CTHRC1 Human, HEK

What is CTHRC1 and what is its basic structure?

CTHRC1 (Collagen Triple Helix Repeat Containing-1) is a secreted glycoprotein that contains a short collagen triple helix repeat NH2-terminal peptide and a COOH-terminal domain . Intracellular CTHRC1 exists as a 26 kDa protein corresponding to the monomeric form, while secreted CTHRC1 can exist in multiple forms consisting of 26, 50, and 75 kDa species, which correspond to monomer, homodimer, and homotrimer sizes respectively . When expressing CTHRC1 in research settings, it's important to note that the protein may form homo- and heterodimeric complexes when secreted into the medium .

What is the cellular localization of CTHRC1?

Based on immunofluorescence assays, ectopically expressed GFP-tagged CTHRC1 is mainly localized in the cytoplasm, whereas endogenous CTHRC1 can be detected in both the nucleus and cytoplasm . This dual localization suggests potential diverse functions depending on its cellular compartmentalization. When conducting localization studies, researchers should consider using both tagged and untagged versions of the protein to confirm that the tag does not interfere with natural localization patterns.

How does CTHRC1 affect normal cellular processes?

CTHRC1 appears to influence cell migration without significantly affecting cell proliferation. In experiments with HepG2 cells, overexpression of CTHRC1 did not affect cell proliferation as measured by MTT assay, nor did it change colony generation ability as assessed by colony formation and soft agar assays . Instead, CTHRC1's primary function seems to be related to promoting cell migration and modulating extracellular matrix remodeling. In vascular contexts, CTHRC1 inhibits collagen I synthesis in rat fibroblasts and promotes cell migration, suggesting a role in tissue repair and vascular remodeling .

How does CTHRC1 contribute to cancer cell invasion and metastasis?

CTHRC1 promotes cancer cell invasion and metastasis through multiple mechanisms:

• Epithelial-Mesenchymal Transition (EMT): CTHRC1 activates the PI3K/Akt/CREB signaling pathway, leading to increased expression of Snail, which induces EMT characterized by decreased epithelial markers (E-cadherin, desmoplakin I/II) and increased mesenchymal markers (fibronectin, vimentin, N-cadherin, α-SMA) .

• Matrix Metalloproteinase Activation: CTHRC1 enhances the expression and activity of matrix metalloproteinases (MMPs), particularly MMP2 and MMP9, which degrade extracellular matrix components and facilitate cancer cell invasion .

• Signaling Pathway Activation: CTHRC1 activates Wnt/PCP signaling through activation of Rac1 in pancreatic cancer, RhoA in HCC, and both Rac1 and RhoA in gastrointestinal stromal tumors .

In vivo studies demonstrate that HepG2 cells stably expressing CTHRC1 form numerous metastatic nodules in lungs of mice following tail vein injection, whereas control cells form few metastatic nodules .

What signaling pathways are activated by CTHRC1 in cancer cells?

CTHRC1 activates multiple signaling pathways in cancer cells:

• PI3K/Akt/ERK Pathway: CTHRC1 overexpression leads to phosphorylation of PI3K, which subsequently activates Akt (at T308, S473, and S474) and ERK .

• CREB/Snail Pathway: Activated Akt and ERK lead to phosphorylation and activation of CREB, which, along with increased Snail expression, modulates the expression of MMPs and EMT-related proteins .

• Wnt/PCP Pathway: CTHRC1 selectively activates the planar cell polarity pathway through stabilizing the Wnt-receptor complex .

• EGFR Pathway: In epithelial ovarian cancer, CTHRC1 promotes invasion by stimulating the EGFR signaling pathway .

The specific pathway activation can be cancer-type dependent, as evidenced by differential activation of Rac1 and RhoA in different cancer types .

How is CTHRC1 expression regulated in cancer cells?

CTHRC1 expression is regulated at multiple levels:

• Growth Factor Regulation: TGF-β1 and EGF upregulate CTHRC1 expression in a dose-dependent manner in HepG2 and SH-J1 cells . The TGF-β1 inhibitor SB431542 efficiently suppresses CTHRC1 expression and inhibits activation of downstream molecules .

• microRNA Regulation: Bioinformatic analyses suggest microRNAs may regulate CTHRC1 expression, though specific microRNAs are not detailed in the search results .

• Transcriptional Regulation: While not explicitly detailed in the search results, the correlation between CTHRC1 mRNA levels and cancer progression suggests transcriptional regulation plays a significant role in its expression .

Understanding these regulatory mechanisms provides potential targets for therapeutic interventions aimed at reducing CTHRC1-mediated cancer invasion and metastasis.

What experimental models are suitable for studying CTHRC1's role in metastasis?

Several experimental models have proven effective for studying CTHRC1's role in metastasis:

• In Vitro Models:

  • Modified Boyden chamber assays to assess cell invasion through matrigel-coated membranes

  • Wound healing assays to evaluate cell migration capacity and wound closure ability

  • 3D culture systems (inferred from typical metastasis research, though not explicitly mentioned in search results)

• In Vivo Models:

  • Tail vein injection models in nude mice to assess metastatic colonization, monitored using bioluminescence imaging of luciferase-expressing cells

  • Orthotopic xenograft models to evaluate more physiologically relevant tumor metastasis

• Genetic Manipulation Approaches:

  • Gain-of-function: Stable transfection or adenoviral delivery of CTHRC1

  • Loss-of-function: Lentiviral delivery of CTHRC1 shRNA

The combination of these models provides complementary evidence for CTHRC1's role in promoting cancer metastasis.

What are the best methods for detecting CTHRC1 expression in cells and tissues?

Multiple complementary methods can be employed to detect CTHRC1 expression:

• Protein Detection:

  • Western blotting: Effective for detecting CTHRC1 protein in cell lysates, tissue samples, and secreted forms in culture supernatants or serum

  • Immunohistochemistry (IHC): Valuable for visualizing CTHRC1 expression in tissue sections, including paraffin-embedded tissues and tissue microarrays

  • Immunofluorescence (IF): Useful for determining the subcellular localization of CTHRC1 in cells

• mRNA Detection:

  • Real-time RT-PCR: Quantifies CTHRC1 mRNA levels with high sensitivity and specificity

• Validation Approaches:

  • Antibody specificity can be confirmed using HEK-293T cells transfected with GFP-tagged or Myc-tagged CTHRC1

  • Reducing and non-reducing SDS-PAGE can distinguish between monomeric and oligomeric forms of CTHRC1

The choice of method depends on the specific research question, sample type, and required sensitivity and specificity.

How can CTHRC1 be effectively overexpressed or knocked down in HEK cells?

Several approaches have been successfully employed for CTHRC1 manipulation:

• Overexpression Strategies:

  • Adenoviral vectors: Adeno-associated virus expressing CTHRC1 for transient transduction with high efficiency

  • Stable transfection: Establishment of cell lines persistently expressing CTHRC1

  • Tagged constructs: GFP-tagged or Myc-tagged CTHRC1 for detection and localization studies

• Knockdown Approaches:

  • Lentiviral shRNA: Lentiviral delivery of CTHRC1 short hairpin RNA for stable knockdown

  • siRNA approaches (inferred from common practices, though not explicitly mentioned in search results)

• Validation Considerations:

  • Confirm expression/knockdown at both mRNA level (RT-PCR) and protein level (Western blot)

  • Assess functional consequences using migration/invasion assays

  • Consider rescue experiments to confirm specificity of observed phenotypes

The selection of approach depends on required duration of expression modulation, target cell type, and experimental design constraints.

What experimental approaches can be used to study CTHRC1's effect on cell migration and invasion?

Multiple complementary approaches provide robust assessment of CTHRC1's effects:

• In Vitro Migration Assays:

  • Wound healing assay (scratch assay): Measures closure rate of an artificial "wound" in a cell monolayer

  • Time-lapse microscopy: Tracks individual cell movement (inferred from standard practices)

• In Vitro Invasion Assays:

  • Modified Boyden chamber assay: Assesses ability of cells to penetrate matrigel-coated membranes

  • 3D matrix invasion assays (inferred from standard practices)

• Morphological Assessment:

  • Phase-contrast microscopy to observe cell morphology changes associated with migratory phenotype

  • Immunofluorescence to visualize cytoskeletal rearrangements (inferred from standard practices)

• In Vivo Metastasis Models:

  • Tail vein injection followed by bioluminescence imaging to monitor metastatic colonization

  • Histological examination of metastatic nodules

A comprehensive experimental approach should include multiple assays to provide complementary evidence for CTHRC1's effects on cell migration and invasion.

How can researchers investigate the signaling pathways activated by CTHRC1?

Several approaches enable detailed characterization of CTHRC1-activated signaling:

• Screening Approaches:

  • Human phospho-kinase arrays to identify phosphorylated proteins following CTHRC1 expression

  • Transcriptomic analyses to identify downstream gene expression changes

• Pathway Validation:

  • Immunoblot analysis to confirm phosphorylation of specific proteins (PI3K, Akt, ERK, CREB)

  • Pharmacological inhibitors to validate pathway involvement:

    • KG501 for CREB inhibition

    • PD98059 for ERK inhibition

    • SB431542 for TGF-β1 inhibition

• Functional Analyses:

  • Knockdown experiments to assess effects of depleting specific pathway components

  • Reporter assays to measure transcriptional activity (e.g., MMP2 and MMP9 promoter activities)

  • Combined inhibitor and CTHRC1 expression studies to establish hierarchical relationships within pathways

These approaches collectively enable detailed mapping of CTHRC1-activated signaling networks and identification of key nodes for potential therapeutic targeting.

What are the common challenges in expressing and purifying CTHRC1 in HEK cells?

Several challenges must be addressed when working with CTHRC1:

• Oligomerization Complexity:

  • Secreted CTHRC1 exists in multiple forms (monomer, homodimer, homotrimer), complicating purification strategies

  • Different oligomeric forms may have distinct functional properties, requiring preservation of native structures

• Post-translational Modification Heterogeneity:

  • As a secreted glycoprotein, CTHRC1 likely undergoes complex post-translational modifications

  • Glycosylation patterns may affect antibody recognition and functional properties

• Subcellular Compartmentalization:

  • CTHRC1's presence in both cytoplasm and nucleus necessitates careful fractionation methods

  • Different subcellular pools may have distinct functions requiring separate analysis

• Secretion Dynamics:

  • Need to capture both intracellular and secreted forms for comprehensive studies

  • Timing of sample collection may significantly affect detection of secreted CTHRC1

These challenges require careful optimization of expression, purification, and detection protocols for successful CTHRC1 research.

How can researchers differentiate between monomeric and oligomeric forms of CTHRC1?

Distinguishing between CTHRC1 forms requires specific technical approaches:

• Electrophoretic Separation:

  • Reducing and non-reducing SDS-PAGE: Intracellular CTHRC1 exists primarily as a 26 kDa monomeric protein under both conditions, while secreted CTHRC1 appears as 26, 50, and 75 kDa species under non-reducing conditions

  • Native PAGE to preserve physiological oligomeric states

• Size-Based Separation:

  • Size exclusion chromatography to separate different oligomeric forms

  • Gradient ultracentrifugation for density-based separation

• Biophysical Characterization:

  • Analytical ultracentrifugation to determine sedimentation coefficients

  • Dynamic light scattering to assess size distribution

• Crosslinking Approaches:

  • Chemical crosslinking followed by SDS-PAGE to stabilize and visualize oligomeric interactions

These methods enable comprehensive characterization of CTHRC1's oligomeric states and their potential functional implications.

What factors influence CTHRC1 expression and secretion in experimental systems?

Several factors significantly impact CTHRC1 expression and secretion:

• Growth Factor Stimulation:

  • TGF-β1 and EGF upregulate CTHRC1 expression in a dose-dependent manner

  • TGF-β1 inhibitor SB431542 efficiently suppresses CTHRC1 expression

• Cell Type Considerations:

  • Expression levels vary significantly across cell lines (highly expressed in most HCC cell lines but weakly expressed in HepG2, HLK-2, and HLK-3 cells)

  • Cell-specific factors may influence post-translational processing and secretion

• Culture Conditions:

  • Serum levels may affect CTHRC1 expression through growth factor content

  • Cell density and extracellular matrix composition may influence expression and secretion

• Differentiation Status:

  • CTHRC1 expression correlates with differentiation grade in HCC, with higher expression in poorly differentiated tumors

Understanding these factors enables optimization of experimental conditions for consistent CTHRC1 expression and secretion studies.

How does CTHRC1 expression correlate with clinical features in cancer patients?

CTHRC1 exhibits significant correlations with multiple clinical parameters:

• Hepatocellular Carcinoma (HCC):

  • Positive correlation with large tumor size (p<0.003)

  • Positive correlation with Edmondson differentiation grade (p<0.0001)

  • Positive correlation with microvessel invasion (p<0.05)

  • Positive correlation with intrahepatic metastasis (p<0.005)

  • Positive correlation with HCC stage (AJCC, p<0.0001)

• Lung Adenocarcinoma (LUAD):

  • High CTHRC1 expression significantly correlates with poor prognosis

• Other Cancers:

  • Overexpression observed in pancreatic cancer, gastric cancer, colorectal cancer, and gastrointestinal stromal tumors

These correlations suggest CTHRC1 as a potential prognostic biomarker across multiple cancer types, with particular relevance to invasion and metastasis-related clinical parameters.

Can serum CTHRC1 levels be used as a biomarker for cancer detection or monitoring?

Evidence supports CTHRC1's potential as a serum biomarker:

• Diagnostic Potential:

  • CTHRC1 is detectable in the serum of HCC patients compared with normal control serum

  • Secreted CTHRC1 exists in multiple forms (monomer, homodimer, homotrimer) in serum

• Monitoring Treatment Response:

  • CTHRC1 levels in the serum of HCC patients decrease after surgery

  • This suggests utility for monitoring disease status and treatment effectiveness

• Technical Considerations:

  • Need for standardized detection methods with appropriate sensitivity and specificity

  • Importance of distinguishing different oligomeric forms in serum

Additional research is needed to establish clinical validity, reference ranges, and standardized detection protocols before clinical implementation.

What methods are most reliable for analyzing CTHRC1 in patient samples?

Multiple complementary methods offer reliable CTHRC1 analysis in clinical samples:

• Tissue Analysis:

  • Immunohistochemistry (IHC): Allows visualization of CTHRC1 expression patterns in tissue sections and tissue microarrays

  • Real-time RT-PCR: Provides quantitative measurement of CTHRC1 mRNA levels with high sensitivity

  • Western blotting: Enables detection of different CTHRC1 forms in tissue lysates

• Liquid Biopsy Approaches:

  • Serum CTHRC1 detection: Can be performed using Western blotting or immunoassays

  • Potential for circulating tumor cell analysis for CTHRC1 expression

• Quality Control Considerations:

  • Proper sample collection and processing to preserve CTHRC1 integrity

  • Inclusion of appropriate positive and negative controls

  • Standardized scoring systems for immunohistochemistry

The method selection should align with specific clinical research questions, available sample types, and required sensitivity and specificity.