Search

Autophagy, one mechanism of programmed cell death, is fundamental to cellular homeostasis. Previous studies have identified autophagy as a novel mechanism by which cytokines control the immune response. However, its precise role in immune-related inflammatory skin diseases such as psoriasis remains unclear. Thus, this study explored the functional role of autophagy in psoriatic inflammation of epidermal keratinocytes. Strong light chain 3 immunoreactivity was observed in epidermal keratinocytes of both human psoriatic lesions and imiquimod-induced mice psoriatic model, and it was readily induced by polycytidylic acid (poly (I:C)), which stimulates Toll-like receptor 3 (TLR3), in human epidermal keratinocytes in vitro. Rapamycin-induced activation of autophagy significantly reduced poly (I:C)-induced inflammatory reaction, whereas, inhibition of autophagy by 3-methyladeine increased that. Our results indicate that the induction of autophagy may attenuate TLR3-mediated immune responses in human epidermal keratinocytes, thus providing novel insights into the mechanisms underlying the development of inflammatory skin diseases including psoriasis., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2020 Xue Mei Li et al.)

Human skin is located at the outermost part of the body, and various cosmetics and chemicals that may come in contact with human skin need to be evaluated for their potential to cause irritation. Until recently, the Draize test was considered the standard method for skin irritation; however, this technique has disadvantages such as the need to sacrifice many rabbits and subjective scoring. Thus, to contribute to the development of an animal-free alternative skin irritation test, we investigated the cytotoxicity and inflammatory response to standard skin irritants in SV40 large T antigen-transformed human epidermal keratinocyte 2 cells (SV-HEK2 cells). In this study, we established an SV-HEK2 cell line immortalized by SV40 large T antigen (SV40 T) and characterized the inherent morphological and cytological properties. We next used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or neutral red uptake (NRU) assays of cell viability to investigate the optimal experimental conditions for determining SV-HEK2 cell viability after exposure to sodium dodecyl sulfate at 6.25×10 -3 % to 1×10 -1 % as a standard skin irritant. We then examined the viability of SV-HEK2 cells in response to five skin irritants (benzalkonium chloride, isopropanol, sodium dodecyl sulfate, Triton X-100 and Tween20) at 5×10 -3 % to 1×10 -1 % by MTT or NRU assay. Finally, we estimated the level of cytokines secretion in response to stimulation by skin irritants in SV-HEK2 cells. The results revealed that SV-HEK2 cells responded well to skin irritants in a concentration-dependent manner and that there was good correlation between irritant concentration and cytotoxicity (or cytokine secretion) when cells were exposed to skin irritants for 10min at room temperature (RT) using an MTT assay. Overall, these findings suggest that SV-HEK2 cells could be a good alternative in vitro model for skin irritation tests., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A20 is a negative regulator of nuclear factor κ-light‑chain-enhancer of activated B cells (NF-κB) signaling, and has been implicated in the pathogenesis of psoriasis through genome-wide association study (GWAS). In the present study, we investigated the putative role of A20 in epidermal keratinocytes. Immunohistochemical analysis showed that A20 was expressed in all layers of the epidermis, with an increasing pattern in the upper layers. In our model of calcium-induced keratinocyte differentiation, A20 expression was increased in a time-dependent manner. To investigate whether A20 affected keratinocyte differentiation, we overexpressed A20 in cultured keratinocytes. As a result, we noted that A20 overexpression did not affect keratinocyte differentiation, suggesting that A20 is not a direct modulator of keratinocyte differentiation. Interestingly, we found that A20 levels were decreased in psoriatic lesional skin compared to non-lesional areas. To investigate whether A20 played a role in the innate immune response of keratinocytes, we overexpressed A20 and then examined poly(I:C)-induced cytokine expression. We noted that A20 significantly inhibited poly(I:C)-induced cytokine production, and this effect was related to the inhibition of NF-κB signaling. These results suggest that the downregulation of A20 increased the susceptibility of keratinocytes to external stimuli, thus contributing to the development of psoriasis.

Imiquimod is known to exert its effects through Toll-like receptor 7 (TLR7) and/or TLR8, resulting in expression of proinflammatory cytokines and chemokines. Keratinocytes have not been reported to constitutively express TLR7 and TLR8, and the action of imiquimod is thought to be mediated by the adenine receptor, not TLR7 or TLR8. In this study, we revealed the expression of TLR7 in keratinocytes after calcium-induced differentiation. After addition of calcium to cultured keratinocytes, the immunological responses induced by imiquimod, such as activation of NF-κB and induction of TNF-α and IL-8, were more rapid and stronger. In addition, imiquimod induced the expression TLR7, and acted synergistically with calcium to induce proinflammatory cytokines. We confirmed that the responses induced by imiquimod were significantly inhibited by microRNAs suppressing TLR7 expression. These results suggest that TLR7 expressed in keratinocytes play key roles in the activation of NF-κB signaling by imiquimod, and that their modulation in keratinocytes could provide therapeutic potential for many inflammatory skin diseases.

In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. In this study, we investigated the role of the POU domain-containing transcription factor Brn2 in keratinocyte differentiation. Immunohistochemical analysis showed that Brn2 is expressed primarily in the upper granular layer. Consistent with its epidermal localization, Brn2 expression was highly induced at 14 days after calcium treatment of cultured normal human epidermal keratinocytes. When Brn2 was overexpressed by adenoviral transduction, Brn2 led to increased expression of the differentiation-related genes involucrin, filaggrin, and loricrin in addition to inhibition of their proliferation. Chromatin immunoprecipitation demonstrated that Brn2 bound to the promoter regions of these differentiation-related genes. We injected the purified Brn2 adenovirus into rat skin, which led to a thickened epidermis with increased amounts of differentiation related markers. The histopathologic features of adenovirus-Brn2 injected skin tissues looked similar to the features of lichen planus, a human skin disease showing chronic inflammation and well-differentiated epidermal changes. Moreover, Brn2 is shown to be expressed in almost all cell nuclei of the thickened epidermis of lichen planus, and Brn2 also attracts T lymphocytes. Our results demonstrate that Brn2 is probably a transcriptional factor playing an important role in keratinocyte differentiation and probably also in the pathogenesis of lichen planus lesions.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. To examine the gene expression profile in calcium-induced keratinocyte differentiation, we constructed a normalized cDNA library using mRNA isolated from these calcium-treated keratinocytes. After sequencing about 10,000 clones, we were able to obtain 4,104 independent genes. They consisted of 3,699 annotated genes and 405 expressed sequence tags (ESTs). Some were the genes involved in constituting epidermal structures and others were unknown genes that are probably associated with keratinocytes. In particular, we were able to identify genes located at the chromosome 1q21, the locus for the epidermal differentiation complex, and 19q13.1, another probable locus for epidermal differentiation-related gene clusters. One EST located at the chromosome 19q13.1 showed increased expression by calcium treatment, suggesting a novel candidate gene relevant to keratinocyte differentiation. These results demonstrate the complexity of the transcriptional profile of keratinocytes, providing important clues on which to base further investigations of the molecular events underlying keratinocyte differentiation.

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.

During a search for keratinocyte differentiation-related genes, we obtained a cDNA fragment from the 5'-untranslated region of a previously identified splicing variant of desmoglein 3 (Dg3). This transcript encodes a protein of 282 amino acids, which corresponds to the N-terminal truncated intracellular domain of Dg3 (deltaNDg3). Northern blot analysis detected a 4.6-kb transcript matching the predicted size of deltaNDg3 mRNA, and Western blot analysis with an antibody raised against the Dg3 C-terminus (H-145) detected a 31-kDa protein. Increased deltaNDg3 expression was observed in differentiating keratinocytes by RT-PCR and Western blot analysis, suggesting that deltaNDg3 is indeed a differentiation-related gene product. In immunohistochemical studies of normal and pathologic tissues, H-145 antibody detected the protein in the cytoplasm of suprabasal layer cells, whereas an antibody directed against the N-terminal region of Dg3 (AF1720) reacted with a membrane protein in the basal layer. In addition, deltaNDg3 transcript and protein were upregulated in psoriatic epidermis, and protein expression appeared to increase in epidermal tumors including Bowen's disease and squamous cell carcinoma. Moreover, overexpression of deltaNDg3 led to increased migration and weakening of cell adhesion. These results suggest that deltaNDg3 have a role in keratinocyte differentiation, and that may be related with tumorigenesis of epithelial origin.

Ceramide is an important constituent of stratum corneum lipids, which act as both physical barriers and signal modulators. We synthesized several ceramide derivatives and investigated their effects on keratinocyte differentiation. RT-PCR and Western blotting showed that the novel synthetic ceramide derivatives K6PC-4 [N-(2,3-dihydroxypropyl)-2-hexyl-3-oxo-decanamide], K6PC-5, [N-(1,3-dihydroxypropyl-2-hexyl-3-oxo-decanamide] and K6PC-9 (N-ethanol-2-hexyl-3-oxo-decanamide) [corrected] These ceramide derivatives elicited a rapid transient increase in intracellular calcium levels, which were measured using laser scanning confocal microscopy. In addition, K6PC-4, K6PC-5, and K6PC-9 stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In a reconstituted epidermis model, K6PC-4, K6PC-5, and K6PC-9 significantly increased keratin 1 expression in the suprabasal layer. These results indicate that these novel synthetic ceramide derivatives have the potential to promote keratinocyte differentiation, suggesting that the lipid molecules are applicable for treating skin diseases involving abnormal keratinocyte differentiation.

We isolated a human gene encoding keratinocyte proline-rich protein (hKPRP). hKPRP gene is located in the region of epidermal differentiation complex on chromosome 1q21, and its approximately 2.5 kb mRNA encodes 579 amino acid protein with high proline content (18%). The mRNA level of hKPRP was markedly increased at both 7 and 14 d after treatment with 1.2 mM calcium in cultured normal human epidermal keratinocytes. In situ hybridization demonstrated that hKPRP was expressed in upper granular layer of normal epidermis with characteristic intermittent pattern. In psoriatic lesion, hKPRP expression was increased as compared with normal skin and showed continuous pattern. Immunohistochemical analysis also confirmed the expression of hKPRP at the protein level. Western blot analysis showed that hKPRP protein of approximately 70 kDa size was significantly increased by calcium in a time-dependent manner. In mouse tissue blot assays, the expression of KPRP was detected in stomach and skin tissues, and began at 17.5 embryonic days. Additionally, hKPRP expression was detected in the periderm of human fetal skin from 16 wk estimated gestational age. Together, these results suggest that hKPRP is an epidermal marker expressed in stratified squamous epithelia and has a potential role in keratinocytes differentiation.

The diverse functions of microtubules (MT) in different cells and tissues may be facilitated by compositional changes in tubulin isotypes. We obtained partial cDNA clones of class II beta-tubulin from a library of differentiating normal human epidermal keratinocytes (NHEK) cells, whereas screening via subtractive hybridization for genes involved in calcium-induced keratinocyte differentiation. Analysis of the isotypic composition of beta-tubulin from NHEK cells revealed elevations in class II beta-tubulin concentrations at both protein and message levels during cell differentiation, resulting in increased rates of incorporation of class II beta-tubulin into MT. Immunohistochemistry of normal and pathologic skin tissues showed that class II beta-tubulin occurred in the granular layer of the epidermis and in differentiated areas of carcinomas. Class II beta-tubulin was, however, not observed in the uppermost granular and cornified layers of normal epidermis. Further experiments showed that MT were likely to decay in the final stage of terminal differentiation during formation of the cornified envelope. Our results suggest that there is differential modulation of MT composition and stability during keratinocyte differentiation.

Adiponectin plays important roles in metabolic function, inflammation and multiple biological activities in various tissues. However, evidence for adiponectin signaling in sebaceous glands is lacking, and its role remains to be clarified. This study investigated the role of adiponectin in lipid production in sebaceous glands in an experimental study of human sebocytes. We demonstrated that human sebaceous glands in vivo and sebocytes in vitro express adiponectin receptor and that adiponectin increased cell proliferation. Moreover, based on a lipogenesis study using Oil Red O, Nile red staining and thin layer chromatography, adiponectin strongly upregulated lipid production in sebocytes. In three-dimensional culture of sebocytes, lipid synthesis was markedly enhanced in sebocytes treated with adiponectin. This study suggested that adiponectin plays a significant role in human sebaceous gland biology. Adiponectin signaling is a promising target in the clinical management of barrier disorders in which sebum production is decreased, such as in atopic dermatitis and aged skin. [ABSTRACT FROM AUTHOR]