Recombinant Human C-C motif chemokine 14 protein (CCL14), partial (Active)

What is the molecular structure of CCL14 and its isoforms?

CCL14 is a C-C motif chemokine that exists in two main isoforms resulting from differential mRNA splicing. After cleavage of a 19 amino acid signal peptide, mature CCL14a (amino acids 20-93) forms a 74 amino acid peptide also known as HCC-1 (Hemofiltrate CC Chemokine-1). The second isoform, CCL14b or HCC-3, is a 90 amino acid peptide (amino acids 20-109) resulting from the insertion of 16 amino acids between residues 7 and 8 of CCL14a .

While CCL14a in its full form is a weak CCR1 agonist, an 8 amino acid N-terminal truncation (amino acids 28-93) significantly enhances its signaling capacity through both CCR1 and CCR5 receptors . This structural modification is crucial for researchers designing functional studies, as the truncated form exhibits substantially different biological activity than the full-length protein.

What are the primary biological functions of CCL14 in normal physiology?

CCL14 functions primarily as a chemokine that promotes chemotaxis of T cells, monocytes, and eosinophils . It is constitutively expressed in multiple organs and plays a role in immune cell trafficking under normal physiological conditions . Unlike many inducible chemokines, CCL14 maintains baseline expression in healthy tissues, suggesting a role in homeostatic immune surveillance.

The protein's activity is regulated through precise N-terminal processing, which dramatically enhances its signaling capacity through CCR1 and CCR5 receptors . This processing-dependent activation mechanism allows for rapid amplification of CCL14 activity during immune responses without requiring de novo protein synthesis, creating an efficient system for modulating immune cell recruitment in response to tissue damage or pathogen invasion.

What are the optimal methods for detecting CCL14 in experimental samples?

For detecting CCL14 in research samples, sandwich immunoassays have proven particularly effective. When implementing such assays, the typical working concentration of anti-CCL14 antibody ranges from 0.5-2.5 μg/mL in the presence of 10 ng/mL recombinant human CCL14a/HCC-1 .

For mRNA quantification, RT-qPCR has been successfully employed to measure CCL14 expression in cell lines, as demonstrated in studies comparing expression between normal lung epithelial cells (BEAS-2B) and lung adenocarcinoma cell lines (NCI-H1299, PC9, and HCC827) . When designing primers, researchers should consider the alternative splicing of CCL14 to ensure specific amplification of the desired transcript variant.

For protein visualization in tissues, immunohistochemistry can effectively reveal differential expression patterns between normal and diseased tissues. The Human Protein Atlas database demonstrates medium CCL14 protein staining in normal lung tissues compared to low staining in lung adenocarcinoma tissues .

How can researchers effectively overexpress CCL14 in cell culture models?

For CCL14 overexpression studies, transfection with miR-CCL14 overexpression vector plasmids has been successfully implemented in lung adenocarcinoma cell lines. Selection of appropriate cell lines is critical - NCI-H1299 and PC9 cells have been identified as suitable models due to their naturally low CCL14 expression levels .

Following transfection, validation of successful CCL14 overexpression is essential and can be performed using RT-qPCR. Studies have shown that transfection with CCL14 overexpression vectors results in significantly increased CCL14 mRNA expression levels in both NCI-H1299 and PC9 cell lines (P < 0.05) .

When designing such experiments, researchers should consider the downstream functional assays that will be performed, as CCL14 overexpression has been demonstrated to significantly reduce growth of LUAD cell lines from 24 to 96 hours post-transfection, as measured by Cell Counting Kit-8 assays .

How does CCL14 expression vary across different cancer types and what are the implications?

CCL14 expression shows significant variability across cancer types, with important prognostic implications. Using the Oncomine database analysis, increased CCL14 expression (compared to normal tissues) has been observed in brain tumors, esophageal cancer, and lymphoma. In contrast, reduced CCL14 expression has been documented in bladder, breast, lung, stomach, liver, and colorectal cancers .

In lung adenocarcinoma specifically, analysis through TIMER and UALCAN databases has confirmed significantly lower CCL14 expression compared to adjacent normal tissues. This differential expression has diagnostic potential, with ROC curve analysis showing an area under the curve (AUC) of 0.905 (95% CI = 0.871–0.939), sensitivity of 0.864, and specificity of 0.837 (P < 0.05) .

Lower CCL14 expression in lung adenocarcinoma correlates with worse prognosis, suggesting its potential role as a tumor suppressor gene. Similar downregulation patterns and prognostic significance have been observed in hepatocellular carcinoma, ovarian cancer, and thyroid cancer, providing a comparative framework for understanding CCL14's role across malignancies .

What functional assays are most informative for studying CCL14's impact on cancer cell behavior?

When investigating CCL14's functional effects on cancer cells, researchers should consider a complementary suite of assays that address different aspects of cancer cell behavior:

• Proliferation assays: The Cell Counting Kit-8 (CCK-8) assay has effectively demonstrated that overexpression of CCL14 significantly reduces the growth of lung adenocarcinoma cell lines (PC9 and NCI-H1299) from 24 to 96 hours post-transfection (P < 0.05) .

• Migration and invasion assays: Transwell and wound healing assays have revealed that enhancing CCL14 expression reduces cell migration and invasion capabilities in vitro .

• Pathway analysis: Gene set enrichment analysis (GSEA) has shown that low CCL14 expression is associated with histone deacetylases, G2/M checkpoints, and Notch signaling pathways, suggesting potential mechanisms through which CCL14 exerts its effects .

When designing these functional studies, inclusion of appropriate controls and time-course analyses are essential for capturing both immediate and delayed effects of CCL14 modulation on cancer cell phenotypes.

How does CCL14 modulate the tumor immune microenvironment?

CCL14 plays a significant role in shaping the tumor immune microenvironment through its interactions with various immune cell populations. Contrary to what might be expected for a chemokine, studies in hepatocellular carcinoma have reported negative correlations between CCL14 expression and the infiltration of B cells, CD4+ and CD8+ T cells, macrophages, neutrophils, and dendritic cells .

To effectively study these interactions, researchers should employ comprehensive immune phenotyping approaches, including multiplexed immunohistochemistry, flow cytometry, or single-cell RNA sequencing, to capture the full spectrum of immune cell populations and their spatial relationships within the tumor microenvironment.

What are the optimal experimental approaches for studying CCL14-mediated chemotaxis?

For investigating CCL14's chemotactic effects, the BaF3 mouse pro-B cell line transfected with human CCR1 provides an excellent model system. Studies have demonstrated that recombinant human CCL14a/HCC-1 induces chemotaxis of these cells in a dose-dependent manner .

Quantification of chemotaxis can be effectively performed using Resazurin to measure the number of cells that migrate through to the lower chemotaxis chamber . When designing neutralization studies, researchers should note that chemotaxis elicited by recombinant human CCL14a/HCC-1 (10 ng/mL) can be neutralized by anti-human CCL14/HCC-1/HCC-3 monoclonal antibody, with an ND50 typically between 0.5-2.5 μg/mL .

For more complex studies examining the interplay between CCL14 and other chemokines or investigating the effects of specific proteolytic processing on chemotactic activity, researchers should consider employing transwell migration assays with primary human immune cells expressing the relevant receptors (CCR1 and CCR5).

How can urinary CCL14 be effectively utilized as a biomarker for acute kidney injury (AKI)?

Urinary CCL14 has emerged as a valuable biomarker for persistent severe acute kidney injury (AKI). Based on the RUBY study cohort (n = 335), the following cutoff values have been established for risk stratification :

• CCL14 ≤ 1.3 ng/mL: Lowest risk of developing persistent severe AKI (n = 124, 37%)

• 1.3 ng/mL < CCL14 ≤ 13 ng/mL: Increased risk of developing persistent severe AKI (n = 157, 47%)

• CCL14 > 13 ng/mL: Highest risk of developing persistent severe AKI (n = 54, 16%)

When implementing CCL14 testing in clinical research, experts recommend developing a comprehensive protocol paired with a treatment plan and clearly defining the target population . CCL14 results can help prioritize AKI management decisions, with levels above the high cutoff (>13 ng/mL) significantly changing the level of concern for modifying the AKI treatment plan (p < 0.001) .

For researchers studying AKI interventions, CCL14 testing can potentially identify patients most likely to benefit from novel therapies by stratifying the risk of persistent injury, thereby enhancing study design and increasing the probability of detecting treatment effects.

What are the methodological considerations for implementing CCL14 testing in AKI research protocols?

When implementing CCL14 testing in AKI research protocols, several methodological considerations should be addressed:

• Sample collection and processing: Standardization of urine collection, storage, and processing is essential to ensure reliable CCL14 measurements across study sites and time points.

• Timing of CCL14 measurement: Testing should be performed in patients with moderate or severe AKI (stage 2/3) to identify those at risk for persistent AKI .

• Integration with clinical data: CCL14 results should be interpreted in the context of other clinical parameters, including traditional AKI markers (serum creatinine, urine output), comorbidities, and concurrent treatments.

• Action protocol development: Research protocols should predefine how CCL14 results will inform clinical decision-making, particularly regarding discussions on renal replacement therapy (RRT) initiation for CCL14 levels >13 ng/mL .

• Outcome assessment: Studies should capture relevant clinical outcomes, including AKI duration, progression to chronic kidney disease, need for RRT, hospital length of stay, and mortality to comprehensively evaluate the impact of CCL14-guided interventions.

By addressing these methodological considerations, researchers can maximize the utility of CCL14 testing in AKI research and generate robust evidence to guide clinical practice.

How can researchers investigate the signaling pathways and molecular mechanisms regulated by CCL14?

To elucidate the signaling pathways and molecular mechanisms regulated by CCL14, researchers should employ a multi-modal approach:

• Gene set enrichment analysis (GSEA): Previous studies have identified associations between low CCL14 expression and histone deacetylases, G2/M checkpoints, and Notch signaling pathways in lung adenocarcinoma . Researchers can expand on these findings by investigating additional pathways and contexts.

• Protein-protein interaction networks: Mapping CCL14's interactions with other proteins can reveal novel regulatory mechanisms and functional complexes.

• Receptor signaling studies: Given CCL14's interactions with CCR1 and CCR5, researchers should investigate downstream signaling events using phosphorylation-specific antibodies, calcium flux assays, and gene expression profiling following receptor activation.

• Proteolytic processing analysis: Since N-terminal processing dramatically enhances CCL14's signaling capacity, researchers should identify the proteases responsible for this modification and characterize how this processing is regulated in different physiological and pathological contexts.

• In vivo models: Developing transgenic or knockout models for CCL14 can provide insights into its systemic functions and tissue-specific effects that cannot be captured in cell culture systems.

What are the most promising translational applications for CCL14 research?

CCL14 research offers several promising translational applications across medical fields:

• Cancer diagnostics and prognostics: The differential expression of CCL14 across cancer types, particularly its downregulation in lung adenocarcinoma with prognostic significance (AUC of 0.905), suggests its potential as a biomarker for early detection and risk stratification .

• Cancer therapeutics: Given CCL14's apparent anti-oncogenic properties in lung adenocarcinoma, where overexpression inhibits proliferation, migration, and invasion, strategies to restore or enhance CCL14 expression or signaling might represent novel therapeutic approaches .

• AKI management: In nephrology, urinary CCL14 testing can identify patients at high risk for persistent severe AKI, potentially guiding more aggressive interventions or earlier initiation of renal replacement therapy in appropriate cases .

• Immunomodulatory therapies: CCL14's role in regulating immune cell infiltration and function in various contexts could be leveraged to develop targeted immunomodulatory therapies for cancer, autoimmune diseases, or inflammatory conditions.

For each of these applications, further mechanistic and clinical validation studies are needed to translate fundamental research findings into practical clinical tools and interventions.