Expression Of Involucrin Research Articles

Transglutaminases, involucrin, and loricrin as markers of epidermal differentiation in skin substitutes derived from human sweat gland cells

In a multi-project research line, we are currently testing whether a morphologically and functionally near normal epidermis can be cultured from human sweat gland (SG) cells and be used as a skin substitute. The present study focuses on the stratum corneum of the epidermis that assumes a vital barrier function for the skin. The main process in the formation of the cornified cell envelope in human epidermis, i.e. crosslinking of proteins and lipids, is catalyzed by several transglutaminases (TG). Therefore, we compared the expression patterns of various TG and their substrates in SG-derived versus keratinocyte-derived epidermal substitutes. Sweat gland cells, keratinocytes, and fibroblasts were isolated from human skin samples and cultivated separately to generate epidermal substitutes. These were transplanted onto the back of athymic rats. After 2 weeks, the transplants were excised and analyzed histologically as well as by indirect immunofluorescence. We looked at the expression of TG1, 3, 5, and their substrates involucrin and loricrin (=markers of epidermal differentiation) in SG-derived and keratinocyte-derived skin substitutes as well as in normal skin. The SG cell-derived epidermis was near normal anatomically, formed a cornified cell envelope and demonstrated TG1, 3, and 5 as well as involucrin and loricrin expression patterns similar to those found in keratinocyte-derived epidermis and normal control skin. These findings support the thesis that SG cells have the potential to form a near normal stratified epidermal analog that might be used as a skin substitute. The expression of TG1 and 3, not normally expressed in human SG, suggests the presence of re-programmed SG cells and/or stem cells capable of both de novo generating and maintaining an epidermis.

Aberrant cytokeratin expression during arsenic-induced acquired malignant phenotype in human HaCaT keratinocytes consistent with epidermal carcinogenesis

Inorganic arsenic is a known human skin carcinogen. Chronic arsenic exposure results in various human skin lesions, including hyperkeratosis and squamous cell carcinoma (SCC), both characterized by distorted cytokeratin (CK) production. Prior work shows the human skin keratinocyte HaCaT cell line, when exposed chronically for >25 weeks to a low level of inorganic arsenite (100 nM) results in cells able to produce aggressive SCC upon inoculation into nude mice. In the present study, CK expression analysis was performed in arsenic-exposed HaCaT cells during the progressive acquisition of this malignant phenotype (0–20 weeks) to further validate this model as relevant to epidermal carcinogenesis induced by arsenic in humans. Indeed, we observed clear evidence of acquired cancer phenotype by 20 weeks of arsenite exposure including the formation of giant cells, a >4-fold increase in colony formation in soft agar and a ∼2.5-fold increase in matrix metalloproteinase-9 secretion, an enzyme often secreted by cancer cells to help invade through the local extra-cellular matrix. During this acquired malignant phenotype, various CK genes showed markedly altered expression at the transcript and protein levels in a time-dependent manner. For example, CK1, a marker of hyperkeratosis, increased up to 34-fold during arsenic-induced transformation, while CK13, a marker for dermal cancer progression, increased up to 45-fold. The stem cell marker, CK15, increased up to 7-fold, particularly during the later stages of arsenic exposure, indicating a potential emergence of cancer stem-like cells with arsenic-induced acquired malignant phenotype. The expression of involucrin and loricrin, markers for keratinocyte differentiation, increased up to 9-fold. Thus, during arsenic-induced acquired cancer phenotype in human keratinocytes, dramatic and dynamic alterations in CK expression occur which are consistent with the process of epidermal carcinogenesis helping validate this as an appropriate model for the study of arsenic-induced skin cancer.

Activators of PPARs and LXR decrease the adverse effects of exogenous glucocorticoids on the epidermis

While glucocorticoids (GC) exert beneficial effects (anti-inflammatory), they also have adverse effects on the epidermis including decreased epidermal differentiation, decreased keratinocyte proliferation, and decreased cutaneous permeability barrier homeostasis. Thus, the purpose of this study was to develop strategies to prevent these GC toxicities using simultaneous topical treatments in clobetasol-treated mice. While a triple-lipid mixture of stratum corneum lipids (ceramide, free fatty acid and cholesterol) was previously shown to reverse the GC-induced abnormality in cutaneous barrier function [J Invest Dermatol, 120 (2003) 456], this lipid mixture did not prevent the GC-induced abnormalities in either keratinocyte proliferation or differentiation. As activators of PPARalpha, beta/delta, gamma and LXR, regulate keratinocyte proliferation and differentiation and improve permeability barrier homeostasis, we next assessed the effects of these activators during concurrent GC treatment. Co-application of either ciglitazone (PPARgamma activator), clofibrate (PPARalpha activator) or 22R (OH) cholesterol (LXR activator) with clobetasol prevented the decrease in involucrin, filaggrin and loricrin expression. By contrast, a PPARbeta/delta activator (GW501516) normalized only the expression of involucrin and filaggrin but not loricrin. Moreover, topical application of PPARalpha, beta/delta or LXR activators partially prevented the decrease in keratinocyte proliferation in GC-treated murine skin, as measured using PCNA, while no effect was seen after co-treatment with PPARgamma activators. Finally, PPARgamma and PPARbeta/delta activators but not PPARalpha and LXR activators improved permeability barrier homeostasis in GC-treated mice. Together, these studies demonstrate that PPAR and LXR activators can prevent several of the adverse effects of topical GC on the epidermis.

Long-Term Culture and Growth Kinetics of Murine Corneal Epithelial Cells Expanded from Single Corneas

To develop a reproducible procedure for the long-term culture of corneal epithelial cells from a single mouse cornea. Corneal limbal explants of C57BL6/J mice were cultured in serum-free, low-Ca(2+) medium supplemented with EGF and cholera toxin. Epithelial cells were subcultured at a 1:3 split until passage (P)4 and at lower densities after P4. Colony-forming efficiency, population-doubling times, and population doublings were determined. The expression of p63, keratin (K)19, K12, and involucrin was analyzed by RT-PCR, immunocytochemistry, and Western blotting. Differentiation potential was examined by switching the medium to serum or high Ca(2+)-containing medium. Stratification ability was analyzed by air-lift culture. Thirty of 32 (93.8%) corneal explants were successfully subcultured to P1. Cultures without cholera toxin did not proliferate past P2 (n = 12), but 55% of cultures supplemented with cholera toxin achieved P4 (n = 20). After P4, cells were stably subcultured over 25 passages. Colony-forming efficiency increased from 9.7% +/- 2.6% at P5 to 29.0% +/- 3.3% at P20. The cells showed cobblestone appearance and expressed p63, K19, and involucrin but were negative for K12. Serum and high Ca(2+) induced differentiation, and cells cultured in DMEM/F12 with serum showed K12 mRNA expression. Stratified epithelium was formed by air-lifting. With this procedure, corneal epithelial cells from a single cornea can be cultured long term and can retain the potential to differentiate and stratify. This procedure can be a powerful tool for studies that require comparison of corneal epithelial cells from normal and transgenic mice in vitro.

Comparative analysis of the expression of cytokeratins (1, 10, 14, 16, 4), involucrin, filaggrin and e‐cadherin in plane warts and epidermodysplasia verruciformis plane wart‐type lesions

Epidermodysplasia verruciformis (EV) is a rare genodermatosis with susceptibility to human papillomavirus (HPV) infection, and high risk of skin cancer considered a model of viral oncogenesis. Fifteen cases of EV plane wart (PW)-type lesions (EV) and 14 cases of PW in healthy individuals were subjected to immunohistochemical technique for cytokeratins (K) 1, 10, 14, 16, 4, involucrin, filaggrin and e-cadherin. K1/10 showed retarded or negative expression in EV, being substituted by K14. Expression of K14 occurred in the basal and suprabasal layers in both groups, but in EV, its expression was observed up to the more superficial layers. Both groups showed positivity for K16 and K4, involucrin expression in lower levels of the spinous layer and unaltered filaggrin expression. E-cadherin expression was diminished at the koilocytotic foci of both lesions, more superficially in EV. Infection by HPV may alter the differentiation status of the epidermis, leading to a major expression of K14, delayed or absent expression of K1/10 and earlier involucrin expression, especially in EV. It also stimulates the expression of K16 and K4. Filaggrin expression is not altered, and e-cadherin is diminished in superficial koilocytotic cells' foci in EV.

Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of basement-membrane type VII collagen

Type VII collagen (ColVII) is the main component of anchoring fibrils, attachment structures within the lamina densa of the basement membrane that are responsible for attachment of the epidermis to the dermis in skin. Mutations in the human ColVII gene, COL7A1, cause the severe inherited blistering disorder recessive dystrophic epidermolysis bullosa (RDEB) affecting skin and mucosae, associated with a greatly increased risk of skin cancer. In this study, we examined the effect of loss of ColVII on squamous cell carcinoma (SCC) tumourigenesis using RNAi in a 3D organotypic skin model. Our findings suggest that loss of ColVII promotes SCC migration and invasion as well as regulating cell differentiation with evidence for concomitant promotion of epithelial-mesenchymal transition (EMT). Immunostaining of RDEB skin and a tissue array of sporadic cutaneous SCCs confirmed that loss of ColVII correlates with decreased involucrin expression in vivo. Gene-expression-array data and immunostaining demonstrated that loss of ColVII increases expression of the chemokine ligand-receptor CXCL10-CXCR3 and downstream-associated PLC signalling, which might contribute to the increased metastatic potential of SCCs with reduced or absent ColVII expression. Together, these findings may explain the aggressive behaviour of SCCs in RDEB patients and may also be relevant to non-RDEB skin cancer, as well as other tumours from organs where ColVII is expressed.

Effects of Propionibacterium acnes on various mRNA expression levels in normal human epidermal keratinocytes in vitro

Propionibacterium acnes is one of the most significant pathogenic factors of acne vulgaris. This bacteria relates to acne by various pathways. It has also been reported that P. acnes influences pro-inflammatory cytokine production in keratinocytes in vitro. However, the influence on the differentiation of keratinocytes by P. acnes has not been studied extensively. We analyzed the expression of keratinocyte differentiation-specific markers, keratins, and pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) exposed to P. acnes in vitro. All P. acnes strains used in this study increased transglutaminase (TGase), keratin 17 (K17) and interleukin (IL) mRNA expression levels in NHEK, and decreased K1 and K10 expression levels. Some P. acnes strains increased involucrin and K6 mRNA expression levels in NHEK and decreased filaggrin, K6 and K16 expression levels in vitro. This experiment clarified that P. acnes influences the differentiation of NHEK in vitro. As a result, P. acnes influenced the expression of not only pro-inflammatory cytokines but also some keratinocyte differentiation-specific markers and keratins in NHEK. Our results suggest that P. acnes relates to acne pathogenesis by not only the induction of inflammation but also in the differentiation of keratinocytes. Moreover, it was considered that the reaction of NHEK to P. acnes may be different depending on the type of bacteria.

Anti-psoriatic effects of indigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component

Indigo naturalis has shown efficacy in treating psoriasis in our previous clinical studies. To investigate the potential effect of indigo naturalis on regulating keratinocyte proliferation and differentiation. Skin samples from six patients were analyzed for proliferating cell nuclear antigen (PCNA) and involucrin expression by immunohistochemical staining. In addition, indigo naturalis extracts from 10 to 500 microg/ml were added to cultured keratinocytes and cell viability determined. Real-time RT-PCR, Western blotting analysis and indirect immunofluorescent labeling were used to investigate the messenger (m)RNA and protein expressions of PCNA and involucrin. Finally, high-performance liquid chromatography (HPLC) was used to identify major components of indigo naturalis and their in vitro effects compared. Immunohistochemical results demonstrated decreased PCNA and increased involucrin in psoriatic lesions after indigo naturalis treatment. Cultured keratinocytes decreased after indigo naturalis treatment, while G(0)/G(1) arrest was observed to dose-dependently increase. Staining revealed decreased PCNA-stained nuclei and increased cytosolic involucrin in treated keratinocytes. Decreased PCNA and increased involucrin at both the mRNA and protein levels were confirmed. Both major components, indirubin and indigo, could cause G(0)/G(1) phase arrest; however, only indirubin modulated the expressions of PCNA and involucrin similar to indigo naturalis. Together, these findings indicate that the anti-psoriatic effects of indigo naturalis are mediated, at least in part, by modulating the proliferation and differentiation of keratinocytes, with indirubin as the major active component.

Cadherin-Mediated Cell–Cell Contact Regulates Keratinocyte Differentiation

Cell-extracellular matrix (ECM) and cell-cell interactions regulate keratinocyte cell fate and differentiation. In the present analysis, we examined the differentiation of primary human keratinocytes cultured on micropatterned substrates that varied the extent of cell-cell contact while maintaining constant cell-ECM areas. Bowtie-shaped micropatterned areas (75-1600 microm(2)) were engineered to either permit or prevent cell-cell contact for pairs of adherent keratinocytes. Cell pairs with direct cell-cell contact exhibited enhanced expression of the differentiation markers involucrin and keratin 10 compared to cells with no cell-cell contact. In contrast, available cell-spreading area, as regulated by pattern size, did not alter keratinocyte involucrin expression. Disruption of E-cadherin binding by either antibody blocking or expression of a dominant-negative receptor diminished the ability of micropattern-regulated cell-cell contact to modulate involucrin expression. These results demonstrate that cadherin-mediated cell-cell contact regulates early keratinocyte differentiation independently from changes in cell shape.

No. 3 Vol. 42, No. 2, pp. 94–101, 2008 Effect of Birch (Betula platyphylla Sukatchev var. japonica Hara) sap on cultured human epidermal keratinocyte differentiation

The beauty of ideal skin texture is closely associated with dermal moisture factors. The key factors of skin moisture are NMF (natural moisturizing factor) and skin normal barrier function. The former keeps dermal surface moisture, and the later protects from excess water loss. So we have searched for the ingredient that improves these factors. Birch sap has been widely used as an effective drink for anti‐fatigue and anti‐stress. However, the effect of birch sap on skin as a cosmetic agent has not been known entirely. In this study, we investigated the effects of birch (Betula platyphylla Sukatchev var. japonica Hara) sap on human skin. Birch sap induced epidermal keratinocyte differentiation properties in vitro. We assessed two epidermal differentiation agents. Filaggrin is a precursor protein of NMF, and involucrin is one of the precursor proteins of the cornified cell envelope (CE), which is related to normal barrier function. We have evaluated the production of these proteins where birch sap was applied to human normal keratinocytes. Birch sap not only increased mRNA expression of filaggrin and involucrin, but also accelerated these proteins production. Otherwise, birch sap did not have any influence for IL‐6 production, which is related to inflammatory and aberrant keratinocyte proliferation. The results of induced differentiation properties on birch sap‐treated keratinocytes are very similar to the differentiation induced by calcium in vitro. This similarity suggested that birch sap has a differentiation inducible property on in vitro cultured keratinocytes. Our study suggested that birch sap is able to control both moisturizing‐ and barrier‐related factor production. From these effects, birch sap provides appropriate epidermal functions and skin homeostasis, and revealed itself as a very useful ingredient in the cosmetic field.

DEK Proto-Oncogene Expression Interferes with the Normal Epithelial Differentiation Program

Overexpression of the DEK gene is associated with multiple human cancers, but its specific roles as a putative oncogene are not well defined. DEK transcription was previously shown to be induced by the high-risk human papillomavirus (HPV) E7 oncogene via E2F and Rb pathways. Transient DEK overexpression was able to inhibit both senescence and apoptosis in cultured cells. In at least the latter case, this mechanism involved the destabilization of p53 and the decreased expression of p53 target genes. We show here that DEK overexpression disrupts the normal differentiation program in a manner that is independent of either p53 or cell death. DEK expression was distinctly repressed upon the differentiation of cultured primary human keratinocytes, and stable DEK overexpression caused epidermal thickening in an organotypic raft model system. The observed hyperplasia involved a delay in keratinocyte differentiation toward a more undifferentiated state, and expansion of the basal cell compartment was due to increased proliferation, but not apoptosis. These phenotypes were accompanied by elevated p63 expression in the absence of p53 destabilization. In further support of bona fide oncogenic DEK activities, we report here up-regulated DEK protein levels in both human papilloma virus-positive hyperplastic murine skin and a subset of human squamous cell carcinomas. We suggest that DEK up-regulation may contribute to carcinoma development at least in part through increased proliferation and retardation of differentiation.

Cholesterol and Lipid Rafts as Regulators of Signaling Through the EGF Receptor in Keratinocytes

The EGF receptor (HER1/EGFR) and its cognate ligands are of considerable interest in epidermal biology and pathology. HER1/EGFR is expressed throughout the entire epidermis and its signaling has been shown involved in the control of keratinocyte proliferation, adhesion, and differentiation. The signaling of HER1/EGFR can be modulated by the receptor's immediate environment, particularly by its localization or not in lipid rafts. These membrane domains are nowadays described as heterogeneous dynamic signaling platforms where lipid molecules, cholesterol especially, are highly ordered and where transmembrane proteins encounter a specialized environment. Cholesterol depletion has been used in order to disorganize lipid rafts in keratinocytes and induces activation of HER1/EGFR (without ligand addition), as well as downstream signaling. The activation of p38 MAPK obtained after cholesterol depletion is responsible for the induction of involucrin expression while keratin expression is suppressed. However in keratinocytes recovering from cholesterol depletion the activation of p38 MAPK induces a strong expression of the heparin-binding EGF-like growth factor, a ligand of HER1/EGFR. Lipid rafts could thus be membrane platforms that regulate cell signaling in keratinocytes in normal, but also pathological conditions.

The CYP26 inhibitor R115866 potentiates the effects of all-transretinoic acid on cultured human epidermal keratinocytes

All-trans retinoic acid (RA) is known to regulate keratinocyte proliferation and differentiation, and retinoids are used as therapeutic agents in certain dermatological disorders, such as psoriasis and acne. Epidermal expression of the heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced by RA treatment and HB-EGF is responsible for RA-mediated epidermal hyperplasia in vivo. RA also induces HB-EGF expression in cultured keratinocytes and alters their differentiating phenotype. R115866 is a specific inhibitor of the cytochrome P450 isoform CYP26, which is involved in the metabolic inactivation pathway of RA. Thereby, R115866 is thought to be able to increase the intracellular levels of endogenous RA. To determine whether or not R115866 potentiates the effect of low concentrations of RA on keratinocytes. We analysed HB-EGF, involucrin and keratin 10 mRNA and protein levels in autocrine human keratinocyte cultures incubated for 18 h with RA or R115866 alone and with RA and R115866 combinations. RA induced HB-EGF and involucrin expression in a concentration-dependent manner, whereas it inhibited keratin 10 expression. R115866 alone had no effect on the expression of these genes. However, when R115866 was combined with low concentrations of RA, HB-EGF and involucrin expression was induced. These results strongly suggest that R115866 potentiates the effects of RA on epidermal keratinocytes when RA is present at low concentrations.

Comparative proteomics analysis of cytokeratin and involucrin expression in lesions from patients with systemic lupus erythematosus

To get a better understanding of the abnormal differentiation or maturation of keratinocytes, we studied the expression and distribution of cytokeratin and involucrin in lesions from systemic lupus erythematosus patients. Two groups of 10 specimens each from systemic lupus erythematosus and normal controls were analyzed by two-dimensional gel electrophoresis, mass spectrometric protein identification, Western blotting and immunohistochemistry. Our results showed that keratin 1 (K1)/K10 together with the new synthesis of K6/K16 were down-regulated and that K5/K14, K2e and involucrin were up-regulated. We found that involucrin was strongly stained in lower epidermal cell layers while K1/10 was weakly stained, particularly when compared with staining in normal epidermis. Additionally, we found that the expression of involucrin was increased. These results imply an aberrant early and terminal differentiation stage in the epidermis of systemic lupus erythematosus, which may be associated with inflammatory cytokines released during the wound healing response of lesion.

Up-regulated expression of Sp1 protein coincident with a viral protein in human and mouse differentiating keratinocytes may act as a cell differentiation marker

Sp1 is a transcription factor that regulates expression of mammalian and viral genes and is involved in different facets of cellular functions in eukaryotic cells. Here, we investigated Sp1 expression in primary mouse and human keratinocyte (KC) culture using quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence microscopy. Expression of Sp1 was post-transcriptionally up-regulated with increasing time in primary KC cultures. Sp1 expression, coincident with expression of human papillomavirus L1 capsid protein and involucrin, was associated with cell differentiation in vitro and in vivo in human and mouse skins. Immunoprecipitation experiments showed that Sp1 and L1 could be bound in a complex. Calcium (Ca 2+) and all- trans retinoic acid are the positive modulators of KC differentiation, which positively and negatively regulated Sp1 and L1 expression. The data suggest that coincident expression of Sp1 with L1 proteins in differentiating KCs is mediated by a calcium-dependent signaling pathway.

Expression of a skin cholesterol sulfotransferase, St2b2, is a trigger of epidermal cell differentiation

1. St2b2, a mouse cytosolic sulfotransferase, predominantly catalyses epidermal cholesterol sulfation. St2b2 was found in the basement layer by immunohistochemical analysis of normal mouse skin. The highest expression level was detected in epidermis from 3-day-old mice and then decreased before maturation. There was a good correlation between expression levels of skin St2b2 and a differentiation marker, involucrin.2. To understand the role of St2b2 in epidermal cell differentiation, recombinant St2b2 was expressed in primary epidermal cells. The expression of St2b2 enhanced the involucrin expression with an increase of cholesterol sulfate. Furthermore, by down-regulation of the St2b2 gene expression, involucrin was decreased in dorsal skin of 1–3-day-old mice by 67% of the control.3. These results strongly suggest a possibility that St2b2 expression plays a trigger of epidermal cell differentiation by controlling cholesterol sulfate level in the cells.

Methods to promote Notch signaling at the biomaterial interface and evaluation in a rafted organ culture model

The Notch signaling pathway is a promising target for controlling cell fate choices at the biomaterial-tissue interface. Building on our previous work in developing Notch-signaling biomaterials, we evaluated various immobilization schemes for Notch ligands, and their effect on human foreskin keratinocytes. A peptide sequence derived from the Jagged-1 DSL-region and immobilized to poly(2-hydroxyethyl methacrylate) (polyHEMA) showed no bioactivity in relation to the Notch-CSL pathway. The full-length Jagged-1 protein immobilized directly to the polyHEMA surface showed activity in signaling the Notch-CSL pathway. However, an indirect affinity immobilization approach yielded a stronger signal. Human keratinocytes plated on bound Jagged-1 showed upregulated involucrin, keratin 10, and loricrin protein expression, with this expression being cell density-dependent. Utilizing a human foreskin rafted organ culture model as a bridge between in vitro and in vivo studies, Jagged-1-modified or control polyHEMA rods were implanted in human foreskin and cultured at the air-medium interface. Keratinocyte proliferation was suppressed and intermediate-stage differentiation promoted in Jagged-1-modified rods compared with control rods. Thus, Notch-signaling biomaterials provide a robust approach to control keratinocyte differentiation and may find application to other progenitor and stem cells.

Insulin‐like growth factor binding protein‐5 enhances the migration and differentiation of gingival epithelial cells

The objective was to define the roles of insulin-like growth factor binding protein-5 (IGFBP-5) in gingival epithelial cells (GEC). Human IGFBP-5 is expressed in many cell types and has diverse biological functions. It stimulates the growth of bone cells and is associated with the impedance of gingival fibroblast apoptosis. In gingival epithelium, IGFBP-5 is expressed in the cells of the differentiated stratum spinosum layer. Recombinant IGFBP-5 protein treatment and knockdown of IGFBP-5 expression using a lentivirus-delivered short hairpin RNA was carried out in human GEC. Proliferation, apoptosis, anoikis, migration, differentiation and gene expression in GEC were analyzed and molecular images were obtained. The IGFBP-5 had no effect on proliferation, but it slightly suppressed apoptosis and anoikis of GEC. It also induced GEC migration and upregulated the expression of involucrin, transglutaminase-1, keratin and focal adhesion kinase. The IGFBP-5 induced migration partly via an insulin-like growth factor-independent mechanism. The knockdown of IGFBP-5 downregulated the expression of involucrin, transglutaminase-1 and focal adhesion kinase. Expression of IGFBP-5 in GEC is associated with anti-apoptosis, migration and differentiation of GEC. These phenotypic effects may be associated with focal adhesion kinase and are advantageous for re-epithelization of GEC and the maintenance of gingival health.

Long-term treatment with moisturizers affects the mRNA levels of genes involved in keratinocyte differentiation and desquamation

In a recent study, we showed that long-term treatment with two different moisturizers affected TEWL in opposite directions. Therefore, we decided to examine the effect of these moisturizers on the cellular and molecular level. In a randomized controlled study on 20 volunteers, epidermal mRNA expression of genes essential for keratinocyte differentiation and desquamation after a 7-week treatment with two moisturizers was analyzed. Treatment with one test moisturizer increased gene expression of involucrin, transglutaminase 1, kallikrein 5, and kallikrein 7, while the other moisturizer affected only expression of cyclin-dependent kinase inhibitor 1A. Thus, moisturizers are able to modify the skin barrier function and change the mRNA expression of certain epidermal genes. Since the type of influence depends on the composition of the moisturizer, these should be tailored in accordance with the requirement of the barrier of each individual patient, which merits further investigations.

PI3-kinase-dependent activation of apoptotic machinery occurs on commitment of epidermal keratinocytes to terminal differentiation

We have investigated the earliest events in commitment of human epidermal keratinocytes to terminal differentiation. Phosphorylated Akt and caspase activation were detected in cells exiting the basal layer of the epidermis. Activation of Akt by retroviral transduction of primary cultures of human keratinocytes resulted in an increase in abortive clones founded by transit amplifying cells, while inhibition of the upstream kinase, PI3-kinase, inhibited suspension-induced terminal differentiation. Caspase inhibition also blocked differentiation, the primary mediator being caspase 8. Caspase activation was initiated by 2 h in suspension, preceding the onset of expression of the terminal differentiation marker involucrin by several hours. Incubation of suspended cells with fibronectin or inhibition of PI3-kinase prevented caspase induction. At 2 h in suspension, keratinocytes that had become committed to terminal differentiation had increased side scatter, were 7-aminoactinomycin D (7-AAD) positive and annexin V negative; they exhibited loss of mitochondrial membrane potential and increased cardiolipin oxidation, but with no increase in reactive oxygen species. These properties indicate that the onset of terminal differentiation, while regulated by PI3-kinase and caspases, is not a classical apoptotic process.