E‐FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro

Epidermal fatty acid‐binding protein (E‐FABP) is a lipid carrier, originally discovered in human epidermis. We show that E‐FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E‐FABP induces overexpression of K10 and involucrin. On the other hand, E‐FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF‐κB and JNK signalling pathways. E‐FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E‐FABP as compared to the same population in normal epidermis. E‐FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high‐calcium conditions, E‐FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E‐FABP plays an important role in keratinocyte differentiation. Moreover, E‐FABP modulates differentiation in psoriatic keratinocytes.     

SMAD inhibition attenuates epithelial to mesenchymal transition by primary keratinocytes in vitro

Epithelial to mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype and contribute directly to fibrotic disease. Recent studies support a role for EMT in cutaneous fibrotic diseases including scleroderma and hypertrophic scarring, although there is limited data on the cytokines and signalling mechanisms regulating cutaneous EMT. We investigated the ability of TGF‐β and TNF‐α, both overexpressed in cutaneous scleroderma and central mediators of EMT in other epithelial cell types, to induce EMT in primary keratinocytes and studied the signalling mechanisms regulating this process. TGF‐β induced EMT in normal human epidermal keratinocytes (NHEK cells), and this process was enhanced by TNF‐α. EMT was characterised by changes in morphology, proteome (down‐regulation of E‐cadherin and Zo‐1 and up‐regulation of vimentin and fibronectin), MMP secretion and COL1α1 mRNA expression. TGF‐β and TNF‐α in combination activated SMAD and p38 signalling in NHEK cells. P38 inhibition with SB203580 partially attenuated EMT, whereas SMAD inhibition using SB431542 significantly inhibited EMT and also reversed established EMT. These data highlight the retained plasticity of adult keratinocytes and support further studies of EMT in clinically relevant in vivo models of cutaneous fibrosis and investigation of SMAD inhibition as a potential therapeutic intervention.     

Enkephalins modulate differentiation of normal human keratinocytes in vitro

Abstract Opioid peptides are a group of neuropeptides which include enkephalins, endorphins and dynorphins. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Previously, we have shown that enkephalins are present in macrophages infiltrating the dermal papillae in involved psoriatic skin and that the amount of enkephalin is significantly increased in involved psoriatic skin. Because enkephalins were detected close to the epidermis, we examined the effects of opioid peptides on the differentiation (transglutaminase type 1 activity and cytokeratin 10 expression) and proliferation (MTT assay) of cultured human keratinocytes. Enkephalins (methionine-enkephalin, leucine-cnkephalin and the synthetic DADL) inhibited cell differentiation dose-dependently, while β-endorphin had no effect. The opioid receptor antagonist naltrexone completely antagonized the inhibitory effect of methionine-enkephalin and leucine-enkephalin. but not that of DADL. Furthermore, methionine-enkephalin had a slight inhibitory effect on the proliferation of keralinocytcs. Enkephalin was detected in unstimulated keratinocyte cultures, and naltrexone alone stimulated keratinocyte differentiation. These results indicate that enkephalins may play a role in the differentiation of epidermal keratinocytes. It remains to be determined whether the enkephalin detected in psoriatic skin are sufficient to affect epidermal differentiation in vivo.     

Increased cholesterol sulfate and cholesterol sulfotransferase activity in relation to the multi-step process of differentiation in human epidermal keratinocytes

In this study the synthesis of cholesterol sulfate is examined in relation to the process of squamous differentiation in normal human epidermal keratinocytes (NHEK) in culture. During the exponential growth phase, NHEK cells exhibit a relatively high colony-forming efficiency and appear undifferentiated on the basis of their morphology and expression of biochemical characteristics. At confluence, the cells undergo terminal differentiation that is characterized by the commitment to terminal cell division (reduction in colony-forming ability) and expression of the differentiated phenotype. An accumulation of cholesterol sulfate accompanies this program of differentiation. This accumulation of cholesterol sulfate parallels the increase in transglutaminase type I activity and the competence to form cross-linked envelopes, whereas it precedes the "spontaneous" formation of cross-linked envelopes. Increased cholesterol sulfotransferase activity appears to account for the increase in cholesterol sulfate. The cholesterol sulfate accumulation, as well as the increase in cholesterol sulfotransferase and transglutaminase activity, are inhibited by retinoids. However, the presence of retinoids does not prevent NHEK cells from undergoing terminal cell division at confluence. Two NHEK cell lines expressing SV40-large T antigen also undergo terminal differentiation at confluence and start to accumulate cholesterol sulfate. Two other, differentiation-defective cell lines do not exhibit an increase in cholesterol sulfate at confluence. These results show that epidermal keratinocytes in culture, like cells in the epidermis, accumulate cholesterol sulfate when undergoing squamous differentiation. This program appears to consist of a retinoid-insensitive step (commitment to terminal cell division) and a retinoid-sensitive step (expression of the squamous differentiated phenotype).     

Control of growth and differentiation in vitro of human keratinocytes cultured in serum-free medium

Normal human proliferative keratinocytes can be serially cultured in serum-free medium. The medium and culture conditions that have been developed support the growth of these cells in the absence of a feeder layer and in the absence of contaminating fibroblasts. Using this system, the nutritional and hormonal factors that affect the growth and differentiation of these cells have been studied in the absence of undefined supplements. The data suggest that the control of proliferation and differentiation in keratinocytes may take place by unique mechanisms. The defined serum-free conditions that have been developed provide an excellent system for the study of nutritional disorders that affect human epidermal cells. In addition, the culture system can be used to propagate large numbers of cells for use in autografts for cases of severe burns.     

Distinctive expression of filaggrin and trichohyalin during various pathways of epithelial differentiation

Filaggrin and trichohyalin are keratin intermediate filament-associated proteins, and are primarily expressed in the granular cells of the epidermis and in the inner root sheath cells of the hair follicles, respectively. These two proteins are, however, occasionally co-expressed in some tissues. To gain more insights into the mechanisms for expression and processing of (pro)filaggrin and trichohyalin during various pathways of epithelial differentiation, we compared their localization by double immunostaining techniques in normal and psoriatic epidermis, tongue filiform papillae and cultured human epidermal keratinocytes. In normal foreskin, trichohyalin immunoreactive cells were observed only occasionally and, when present, they always co-expressed filaggrin. Trichohyalin expression occurred, however, in filaggrin-negative cells as well as in filaggrin-positive cells in the psoriatic epidermis, tongue papillae and cultured keratinocytes. Filaggrin and trichohyalin were induced independently at different times following calcium shift in cultured keratinocytes. Immunoelectron microscopy demonstrated the distinct intracellular distribution of filaggrin and trichohyalin. Some filaggrin granules were observed in the cells where trichohyalin was diffusely distributed. Likewise some trichohyalin granules were found in the cells with diffuse filaggrin labelling. These results suggest the existence of distinct expression/processing mechanisms of filaggrin and trichohyalin in differentiating keratinocytes.     

Effects of diisopropylamine dichloroacetate on proliferation and differentiation of normal human keratinocytes in vitro.

We found that diisopropylamine dichloroacetate (DADA), known as a vasodilator, enhanced growth of keratinocytes in 4 days culture at 1-30 microg/ml, and such promoting effects of cell proliferation were reconfirmed by measuring DNA synthesis using [(3)H]thymidine incorporation. On the other hand, this substance enhanced synthesis of keratin K1, a potent marker of differentiation in keratinocytes, at 1-100 microg/ml in low calcium (0.1 mM) or high calcium medium (1. 25 mM). Moreover, the formation of cornified envelope, another potent marker of differentiation in keratinocytes, was also promoted by DADA at a concentration of 0.1-10 mM which includes valid concentration of DADA for the enhancement of keratin K1 formation (1-100 microg/ml: 0.05-0.5 mM DADA). These results indicate that DADA has a double function, enhancement of both proliferation and differentiation of cells, which could be linked to the turnover of skin epidermis. Furthermore, in order to analyze the effect of DADA on keratinocytes, we examined the effects of each component of this substance, diisopropylamine (DIA) and dichloroacetate (DCA), on keratinocytes. As the result of these investigations, evidence was found that DCA was effective on enhancement of cell growth, but DIA was ineffective. Moreover, we found that DCA was effective on keratinocyte differentiation by evaluating the enhancement of a differentiation marker, formation of cornified envelopes, within 10 mM, while DIA was not effective. Therefore, we concluded that only DCA was an active component of the DADA molecule for the proliferation and the differentiation of keratinocytes in vitro.     

Effect of recombinant alpha A-interferon on DNA synthesis and differentiation of human keratinocytes in vitro

Interferons are known to protect cells against virus infection, to inhibit cell growth in a number of cell lines and also to modulate cell differentiation. Their possible clinical application has now entered its final phase. The aim of this investigation was to study the effect of recombinant alpha A-interferon (r IFN-alpha A) on human keratinocyte DNA synthesis and differentiation in vitro. [3H]-TdR incorporation into DNA was used to quantify DNA synthesis in keratinocyte cultures at varying concentrations of rIFN-alpha A. Cell differentiation was determined using polyacrylamide gel electrophoresis of six specific protein fractions extracted from the keratinocytes. Our results indicate that rIFN-alpha A is a potent inhibitor of keratinocyte DNA synthesis in vitro; it also decreases significantly the amount and synthesis of non-covalently bound keratins while increasing the disulphide cross-linked keratins. No qualitative changes in keratinocyte proteins were observed.     

The effect of vitamin A on growth and differentiation of human keratinocytes in vitro.

Epithelial outgrowths (keratinocytes) from normal human skin in vitro were exposed daily for 30 min to vitamin A alcohol for periods up to 5 weeks. There was a markedly decreased number of keratohyaline granules in treated cultures, indicating an effect on the differentiation process, but there was no evidence for mucous metaplasia. The area of vitamin A-treated outgrowths was greater than that of controls at all times. In addition, there was a higher mitotic index, higher labeling index, and larger growth fraction in treated cultures. The combination of altered differentiation and enhanced proliferation of keratinocytes would appear to account for the larger outgrowth area found in vitamin A-treated cultures.     

Oligonucleotides suppress IL-8 in skin keratinocytes in vitro and offer anti-inflammatory properties in vivo

DNA codes for genetic information. Furthermore, recent findings suggest that DNA offers additional function, particularly in the recognition of microorganisms. In this study, we investigated two classes of oligodeoxynucleotides (ODN) in skin keratinocytes; namely, an ODN comprising two cytidine-phosphate-guanosine (CpG) motifs (CpG-1-phosphorothioate (PTO)) and a poly-cytidine (Non-CpG-5-PTO) as control. Both fluorescence-tagged ODN were rapidly taken up by cells and accumulated already after 5 minutes in perinuclear compartments. In order to test whether ODN convey immunological effects in keratinocytes, secretion of IL-8 was measured. Interestingly, both CpG-1-PTO and Non-CpG-5-PTO suppressed basal and tumor necrosis factor alpha-induced IL-8 levels measured in cell culture supernatants. Experiments using deletion mutant revealed a critical length of approximately 16 nucleotides conveying IL-8 suppression. Studies regarding the ODN backbone offered that PTO bondings are critical for significant IL-8 suppression. In order to substantiate the anti-inflammatory response, a contact hypersensitivity mouse model was utilized. Topical application of Non-CpG-5-PTO-containing ointments reduced ear thickness in sensitized mice. Taken together, these findings suggest an anti-inflammatory effect of ODN in epithelial cells in vitro and in vivo, indicating that DNA molecules offer distinct biological activities restricted to the physiological compartment applied. This effect seems to be independent from Toll-like receptor 9.     

Ergocalciferol promotes in vivo differentiation of keratinocytes and reduces photodamage caused by ultraviolet irradiation in hairless mice

BACKGROUND: Ergocalciferol (VD(2)) is usually administered orally and it is metabolized to produce its biologically active metabolites in the liver and kidney. Active vitamin D is a well-known potent regulator of cell growth and differentiation. PURPOSE: Active vitamin D such as 1,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) prevents photodamage, including wrinkles and morphologic alterations. However, its clinical and cosmetic use is limited because of its potent, associated effect on calcium metabolism. We examined the efficacy of vitamin D analogues with few adverse effects for preventing skin photodamage. METHOD: Topical application of VD(2) to hairless mouse dorsal skin, and exposure to solar-simulating ultraviolet (UV) radiation at a dose of 10.8 J/cm(2) (UVA) were performed for 15 weeks, five times a week on weekdays. At the end of the final irradiation, histological and analytical studies were performed. RESULTS: Topical application of VD(2) significantly prevented wrinkle formation and abnormal accumulation of extracellular matrix components. In addition, VD(2) suppressed excessive secretion of IL-6 induced by UV irradiation in cultured human normal keratinocytes, in a dose-dependent manner. CONCLUSION: VD(2) promoted keratinocytes differentiation in the epidermis and showed diverse physiological effects, the same as the active form of VD(3). The results suggested that the suppression of skin photodamage involved the promotion of keratinocytes differentiation and suppression of IL-6 secretion induced by exposure to UV. Topical application of VD(2) may become an effective means to suppress solar UV-induced human skin damage.     

Markers for Ca++-induced terminal differentiation of keratinocytes in vitro under defined conditions

Differentiation of normal human keratinocytes (NHK) grown in vitro as a monolayer to confluency can be triggered with an acute increase in concentration of extracellular Ca⁺⁺. Over several days, induced by Ca⁺⁺, the cells form pseudostratified sheets that somewhat resemble the basic organization of the intact skin. This experimental system is widely used in studies of keratinocyte biology and skin pathology. However, expression pattern of the genes considered as markers for cells in specific layers of epidermis in vivo does not always match the specific pattern observed in vitro and might lead to misinterpretation of data. Here, we demonstrate that among 18 markers of terminally differentiated keratinocytes of stratum granulosum (SG) and stratum corneum (SC) in vivo, only four (CDSN, KPRP, LCE1C and SPRR4) have reproduced their expression pattern in vitro. Our data suggest that findings based on two-dimensional (2D) Ca⁺⁺-induced terminal differentiation of NHK in vitro should be subjected to additional scrutiny before conclusions could be made and, if possible, verified in other experimental system that might more faithfully represent the in vivo microenvironment.     

Extracellular vesicles derived from Malassezia furfur stimulate IL-6 production in keratinocytes as demonstrated in in vitro and in vivo models

Background

Malassezia is one of the commensal microorganisms colonized on human skin and has been shown to be related to several inflammatory cutaneous disorders. Previous studies indicated that Malassezia. sympodialis (M. sympodialis) can produce extracellular vesicles, however, the immunoregulatory function of Malassezia extracellular vesicles on keratinocytes has not been studied.