KRT14 Human, His

What is KRT14 and what is its role in human tissues?

KRT14 (Keratin 14) is a type I intermediate filament protein encoded by the KRT14 gene, which is 4615 base pairs long and contains eight exons. The protein is 472 amino acids in length and features four helical domains flanked by non-helical head and tail domains . KRT14 partners with Keratin 5 (encoded by KRT5) to form heterodimeric complexes that provide critical structural support in the basal keratinocytes of the epidermis . Additionally, these keratin filaments regulate the distribution of melanin in the skin.

Beyond the epidermis, KRT14 is expressed in a subpopulation of basal cells in the urothelium (bladder epithelium), where it marks cells with self-renewal capacity that can give rise to all cell types during tissue regeneration . These KRT14-positive cells appear to be urothelial progenitors with pivotal roles in both natural and injury-induced bladder regeneration.

How does the addition of a histidine tag affect KRT14 protein structure and function?

The addition of a histidine tag (typically 6-10 histidine residues) to KRT14 is primarily designed for protein purification purposes rather than functional modification. Methodologically, researchers should consider:

For critical functional studies, comparing tagged and untagged protein behavior is recommended to ensure tag neutrality.

What are the expression patterns of KRT14 in different human tissues?

KRT14 shows a tissue-specific expression pattern that varies significantly across different epithelial tissues:

In the bladder, KRT14-positive cells represent a distinct progenitor population. Upon injury (such as chemical exposure to cyclophosphamide), the percentage of KRT14-positive/KRT5-positive cells increases significantly from ~3.89% to 17.33% after single exposure, and up to 24.87% after multiple exposures . This suggests KRT14-positive cells play a crucial role in tissue regeneration following injury.

What mutations in the KRT14 gene are associated with human diseases?

The KRT14 gene harbors various mutation types that cause different clinical phenotypes:

A novel large intragenic deletion in KRT14 was recently reported in a Chinese family with autosomal-dominant EBS presenting with generalized hyperpigmentation . This case highlights that not only point mutations but also larger genomic rearrangements in KRT14 can cause disease phenotypes.

The relationship between mutation location and disease severity follows specific patterns: mutations in highly conserved regions of the helical domains typically cause more severe phenotypes due to greater disruption of filament assembly.

How do KRT14-positive cells contribute to bladder cancer development?

KRT14-positive cells appear to be cells of origin for bladder cancer, contributing to tumorigenesis through the following mechanisms:

• Expansion during carcinogenesis: Upon exposure to bladder carcinogens such as N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), there is a marked increase in KRT14-positive cell numbers after 4 months .

• Tumor initiation capacity: Lineage tracing experiments have demonstrated that KRT14-positive cells can give rise to invasive tumors. After 6 months of BBN exposure, animals developed invasive tumors that expressed both KRT5 and KRT14 .

• Enrichment in aggressive tumors: In human bladder cancer, KRT14-positive cells are considered the most primitive population and are enriched following chemotherapy, suggesting a role in therapeutic resistance .

• Role in STAT3-driven cancer: KRT14-positive cells are preferentially amplified upon STAT3 overexpression in mouse models of invasive bladder cancer .

These findings implicate KRT14-positive cells as critical elements in bladder cancer initiation, progression, and potentially resistance to therapy.

What methodological approaches are used to study KRT14 mutations in epidermolysis bullosa?

When investigating KRT14 mutations in epidermolysis bullosa, researchers typically employ a multi-tiered methodological approach:

• Clinical assessment and sample collection:

  • Detailed clinical phenotyping including blister distribution, age of onset, and additional features

  • Skin biopsy from affected and unaffected areas

  • Blood samples for DNA extraction

• Mutation identification:

  • PCR amplification of KRT14 exons and exon-intron boundaries

  • Sanger sequencing of PCR products

  • Next-generation sequencing for comprehensive gene panels

  • Copy number variation analysis for large deletions/duplications

• Functional validation:

  • Generation of patient-derived keratinocyte cultures

  • Creation of isogenic cell lines using CRISPR/Cas9

  • Assessment of keratin filament formation using immunofluorescence

  • Protein stability analysis using cycloheximide chase assays

• Disease modeling:

  • Patient-derived induced pluripotent stem cells (iPSCs)

  • 3D skin equivalents

  • Mouse models using knock-in of specific mutations

For novel mutations, such as the recently reported large deletion in a Chinese family , additional analyses may include breakpoint characterization and transcript analysis to understand the precise molecular consequences.

What are the optimal techniques for purifying His-tagged KRT14 protein?

The purification of His-tagged KRT14 presents several challenges due to its propensity to form insoluble intermediate filaments. A methodological optimization approach includes:

• Expression system selection:

  • Bacterial systems (E. coli BL21(DE3)): High yield but requires refolding

  • Insect cells (Sf9): Better folding but lower yield

  • Mammalian cells (HEK293): Best folding but most expensive and lowest yield

• Optimal purification protocol:

  • Lysis buffer: 8M urea, 50mM NaH₂PO₄, 300mM NaCl, pH 8.0

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA

  • Step gradient elution with imidazole (50mM, 100mM, 250mM)

  • Size exclusion chromatography for higher purity

• Refolding strategy for bacterial expression:

  • Dialysis against decreasing concentrations of urea

  • Addition of oxidized and reduced glutathione (3:1 ratio)

  • Presence of KRT5 for heterodimer formation improves stability

• Quality control:

  • SDS-PAGE and western blot analysis

  • Circular dichroism to confirm secondary structure

  • Negative stain electron microscopy to assess filament formation

Typical yield from optimal bacterial expression is 5-10mg of purified KRT14 per liter of culture, with >90% purity achievable using the above protocol.

What gene-targeting strategies are most effective for KRT14 modification in human cells?

Several gene-targeting approaches have been utilized for KRT14 modification, with varying efficiencies:

• AAV-mediated homologous recombination:

  • Demonstrated high efficiency in targeting KRT14 exon 3

  • All analyzed clones (25/25) contained targeted insertions

  • Minimal off-target integration when using appropriate selection methods

• CRISPR/Cas9-mediated gene editing:

  • Design considerations for sgRNAs targeting KRT14:

    • Target conserved exons (exons 1 and 7 show highest efficiency)

    • Avoid regions with secondary structures

    • Score >85 on standard prediction algorithms

• HDR template design for precise modifications:

  • Homology arms: 800bp for optimal efficiency

  • Silent mutations in PAM site to prevent re-cutting

  • Selection markers: Puromycin resistance or fluorescent proteins

• Clone verification strategies:

  • Southern blot analysis using KRT14-specific probes

  • PCR across integration junctions

  • Whole-genome sequencing to detect possible off-target effects

The efficiency of KRT14 targeting varies by cell type, with keratinocytes showing targeting frequencies of ~0.5% using AAV vectors . This efficiency can be increased through optimization of delivery methods and selection strategies.

How can researchers effectively trace KRT14-positive cell lineages in tissue regeneration studies?

Lineage tracing of KRT14-positive cells requires sophisticated genetic approaches:

• Generation of knockin Cre recombinase lines:

  • CreERT2 recombinase inserted into the KRT14 locus creates a null allele

  • Administration of tamoxifen in KRT14CreERT2/+; R26tdTomato/+ mice allows temporal control

  • Validation of specificity through co-localization of KRT14 and tdTomato

• Experimental design considerations:

  • Pulse-chase approach: Brief tamoxifen administration followed by long-term monitoring

  • Single vs. multiple tamoxifen doses to distinguish between labeling efficiency and true lineage expansion

  • Controls must include vehicle-treated mice to assess "leaky" recombination

• Injury models to assess regenerative capacity:

  • Chemical injury (cyclophosphamide treatment)

  • Mechanical injury (catheterization)

  • Carcinogen exposure (BBN)

• Analysis methods:

  • Quantification metrics: Percentage of tdTomato-positive cells co-expressing specific markers

  • 3D reconstruction of tissue architecture

  • Single-cell sequencing of labeled populations

In published studies, KRT14-positive cells showed significant expansion following injury, increasing from 3.89% to 17.33% after single cyclophosphamide exposure . These cells demonstrated multipotency by giving rise to all urothelial cell layers during regeneration.

How do KRT14-KRT5 interactions influence intermediate filament assembly and function?

The interaction between KRT14 and KRT5 follows a hierarchical assembly process that determines cytoskeletal integrity:

• Molecular basis of interaction:

  • Obligate heterodimer formation through coiled-coil domains

  • Alignment in register with parallel orientation

  • Stabilized by hydrophobic interactions and ionic bonds

• Assembly hierarchy:

  • Heterodimer formation (KRT14-KRT5)

  • Tetramer assembly (two heterodimers)

  • Protofilament formation (8 tetramers)

  • Mature 10nm filament (4 protofilaments)

• Regulatory mechanisms:

  • Phosphorylation of head domain serines increases solubility

  • Glycosylation alters filament bundling properties

  • Proteolytic processing modulates network turnover

• Functional consequences of disruption:

  • Mutations at heterodimerization interfaces cause most severe disease phenotypes

  • Altered assembly kinetics affect keratinocyte resilience to mechanical stress

  • Changes in network density influence cell signaling pathways

Advanced imaging techniques like super-resolution microscopy and cryo-electron microscopy have revealed that the KRT14-KRT5 filament network is highly dynamic, with continuous remodeling in response to mechanical stimuli.

What role do KRT14-positive cells play in regenerative medicine applications?

KRT14-positive cells show considerable promise for regenerative medicine:

• Stem cell properties:

  • Self-renewal capacity demonstrated through clonogenic assays

  • Multipotency evidenced by lineage tracing experiments

  • Persistence through multiple rounds of injury and repair

• Tissue engineering applications:

  • Skin equivalents: Human skin equivalents made from KRT14-targeted keratinocytes function normally after transplantation

  • Bladder reconstruction: KRT14-positive cells could potentially serve as a progenitor population for bladder tissue engineering

  • Corneal repair: KRT14-expressing limbal stem cells for corneal regeneration

• Methodological considerations for clinical translation:

  • Isolation methods: Fluorescence-activated cell sorting based on surface markers co-expressed with KRT14

  • Expansion protocols: Colony-forming efficiency assays show highest regenerative potential in holoclone-forming cells

  • Scaffold compatibility: Preferential attachment to specific extracellular matrix proteins

• Genetic modification for therapy:

  • Demonstration of functional correction in EBS patient cells through gene editing

  • Assessment of off-target effects crucial for safety

  • Long-term engraftment potential of corrected cells

Research has demonstrated that gene-targeted keratinocyte populations can be expanded sufficiently to generate clinically significant skin grafts , supporting the therapeutic potential of modified KRT14-positive cells.

How does ablation of KRT14-positive cells affect tissue homeostasis and regeneration?

Conditional ablation studies have provided crucial insights into the necessity of KRT14-positive cells:

• Experimental ablation approaches:

  • Genetic: KRT14CreERT2/+; R26DTR/+ mice allow diphtheria toxin (DT)-mediated ablation

  • Pharmacological: Specifically designed antibody-toxin conjugates

  • Physical: Laser ablation of fluorescently labeled cells

• Immediate consequences of ablation:

  • In bladder explant cultures, DT-mediated ablation of KRT14-positive cells completely prevented explant growth

  • In vivo, despite obvious tissue damage, no proliferation was observed when KRT14-positive cells were ablated

  • The KRT14-positive cell pool appears to be regenerated exclusively from existing KRT14-positive cells

• Long-term effects on tissue function:

  • Complete regeneration requires KRT14-positive cells

  • No compensatory mechanisms were observed that could replace KRT14-positive progenitor function

  • The hierarchical relationship between KRT14-positive cells and other cell types remains stable even after injury

• Implications for disease understanding:

  • In cancer, targeting KRT14-positive cells could potentially eliminate tumor-initiating cells

  • In genetic disorders, reconstitution with genetically corrected KRT14-positive cells may provide long-term therapeutic benefit

These findings establish KRT14-positive cells as essential progenitors in epithelial tissue homeostasis and regeneration, with no redundant cellular mechanisms capable of compensating for their absence.

What are the primary challenges in developing specific antibodies for human KRT14 detection?

Developing specific antibodies against human KRT14 presents several technical challenges:

• Structural homology issues:

  • High sequence similarity (~60%) between KRT14 and other type I keratins

  • Conserved rod domains across keratin family members

  • Need to target unique epitopes in head or tail domains

• Optimal immunization strategies:

  • Recombinant protein fragments vs. synthetic peptides

  • Selection of species for immunization (rabbit provides best specificity)

  • Adjuvant selection affects epitope presentation

• Validation requirements:

  • Western blot on samples with variable KRT14 expression

  • Immunohistochemistry on tissues with known KRT14 distribution

  • Testing on cells with CRISPR-mediated KRT14 knockout as negative controls

  • Cross-reactivity assessment against related keratins

• Application-specific optimization:

  • Fixation compatibility (formaldehyde vs. methanol)

  • Epitope retrieval methods (heat-induced vs. enzymatic)

  • Blocking agent selection to minimize background

Researchers should prioritize antibodies targeting the C-terminal region of KRT14 for highest specificity, with monoclonal antibodies generally providing better reproducibility than polyclonal preparations.

How can contradictions in KRT14 lineage tracing data be reconciled across different experimental systems?

Contradictions in KRT14 lineage tracing studies can be systematically addressed through methodological analysis:

• Sources of experimental variability:

  • Promoter activity: Different versions of KRT14 promoters vary in specificity and strength

  • Recombination efficiency: CreERT2 systems show variable tamoxifen sensitivity

  • Reporter lines: Different fluorescent reporters have varying detection thresholds

  • Tissue context: KRT14 expression patterns differ between species and organs

• Reconciliation strategies:

  • Use multiple independent lineage tracing systems

  • Combine genetic lineage tracing with alternative approaches:

    • Single-cell RNA sequencing with computational lineage inference

    • Clonal analysis using multicolor reporters

    • In vitro fate mapping with time-lapse imaging

• Specific contradictions and explanations:

  • Regarding bladder regeneration, some studies suggest distinct progenitors for different urothelial layers, while others indicate KRT14-positive cells can generate all layers

  • Potential explanation: Injury severity and type may determine whether specialized or multipotent progenitors are activated

• Critical controls to resolve contradictions:

  • Pulse-chase experiments with varying chase periods

  • Dose-response studies for tamoxifen induction

  • Single-cell validation of marker co-expression

  • Cross-validation between in vitro and in vivo systems

The current consensus based on multiple experimental approaches suggests that KRT14-positive cells represent a truly multipotent progenitor population in the bladder urothelium, but their contribution to homeostasis versus regeneration may follow different rules .

What emerging technologies will advance understanding of KRT14 function in tissue homeostasis?

Several cutting-edge technologies are poised to transform KRT14 research:

• Single-cell multi-omics approaches:

  • Integrated single-cell RNA-seq, ATAC-seq, and proteomics of KRT14-positive populations

  • Spatial transcriptomics to map KRT14-positive cell niches and their microenvironments

  • Live-cell proteomics to track KRT14 interaction partners during differentiation

• Advanced imaging techniques:

  • Light sheet microscopy for whole-organ imaging of KRT14-positive cell distribution

  • Super-resolution microscopy of keratin filament dynamics

  • Intravital imaging with photoconvertible reporters to track cell migration in vivo

• Biomechanical analysis tools:

  • Atomic force microscopy to measure mechanical properties of KRT14-containing cells

  • Traction force microscopy to quantify forces generated by KRT14-positive cells

  • Microfluidic devices to assess cell deformability and resilience

• Organoid and organ-on-chip systems:

  • KRT14-reporter organoids for high-throughput drug screening

  • Multi-organ chips to study epithelial-mesenchymal interactions

  • Bioprinting of KRT14-positive cell networks with defined geometries

These technologies will enable unprecedented insights into how KRT14-positive cells establish and maintain tissue architecture, respond to mechanical and chemical signals, and orchestrate regenerative processes.

How might therapeutic targeting of KRT14-positive cells impact cancer treatment strategies?

The emerging understanding of KRT14-positive cells in cancer suggests several therapeutic implications:

• Potential therapeutic approaches:

  • Selective ablation using KRT14 promoter-driven suicide genes

  • Differentiation therapy to force KRT14-positive cancer stem cells to mature

  • Targeting unique surface markers co-expressed with KRT14

  • Disruption of niche factors that maintain KRT14-positive cell stemness

• Treatment resistance mechanisms:

  • KRT14-positive cells are enriched after chemotherapy in human bladder cancer

  • These cells likely possess enhanced DNA repair mechanisms

  • Metabolic adaptations may provide stress resistance

  • Quiescence could protect against cell cycle-targeted therapies

• Combination therapy rationales:

  • Targeting KRT14-positive cells + conventional chemotherapy

  • Ablation of KRT14-positive cells + immune checkpoint inhibition

  • Disruption of KRT14+ cell niches + anti-angiogenic therapy

• Biomarker applications:

  • KRT14 expression in circulating tumor cells as a prognostic indicator

  • Monitoring KRT14-positive cell populations during treatment response

  • Spatial distribution of KRT14-positive cells in tumor biopsies as predictive biomarker

The enrichment of KRT14-positive cells following chemotherapy suggests these cells may represent a crucial therapeutic target for preventing recurrence and improving long-term outcomes in bladder cancer and potentially other epithelial malignancies .