miR-181a/b-5p negatively regulates keratinocytes proliferation by targeting MELK.

Accumulating evidence indicates that microRNAs (miRNAs) have a vital effect on the pathogenesis of psoriasis. This study is conducted to investigate the potential involvement of miR-181a-5p and miR-181b-5p in the proliferation of HaCaT keratinocytes. Cell viability and proliferation were evaluated respectively in this study using the CCK-8 and the 5-ethynyl-2'-deoxyuridine (EdU) assays. The expression of Maternal Embryonic Leucine Zipper Kinase (MELK) and Keratin 16 (KRT16) mRNA and protein in tissues and cells was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The Luciferase reporter system analyzes the connection between miR-181a-5p/miR-181b-5p and MELK. The results showed that miR-181a/b-5p expression was downregulated in the psoriasis lesions and negatively regulated the proliferation of keratinocytes. MELK was directly targeted by miR-181a-5p/miR-181b-5p. In addition, HaCaT keratinocytes proliferation was inhibited by knockdown of MELK while promoted dramatically by MELK overexpression. Notably, miR-181a/b-5p mimics could attenuate the effects of MELK in keratinocytes. In conclusion, our research findings suggested miR-181a-5p and miR-181b-5p negatively regulate keratinocyte proliferation by targeting MELK, providing potential diagnostic biomarkers and therapeutic targets for psoriasis.

BarH-like homeobox 2 represses the transcription of keratin 16 and affects Ras signaling pathway to suppress nasopharyngeal carcinoma progression.

Nasopharyngeal carcinoma (NPC) refers to a malignancy initiating from the superior mucosal epithelium of the nasopharynx. Optimal therapies for NPC are still needed. In this investigation, we attempted to explore whether BarH-like homeobox 2 (BARX2), a well-known tumor suppressor, had anti-cancer properties on NPC, and the possible mechanisms. After searching for NPC-related databases, we determined BARX2 as one of the core genes in NPC. The results of RT-qPCR and immunohistochemistry or Western blot demonstrated that BARX2 was reduced in NPC patients and cells. Ectopic expression of BARX2 reverted the malignant phenotype of NPC cells. Mechanistically, BARX2 bound to the keratin 16 (KRT16) promoter to downregulate its expression. In addition, BARX2 was found to reduce the phosphorylation levels of MEK and ERK. Further KRT16 upregulation in cells overexpressing BARX2 promoted malignant aggressiveness of C666-1 and HNE3 cells and activated the Ras signaling pathway. BARX2 inhibited the growth and metastasis of tumors and suppressed the Ras signaling pathway in vivo. In conclusion, our findings indicate that BARX2 reverts malignant phenotypes of NPC cells by downregulating KRT16 in a Ras-dependent fashion. BARX2 might act as a possible therapeutic regulator for NPC.

Genotype‒Structurotype‒Phenotype Correlations in Patients with Pachyonychia Congenita.

Pachyonychia congenita (PC) is a genetic disorder of keratin that presents with nail dystrophy, painful palmoplantar keratoderma, and other clinical manifestations. We investigated the genotype‒structurotype‒phenotype correlations seen with mutations in keratin genes (keratin [K]6A, K6B, K6C, K16, K17) and utilized protein structure modeling of high-frequency mutations to examine the functional importance of keratin structural domains in PC pathogenesis. Participants of the International PC Research Registry underwent genetic testing and completed a standardized survey on their symptoms. Our results support previous reports associating oral leukokeratosis with K6A mutations and cutaneous cysts, follicular hyperkeratosis, and natal teeth with K17 mutations. Painful keratoderma was prominent with K6A and K16 mutations. Nail involvement was most common in patients with K6A mutation and least common in those with K6C mutation. Across keratin subtypes, patients with coil 2B mutations had the greatest impairment in ambulation, and patients with coil 1A mutations reported more emotional issues. Molecular modeling demonstrated that hotspot missense mutations in PC largely disrupted hydrophobic interactions or surface charge. The former may destabilize keratin dimers/tetramers, whereas the latter likely interferes with higher-order keratin filament formation. Understanding the pathologic alterations in keratin structure improves our knowledge of how PC genotype correlates with clinical phenotype, advancing insight into disease pathogenesis and therapeutic development.

LncRNA NORAD engages in psoriasis by binding to miR-26a to regulate keratinocyte proliferation.

BACKGROUND: Psoriasis is a chronic, inflammatory skin disease. It was reported that lncRNA Non-coding RNA-activated by DNA damage (NORAD) has potential regulatory effects on skin diseases. Our previous studies found that lncRNA NORAD was highly expressed and its potential target miR-26a was down-regulated in psoriasis model mice. Here, we aimed to investigate the role of NORAD in the development of psoriasis. METHODS: IL-22/LPS (interleukin-22/lipopolysaccharide)-stimulated HaCaT (human immortalized keratinocytes) cell model and imiquimod-induced mouse model were established. Keratin 6 (K6), Keratin 16 (K16), Keratin 17 (K17), and Cell division cycle 6 (CDC6) levels were detected by western blot. Cell activity was detected by CCK-8, MTT, and EdU assays. Quantitative real-time PCR was performed to examine the levels of NORAD, miR-26a, CDC6, K6, K16, and K17. Haematoxylin-eosin staining was applied to observe the degree of skin thickening and hyperplasia. Fluorescence in situ hybridization detects the location of NORAD. RNA immunoprecipitation, RNA pull-down, and Luciferase test were performed to detect the interaction between NORAD and miR-26a. RESULTS: In IL-22/LPS-stimulated HaCaT cells, NORAD, CDC6, and keratinocyte proliferation-related proteins (K6, K16, and K17) were up-regulated and miR-26a was down-regulated. Cell survival and proliferation were also increased. However, the results were reversed after interference with NORAD. Also, in vitro experiments revealed that NORAD negatively regulated miR-26a. In IL-22/LPS-stimulated HaCaT cells and skin of imiquimod-induced mice, we found that lower NORAD resulted in an increase of miR-26a and a decrease of CDC6, further decreased levels of keratinocyte proliferation-related proteins (K6, K16, and K17).

A role for keratins in supporting mitochondrial organization and function in skin keratinocytes.

Mitochondria fulfill essential roles in ATP production, metabolic regulation, calcium signaling, generation of reactive oxygen species (ROS), and additional determinants of cellular health. Recent studies have highlighted a role for mitochondria during cell differentiation, including in skin epidermis. The observation of oxidative stress in keratinocytes from Krt16 null mouse skin, a model for pachyonychia congenita (PC)-associated palmoplantar keratoderma, prompted us to examine the role of Keratin (K) 16 protein and its partner K6 in regulating the structure and function of mitochondria. Electron microscopy revealed major anomalies in mitochondrial ultrastructure in late stage, E18.5, Krt6a/Krt6b null embryonic mouse skin. Follow-up studies utilizing biochemical, metabolic, and live imaging readouts showed that, relative to controls, skin keratinocytes null for Krt6a/Krt6b or Krt16 exhibit elevated ROS, reduced mitochondrial respiration, intracellular distribution differences, and altered movement of mitochondria within the cell. These findings highlight a novel role for K6 and K16 in regulating mitochondrial morphology, dynamics, and function and shed new light on the causes of oxidative stress observed in PC and related keratin-based skin disorders.

Pathophysiology of pachyonychia congenita-associated palmoplantar keratoderma: new insights into skin epithelial homeostasis and avenues for treatment.

BACKGROUND: Pachyonychia congenita (PC), a rare genodermatosis, primarily affects ectoderm-derived epithelial appendages and typically includes oral leukokeratosis, nail dystrophy and very painful palmoplantar keratoderma (PPK). PC dramatically impacts quality of life although it does not affect lifespan. PC can arise from mutations in any of the wound-repair-associated keratin genes KRT6A, KRT6B, KRT6C, KRT16 or KRT17. There is no cure for this condition, and current treatment options for PC symptoms are limited and palliative in nature. OBJECTIVES: This review focuses on recent progress made towards understanding the pathophysiology of PPK lesions, the most prevalent and debilitating of all PC symptoms. METHODS: We reviewed the relevant literature with a particular focus on the Krt16 null mouse, which spontaneously develops footpad lesions that mimic several aspects of PC-associated PPK. RESULTS: There are three main stages of progression of PPK-like lesions in Krt16 null mice. Ahead of lesion onset, keratinocytes in the palmoplantar (footpad) skin exhibit specific defects in terminal differentiation, including loss of Krt9 expression. At the time of PPK onset, there is elevated oxidative stress and hypoactive Keap1-Nrf2 signalling. During active PPK, there is a profound defect in the ability of the epidermis to maintain or return to normal homeostasis. CONCLUSIONS: The progress made suggests new avenues to explore for the treatment of PC-based PPK and deepens our understanding of the mechanisms controlling skin tissue homeostasis. What's already known about this topic? Pachyonychia congenita (PC) is a rare genodermatosis caused by mutations in KRT6A, KRT6B, KRT6C, KRT16 and KRT17, which are normally expressed in skin appendages and induced following injury. Individuals with PC present with multiple clinical symptoms that usually include thickened and dystrophic nails, palmoplantar keratoderma (PPK), glandular cysts and oral leukokeratosis. The study of PC pathophysiology is made challenging because of its low incidence and high complexity. There is no cure or effective treatment for PC. What does this study add? This text reviews recent progress made when studying the pathophysiology of PPK associated with PC. This recent progress points to new possibilities for devising effective therapeutics that may complement current palliative strategies.

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.

Silencing KRT16 inhibits keratinocyte proliferation and VEGF secretion in psoriasis via inhibition of ERK signaling pathway.

Psoriasis is a multisystem disease affecting about 2% of the population, while keratin16 (KRT16) has been reported to participate in psoriasis. However, the specific mechanism of KRT16 in psoriasis was inadequately investigated. The objective of the study was to elucidate the mechanism by which siRNA-mediated silencing of KRT16 affects keratinocyte proliferation and vascular endothelial growth factor (VEGF) secretion in psoriasis through the extracellular signal-related kinase (ERK) signaling pathway. Psoriasis-related core gene KRT16 was screened out. Then, the expression of KRT16, VEGF, and ERK signaling pathway-related genes was detected in psoriatic patients. To further investigate the mechanism of KRT16, keratinocytes in psoriatic patients were treated with KRT16 siRNA or/and ERK inhibitor (PD98059) to detect the changes in related gene expression and cell survival. KRT16 was involved in psoriasis development. The expression levels of KRT16, p-ERK1/2, and VEGF in lesion tissues are significantly elevated. Keratinocytes treated with KRT16-siRNA and KRT16-siRNA + PD98059 exhibited reduced KRT16, p-ERK1/2, and VEGF expression. The cell survival rate in cells treated with KRT16-siRNA, PD98059, and KRT16-siRNA + PD98059 reduced significantly. These findings indicate that silencing KRT16 inhibits keratinocyte proliferation and VEGF secretion in psoriasis via inhibition of ERK signaling pathway, which provides a basic theory in the treatment of psoriasis.

A KRT16 mutation in the first Chinese pedigree with Pachyonychia congenita and review of the literatures.

BACKGROUND: Pachyonychia congenita (PC), a rare autosomal dominant disorder, is featured by significant hypertrophic nail, palmoplantar keratoderma, and plantar pain. It is caused by the mutation of KRT6A, KRT6B, KRT6C, KRT16, or KRT17. AIMS: To identify the gene mutation caused the PC in a Chinese family. PATIENTS/METHODS: Genomic DNA was extracted from peripheral blood samples of five patients and six healthy individuals. Genomic DNA of three patients was sequenced by whole-exome sequencing (WES). Then, exons 6 of KRT16 of all samples were amplified by polymerase chain reaction (PCR), and PCR products were sequenced to identify potential mutations. RESULTS: We identified the proline substitution mutation p.Leu421Pro (c.1262T>C) in the 2B domain of K16 that is associated with PC in a Chinese family. The same mutation was not found in the six healthy individuals of the family. CONCLUSIONS: The mutation found in this study is the first report in China. So far, 25 mutations in KRT16 have been reportedly associated with PC. Twenty-one mutations are located on exon 1, and four mutations on exon 6.

A novel miR-365-3p/EHF/keratin 16 axis promotes oral squamous cell carcinoma metastasis, cancer stemness and drug resistance via enhancing ß5-integrin/c-met signaling pathway.

BACKGROUND: Targeting the c-Met signaling pathway has become a therapeutic strategy in multiple types of cancer. We unveiled a novel c-Met regulating mechanism that could be applied as a modality for oral squamous cell carcinoma (OSCC) therapy. METHODS: Upregulation of keratin 16 (KRT16) was found by comparing isogenic pairs of low and high invasive human OSCC lines via microarray analysis. OSCC cells with ectopic expression or silencing of KRT16 were used to scrutinize functional roles and associated molecular mechanisms. RESULTS: We observed that high KRT16 expression significantly correlated with poorer pathological differentiation, advanced stages, increased lymph nodes metastasis, and decreased survival rate from several Taiwanese OSCC patient cohorts. We further revealed that miR-365-3p could target ETS homologous factor (EHF), a KRT16 transcription factor, to decrease migration, invasion, metastasis and chemoresistance in OSCC cells via inhibition of KRT16. Under confocal microscopic examination, c-Met was found possibly partially associates with KRT16 through ß5-integrin. Colocalization of these three proteins may facilitate c-Met and ß5-integrin-mediated signaling in OSCC cells. Depletion of KRT16 led to increased protein degradation of ß5-integrin and c-Met through a lysosomal pathway leading to inhibition of their downstream Src/STAT3/FAK/ERK signaling in OSCC cells. Knockdown of KRT16 enhanced chemosensitivity of OSCC towards 5-fluorouracil (5-FU). Various combination of c-Met inhibitor (foretinib), protein tyrosine kinase inhibitor (genistein), ß5-integrin antibody, and 5-FU markedly augmented cytotoxic effects in OSCC cells as well as tumor killing effects in vitro and in vivo. CONCLUSIONS: Our data indicate that targeting a novel miR-365-3p/EHF/KRT16/ß5-integrin/c-Met signaling pathway could improve treatment efficacy in OSCC.

Lupeol, a Pentacyclic Triterpene, Promotes Migration, Wound Closure, and Contractile Effect In Vitro: Possible Involvement of PI3K/Akt and p38/ERK/MAPK Pathways.

Skin wound healing is a dynamic and complex process involving several mediators at the cellular and molecular levels. Lupeol, a phytoconstituent belonging to the triterpenes class, is found in several fruit plants and medicinal plants that have been the object of study in the treatment of various diseases, including skin wounds. Various medicinal properties of lupeol have been reported in the literature, including anti-inflammatory, antioxidant, anti-diabetic, and anti-mutagenic effects. We investigated the effects of lupeol (0.1, 1, 10, and 20 µg/mL) on in vitro wound healing assays and signaling mechanisms in human neonatal foreskin keratinocytes and fibroblasts. Results showed that, at high concentrations, Lupeol reduced cell proliferation of both keratinocytes and fibroblasts, but increased in vitro wound healing in keratinocytes and promoted the contraction of dermal fibroblasts in the collagen gel matrix. This triterpene positively regulated matrix metalloproteinase (MMP)-2 and inhibited the NF-κB expression in keratinocytes, suggesting an anti-inflammatory effect. Lupeol also modulated the expression of keratin 16 according to the concentration tested. Additionally, in keratinocytes, lupeol treatment resulted in the activation of Akt, p38, and Tie-2, which are signaling proteins involved in cell proliferation and migration, angiogenesis, and tissue repair. These findings suggest that lupeol has therapeutic potential for accelerating wound healing.

Gallic acid inhibits the expression of keratin 16 and keratin 17 through Nrf2 in psoriasis-like skin disease.

Gallic acid (GA), a natural small molecule found in Radix Paeoniae Rubra, has a variety of favorable biological activities. However, the anti-psoriasis effect of GA has never been explored up to now. This study evaluates the protective effect of GA on psoriasis-like skin disease both in vitro and in vivo and explores the underlying mechanism. The results show that GA significantly decreases the mRNA and protein expression of keratin 16 and keratin 17 which are the markers of psoriasis. Additionally, GA obviously ameliorates psoriasis area and severity index scores and decreases the epidermal hyperplasia of psoriasis-like disease mice. The activity of Nrf2 which targets keratin 16 and keratin 17 is significantly downregulated by GA. Furthermore, the downregulation of keratin 16 and keratin 17 induced by GA was abolished by the Nrf2-overexpression in vitro. This study initially elucidates the anti-psoriasis effect and mechanism of GA which hints that GA would be a potential candidate for the treatment of psoriasis.

Effect of short-term liver X receptor activation on epidermal barrier features in mild to moderate atopic dermatitis: A randomized controlled trial.

BACKGROUND: Liver X receptors (LXRs) are involved in maintaining epidermal barrier and suppressing inflammatory responses in model systems. The LXR agonist VTP-38543 showed promising results in improving barrier function and inflammatory responses in model systems. OBJECTIVE: To assess the safety, tolerability, cellular and molecular changes, and clinical efficacy of the topical VTP-38543 in adults with mild to moderate atopic dermatitis (AD). METHODS: A total of 104 ambulatory patients with mild to moderate AD were enrolled in this randomized, double-blind, vehicle-controlled trial between December 2015 and September 2016. VTP-38543 cream in 3 concentrations (0.05%, 0.15%, and 1.0%) or placebo was applied twice daily for 28 days. Pretreatment and posttreatment skin biopsy specimens were obtained from a subset of 33 patients. Changes in SCORing of Atopic Dermatitis, Eczema Area and Severity Index, Investigator's Global Assessment, and tissue biomarkers (by real-time polymerase chain reaction and immunostaining) were evaluated. RESULTS: Topical VTP-38543 was safe and well tolerated. VTP-38543 significantly increased messenger RNA (mRNA) expression of epidermal barrier differentiation (loricrin and filaggrin, P = .02) and lipid (adenosine triphosphate-binding cassette subfamily G member 1 and sterol regulatory element binding protein 1c, P < .01) measures and reduced epidermal hyperplasia markers (thickness, keratin 16 mRNA). VTP-38543 nonsignificantly suppressed cellular infiltrates and down-regulated mRNA expression of several TH17/TH22-related (phosphatidylinositol 3, S100 calcium-binding protein A12) and innate immunity (interleukin 6) markers. CONCLUSION: Topical VTP-38543 is safe and well tolerated. Its application led to improvement in barrier differentiation and lipids. Longer-term studies are needed to clarify whether a barrier-based approach can induce meaningful suppression of immune abnormalities. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02655679.

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.

Implication of KRT16, FAM129A and HKDC1 genes as ATF4 regulated components of the integrated stress response.

The ATF4 transcription factor is a key regulator of the adaptive integrated stress response (ISR) induced by various stresses and pathologies. Identification of novel transcription targets of ATF4 during ISR would contribute to the understanding of adaptive networks and help to identify novel therapeutic targets. We were previously searching for genes that display an inverse regulation mode by the transcription factors ATF4 and p53 in response to mitochondrial respiration chain complex III inhibition. Among the selected candidates the human genes for cytokeratine 16 (KRT16), anti-apoptotic protein Niban (FAM129A) and hexokinase HKDC1 have been found highly responsive to ATF4 overexpression. Here we explored potential roles of the induction of KRT16, FAM129A and HKDC1 genes in ISR. As verified by RT-qPCR, a dysfunction of mitochondrial respiration chain and ER stress resulted in a partially ATF4-dependent stimulation of KRT16, FAM129A and HKDC1 expression in the HCT116 colon carcinoma cell line. ISRIB, a specific inhibitor of ISR, was able to downregulate the ER stress-induced levels of KRT16, FAM129A and HKDC1 transcripts. An inhibition of ATF4 by RNAi attenuated the induction of KRT16, FAM129A and HKDC1 mRNAs in response to ER stress or to a dysfunctional mitochondrial respiration. The similar induction of the three genes was observed in another tumor-derived cervical carcinoma cell line HeLa. However, in HaCaT and HEK293T cells that display transformed phenotypes, but do not originate from patient-derived tumors, the ER stress-inducing treatments resulted in an upregulation of FAM129A and HKDC1, but not KRT16 transcripts, By a luciferase reporter approach we identified a highly active ATF4-responsive element within the upstream region of the KRT16 gene. The results suggest a conditional regulation of KRT16 gene by ATF4 that may be inhibited in normal cells, but engaged during cancer progression. Potential roles of KRT16, FAM129A and HKDC1 genes upregulation in adaptive stress responses and pathologies are discussed.

Nrf2 Promotes Keratinocyte Proliferation in Psoriasis through Up-Regulation of Keratin 6, Keratin 16, and Keratin 17.

Psoriasis is a chronic inflammatory skin disease characterized by keratinocyte hyperproliferation of epidermis. Although hyperproliferation-associated keratins K6, K16, and K17 are considered to be the hallmarks of psoriasis, the molecular basis underlying the overexpression of these keratins remains unclear. Nrf2 regulates cell proliferation. Therefore, we investigated whether Nrf2 regulates keratinocyte proliferation via promoting expression of K6, K16, and K17 in psoriasis. We initially found that psoriatic epidermis exhibited elevated expression of Nrf2. Furthermore, Nrf2 promoted expression of K6, K16, and K17 in both HaCaT cells and primary human keratinocytes by binding to the ARE domains located in the promoter of these genes. Additionally, upon stimulation with IL-17 or IL-22, Nrf2 translocated to the nucleus and initiated expression of targeted keratins. In mice of imiquimod-induced psoriasis-like dermatitis, topical application of Nrf2 small interfering RNA alleviated the epidermal hyperplasia with reduced expression of these keratins. More importantly, Nrf2 promoted the proliferation of human keratinocytes through up-regulation of K6, K16, or K17. These data suggested that inflammatory cytokines promoted Nrf2 nuclear translocation in psoriatic epidermis, which led to elevated expression of K6, K16, and K17, thus promoting keratinocyte proliferation and contributing to the pathogenesis of psoriasis.