GP37 expression in normal and diseased human epidermis: a marker for keratinocyte differentiation

An antiserum prepared against a glycoprotein (GP37) extracted from the upper epidermal layers, was used to stain frozen sections of human oral mucosa, normal and abnormal skin by an indirect immunofluorescence technique. On normal human epidermis, this antiserum mainly reacted with the cytoplasm of granular cells, whereas on buccal mucosa the recognized antigen was observed as scattered dots limited to the upper epithelial layers. In epidermal diseases, alterations in the staining pattern were observed. In psoriasis, the labelling was markedly diminished; in contrast, in lichen planus it was intense and present on the 3-6 uppermost cellular layers. Basal cell epitheliomas were almost negative, except around horn cysts. In Bowen's disease dyskeratotic cells were strongly labelled. In squamous cell carcinomas a clear-cut staining was observed in squamous nests. On cultures, GP37 expression could be induced by growing epidermal cells in vitamin A-depleted medium. The biological significance of the observed staining patterns remains to be precised. Nevertheless, GP37 represents a sensitive marker of epidermal differentiation and may be useful in skin pathology and in in vitro studies.     

Neutrophil gelatinase-associated lipocalin is a marker for dysregulated keratinocyte differentiation in human skin

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa protein initially isolated from the specific granules of human neutrophils. It is a member of the highly heterogeneous lipocalin protein family, which shares a common tertiary structure. Its synthesis is induced in gastrointestinal epithelium in association with inflammation and malignancy. To gain insight into its potential role in other epithelia we have investigated the expression of NGAL in human skin embryonic development, in normal adult skin, and in skin associated with inflammation and neoplastic transformation. In the present study we report that the embryonic expression of NGAL appears to be regulated in a spatio-temporal pattern. It was induced in the interfollicular epidermis at 20-24 weeks of gestational age but thereafter progressively receded towards the hair follicles. In normal adult skin, NGAL was detected solely in association with hair follicles. However, strong induction of NGAL in the epidermis was seen in a variety of skin disorders characterized by dysregulated epithelial differentiation such as psoriasis, pityriasis rubra and squamous cell carcinoma. In these tissues production of NGAL was confined to spatially distinct subpopulations of keratinocytes underlying areas of parakeratosis, whereas skin samples lacking parakeratotic epithelium such as lichen ruber planus, acute contact eczema and basal cell carcinoma were negative for NGAL. Consistent with being a marker for disturbed terminal differentiation, NGAL immunoreactivity showed an inverse pattern when compared with that of the differentiation marker filaggrin. The biologic functions of NGAL in epithelia are not fully known, although an immunomodulatory role in host defense has been proposed. In addition, the transient interfollicular NGAL expression during skin embryogenesis along with the induction of NGAL in adult parakeratotic epidermis suggests it play a role in epithelial differentiation pathways.     

Calmodulin-like skin protein: a new marker of keratinocyte differentiation.

The expression of the calmodulin-like skin protein, a recently discovered new skin-specific calcium binding protein, was studied in cultured keratinocytes, reconstructed human epidermis, and normal human skin. Using a calmodulin-like skin protein specific polyclonal antibody and Western blot analysis we could show that in cultured keratinocytes calmodulin-like skin protein expression is strongly induced after stimulating cell differentiation by increasing the medium calcium concentration. Known modulators of epidermal differentiation such as sodium butyrate and the synthetic retinoid CD 367 strongly affected calmodulin-like skin protein expression. A more than 10-fold increase was observed in the presence of sodium butyrate, whereas CD 367 abolished almost completely calmodulin-like skin protein expression already at nanomolar concentrations. Calmodulin, another calcium binding protein that is expressed throughout the living layers of the epidermis, is not affected by these modulators. In normal human skin, calmodulin-like skin protein expression is restricted to the stratum granulosum and the lower layers of the stratum corneum. From these results we conclude that calmodulin-like skin protein is a new marker of late keratinocyte differentiation with a role distinct from calmodulin.     

Involucrin expression in skin appendage tumours

The expression of involucrin was examined in 23 skin tumours of hair follicle origin, 17 tumours of sweat gland origin and three tumours of unknown origin, using an immunoperoxidase technique. All tumours from the hair follicle showed a positive reaction for involucrin. In particular keratoacanthoma and the squamous eddies in various tumours stained strongly. Trichofolliculoma, trichilemmoma and pilomatrixoma exhibited characteristic staining patterns which resembled those in the normal hair follicle. On the other hand the majority of the tumours of sweat gland origin did not stain, with restricted positive reactions in areas showing lumen formation or squamous metaplasia. In contrast to the lack of staining in syringoma, a positive reaction was observed in desmoplastic trichoepithelioma, which is histologically similar to syringoma. Clear cell acanthoma, the origin of which is still controversial, showed a staining pattern which indicated that its origin may not be in the sweat gland. These results suggest that testing for involucrin in skin appendage tumours may be very useful for understanding the kinetics of maturation as well as in determining the origin of the tumours.     

Minoxidil sulfotransferase, a marker of human keratinocyte differentiation.

The sulfation of minoxidil is catalyzed by a sulfotransferase activity in a number of tissues including skin. To investigate further the nature of the minoxidil sulfotransferase activity in epithelial tissue and to compare this activity to that of cholesterol sulfotransferase, which has already been shown to be induced during the differentiation of epithelial cells, we cultured normal human epidermal keratinocytes in a keratinocyte growth medium for 4 d, after which the media were replaced with either the same growth media or media with increasing Ca++ concentrations. Cholesterol sulfotransferase, minoxidil sulfotransferase, and transglutaminase were determined during the differentiation of the cells in the three media. Time-activity curves that suggested two different sulfotransferase activities were induced during the differentiation process. U-77581, a competitive inhibitor of minoxidil sulfotransferase activity, inhibited the sulfation of minoxidil sulfotransferase activity in the keratinocyte homogenates, but it did not inhibit the sulfation of cholesterol. These data indicate that at least two sulfotransferase activities are induced during the differentiation of epithelial keratinocytes and minoxidil sulfotransferase is an early marker of that differentiation.     

Carbohydrate expression and modification during keratinocyte differentiation in normal human and reconstructed epidermis

Using fluorescein isothiocyanate (FITC)-labeled lectins we were able to demonstrate the presence of specific carbohydrate moieties in normal human and reconstructed epidermis. Evidence is provided that in both cases the strongly reduced lectin staining at the level of the stratum corneum is the result of a hindered accessibility of the lectins in this lipid-rich hydrophobic environment. Isolated corneocytes and purified cornified envelopes (CEs) exhibited clearly glycosylated structures reacting with distinct lectins. The presence of glycosidase activity, particularly in the upper layers of the epidermis characterized by an acidic environment (pH 5.5), indicates that modifications of the sugar residues might be important in epidermal homeostasis, barrier behavior and desquamation. Absent or strongly reduced glycosidase activity in the stratum corneum of reconstructed epidermis with an impaired pH gradient could be in part responsible for the reduced barrier function and the lack of desquamation in this model.     

Expression of gamma-glutamyl transpeptidase in normal and neoplastic epithelial cells of human skin: a marker for distinguishing malignant epithelial tumours

The distribution of gamma-glutamyl transpeptidase (GGT) activity was studied histochemically in benign and malignant epithelial tumours of human skin. We found that GGT activity in normal skin was confined to the secretory portion of the eccrine and apocrine glands and to the inner root sheath of the hair follicles. In Bowen's disease and actinic keratosis, GGT activity was noted focally in areas where atypical cells were observed. In extramammary Paget's disease, GGT activity was found only in large round cells scattered among GGT negative epidermal cells. No GGT activity was observed in basal cell epitheliomas or benign epithelial tumours, while squamous cell carcinoma and eccrine porocarcinoma exhibited intense GGT activity. Our study suggests that GGT may be useful as a histochemical marker for distinguishing malignant tumours from benign epithelial tumours in human skin.     

Premature keratinocyte death and expression of marker proteins of apoptosis in human skin after UVB exposure

Epidermal keratinocytes undergo a process of terminal differentiation or cornification that in many aspects resembles apoptosis. It is characterized by the elimination of cell nuclei within the granular layer, whereas the cytoplasm is transformed into horn cells. Premature death of keratinocytes can be induced by extrinsic factors such as UV irradiation. We investigated the time-dependent expression of apoptotic marker proteins in the skin of one healthy human volunteer after irradiation with a fourfold minimal erythema dose (MED) of UVB. The data were supplemented by including healthy skin areas of biopsies from patients UVB-irradiated for therapeutic reasons. Punch biopsies were analysed by in situ end-labelling (ISEL) for DNA strand breaks and by immunohistochemistry for expression of p53, bcl-2, active caspase-3 and its proform, and deoxyribonuclease I (DNase I). Keratinocytes with pyknotic nuclei were first detected 6 h after UVB exposure, and apoptotic keratinocytes (sunburn cells) 12 h after exposure. These aggregated to sunburn bodies after 24 h. In control skin, nuclei with DNA strand breaks were only occasionally detected in the granular layer but 6 h after UVB irradiation in the spinous layer. After 12 h, many sunburn cells were ISEL-positive and positively stained for active caspase-3, P53, and DNase I. Morphometric evaluation of the immunohistochemical data demonstrated that maximal upregulation of P53, DNase I and activation of caspase-3 occurred 12 h after irradiation and in advance of the peak of apoptotic cell death reached after 24 h as verified by ISEL. In contrast, strong Bcl-2 immunostaining appeared restricted to presumed melanocytes and basal cells but was not increased after UVB irradiation.     

The mixed lineage kinase leucine-zipper protein kinase exhibits a differentiation-associated localization in normal human skin and induces keratinocyte differentiation upon overexpression

Leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase is a recently described protein serine/threonine kinase which belongs to the mixed lineage kinase family. The overall pattern of expression of the leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase gene in embryonic and adult mouse tissues suggested that this kinase could be involved in the regulation of epithelial cell proliferation and differentiation. In order to get more insights into the potential role of leucine-zipper protein kinase in these cellular processes, we characterized its expression in normal human skin, both at the mRNA and protein levels. In situ hybridization, western blotting, and indirect immunofluorescence studies were conducted to localize leucine-zipper protein kinase on various human skin tissues. This is one of the first reports that leucine-zipper protein kinase has a very precise pattern of expression in human skin epithelia, as both mRNA and protein are restricted to the granular layer of the epidermis and inner root sheath of hair follicles. Detection of leucine-zipper protein kinase protein on skin from various body sites, donors of different ages as well as on reconstructed human skin always reveals that leucine-zipper protein kinase is present only in the very differentiated keratinocytes of epidermis and hair follicles. To determine directly whether leucine-zipper protein kinase exhibits any effect on cell growth and differentiation, keratinocytes were transfected with an expression vector harboring the leucine-zipper protein kinase cDNA. The presence of this construct in keratinocytes results in growth arrest together with a concomitant increase in filaggrin expression. Collectively, our results indicate that leucine-zipper protein kinase plays an active part in cellular processes related to terminal differentiation of epidermal keratinocytes.     

Functional thrombomodulin expression on epithelial skin tumours as a differentiation marker for suprabasal keratinocytes

Summary In normal human skin, immtmoreactive thrombomodulin (TM) is expressed in a strict differentiation related pattern. solely in suprabasal spinous layer keratinocytes. To evaluate the polential application of TM as a differentiation marker for keratinocyte-derived skin tumours, we have studied immunohistopathological, biochemical and functional TM activities in various skin tumours. Immunoreactive, full sized and enzymatically active TM was expressed in keratinocyte-derived skin tumours (squamous cell carcinoma, seborrhoeic keratosis and partly Bowen's disease), as well as normal epidermal keratinoeytes and endothelial cells. However, no TM was detected in basal cell carcinotnas, senile keratosis or non-squanious epithelial tumours such as malignant melanoma, naevus pigmentosus and Paget's disease. Interestingly, decreased expression was observed in verruca vulgaris. These findings suggested that differentiation-dependent TM expression was restricted to epithelial skin tumours and undetectable on neural crest derived tumours. TM is a differentiation marker for spinous layer keratinocytes and is a useful tool in histopathological study of epithelial tumours.     

Intra-epidermal nerve endings progress within keratinocyte cytoplasmic tunnels in normal human skin

Intra-epidermal nerve endings, responsible for cutaneous perception of temperature, pain and itch, are conventionally described as passing freely between keratinocytes, from the basal to the granular layers of the epidermis. However, the recent discovery of keratinocyte contribution to cutaneous nociception implies that their anatomical relationships are much more intimate than what has been described so far. By studying human skin biopsies in confocal laser scanning microscopy, we show that intra-epidermal nerve endings are not only closely apposed to keratinocytes, but can also be enwrapped by keratinocyte cytoplasms over their entire circumference and thus progress within keratinocyte tunnels. As keratinocytes must activate intra-epidermal nerve endings to transduce nociceptive information, these findings may help understanding the interactions between the keratinocytes and nervous system. The discovery of these nerve portions progressing in keratinocyte tunnels is a strong argument to consider that contacts between epidermal keratinocytes and intra-epidermal nerve endings are not incidental and argue for the existence of specific and rapid paracrine communication from keratinocytes to sensory neurons.     

A monoclonal antibody labelling the keratinocyte membrane: a marker of epidermal differentiation

A murine hybridoma secreting an IgM monoclonal antibody (KL3) was produced by cell fusion of mouse myeloma cells with spleen cells from mice immunized with human epidermal keratins. On normal human epidermis KL3 stained the intercellular spaces from the stratum germinatum to the stratum granulosum with a fluorescence intensity increasing from the basal layer to the upper layers. Basal cells were not stained on the side facing the basement membrane. About 90% of free keratinocytes isolated after trypsinization were labelled by KL3 in a punctate staining. Immunoelectron microscopy allowed us to show that the antigen recognized by KL3 was exclusively localized on the keratinocyte membrane especially in the desmosomal plaques. KL3 reactivity was not modified by preincubation of skin sections with lectins showing a selective intercellular labelling of upper layers of epidermis or pemphigus antisera, nor by adsorption of the antibody on NP40 soluble proteins of the epidermis. Though KL3 reactivity was completely abolished after adsorption of purified keratins, no immunological reactivity of KL3 was detected with epidermal keratin polypeptides blotted on nitrocellulose paper. In psoriatic epidemis and epidermal tumors KL3 reactivity was drastically modified. These results suggest that KL3 recognized a keratinocyte membrane antigen implied in the epidermal differentiation process.     

Chemical peeling by SA-PEG remodels photo-damaged skin: suppressing p53 expression and normalizing keratinocyte differentiation

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG), which specifically acts on the stratum corneum, suppresses the development of skin tumors in UVB-irradiated hairless mice. To elucidate the mechanism through which chemical peeling with SA-PEG suppresses skin tumor development, the effects of chemical peeling on photodamaged keratinocytes and cornified envelopes (CEs) were evaluated in vivo. Among UVB-irradiated hairless mice, the structural atypia and expression of p53 protein in keratinocytes induced by UVB irradiation were intensely suppressed in the SA-PEG-treated mice 28 days after the start of weekly SA-PEG treatments when compared to that in the control UVB-irradiated mice. Incomplete expression of filaggrin and loricrin in keratinocytes from the control mice was also improved in keratinocytes from the SA-PEG-treated mice. In photo-exposed human facial skin, immature CEs were replaced with mature CEs 4 weeks after treatment with SA-PEG. Restoration of photodamaged stratum corneum by treatment with SA-PEG, which may affect remodeling of the structural environment of the keratinocytes, involved the normalization of keratinocyte differentiation and suppression of skin tumor development. These results suggest that the stratum corneum plays a protective role against carcinogenesis, and provide a novel strategy for the prevention of photo-induced skin tumors.     

Communication: expression of the novel inhibitor of apoptosis survivin in normal and neoplastic skin.

Apoptosis plays a fundamental part in epidermal homeostasis, and apoptotic cells have been detected in normal and diseased skin. Little is known, however, on the inhibitory mechanisms of apoptosis at the skin level. In addition to bcl-2, a novel inhibitor of apoptosis designated survivin and structurally analogous to IAP apoptosis inhibitors has been recently identified. The expression of survivin in normal and pathologic skin was investigated. Immunohistochemical studies revealed that survivin is expressed in basal keratinocytes, but not in suprabasal epidermal layers, with a pattern similar to bcl-2. In western blots, the anti-survivin antibody recognized a single band of 16.5 kDa in protein extracts from normal human keratinocytes in culture, in agreement with the predicted size of survivin. In addition, survivin immunoreactivity was detected in benign and malignant melanocytic lesions, with strong expression in invasive lesions of melanomas. Whereas survivin staining was undetectable in benign epithelial tumors, such as seborrheic keratoses, it was observed in all epidermal layers in Bowen's disease. Interestingly, at variance with bcl-2, survivin was markedly expressed in squamous cell carcinoma, but virtually lacking in basal cell carcinoma, suggesting that these two apoptosis inhibitors may act through different anti-apoptotic pathways. Deregulation of survivin may influence both epidermal homeostasis and the development of melanoma and nonmelanoma skin cancer.     

Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation

Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers—K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.     

Vitamin A esterification in human epidermis: a relation to keratinocyte differentiation

Keratinocytes from three different layers of epidermis (stratum basale, stratum spinosum, and stratum granulosum/corneum) were shown by high-performance liquid chromatography to contain retinol, 3,4-didehydroretinol and several fatty acyl esters thereof. The concentration of unesterified congeners increased 1.8-2.8 times from the inner to the outer layers of epidermis, while the corresponding increase in fatty acyl esters was 4.0-6.5 times. Together the esters represented 71% of the total vitamin A content in stratum granulosum/corneum as compared to 54% in stratum basale. The in situ synthesis of fatty acyl esters of retinol and 3,4-didehydroretinol (vitamin A2) was studied by addition of [3H]retinol to organ-cultured human breast skin. The radioactive compounds appearing in the epidermis after 48 h were, in order of abundance, retinyl esters, retinol, 3,4-didehydroretinyl esters, and 3,4-didehydroretinol. Studies at the subcellular level demonstrated the highest esterifying activity in the microsomal fraction. The enzyme catalyzing the reaction, acyl CoA:retinol acyltransferase (ARAT; EC 2.3.1.76), had a pH optimum of 5.5-6.0, which differs from that of ARAT in other tissues. ARAT activities in microsomes from different layers of epidermis were similar, but, owing to a presumed pH gradient in upper epidermis, the in vivo esterification of vitamin A may be enhanced in terminally differentiating keratinocytes. The mean ARAT activities in basal cell carcinomas and squamous cell carcinomas were less than 50% of the control values, and the relative amounts of retinyl esters were significantly lower than normal. We suggest that the esterification of vitamin A may also be of importance in relation to pathologic keratinocyte differentiation.