Recombinant Human C-X-C motif chemokine 14 (CXCL14) (Active)

What is the molecular structure and expression pattern of CXCL14?

CXCL14, also known as BRAK (breast and kidney-expressed chemokine), is a 77-amino acid chemokine with the critical C-X-C motif in exon 2 that is essential for receptor binding and stabilization of chemokine-receptor complexes . The recombinant human form typically covers the 35-111aa expression region of the full-length mature protein . Unlike most chemokines that show inducible expression, CXCL14 is constitutively and abundantly expressed in normal tissues, particularly epithelial cells, but is frequently downregulated in various tumor types, suggesting a tumor-suppressive function . In the developing brain, CXCL14 is specifically expressed by single-bouquet cells (SBCs) in layer I of the somatosensory cortex, where its expression is activity-dependent and can be markedly decreased by sensory deprivation during neonatal stages .

What experimental methods are commonly used to measure CXCL14 levels in biological samples?

Enzyme-Linked Immunosorbent Assay (ELISA) is the standard method for quantifying CXCL14 in plasma and urine samples. Commercial sandwich ELISA kits (e.g., Human CXCL14/BRAK DuoSet ELISA) with a sensitivity range of 31.25 to 4000 pg/ml are typically employed . For tissue expression analysis, Fluorescent In Situ Hybridization (FISH) with probes specifically targeting CXCL14 exons can demonstrate CXCL14 expression patterns, particularly when validating knockout models . Immunohistochemical (IHC) and immunofluorescence (IF) staining of formalin-fixed paraffin-embedded tissues are widely used to detect CXCL14 protein expression in tumor versus non-tumor regions . For broader proteomic profiling, techniques such as ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS) combined with multivariate statistical analysis approaches like principal component analysis (PCA) can be employed to analyze CXCL14 in complex biological matrices .

How does recombinant CXCL14 protein demonstrate biological activity in experimental settings?

Recombinant CXCL14 protein activity is primarily assessed through calcium flux assays using prostaglandin E2-treated THP1 human acute monocytic leukemia cells, with effective doses (ED50) typically ranging from 1-10 ng/mL . Additionally, researchers evaluate CXCL14's chemotactic functions on monocytes and dendritic cells through migration assays. In neuronal contexts, electrophysiological analyses can detect changes in neuronal excitability and complexity upon CXCL14 application or deletion . Flow cytometry coupled with appropriate antibody panels is commonly used to evaluate CXCL14's effects on immune cell responses, particularly in tumor microenvironments . For receptor binding studies, G protein-dependent assays and β-arrestin recruitment assays can demonstrate CXCL14's interaction with receptors such as MAS-related G protein-coupled receptor X2 (MRGPRX2) .

What are the optimal experimental designs for investigating CXCL14's dual role in tumor suppression versus progression?

Investigating CXCL14's context-dependent roles in cancer requires comprehensive experimental approaches. Researchers should design studies that account for CXCL14 concentration variations, as high concentrations (300 nM, approximately 3000 times higher than physiological blood levels) can produce effects contradictory to those seen at physiological concentrations . The experimental design should include:

• Comparative assays using both recombinant CXCL14 protein and CXCL14-overexpressing cell lines (e.g., NIH-CXCL14)

• Analysis of co-occurring factors like CXCL12, as CXCL14 can modulate CXCL12 activity through binding to CXCR4

• In vivo models that permit assessment of both direct tumor effects and tumor microenvironment modifications

• Multi-omics approaches to detect molecular changes beyond direct CXCL14 signaling

A critical consideration is the potential confounding effect of other chemokines, particularly CXCL12, which has tumorigenic and angiogenic properties that may be modulated by CXCL14 . Researchers should examine CXCL14's effects on tumor cells in isolation and then in the presence of CXCL12 or other relevant chemokines to understand potential synergistic or antagonistic interactions.

How can researchers effectively validate CXCL14 knockout models for neuronal development studies?

Validation of CXCL14 knockout models requires multiple complementary approaches to ensure specific and efficient gene targeting. Based on successful strategies in the field, researchers should implement:

• PCR validation with custom-designed primer sets flanking the conditional exon (e.g., exon 2 containing the C-X-C motif)

• Fluorescent In Situ Hybridization (FISH) with probes specifically against the targeted exon to demonstrate loss of signal in the tissue of interest

• Confirmation of recombination specificity by evaluating CXCL14 expression in Cre-negative tissues

• Functional validation through electrophysiological analyses to detect predicted phenotypes (e.g., increased intrinsic excitability in specific neuronal populations)

For conditional knockout strategies targeting specific neuronal populations, researchers should consider crossing CXCL14 floxed mice (CXCL14 fl/fl) with appropriate Cre driver lines (e.g., 5HT3aR.Cre for targeting superficial interneurons) . Breeding strategies should be designed to produce littermates of multiple genotypes (CXCL14+/+, CXCL14+/-, CXCL14-/-) to control for genetic background effects . Researchers must verify knockout efficiency at both mRNA and protein levels before proceeding with phenotypic analyses.

What methodological approaches can resolve contradictory findings regarding CXCL14's effects on angiogenesis?

CXCL14 has been characterized as both angiostatic (inhibiting angiogenesis) and, under certain conditions, pro-angiogenic. To resolve these contradictions, researchers should employ multiparametric approaches that account for experimental context:

• Compare purified recombinant CXCL14 protein versus CXCL14-expressing cell systems (e.g., NIH-CXCL14 cells) in identical angiogenesis assays

• Implement dose-response studies across a wide concentration range (physiological to supraphysiological)

• Systematically evaluate the presence of additional factors (particularly CXCL12) that may modulate CXCL14 activity

• Utilize receptor-blocking antibodies to determine which effects are receptor-dependent versus independent

The contradictory findings may be explained by concentration-dependent effects or the co-presence of modulatory molecules. At high concentrations (300 nM), CXCL14 can bind to CXCR4 (the receptor for CXCL12) and alter the receptor's three-dimensional structure, potentially enhancing CXCL12 binding and activity . Therefore, experimental designs must control for CXCL12 levels and include CXCR4 blockade controls to distinguish direct CXCL14 effects from indirect effects mediated through CXCR4 modulation.

How can CXCL14 be effectively evaluated as a biomarker for lung cancer diagnosis?

To evaluate CXCL14 as a diagnostic biomarker for lung cancer, researchers should implement a comprehensive validation framework:

• Large-scale case-control studies with adequate statistical power, including patients with various stages of lung cancer and appropriate healthy controls

• Standardized specimen collection and processing protocols to minimize preanalytical variability

• Robust analytical methods using validated ELISA kits with established performance characteristics

• Advanced statistical analyses including Receiver Operating Characteristic (ROC) curves to determine optimal cutoff values

Prior research has shown promising results, with plasma CXCL14 achieving an Area Under the Curve (AUC) of 0.9464 (95% CI, 0.9209–0.9719) at a cutoff point of 746.0 pg/ml for diagnosis of lung cancer, demonstrating 87.4% sensitivity and 85.0% specificity . For early-stage (Stage I) lung cancer, plasma CXCL14 achieved an AUC of 0.9353 (95% CI, 0.9034–0.9672) at a cutoff point of 840.3 pg/ml, with 81.02% sensitivity and 92.5% specificity .

Researchers should also conduct longitudinal studies to determine whether CXCL14 levels correlate with disease progression or response to therapy, potentially enhancing its clinical utility beyond initial diagnosis.

What are the optimal expression systems and purification strategies for producing high-quality recombinant CXCL14?

For producing functional recombinant CXCL14, Escherichia coli (E. coli) expression systems have been successfully employed to generate tag-free protein covering the 35-111aa expression region of the full-length mature protein . The recombinant protein should undergo rigorous quality control including:

• SDS-PAGE and HPLC analysis to confirm >95% purity

• Endotoxin testing using the LAL method to ensure levels below 1.0 EU/μg

• Functional validation through calcium flux assays with prostaglandin E2-treated THP1 cells

• Mass spectrometry to verify protein identity and detect potential post-translational modifications

The lyophilized protein preparation should facilitate straightforward reconstitution with sterile water or appropriate buffer, depending on experimental requirements . For long-term storage stability, researchers should validate protein activity after various storage conditions and freeze-thaw cycles to establish optimal handling protocols.

How should researchers design experiments to investigate CXCL14's role in activity-dependent cortical development?

To investigate CXCL14's role in activity-dependent cortical development, researchers should implement multifaceted experimental approaches:

• Sensory deprivation paradigms: Implement whisker trimming or plucking in rodent models during critical developmental periods to modulate sensory inputs and measure consequent changes in CXCL14 expression

• Longitudinal in vivo imaging: Utilize two-photon microscopy with appropriate genetic reporters to track CXCL14-expressing neurons during development and after sensory manipulation

• Electrophysiological analyses: Perform whole-cell patch-clamp recordings to assess changes in intrinsic excitability and network recruitment following CXCL14 manipulation

• Anatomical analyses: Quantify neuronal complexity, dendritic branching, and spine density in wild-type versus CXCL14-deficient neurons

The experimental design should include appropriate genetic models (conditional and constitutive knockouts) and age-matched controls. Given CXCL14's expression in specific neuronal populations like single-bouquet cells in layer I of the somatosensory cortex, cell-type specific manipulations are crucial for dissecting its function in circuit development . Researchers should also implement rescue experiments, reintroducing CXCL14 into knockout models to determine whether developmental defects can be reversed.

What analytical approaches are recommended for studying CXCL14's interaction with the MRGPRX2 receptor?

To comprehensively characterize CXCL14's interaction with the MAS-related G protein-coupled receptor X2 (MRGPRX2), researchers should implement:

• G protein-dependent assays to measure receptor activation upon CXCL14 binding

• β-arrestin recruitment assays to assess non-canonical signaling pathways

• Truncation and mutagenesis studies to identify the pharmacophoric sequence of CXCL14 responsible for receptor interaction

• Computational modeling to predict binding interfaces between CXCL14 and MRGPRX2

Prior research has demonstrated that C-terminal domain sequences of CXCL14 consisting of just 4 to 11 amino acids can display similar or even increased potency and efficacy compared to the full 77-amino acid CXCL14 sequence in activating MRGPRX2 . This suggests that truncated peptides derived from CXCL14 might serve as more potent and specific agonists for MRGPRX2, with potential therapeutic applications in conditions like idiopathic pulmonary fibrosis where CXCL14 is upregulated .

Researchers should utilize selective MRGPRX2/B2 antagonists as experimental controls to confirm specificity of interactions and employ cross-species analyses (comparing human MRGPRX2 with mouse MRGPRB2) to facilitate translational research from animal models to human applications.