The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.

Inherited ichthyosis comprises a series of heterogeneous dermal conditions; it mainly manifests as widespread hyperkeratosis, xerosis and scaling of the skin. At times, overlapping symptoms require differential diagnosis between ichthyosis and several other similar disorders. The present study reports seven patients with confirmed or suspected to be associated with ichthyosis by conducting a thorough clinical and genetic investigation. Genetic testing was conducted using whole‑exome sequencing, with Sanger sequencing as the validation method. The MEGA7 program was used to analyze the conservation of amino acid residues affected by the detected missense variants. The enrolled patients exhibited ichthyosis‑like but distinct clinical manifestations. Genetic analysis identified diagnostic variations in the FLG, STS, KRT10 and SERPINB7 genes and clarified the carrying status of each variant in the respective family members. The two residues affected by the detected missense variants remained conserved across multiple species. Of note, the two variants, namely STS: c.452C>T(p.P151L) and c.647_650del(p.L216fs) are novel. In conclusion, a clear genetic differential diagnosis was made for the enrolled ichthyosis‑associated patients; the study findings also extended the mutation spectrum of ichthyosis and provided solid evidence for the counseling of the affected families.

Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of Keratin-10 (K10) nascent RNA, which marks the onset of stem cell differentiation, we find that Keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.

Reference genes are crucial in molecular biological studies as an internal control for gene re-search as they exhibit consistent expression patterns across many tissue types. In canines, radiation therapy is the most important therapeutic tool to cure various diseases like cancer. However, when using radiation for therapeutic strategy, radiation exposure to healthy tissues leads to some possible side effects such as acute radiation-induced skin injury and alters gene expression. Therefore, the analysis of a change in reference gene expression during the skin recovery process after radiation therapy is essential in healthy canine tissue. In the present study, we analyzed eight reference genes (ACTB, GAPDH, YWHAZ, GUSB, HPRT1, RPL4, RPS5, and TBP) in canine dermal tissues at 0, 1, 2, 3, 4, 5, 7, and 9 weeks of radiation exposure that affected the skin condition of canines. The stability of reference genes is determined by evaluating radiation therapy\'s effect on healthy canine dermal tissue. Epidermal marker, Keratin 10 expression varies each week after irradiation, and HPRT1 is found to be the most suitable for normalization of mRNA expression in radiation-exposed canine dermal tissues. Changes in the gene expression level were evaluated by using a reliable tool such as quantitative real-time polymerase chain reaction (qRT-PCR). In order to achieve a valid qRT-PCR result, the most stable reference genes used for normalization after the radiation exposure process are important. Therefore, the current study was designed to evaluate the most stable reference gene for the post-irradiation canine tissues. After radiation exposure, the alternation of reference gene expression was estimated by three algorithms (geNorm, Normfinder, and Bestkeeper). The RG validation programs (GeNorm and NormFinder) suggested that HPRT1, RPL4, and TBP were suitable for normalization in qRT-PCR. Furthermore, three algorithms suggested that HPRT1 was the most stable reference gene for normalization with qRT-PCR results, regardless of before and after radiation exposure. Whereas GAPDH was found to be the most unstable reference gene. In addition, the use of stable or unstable reference genes for the normalization of Keratin 10 expression showed statistical differences. Therefore, we observed that, to obtain accurate and suitable PCR results of the canine tissues with and without radiation exposure, the HPRT1 reference gene is recommended for normalization with its high stability. Additionally, the use of RGs such as HPRT1, RPL4, and TBP for normalization in qRT-PCR experiments is recommended for post-radiation canine tissues to generate more accurate and reliable data. These results will provide fundamental information regarding internal controls for gene expression studies and can be used for the analysis of gene patterns in regenerative medicine.

A 3-months old Chinese shar-pei puppy with ichthyosis was investigated. The dog showed generalized scaling, alopecia and footpad lesions. Histopathological examinations demonstrated a non-epidermolytic hyperkeratosis. The parents of the affected puppy did not show any skin lesions. A trio whole genome sequencing analysis identified a heterozygous de novo 3 bp deletion in the KRT1 gene in the affected dog. This variant, NM_001003392.1:c.567_569del, is predicted to delete a single asparagine from the conserved coil 1A motif within the rod domain of KRT1, NP_001003392.1:p.(Asn190del). Immunohistochemistry demonstrated normal levels of KRT1 expression in the epidermis and follicular epithelia. This might indicate that the variant possibly interferes with keratin dimerization or another function of KRT1. Missense variants affecting the homologous asparagine residue of the human KRT1 cause epidermolytic hyperkeratosis. Histologically, the investigated Chinese shar-pei showed a non-epidermolytic ichthyosis. The finding of a de novo variant in an excellent functional candidate gene strongly suggests that KRT1:p.Asn190del caused the ichthyosis phenotype in the affected Chinese shar-pei. To the best of our knowledge, this is the first description of a KRT1-related non-epidermolytic ichthyosis in domestic animals.

The Runt-related transcription factor (Runx) family has been suggested to play roles in stem cell regulation, tissue development, and oncogenesis in various tissues/organs. In this study, we investigated the possible functions of Runx1 and Runx3 in keratinocyte differentiation. Both Runx1 and Runx3 proteins were detected in primary cultures of mouse keratinocytes. Proteins were localized in the nuclei of undifferentiated keratinocytes but translocated to the cytoplasm of differentiated cells. The siRNA-mediated inhibition of Runx1 and Runx3 expression increased expression of keratin 1 and keratin 10, which are early differentiation markers of keratinocytes. In contrast, overexpression of Runx1 and Runx3 suppressed keratin 1 and keratin 10 expression. Endogenous Runx1 and Runx3 proteins were associated with the promoter sequences of keratin 1 and keratin 10 genes in undifferentiated but not differentiated keratinocytes. In mouse skin, the inhibition of Runx1 and Runx3 expression by keratinocyte-specific gene targeting increased the ratios of keratin 1- and keratin 10-positive cells in the basal layer of the epidermis. On the other hand, inhibition of Runx1 and Runx3 expression did not alter the proliferation capacity of cultured or epidermal keratinocytes. These results suggest that Runx1 and Runx3 likely function to directly inhibit differentiation-induced expression of keratin 1 and keratin 10 genes but are not involved in the regulation of keratinocyte proliferation.

Human epithelial keratin is an intermediate filament protein that serves as a backbone to maintain the stability of the cell nucleus and mechanical stability of the whole cells. The present study focused on two point mutations, F231L and S233L, of the 1B domain of keratin K 1/10 related to the rare genetic skin disease palmoplantar keratoderma (PPK). We used molecular dynamics simulation to study the effects of the mutations on various hierarchical structures, including heterodimers, tetramers, and octamers of the K1/10 1B domain at the atomic scale. The initial results demonstrated that the wild type and mutant proteins were highly similar at the dimer level but had different microstructures and mechanics at a higher-level assembly. A decrease in the hydrophobic interactions and hydrogen bonds at the terminus resulted in weakened mechanical properties of the tetramer and octamer of the F231L mutant. The asymmetrical structure of the S233L tetramer with an uneven distribution of the hydrogen bonds decreased its mechanical properties. However, the S233L mutation provided extra hydrophobic interactions between these mutated amino acid residues in the octamer, leading to improved mechanical properties. The results of the present study provided a deeper understanding of how the differences in point mutations induced the changes in the configuration and mechanical properties at the molecular scale. The differences in these properties may influence keratin assembly at the microscopic scale and ultimately cause diseases at the macroscopic scale.

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is characterized by diffuse hyperkeratosis affecting palms and soles with suprabasal epidermolysis or vacuolar degeneration histopathologically. The disorder is caused by heterozygous mutations in KRT9 or KRT1. Dominant-negative mutations in KRT1 could also result in epidermolytic ichthyosis with EPPK, a more severe entity affecting the entire body.
OBJECTIVE: To investigate the genetic basis and pathogenesis of two unrelated patients with EPPK and knuckle pads, both of whom were born to consanguineous parents of Chinese origin.
METHODS: Next-generation sequencing was applied to the two patients using genomic DNA extracted from peripheral blood. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining and Western blot (WB) were employed to evaluate mRNA and protein expression level. Ultrastructural changes of skin lesion were analysed using transmission electron microscopy.
RESULTS: Two novel homozygous mutations, c.457C>T (p.Gln153*) and c.33C>G (p.Tyr11*) in KRT1, were identified in patients 1 and 2 respectively. The nonsense mutations were predicted to result in nonsense-mediated mRNA decay and absence of keratin 1, which was confirmed in the skin lesions from patient 1. Upregulated keratin 2 was detected both in the affected and unaffected skin samples from patient 1, while the protein abundance and distribution pattern of keratin 10 remained unchanged. An aberrant and clumped staining pattern of keratin 9 was noted in the palmar skin of patient 1.
CONCLUSIONS: Homozygous \'knockout\' mutations in KRT1 resulted in EPPK with knuckle pads rather than epidermolytic ichthyosis. We speculated that sparing of non-acral skin might be due to compensatory effect of keratin 2 upregulation by forming heterodimer with keratin 10.

暂无摘要。

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.