KRT16 Human

What is KRT16 and where is it expressed in human skin?

KRT16 (Keratin 16) is a type I intermediate filament gene that encodes the K16 protein. It is constitutively expressed in ectoderm-derived appendages and palmar/plantar epidermis, and is robustly induced when the epidermis experiences chemical, mechanical, or environmental stress . Spatial analysis reveals that KRT16 is strongly expressed in the suprabasal layers of the epidermis in lesional skin, contrasting with the pattern of keratins K14 and K5, which are predominantly expressed in the basal layer .

Methodological approach: To investigate KRT16 expression patterns, researchers can employ immunofluorescence staining with K16-specific antibodies, RNA in situ hybridization, single-cell RNA sequencing, and spatial transcriptomics. These techniques allow for precise mapping of KRT16 expression within the tissue architecture and can reveal relationships with other markers.

What mutations in KRT16 are associated with human disease and what are their phenotypic consequences?

Missense mutations in the KRT16 locus can cause pachyonychia congenita (PC, OMIM:167200) or focal non-epidermolytic palmoplantar keratoderma (FNEPPK, OMIM:613000), which both involve painful calluses on palmar and plantar skin . Specific mutations identified in human patients include K16-R127C and K16-R127G .

Methodological approach: To study these mutations, researchers typically use:

• DNA extraction and sequencing to identify mutations

• Patient-derived keratinocyte cultures

• CRISPR-Cas9 gene editing to introduce specific mutations in cell models

• Transgenic mouse models expressing mutant KRT16

• Comparative transcriptomic analysis between wild-type and mutant tissues

How does KRT16 expression change in inflammatory skin conditions?

KRT16 is extensively used as a biomarker for psoriasis, hidradenitis suppurativa, atopic dermatitis, and other inflammatory disorders . In these conditions, KRT16 expression is significantly upregulated in the suprabasal layers of the epidermis.

Methodological approach: To study KRT16 changes in inflammatory conditions, researchers can:

• Perform immunohistochemistry on biopsy samples

• Compare KRT16 mRNA levels using RT-qPCR in affected versus unaffected skin

• Conduct single-cell RNA sequencing to identify cell populations with altered KRT16 expression

• Use spatial transcriptomics to map KRT16 expression patterns in relation to inflammatory infiltrates

• Correlate KRT16 expression levels with clinical severity scores

What animal models are available for studying KRT16-related disorders?

Krt16-null mice develop footpad lesions that mimic PC-associated palmoplantar keratoderma (PPK) . These models show similar transcriptional changes to human PC cases, with significant overlap in differentially expressed genes.

Methodological approach: When working with Krt16-null mouse models, researchers should:

• Perform time-course analyses to track disease progression

• Use histopathological assessment to characterize tissue architecture changes

• Conduct transcriptomic profiling at different disease stages

• Compare findings with human patient samples for translational relevance

• Consider developing conditional knockout models for temporal control of gene deletion

What are the standard methods for detecting and quantifying KRT16 expression?

Methodological approach: Multiple complementary techniques should be used:

• Protein level detection:

  • Immunohistochemistry or immunofluorescence on tissue sections

  • Western blotting for semi-quantitative analysis

  • Flow cytometry for cell-level quantification

• mRNA level detection:

  • RT-qPCR for targeted quantification

  • RNA-seq for genome-wide expression analysis

  • In situ hybridization for spatial localization

• Single-cell approaches:

  • Single-cell RNA-seq for cellular heterogeneity

  • CyTOF for protein expression at single-cell resolution

  • Spatial transcriptomics for preserving tissue context

How does KRT16 regulate innate immune responses in skin?

Research shows that K16 negatively regulates type-I interferon (IFN) signaling and innate immune responses . In mouse skin, loss of Krt16 leads to exacerbation of imiquimod-induced psoriasiform disease and heightened recruitment of neutrophils in phorbol ester-induced acute sterile inflammation models .

Methodological approach: To investigate this regulatory function, researchers should:

• Use co-immunoprecipitation followed by mass spectrometry to identify K16 interaction partners

• Employ proximity ligation assays to detect protein-protein interactions in situ

• Compare inflammatory responses in wild-type versus Krt16-null cells/tissues

• Analyze activation of immune signaling pathways using phospho-specific antibodies

• Perform ChIP-seq to identify transcription factors regulated by K16-dependent pathways

What is the molecular mechanism by which KRT16 inhibits type I interferon signaling?

K16 interacts with effectors of the RIG-I-like receptor (RLR) pathway, including 14-3-3ɛ, and inhibits the 14-3-3ɛ:RIG-I interaction upstream of IFN activation both in vivo and ex vivo . In KRT16 null human keratinocytes, loss of K16 amplifies IFN signaling including phospho-IRF7 and ISG15 after treatment with synthetic dsRNA poly(I:C) .

Methodological approach: To elucidate this mechanism:

• Use CRISPR-Cas9 to generate KRT16 knockout in human keratinocytes

• Treat cells with poly(I:C) to stimulate IFN pathways

• Analyze phosphorylation status of key signaling molecules (IRF3, IRF7, STAT1)

• Perform domain mapping to identify regions of K16 critical for interaction with 14-3-3ɛ

• Reconstitute knockout cells with wild-type or mutant K16 to assess rescue effects

How do alterations in KRT16 affect keratinocyte differentiation programs?

Research indicates that altered keratinocyte differentiation is an early driver of keratin mutation-based palmoplantar keratoderma . In Krt16-null mouse models, decreased Krt9 expression precedes the onset of lesions in paw skin, suggesting differentiation defects are an early event in pathogenesis .

Methodological approach: To study differentiation effects:

• Perform time-course analysis of differentiation markers in Krt16-null versus wild-type models

• Use 3D organotypic cultures to recapitulate differentiation in vitro

• Apply single-cell RNA-seq to identify altered cell state transitions

• Conduct calcium-shift assays to induce differentiation in cultured keratinocytes

• Analyze expression of differentiation-associated genes using RT-qPCR and immunostaining

What is the relationship between KRT16 and other keratin genes in stress responses?

The relationship between different keratin genes during stress responses reveals complex regulatory mechanisms. In Krt16-null paw skin, Krt6a mRNA is significantly increased at 2, 4, and especially in 8-week-old mice relative to wild-type footpad skin . This induction increases over time and reaches very high levels in established PPK-like lesions .

Methodological approach: To investigate keratin gene relationships:

• Perform comprehensive keratin profiling using RNA-seq in different conditions

• Use ChIP-seq to identify shared regulatory elements between keratin genes

• Apply CRISPR interference or activation to modulate expression of individual keratins

• Analyze compensatory responses in knockout models

• Study promoter activities using reporter assays

How can spatial transcriptomics enhance our understanding of KRT16 in inflammatory skin conditions?

Spatial transcriptomics reveals that KRT16 is strongly expressed in the suprabasal layers in the epidermis of lesional skin, while IFN responsive genes like IFITM1, IFITM3, and CD74 are spatially restricted to the basal layer . This spatial separation appears critical for maintaining immune homeostasis.

Methodological approach: For spatial analysis:

• Apply spatial transcriptomics platforms (e.g., 10x Visium) to skin biopsies

• Use computational analysis to identify spatially co-regulated gene modules

• Validate key findings with multiplexed RNA in situ hybridization

• Integrate spatial data with single-cell RNA-seq for cell type resolution

• Correlate spatial patterns with histopathological features

How do transcriptional profiles compare between KRT16-null models and human PC patients?

Transcriptomic comparisons show significant overlap between Krt16-null mouse footpad lesions and human PC cases. Of the 391 upregulated genes in mouse data, 22 are shared with KRT16 human cases (p = 5.0e-19) and 32 with KRT6 human cases (p = 1.4e-25) . Similarly, of 564 downregulated genes, 30 are shared with KRT16 human cases (p = 1.1e-16) and 9 with KRT6 human cases (p = 1.2e-6) .

Methodological approach: For comparative transcriptomics:

• Use standardized RNA isolation and sequencing protocols

• Apply robust bioinformatic pipelines for cross-species comparisons

• Validate key differentially expressed genes by RT-qPCR

• Perform pathway enrichment analysis to identify shared biological processes

• Use network analysis to identify conserved regulatory hubs

What are optimal 3D skin models for studying KRT16 function in human keratinocytes?

The N/TERT human keratinocyte cell line can be adapted to generate a post-confluent, uniformly bilayered epithelium in submerged culture under standard KSFM growth medium, without requiring 3D growth at the liquid-air interface . This system allows for the study of KRT16 function in a physiologically relevant context.

Methodological approach: For 3D model development:

• Optimize culture conditions (media, substrate, cell density)

• Validate model using immunostaining for differentiation markers

• Apply CRISPR-Cas9 to generate KRT16 knockout or mutant lines

• Test model responses to inflammatory stimuli (e.g., poly(I:C))

• Compare findings with primary human skin samples for validation