Ultrastructural localization of basigin in normal human epidermis

Basigin is a glycosylated transmembrane protein belonging to the immunoglobulin superfamily. It is thought to play roles in intercellular recognition involved in cell differentiation. We previously demonstrated at the light microscope level a correlation between basigin expression and epidermal differentiation. In the present study, the ultrastructural localization of basigin in normal human epidermal keratinocytes was investigated by immunoelectron microscopy. The basigin labeling was strongest on membranes of basal cells, weaker on prickle cells, and absent in granular and horny cells. On the membrane of basal cells, labeling was observed on the apical and lateral sides but not on the dermal side. Gold particles were mostly observed on the surface of microvilli, especially on their tips. There were fewer on the intermicrovillous membrane and they were absent on the desmosome. These results are consistent with our previous report that basigin expression is correlated with differentiation of epidermal keratinocytes. Microvilli on basal and suprabasal keratinocytes might play roles in the differentiation of keratinocytes through basigin on the tips of microvilli.     

Interleukin 1 is present in normal human epidermis

We investigated the presence of interleukin 1 (IL 1)-like molecules in normal unstimulated human epidermal tissue. Epidermis from 21 healthy individuals that was prepared by two different methods showed prostaglandin E2 (PGE2) and collagenase stimulating activity for human dermal fibroblasts. All epidermal extracts tested were positive for thymocyte comitogenic activity (lymphocyte activating factor; LAF). Removal of the horny layer decreased epidermal IL 1-like activity. In contrast to epidermal tissue, freshly isolated peripheral blood mononuclear cells (PBMC) contained no detectable PGE2 stimulatory activity. They could, however, produce PGE2 stimulatory activity after culture and stimulation with phytohemagglutinin (PHA) and concanavalin A (Con A). Little membranous IL 1-like activity could be detected in epidermal extracts when using a method that has previously rendered membranous IL 1 from murine proteose peptone-elicited peritoneal macrophages. Gel filtration chromatography yielded double peaks at m.w. approximately 30,000 and approximately 17,000 for all three activities. High pressure liquid chromatography (HPLC) analysis identified two species with a m.w. of approximately 17,000, and one approximately 30,000 species nondissociable in detergent, all having superposable PGE2 and collagenase stimulatory as well as LAF activity. These results establish the existence of IL 1-like molecules, together with a possible precursor, in normal human epidermis. The release of these preformed epidermal IL 1 stores might be important in vivo.     

Role of LIM kinases in normal and psoriatic human epidermis

We present evidence that LIM kinases can control cell adhesion and compaction in human epidermis. LIMK2 is expressed in the epidermal basal layer and signals downstream of the GTPase Rac1 to promote extracellular matrix adhesion and inhibit terminal differentiation. Conversely, LIMK1 is expressed in the upper granular layers and phosphorylates and inhibits cofilin. Expression of LIMK1 is lost in psoriatic lesions and other skin disorders characterized by lack of cell compaction in the differentiating cell layers. In psoriatic lesions down-regulation of LIMK1 correlates with up-regulation of Myc. Expression of constitutively active cofilin or Myc in reconstituted human epidermis blocks cell compaction. Overexpression of LIMK1 leads to down-regulation of Myc, whereas inhibition of Rho kinase, an upstream activator of LIMK1, stimulates Myc expression. Inhibition of Myc by LIMK1 is via inhibition of Stat3 phosphorylation, because constitutively active cofilin or inhibition of Rho kinase results in Stat3 phosphorylation and increased Myc levels, whereas dominant negative Stat3 abolishes the effect. In conclusion, we have uncovered a novel antagonistic relationship between the LIMK1/phosphocofilin and Myc/Stat3 pathways in the differentiating layers of human epidermis and propose that down-regulation of LIMK1 contributes to one of the pathological features of psoriatic epidermal lesions.     

Protein kinase C mRNA levels and activity in reconstituted normal human epidermis: relationships to cell differentiation.

Although keratinocytes are a major target of phorbol ester actions, the activity and the expression of the eight cloned protein kinase C (PKC) isoenzymes have not been studied in detail in human epidermis. Starting from normal human keratinocytes, we reconstituted in culture a multilayered epithelial tissue which presents many hystological, biochemical, and molecular features of the authentic epidermis and we used it as a model to investigate the PKC activity and mRNA levels. We found that i) PKC activity is higher in differentiated than in non-differentiated cells; ii) the mRNA levels of PKC delta and -eta/L, while are differently affected by spontaneous keratinocyte differentiation, are down-regulated during phorbol esters-induced cell differentiation. Our findings could represent a basis to investigate the involvement of PKC isoforms in the keratinocyte differentiation process.     

Fibromodulin gene is expressed in human epidermal keratinocytes in culture and in human epidermis in vivo

Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-β biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family.     

Differential extraction of keratin subunits and filaments from normal human epidermis

We have investigated keratin interactions in vivo by sequentially extracting water-insoluble proteins from normal human epidermis with increasing concentrations of urea (2, 4, 6, and 9.5 M) and examining each extract by one- and two-dimensional gel electrophoresis, immunoblot analysis using monoclonal anti-keratin antibodies, and EM. The viable layers of normal human epidermis contain keratins K1, K2, K5, K10/11, K14, and K15, which are sequentially expressed during the course of epidermal differentiation. Only keratins K5, K14, and K15, which are synthesized by epidermal basal cells, were solubilized in 2 M urea. Extraction of keratins K1, K2, and K10/11, which are expressed only in differentiating suprabasal cells, required 4-6 M urea. Negative staining of the 2-M urea extract revealed predominantly keratin filament subunits, whereas abundant intermediate-sized filaments were observed in the 4-urea and 6-M urea extracts. These results indicate that in normal human epidermis, keratins K5, K14, and K15 are more soluble than the differentiation-specific keratins K1, K2, and K10/11. This finding suggests that native keratin filaments of different polypeptide composition have differing properties, despite their similar morphology. Furthermore, the observation of stable filaments in 4 and 6 M urea suggests that epidermal keratins K1, K2, and K10/11, which ultimately form the bulk of the protective, nonviable stratum corneum, may comprise filaments that are unusually resistant to denaturation.     

Repeated injection (continuous labelling) experiments in mouse epidermis

Theoretical labelling index curves for epidermis have been generated under conditions of repeated tritiated thymidine injection. These curves take into account different injection intervals, circadian fluctuations in labelling and two different models for epidermal proliferation; one based on a homogeneous basal layer with “random” loss initially (later, loss was restricted to late G₁), and the other based on a programmed sequential aging of proliferative cells in a compartment derived from a minority class of stem cells. These curves have been compared with previously published experimental results and with results from some new experiments. Both models fit the data to some extent provided a mean value of T_c of about 140 h is assumed. However, the sequential aging model provides a slightly better overall fit. A further conclusion is that it is impossible to make any accurate statements on the epidermal growth fraction from repeated labelling data.     

Exposure to naphthalene induces naphthyl-keratin adducts in human epidermis in vitro and in vivo

We observed naphthyl-keratin adducts and dose-related metabolic enzyme induction at the mRNA level in reconstructed human epidermis in vitro after exposure to naphthalene. Immunofluorescence detection of 2-naphthyl-keratin-1 adducts confirmed the metabolism of naphthalene and adduction of keratin. We also observed naphthyl-keratin adducts in dermal tape-strip samples collected from naphthalene-exposed workers at levels ranging from 0.004 to 6.104 pmol adduct µg^?1 keratin. We have demonstrated the ability of the human skin to metabolize naphthalene and to form naphthyl-keratin adducts both in vitro and in vivo. The results indicate the potential use of keratin adducts as biomarkers of dermal exposure.     

CD44 standard and variant isoform expression in normal human skin appendages and epidermis

 CD44 isoforms have been implicated in tumor progression and metastasis formation. This study presents a thorough immunohistochemical analysis of CD44 standard and isoform expression in normal human skin appendages and epidermis applying monoclonal antibodies against CD44s, CD44v3, -v4, -v5, -v6, and -v9. An improved immunohistochemical protocol with microwave-based antigen retrieval in paraffin sections and heavy metal amplification of the diaminobenzidine reaction product provided enhanced resolution and sensitivity as compared to studies on frozen sections. The hair follicle, the seborrheic and eccrine sweat glands were strongly positive for all CD44 isoforms studied. In the latter, the clear cells but not the dark (intercalated) cells were positive. The sudoriferous ducts adjacent to the glands were weakly positive for all CD44 isoforms and strongly positive near the skin surface. In the apocrine glands, the basal cells showed only a moderate positivity. The myoepithelial cells expressed only CD44s. In the epidermis, all CD44 isoforms were detectable, with strongest CD44 immunostaining in the lower third of the stratum spinosum and weaker staining in the stratum basale and the upper two-thirds of the stratum granulosum. The stratum granulosum and corneum were unreactive. Thus, a regional and cell type-specific CD44 expression was revealed. Accepted: 10 May 1996