Revisiting the significance of keratin expression in complex epithelia.

A large group of keratin genes (n=54 in the human genome) code for intermediate filament (IF)-forming proteins and show differential regulation in epithelial cells and tissues. Keratin expression can be highly informative about the type of epithelial tissue, differentiation status of constituent cells and biological context (e.g. normal versus diseased settings). The foundational principles underlying the use of keratin expression to gain insight about epithelial cells and tissues primarily originated in pioneering studies conducted in the 1980s. The recent emergence of single cell transcriptomics provides an opportunity to revisit these principles and gain new insight into epithelial biology. Re-analysis of single-cell RNAseq data collected from human and mouse skin has confirmed long-held views regarding the quantitative importance and pairwise regulation of specific keratin genes in keratinocytes of surface epithelia. Furthermore, such analyses confirm and extend the notion that changes in keratin gene expression occur gradually as progenitor keratinocytes commit to and undergo differentiation, and challenge the prevailing assumption that specific keratin combinations reflect a mitotic versus a post-mitotic differentiating state. Our findings provide a blueprint for similar analyses in other tissues, and warrant a more nuanced approach in the use of keratin genes as biomarkers in epithelia.

A role for keratin 17 during DNA damage response and tumor initiation.

High levels of the intermediate filament protein keratin 17 (K17) are associated with poor prognoses for several human carcinomas. Studies in mouse models have shown that K17 expression is positively associated with growth, survival, and inflammation in skin and that lack of K17 delays onset of tumorigenesis. K17 occurs in the nucleus of human and mouse tumor keratinocytes where it impacts chromatin architecture, gene expression, and cell proliferation. We report here that K17 is induced following DNA damage and promotes keratinocyte survival. The presence of nuclear K17 is required at an early stage of the double-stranded break (DSB) arm of the DNA damage and repair (DDR) cascade, consistent with its ability to associate with key DDR effectors, including γ-H2A.X, 53BP1, and DNA-PKcs. Mice lacking K17 or with attenuated K17 nuclear import showed curtailed initiation in a two-step skin carcinogenesis paradigm. The impact of nuclear-localized K17 on DDR and cell survival provides a basis for the link between K17 induction and poor clinical outcomes for several human carcinomas.

Keratin 17 regulates nuclear morphology and chromatin organization.

Keratin 17 (KRT17; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wild-type K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17-null cells. Analyses of primary cultures of skin keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization and RNA processing. Key histone modifications and LAP2ß (an isoform encoded by TMPO) localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells.This article has an associated First Person interview with the first author of the paper.

Emerging role for the cytoskeleton as an organizer and regulator of translation.

The cytoskeleton is an intricate and dynamic fibrous network that has an essential role in the generation and regulation of cell architecture and cellular mechanical properties. The cytoskeleton also evolved as a scaffold that supports diverse biochemical pathways. Recent evidence favours the hypothesis that the cytoskeleton participates in the spatial organization and regulation of translation, at both the global and local level, in a manner that is crucial for cellular growth, proliferation and function.

Altered keratinocyte differentiation is an early driver of keratin mutation-based palmoplantar keratoderma.

The type I intermediate filament keratin 16 (KRT16 gene; K16 protein) is constitutively expressed in ectoderm-derived appendages and in palmar/plantar epidermis and is robustly induced when the epidermis experiences chemical, mechanical or environmental stress. Missense mutations at the KRT16 locus can cause pachyonychia congenita (PC, OMIM:167200) or focal non-epidermolytic palmoplantar keratoderma (FNEPPK, OMIM:613000), which each entail painful calluses on palmar and plantar skin. Krt16-null mice develop footpad lesions that mimic PC-associated PPK, providing an opportunity to decipher its pathophysiology, and develop therapies. We report on insight gained from a genome-wide analysis of gene expression in PPK-like lesions of Krt16-null mice. Comparison of this data set with publicly available microarray data of PPK lesions from individuals with PC revealed significant synergies in gene expression profiles. Keratin 9 (Krt9/K9), the most robustly expressed gene in differentiating volar keratinocytes, is markedly downregulated in Krt16-null paw skin, well-ahead of lesion onset, and is paralleled by pleiotropic defects in terminal differentiation. Effective prevention of PPK-like lesions in Krt16-null paw skin (via topical delivery of the Nrf2 inducer sulforaphane) involves the stimulation of Krt9 expression. These findings highlight a role for defective terminal differentiation and loss of Krt9/K9 expression as additional drivers of PC-associated PPK and highlight restoration of KRT9 expression as a worthy target for therapy. Further, we report on the novel observation that keratin 16 can localize to the nucleus of epithelial cells, implying a potential nuclear function that may be relevant to PC and FNEPPK.

The incidental pore: CaV1.2 and stem cell activation in quiescent hair follicles.

The hair follicle undergoes a lifelong developmental cycle that depends on the integration between activating and inhibitory signals acting to regulate and guide the proliferation and differentiation of pluripotent epithelial stem cells. The effectors and mechanisms responsible for re-entry of quiescent telogen hair follicles into the hair-producing anagen stage in mature skin remain incompletely understood. In the June 1, 2013, issue of Genes & Development, Yucel and colleagues (pp. 1217-1222) reported the unexpected finding that CaV1.2, the pore-forming subunit in a well-characterized voltage-gated, L-type calcium channel, is expressed in hair follicle stem cells and contributes to anagen re-entry but does so in a calcium flux-independent fashion.

Oxidative stress management in the hair follicle: Could targeting NRF2 counter age-related hair disorders and beyond?

Widespread expression of the transcription factor, nuclear factor (erythroid-derived 2)-like 2 (NRF2), which maintains redox homeostasis, has recently been identified in the hair follicle (HF). Small molecule activators of NRF2 may therefore be useful in the management of HF pathologies associated with redox imbalance, ranging from HF greying and HF ageing via androgenetic alopecia and alopecia areata to chemotherapy-induced hair loss. Indeed, NRF2 activation has been shown to prevent peroxide-induced hair growth inhibition. Multiple parameters can increase the levels of reactive oxygen species in the HF, for example melanogenesis, depilation-induced trauma, neurogenic and autoimmune inflammation, toxic drugs, environmental stressors such as UV irradiation, genetic defects and aging-associated mitochondrial dysfunction. In this review, the potential mechanisms whereby NRF2 activation could prove beneficial in treatment of redox-associated HF disorders are therefore discussed.

The keratin 16 null phenotype is modestly impacted by genetic strain background in mice.

The type I intermediate filament keratin 16 (K16) is constitutively expressed in ectoderm-derived appendages and is inducibly expressed in the epidermis upon barrier-compromising challenges. Dominantly acting missense alleles in KRT16 are causative for pachyonychia congenita (PC), a genodermatosis involving debilitating palmoplantar keratoderma (PPK), nail dystrophy, oral lesions and, frequently, alterations in glands and hair. C57Bl/6;Krt16-/- mice develop oral lesions early after birth and PC-like PPK lesions as young adults. These PPK lesions have a marked dysregulation of skin barrier-related genes and innate immunity effectors (eg danger-associated molecular patterns) and are preceded by oxidative stress secondary to hypoactive Nrf2 signalling. These molecular features are present in PPK lesions of PC patients. Here, we report that all components of the C57Bl/6;Krt16-/- mouse phenotype occur as well in the FVB strain background, albeit less severely so, a significant observation in the light of variations in the clinical presentation of individuals harbouring disease-causing mutations in the KRT16 gene.

Randomized, split-body, single-blinded clinical trial of topical broccoli sprout extract: Assessing the feasibility of its use in keratin-based disorders.

BACKGROUND: Epidermolysis bullosa simplex is a skin-blistering disorder caused by mutations in keratin (K)14 or K5. Treatment with nuclear factor (erythroid-derived 2)-like 2 inducer sulforaphane ameliorated skin blistering in Krt14-null mice, correlating with induction of K17. To be therapeutically useful for epidermolysis bullosa simplex, topical broccoli sprout extract (BSE), enriched for sulforaphane, would ideally induce the expression of homologous keratins (eg, K6, K17, K16) in the basal layer of human epidermis without impacting expression of defective keratins (K5/K14). OBJECTIVE: The purpose of this 1-week, randomized, split-body, single-blinded, placebo-controlled trial was to assess the impact of BSE on keratin expression. METHODS: Five subjects (34-71 years old) applied BSE (500 nmol of sulforaphane/mL) or vehicle alone to the inner aspect of the arm daily. Expression of keratin, nuclear factor (erythroid-derived 2)-like 2, and other markers was assessed using reverse transcription-polymerase chain reaction and indirect immunofluorescence. RESULTS: One subject (age 71 years) was excluded a posteriori because of poor tissue quality. Topical BSE activated nuclear factor (erythroid-derived 2)-like 2 and up-regulated K17 in the epidermis of all subjects, had variable effects on K16 and K6 expression, and did not alter expression of K14 or K5. LIMITATIONS: Small sample size is a limitation. CONCLUSION: BSE represents an attractive therapeutic candidate for K14-associated epidermolysis bullosa simplex.

Complementary roles of specific cysteines in keratin 14 toward the assembly, organization, and dynamics of intermediate filaments in skin keratinocytes.

We recently showed that inter-keratin disulfide bonding plays an important role in the assembly, organization, and dynamics of keratin intermediate filaments in skin keratinocytes. In particular, cysteine 367 located in the central α-helical rod domain of keratin 14 is necessary for the formation of a stable perinuclear network of keratin filaments (with type II partner keratin 5) in skin keratinocytes analyzed by static and live cell imaging. Here, we show that two additional cysteine residues located in the non-helical head domain of K14, Cys-4 and Cys-40, also participate in inter-keratin disulfide bonding and tandemly play a key role complementary to that of Cys-367 in the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in primary culture. Analysis of K14 variants with single or multiple substitutions of cysteine residues points to a spatial and temporal hierarchy in how Cys-4/Cys-40 and Cys-367 regulate keratin assembly in vitro and filament dynamics in live keratinocytes in culture. Our findings substantiate the importance and complexity of a novel determinant, namely inter-keratin disulfide bonding, for the regulation of several aspects of keratin filaments in surface epithelia.

Keratin 16 regulates innate immunity in response to epidermal barrier breach.

Mutations in the type I keratin 16 (Krt16) and its partner type II keratin 6 (Krt6a, Krt6b) cause pachyonychia congenita (PC), a disorder typified by dystrophic nails, painful hyperkeratotic calluses in glabrous skin, and lesions involving other epithelial appendages. The pathophysiology of these symptoms and its relationship to settings in which Krt16 and Krt6 are induced in response to epidermal barrier stress are poorly understood. We report that hyperkeratotic calluses arising in the glabrous skin of individuals with PC and Krt16 null mice share a gene expression signature enriched in genes involved in inflammation and innate immunity, in particular damage-associated molecular patterns. Transcriptional hyper-activation of damage-associated molecular pattern genes occurs following de novo chemical or mechanical irritation to ear skin and in spontaneously arising skin lesions in Krt16 null mice. Genome-wide expression analysis of normal mouse tail skin and benign proliferative lesions reveals a tight, context-dependent coregulation of Krt16 and Krt6 with genes involved in skin barrier maintenance and innate immunity. Our results uncover a role for Krt16 in regulating epithelial inflammation that is relevant to genodermatoses, psoriasis, and cancer and suggest a avenue for the therapeutic management of PC and related disorders.

Directed expression of a chimeric type II keratin partially rescues keratin 5-null mice.

The crucial role of structural support fulfilled by keratin intermediate filaments (IFs) in surface epithelia likely requires that they be organized into cross-linked networks. For IFs comprised of keratins 5 and 14 (K5 and K14), which occur in basal keratinocytes of the epidermis, formation of cross-linked bundles is, in part, self-driven through cis-acting determinants. Here, we targeted the expression of a bundling-competent KRT5/KRT8 chimeric cDNA (KRT8bc) or bundling-deficient wild type KRT8 as a control to the epidermal basal layer of Krt5-null mice to assess the functional importance of keratin IF self-organization in vivo. Such targeted expression of K8bc rescued Krt5-null mice with a 47% frequency, whereas K8 completely failed to do so. This outcome correlated with lower than expected levels of K8bc and especially K8 mRNA and protein in the epidermis of E18.5 replacement embryos. Ex vivo culture of embryonic skin keratinocytes confirmed the ability of K8bc to form IFs in the absence of K5. Additionally, electron microscopy analysis of E18.5 embryonic skin revealed that the striking defects observed in keratin IF bundling, cytoarchitecture, and mitochondria are partially restored by K8bc expression. As young adults, viable KRT8bc replacement mice develop alopecia and chronic skin lesions, indicating that the skin epithelia are not completely normal. These findings are consistent with a contribution of self-mediated organization of keratin IFs to structural support and cytoarchitecture in basal layer keratinocytes of the epidermis and underscore the importance of context-dependent regulation for keratin genes and proteins in vivo.

Keratin function in skin epithelia: a broadening palette with surprising shades.

Keratins make up the largest subgroup of intermediate filament (IF) proteins and form a dynamic network of 10-12 nm filaments, built from type I/type II heterodimers, in the cytoplasm of epithelial cells. A major function of keratin IFs is to protect epithelial cells from mechanical and non-mechanical stresses that cause cell rupture and death. Interference with this role is the root cause of a large number of inherited epithelial fragility conditions. Additional functions, non-mechanical in nature, are manifested in a way that depends on the specific keratin and on the epithelial context. The recent discovery of unusual mutations affecting keratin proteins has uncovered a novel dimension of their mechanical support function, and has synergized with mouse genetics to reveal a role in skin pigmentation. Other studies extended the role of keratin proteins in regulating the response to pro-apoptotic signals, and revealed their ability to modulate protein synthesis and cell size in epithelial cells challenged to grow.

Nuclear roles for non-lamin intermediate filament proteins.

The nuclear-localized lamins have long been thought to be the only intermediate filaments (IFs) with an impact on the architecture, properties, and functions of the nucleus. Recent studies, however, uncovered significant roles for IFs other than lamins (here referred to as "non-lamin IFs") in regulating key properties of the nucleus in various cell types and biological settings. In the cytoplasm, IFs often occur in the perinuclear space where they contribute to local stiffness and impact the shape and/or the integrity of the nucleus, particularly in cells under stress. In addition, selective non-lamin IF proteins can occur inside the nucleus where they partake in fundamental processes including nuclear architecture and chromatin organization, regulation of gene expression, cell cycle progression, and the repair of DNA damage. This text reviews the evidence supporting a role for non-lamin IF proteins in regulating various properties of the nucleus and highlights opportunities for further study.

Significance of stress keratin expression in normal and diseased epithelia.

A group of keratin intermediate filament genes, the type II KRT6A-C and type I KRT16 and KRT17, are deemed stress responsive as they are induced in keratinocytes of surface epithelia in response to environmental stressors, in skin disorders (e.g., psoriasis) and in carcinomas. Monitoring stress keratins is widely used to identify keratinocytes in an activated state. Here, we analyze single-cell transcriptomic data from healthy and diseased human skin to explore the properties of stress keratins. Relative to keratins occurring in healthy skin, stress-induced keratins are expressed at lower levels and show lesser type I-type II pairwise regulation. Stress keratins do not "replace" the keratins expressed during normal differentiation nor reflect cellular proliferation. Instead, stress keratins are consistently co-regulated with genes with roles in differentiation, inflammation, and/or activation of innate immunity at the single-cell level. These findings provide a roadmap toward explaining the broad diversity and contextual regulation of keratins.

Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation.

Acute inflammation, characterized by a rapid influx of neutrophils, is a protective response that can lead to chronic inflammatory diseases when left unresolved. Secretion of LTB 4 -containing exosomes is required for effective neutrophil infiltration during inflammation. In this study, we show that neutrophils release nuclear DNA in a non-lytic, rapid, and repetitive manner, via a mechanism distinct from suicidal NET release and cell death. The packaging of nuclear DNA occurs in the lumen of nuclear envelope (NE)-derived multivesicular bodies (MVBs) that harbor the LTB 4 synthesizing machinery and is mediated by the lamin B receptor (LBR) and chromatin decondensation. Disruption of secreted exosome-associated DNA (SEAD) in a model of sterile inflammation in mouse skin amplifies and prolongs the presence of neutrophils, impeding the onset of resolution. Together, these findings advance our understanding of neutrophil functions during inflammation and the physiological significance of NETs, with implications for novel treatments for inflammatory disorders.

Pachyonychia Congenita: A Research Agenda Leading to New Therapeutic Approaches.

Pachyonychia congenita (PC) is a dominantly inherited genetic disorder of cornification. PC stands out among other genodermatoses because despite its rarity, it has been the focus of a very large number of pioneering translational research efforts over the past 2 decades, mostly driven by a patient support organization, the Pachyonychia Congenita Project. These efforts have laid the ground for innovative strategies that may broadly impact approaches to the management of other inherited cutaneous and noncutaneous diseases. This article outlines current avenues of research in PC, expected outcomes, and potential hurdles.

Identification of novel interaction between annexin A2 and keratin 17: evidence for reciprocal regulation.

Keratins are cytoplasmic intermediate filament proteins providing crucial structural support in epithelial cells. Keratin expression has diagnostic and even prognostic value in disease settings, and recent studies have uncovered modulatory roles for select keratin proteins in signaling pathways regulating cell growth and cell death. Elevated keratin expression in select cancers is correlated with higher expression of EGF receptor (EGFR), whose overexpression and/or mutation give rise to cancer. To explore the role of keratins in oncogenic signaling pathways, we examined the regulation of epithelial growth-associated keratin 17 (K17) in response to EGFR activation. K17 is specifically up-regulated in detergent-soluble fraction upon EGFR activation, and immunofluorescence analysis revealed alterations in K17-containing filaments. Interestingly, we identified AnxA2 as a novel interacting partner of K17, and this interaction is antagonized by EGFR activation. K17 and AnxA2 proteins show reciprocal regulation. Modulating expression of AnxA2 altered K17 stability, and AnxA2 overexpression delays EGFR-mediated change in K17 detergent solubility. Down-regulation of K17 expression, in turn, results in decreased AnxA2 phosphorylation at Tyr-23. These findings uncover a novel interaction involving K17 and AnxA2 and identify AnxA2 as a potential regulator of keratin filaments.

Keratin 17- and PKCα-dependent transient amplification of neutrophil influx after repeated stress to the skin.

Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, a scaffold essential for PKCα activity. Finally, analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.

Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized. RESULTS: Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs and showed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs bound to the 3' untranslated region (3'UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, these mRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO). CONCLUSIONS: This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling the subcellular localization of RNA-binding proteins to support pathways involved in cancer progression.