SPINT2 Human, Sf9

What is SPINT2 and what are its primary biological functions?

SPINT2, also known as hepatocyte growth factor activator inhibitor type-2, belongs to the Kunitz family of serine protease inhibitors. It functions primarily as a potent inhibitor of hepatocyte growth factor activator (HGFA) . This inhibition is significant as SPINT2 effectively blocks the conversion of inactive pro-HGF/SF (hepatocyte growth factor/scatter factor) into bioactive HGF/SF . Through this mechanism, SPINT2 regulates multiple cellular processes including:

• Cell proliferation and viability

• Cell migration and invasion

• Extracellular matrix (ECM) degradation

• Cellular phenotypic modulation

SPINT2 achieves these regulatory functions by modulating key signaling pathways, particularly those involving HGF/Met signaling, which plays prominent roles in cell migration, survival, growth, and cardiovascular remodeling following tissue injury .

Why are Sf9 cells suitable for human SPINT2 expression studies?

Spodoptera frugiperda (Sf9) cells provide an advantageous platform for SPINT2 expression studies for several reasons:

• High protein expression yields compared to mammalian systems

• Post-translational processing capabilities that maintain protein functionality

• Cost-effectiveness for large-scale protein production

• Well-established baculovirus expression vector systems

As demonstrated in research utilizing similar expression systems, Sf9 cells can be effectively transduced with vectors like pAcGP67A for preparing baculovirus stocks that express proteins of interest . This approach has been successfully employed for complex proteins including cytokine mimetics, suggesting its potential suitability for SPINT2 expression.

How does SPINT2 expression vary across normal and pathological tissues?

SPINT2 expression demonstrates notable variation between normal and pathological tissues:

• Downregulation in thoracic aortic dissection (TAD) specimens compared to normal aortic tissues

• Epigenetic silencing in several human cancer types

• Significantly higher expression in ovarian cancer cell lines (including SK-OV-3) compared to normal ovarian epithelial cells (IOSE80)

This differential expression pattern suggests context-dependent roles for SPINT2 in different tissue types and disease states. Interestingly, while SPINT2 appears downregulated in aortic dissection, suggesting a potential protective role in vascular integrity, it shows elevated expression in certain cancer types, where it may promote tumor progression.

What experimental approaches are used to study SPINT2 function in smooth muscle cell phenotypic switching?

Researchers investigating SPINT2's role in smooth muscle cell (SMC) phenotypic switching employ several methodological approaches:

• In vitro phenotypic modulation induction: Treatment of isolated primary mouse aortic SMCs with platelet-derived growth factor BB (PDGF-BB) at different concentrations (0, 10, 20, and 40 ng/ml) for 24 hours to induce phenotypic switching from contractile to synthetic phenotype .

• SPINT2 overexpression: Utilization of adenoviral vectors carrying the SPINT2 sequence to establish SPINT2-overexpressing cell lines .

• Phenotypic marker assessment: Western blotting analysis to measure expression levels of:

  • Synthetic phenotype markers: vimentin and collagen I

  • Contractile phenotype markers: α-smooth muscle actin (α-SMA) and smooth muscle protein 22-α (SM22α)

• Signaling pathway analysis: Examination of ERK signaling pathway activation, using specific ERK agonists like 12-O-tetradecanoylphorbol-13-acetate to verify mechanisms .

This integrated approach allows for comprehensive evaluation of SPINT2's effects on the phenotypic plasticity of SMCs, which has important implications for understanding vascular pathologies.

How can researchers effectively study SPINT2's impact on cell proliferation and migration?

To investigate SPINT2's effects on cellular proliferation and migration, researchers can employ these validated methodologies:

For proliferation assessment:

• MTT assay to detect cell viability following SPINT2 manipulation

• Immunofluorescence staining for Ki-67-positive cells to directly measure proliferating cells

• Colony formation assays, as demonstrated in ovarian cancer cell studies

For migration analysis:

• Wound healing assays to quantify migratory capacity after SPINT2 overexpression or knockdown

• Transwell migration assays for more quantitative assessment

• Invasion assays using Matrigel-coated chambers to evaluate invasive potential

For molecular mechanism investigation:

• ELISA and western blotting to measure MMP-2 and MMP-9 expression and activity levels

• Analysis of signaling pathway components, particularly ERK activation status

These complementary approaches provide robust assessment of SPINT2's functional impact on cellular behavior and underlying mechanisms.

What strategies are effective for SPINT2 knockdown or overexpression in experimental models?

Researchers have successfully employed several approaches for modulating SPINT2 expression:

For SPINT2 overexpression:

• Adenoviral vector systems carrying the SPINT2 sequence (Ad-SPINT2)

• Infection of target cells at multiplicity of infection of 100 for 24 hours

• Verification of overexpression by RT-qPCR and western blotting

For SPINT2 knockdown:

• Short hairpin RNA (shRNA) approaches as demonstrated in studies with ovarian cancer cell lines

• Multiple shRNA constructs (e.g., shSPINT2-1# and shSPINT2-2#) to control for off-target effects

• Validation of knockdown efficiency by western blotting

The choice between these approaches depends on research objectives, with overexpression models providing insight into protective or therapeutic potential, while knockdown approaches help elucidate endogenous functions.

What is SPINT2's role in thoracic aortic dissection pathogenesis?

SPINT2 appears to play a protective role against thoracic aortic dissection (TAD) development through several mechanisms:

• SPINT2 expression is significantly downregulated in TAD specimens compared to normal aortic tissues, suggesting its loss contributes to disease progression

• SPINT2 overexpression inhibits PDGF-BB-induced SMC proliferation and migration, key processes in TAD pathogenesis

• SPINT2 suppresses the phenotypic switching of SMCs from contractile to synthetic type by:

  • Decreasing expression of synthetic markers (vimentin and collagen I)

  • Increasing expression of contractile markers (α-SMA and SM22α)

• SPINT2 reduces the expression and activity of matrix metalloproteinases (MMP-2 and MMP-9), which contribute to extracellular matrix degradation in aortic dissection

• SPINT2's protective effects appear mediated through inhibition of ERK pathway activation

These findings suggest SPINT2 as a potential therapeutic target for TAD, with restoration of SPINT2 expression potentially limiting disease progression.

How does SPINT2 influence tumor progression and immune cell infiltration in cancer?

SPINT2 demonstrates context-dependent roles in cancer, with evidence suggesting both tumor-suppressive and tumor-promoting functions:

Potential tumor-suppressive roles:

• SPINT2 is epigenetically silenced in several human cancers, contributing to malignant phenotype development, particularly enhanced cell migration and invasion

• SPINT2 can suppress pro-HGF activation, ECM degradation, and cancer cell invasion by inhibiting proteolytic enzymes

• SPINT2 may reduce cell migration and invasion via downregulation of MMP-2 expression and activity

Potential tumor-promoting roles in specific contexts:

• High SPINT2 expression is associated with poorer prognosis in breast cancer and potentially in ovarian cancer

• SPINT2 plays a key role in macrophage infiltration in ovarian cancer

• SPINT2 knockdown inhibits the migration of M2 macrophages and blocks macrophage polarization from M0 to M2 phenotype, suggesting a role in promoting tumor-associated macrophage (TAM) activity

The correlation analysis indicates SPINT2 expression is significantly associated with macrophage infiltration (r = 0.219, p = 1.29 × 10^-6) and to a lesser extent with neutrophil infiltration (r = 0.137, p = 2.57 × 10^-3) in ovarian cancer .

What molecular pathways mediate SPINT2's effects on cell behavior?

SPINT2 modulates cellular behavior through several interconnected molecular pathways:

• HGF/Met signaling pathway:

  • SPINT2 inhibits HGFA, impairing conversion of inactive pro-HGF/SF to bioactive HGF/SF

  • This regulation impacts the HGF/Met pathway, which mediates cell migration, survival, growth, and tissue remodeling

• Matrix metalloproteinase regulation:

  • SPINT2 reduces expression and activity of MMP-2 and MMP-9

  • This regulation affects extracellular matrix degradation and remodeling

• ERK signaling pathway:

  • SPINT2 overexpression inhibits ERK activation

  • ERK pathway inhibition mediates SPINT2's effects on SMC migration and phenotypic switching

  • ERK pathway activation via 12-O-tetradecanoylphorbol-13-acetate reverses SPINT2-mediated inhibition of SMC migration and phenotypic switching

• Macrophage polarization:

  • SPINT2 appears to influence M0 to M2 macrophage polarization

  • This effect has implications for tumor microenvironment modulation

Understanding these pathways provides potential targets for therapeutic intervention in SPINT2-associated pathologies.

What cellular models are most appropriate for studying SPINT2 function?

Researchers have successfully employed several cellular models to investigate SPINT2 function:

For vascular disease research:

• Primary mouse aortic smooth muscle cells (SMCs)

• SMCs treated with PDGF-BB (20 ng/ml) to induce phenotypic modulation, creating an in vitro model of vascular pathology

For cancer research:

• Ovarian cancer cell lines such as SK-OV-3, which show high endogenous SPINT2 expression

• Normal ovarian epithelial cell line IOSE80 as a control with lower SPINT2 expression

• Co-culture systems with macrophages to study SPINT2's role in immune cell interactions

For protein expression:

• Spodoptera frugiperda (Sf9) insect cells for recombinant protein production

• 293A cells for adenoviral packaging in overexpression studies

The choice of model system should align with specific research objectives and the aspect of SPINT2 biology under investigation.

How can researchers effectively validate SPINT2 expression and function?

Comprehensive validation of SPINT2 expression and function requires multiple complementary approaches:

For expression validation:

• Reverse transcription-quantitative PCR (RT-qPCR) to measure mRNA levels

• Western blotting for protein expression quantification

• Bioinformatics analysis of gene expression data from databases like GEO (e.g., dataset GSE52093)

For functional validation:

• Cell viability and proliferation assays (MTT, Ki-67 staining)

• Migration assays (wound healing)

• Invasion assays for cancer cell studies

• Analysis of downstream molecular markers:

  • MMPs (MMP-2, MMP-9) expression and activity

  • SMC phenotypic markers (vimentin, collagen I, α-SMA, SM22α)

  • Signaling pathway components (e.g., phosphorylated ERK)

For interaction studies:

• Co-culture experiments (e.g., cancer cells with macrophages)

• Immune infiltration analysis using computational tools like TIMER

This multi-faceted approach ensures robust validation of both expression patterns and functional consequences of SPINT2 manipulation.