function across different studies:

• Contextual considerations:

  • Tissue-specific effects: CYTL1 functions differently in cartilage, bone, vascular tissue, and various cancer types.

  • Experimental systems: Results may vary between in vitro cell lines, primary cells, and in vivo models.

• Methodological approaches:

  • When comparing studies, carefully evaluate differences in:

    • CYTL1 protein sources (recombinant vs. endogenous)

    • Expression systems and post-translational modifications

    • Experimental readouts and timelines

    • Genetic backgrounds of model organisms

• Analytical strategies:

  • Meta-analysis of multiple studies can help identify consistent patterns

  • Pathway-focused analysis may reconcile seemingly contradictory effects by identifying common molecular mechanisms

  • Multi-omics integration can provide a more comprehensive understanding of CYTL1's complex effects

• Validation experiments:

  • Direct comparison studies using standardized protocols across multiple cell types or tissues

  • Confirmation of findings using complementary methodologies

  • Careful dose-response analyses to identify concentration-dependent effects

• Consideration of splice variants or post-translational modifications:

  • Investigate whether different CYTL1 isoforms or modifications explain divergent findings

  • Assess the impact of O-glycosylation on CYTL1 function across different experimental systems
    By systematically addressing these considerations, researchers can better understand the context-dependent nature of CYTL1 function and develop more accurate models of its role in health and disease.

What are the key considerations for analyzing CYTL1 expression data across different datasets?

When analyzing CYTL1 expression data across different datasets, researchers should consider several important factors:

• Normalization approaches:

  • Different normalization methods (FPKM, TPM, counts per million) can affect interpretation

  • When comparing datasets, ensure consistent normalization or apply appropriate transformations

  • Consider using relative expression values when absolute values may not be comparable

• Platform-specific biases:

  • Microarray vs. RNA-seq data may show systematic differences

  • Probe design (for microarrays) may affect detection efficiency

  • Sequencing depth and coverage (for RNA-seq) impact detection sensitivity

• Reference datasets:

  • Use appropriate control tissues (e.g., GTEx for normal tissue expression)

  • Match for demographic factors when possible (age, sex, ethnicity)

  • Consider tissue heterogeneity and single-cell vs. bulk sequencing approaches

• Statistical considerations:

  • Apply appropriate statistical tests based on data distribution

  • Adjust for multiple comparisons (e.g., using FDR correction)

  • Consider the impact of outliers and small sample sizes

• Validation strategies:

  • Verify key findings in independent datasets

  • Confirm RNA-level expression changes at the protein level when possible

  • Consider functional validation of expression differences

• Contextual interpretation:

  • Interpret expression changes in light of pathway analysis

  • Consider co-expression patterns with known interacting partners

  • Evaluate potential technical vs. biological sources of variation
    By addressing these considerations, researchers can enhance the reliability and interpretability of CYTL1 expression analyses across diverse datasets and experimental contexts.

How can researchers design experiments to definitively establish CYTL1's role in specific biological processes?

To definitively establish CYTL1's role in specific biological processes, researchers should consider comprehensive experimental designs:

• Loss and gain of function approaches:

  • Generate both knockout models (Cytl1−/−) and overexpression systems

  • Use inducible or tissue-specific expression systems to control timing and location

  • Apply CRISPR-Cas9 gene editing for precise genetic manipulation

  • Complement with neutralizing antibodies or recombinant protein treatment

• Mechanistic dissection:

  • Identify direct interaction partners through techniques like co-immunoprecipitation, proximity labeling, or yeast two-hybrid screening

  • Map signaling pathways using phospho-proteomics and pathway inhibitors

  • Perform receptor binding studies with mutant variants to identify critical interaction domains

  • Use live cell imaging to track CYTL1 dynamics and localization

• Physiological relevance:

  • Design experiments in primary cells rather than relying solely on immortalized cell lines

  • Validate findings in appropriate animal models

  • Consider species differences when translating between mouse and human studies

  • Examine effects under physiological stress conditions (e.g., inflammation, hypoxia)

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Perform ChIP-seq to identify transcriptional effects

  • Use spatial transcriptomics to capture tissue-specific contexts

  • Apply single-cell approaches to resolve cellular heterogeneity

• Translational validation:

  • Correlate experimental findings with clinical samples

  • Establish robust biomarkers based on CYTL1 expression or activity

  • Develop therapeutic strategies based on mechanistic insights

  • Assess potential off-target effects by examining multiple biological systems By implementing these comprehensive experimental strategies, researchers can establish definitive evidence for CYTL1's specific roles across different biological contexts and resolve current contradictions in the literature.