Ulrike Lichti

Identification of a major keratinocyte cell envelope protein, loricrin

During epidermal cell cornification, the deposition of a layer of covalently cross-linked protein on the cytoplasmic face of the plasma membrane forms the cell envelope. We have isolated and characterized cDNA clones encoding a major differentiation product of mouse epidermal cells, which has an amino acid composition similar to that of purified cell envelopes. Transcripts of this gene are restricted to the granular layer and are as abundant as the differentiation-specific keratins, K1 and K10. An antiserum against a C-terminal peptide localizes this protein in discrete granules in the stratum granulosum and subsequently at the periphery of stratum corneum cells. Immunofluorescence and immunoelectron microscopy detect this epitope only on the inner surface of purified cell envelopes. Taken together, these results suggest that it is a major component of cell envelopes. On the basis of its presumed function, this protein is named loricrin.

Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice.

Protocols for preparing and culturing primary keratinocytes from newborn and adult mouse epidermis have evolved over the past 35 years. This protocol is now routinely applied to mice of various genetic backgrounds for in vitro studies of signaling pathways in differentiation and cell transformation, and for assessing the in vivo phenotype of altered keratinocytes in grafts of cells on immunodeficient mice. Crucial in the development and application of the procedure was the observation that keratinocytes proliferate in media of low calcium concentration, but rapidly commit to differentiation at calcium concentrations >0.07 mM after the initial attachment period. Preparing primary keratinocytes from ten newborn mice requires 2–3 h of hands-on time. Related procedures are also provided: preparing immature hair follicle buds, developing dermal hair follicles and fibroblasts from newborn mice, preparing primary keratinocytes from adult mice and grafting cell mixtures on athymic nude mice.

In vivo regulation of murine hair growth: Insights from grafting defined cell populations onto nude mice

The nude mouse graft model for testing the hair-forming ability of selected cell populations has considerable potential for providing insights into factors that are important for hair follicle development and proper hair formation. We have developed a minimal component system consisting of immature hair follicle buds from newborn pigmented C57BL/6 mice and adenovirus E1A-immortalized rat vibrissa dermal papilla cells. Hair follicle buds contribute to formation of hairless skin when grafted alone or with Swiss 3T3 cells, but produce densely haired skin when grafted with a fresh dermal cell preparation. The fresh dermal cell preparation represents the single cell fraction after hair follicles have been removed from a collagenase digest of newborn mouse dermis. It provides dermal papilla cells, fibroblasts, and possibly other important growth factor-producing cell types. Rat vibrissa dermal papilla cells supported dense hair growth at early passage in culture but progressively lost this potential during repeated passage in culture. Of 19 E1A-immortalized, clonally derived rat vibrissa dermal papilla cell lines, the four most positive clones supported hair growth to the extent of approximately 200 to 300 hairs per 1-2 cm2 graft area. The remaining clones were moderately positive (five clones), weakly positive (three clones), or negative (seven clones). Swiss 3T3 cells prevented contraction of the graft area but did not appear to affect the number of hairs in the graft site produced by dermal papilla cells plus hair follicle buds alone. The relatively low hair density (estimated 1-5% of normal) resulting from grafts of hair follicle buds with the most positive of the immortalized dermal papilla cell clones compared to fresh dermal cells suggests that optimal reconstitution of hair growth requires some function of dermal papilla cells partially lost during the immortalization process and possibly the contribution of other cell types present in the fresh dermal cell preparation, which is not supplied by the Swiss 3T3 cells. The current graft system, comprising hair follicle buds and immortalized dermal papilla cell clones, provides an assay for positive or negative influences on hair growth exerted by added selected cell types, growth factors, or other substances. Characterization of the phenotype of the dermal papilla cell lines, which differ in their ability to support hair growth when grafted with hair follicle buds, may provide insight into specific dermal papilla cell properties important for their function in this system.

Phorbol ester tumor promoters induce epidermal transglutaminase activity

Abstract Epidermal basal cells in culture have low levels of epidermal transglutaminase, the enzyme responsible for the formation of the cross-linked envelope in differentiated cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and other active (but not inactive) phorbol ester skin tumor promoters induce transglutaminase activity. Sloughing of differentiated cells accompanies the rise in transglutaminase activity. Phorbol esters do not affect transglutaminase activity when added directly to cell lysates. Corticosteroids have little influence on transglutaminase induction by phorbol esters. Retinoic acid induces transglutaminase activity, but activity does not further increase when basal cells are treated with both retinoic acid and 12-O-tetradecanoylphorbol-13-acetate.

Keratinocytes blocked in phorbol ester-responsive early stage of terminal differentiation by sarcoma viruses

It has been suggested that the initiation step in mouse skin carcinogenesis involves an alteration in epidermal differentiation, as mouse basal keratinocytes exposed to initiators resist the arrest of cell growth that is normally associated with the induction of terminal differentiation by calcium ions1–3. The growth of epidermal basal cells infected by Kirsten (Ki) or Harvey (Ha) sarcoma viruses is, however, arrested in response to calcium ions, although the cells do not progress through their entire maturation programme when a functioning ras gene of those viruses is expressed4. If continuous proliferation in the differentiating cell layers is a requirement for tumour formation in skin3,5, the response of sarcoma virus-infected cells seems inconsistent with the suggestion that an activated ras gene is sufficient to initiate skin carcinogenesis6. We now show that sarcoma virus-infected keratinocytes, when induced to differentiate, are blocked at an early, reversible stage of maturation. Furthermore, the cells respond to phorbol ester tumour promoters by undergoing a phenotypic reversion to a less mature stage. These results suggest that activation of a ras gene can produce conditionally initiated cells, in which the full expression of tumorigenicity depends on exposure to tumour promoters.

Fluocinolone acetonide: a potent inhibitor of mouse skin tumor promotion and epidermal DNA synthesis.

Abstract The relationship between the inhibition of mouse skin tumor promotion and the inhibition of epidermal DNA synthesis by the steroidal anti-inflammatory agent, fluocinolone acetonide (FA), was investigated. Simultaneous doses of either 10, 1, or 0.1 μg of FA and phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in an almost complete inhibition of promotion, whereas 0.01 and 0.001 μg of FA resulted in inhibition rates of 82% and 15%, respectively. Likewise, simultaneous doses of 10 or 1 μg of fluclorolone acetonide (FCA) and TPA caused a nearly complete inhibition of promotion, whereas 0.1 μg of FCA decreased promotion by 62%. In general, as the dose of both steroids was increased, an increase in the tumor latency period was observed. With the exception of the borderline effect of 0.001 μg of FA, the above doses of FA inhibited epidermal DNA synthesis by at least 60% for a 24-h period. Topical treatment with 10 μg of FA resulted in an almost complete inhibition of DNA synthesis for 6 days. The administration of 10 μg of FA 24 h after TPA treatment brought about a maximal inhibition of DNA synthesis of 65%, as compared with a 98% inhibition in control mice whose DNA synthesis had not been prestimulated. That is, FA was not quite as effective on S-phase cells as on G-1 cells. There appears to be a relationship between the inhibition of tumor promotion and epidermal DNA synthesis.

Selenoproteins Are Essential for Proper Keratinocyte Function and Skin Development

Dietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec). Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp) ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14) expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development.

Gene from a psoriasis susceptibility locus primes the skin for inflammation.

Psoriasis candidate genes promote susceptibility to skin inflammation and provide a treatment approach for psoriasis-related inflammation. Tipping the Scales Fashion models and the masses alike crave flawless skin achieved without the help of Photoshop. Yet, for sufferers of psoriasis, an autoimmune disease of the skin, this dream is far out of reach. Psoriasis, which frequently manifests as red or white scaly plaques on the skin, currently has no cure, although the symptoms may be treated by inhibiting either T cells or inflammatory cytokines. Wolf et al. bring us one step closer to a cure by fashioning a mouse model that mimics psoriasis in patients and can be used to develop therapies for the disease. Both immune cells and epidermal cells of the skin contribute to pathogenesis in psoriatic lesions; however, the relative contribution of these systems to disease development remains unknown. To investigate this contribution, Wolf et al. generated a mouse model of psoriasis by conditionally overexpressing the inflammation-associated protein mS100a7a15 in keratinocytes. The related human S100A7 and S100A15 proteins are encoded in a psoriasis susceptibility locus and are involved in cellular signaling during epithelial host defense. This new mouse model is especially relevant to the human disease because the human homologs of mS100a7a15 (hS100A7/S100A15) are overexpressed in the epidermis of inflammatory lesions from psoriasis patients. The model by Wolf et al. mimicked the disease in humans, inducing an inflammatory response after abrasion that consisted of elevated amounts of proinflammatory T cells and cytokines. These effects were mediated by the mS100a7a15 receptor RAGE (receptor of advanced glycation end products), suggesting the S100a7a15-RAGE axis as a therapeutic target for psoriasis. Thus, this model not only links the epidermal and immune contributions of psoriasis but also may tip the scales toward fewer scales (and flawless skin) for psoriasis patients. Psoriasis is a common complex genetic disease characterized by hyperplasia and inflammation in the skin; however, the relative contributions of epidermal cells and the immune system to disease pathogenesis remain unclear. Linkage studies have defined a psoriasis susceptibility locus (PSORS4) on 1q21, the epidermal differentiation complex, which includes genes for small S100 calcium-binding proteins. These proteins are involved in extracellular and intracellular signaling during epithelial host defense, linking innate and adaptive immunity. Inflammation-prone psoriatic skin constitutively expresses elevated concentrations of S100A7 (psoriasin) and S100A15 (koebnerisin) in the epidermis. Here, we report that genetically modified mice expressing elevated amounts of doxycycline-regulated mS100a7a15 in skin keratinocytes demonstrated an exaggerated inflammatory response when challenged by exogenous stimuli such as abrasion (Koebner phenomenon). This immune response was characterized by immune cell infiltration and elevated concentrations of T helper 1 (TH1) and TH17 proinflammatory cytokines, which have been linked to the pathogenesis of psoriasis and were further amplified upon challenge. Both inflammation priming and amplification required mS100a7a15 binding to the receptor of advanced glycation end products (RAGE). mS100a7a15 potentiated inflammation by acting directly as a chemoattractant for leukocytes, further increasing the number of inflammatory cells infiltrating the skin. This study provides a pathogenetic psoriasis model using a psoriasis candidate gene to link the epidermis and innate immune system in inflammation priming, highlighting the S100A7A15-RAGE axis as a potential therapeutic target.

Regulation of hair follicle development : an in vitro model for hair follicle invasion of dermis and associated connective tissue remodeling

During embryonic development presumptive hair follicle cells of epithelial and mesenchymal origin are determined in defined body locations. This is followed by rapid proliferation of epithelial cells and associated penetration into the dermis in response to as yet undetermined signals. A collagen matrix culture system, which maintains the three-dimensional relationships of hair follicle cells to each other, was developed to study the regulation of the enlargement of immature hair follicles and the accompanying remodeling of the dermis. In studies with a heterogeneous dermis-derived preparation of murine hair follicles, ranging in size from the earliest down-growing budding cell mass to hair-forming follicles, we had previously shown that cell proliferation was stimulated by cholera toxin and epidermal growth factor, but only the epidermal growth factor-stimulated proliferation was accompanied by digestion of the collagen matrix due to release of collagenolytic enzymes. Further studies revealed that transforming growth factor-alpha also stimulated hair follicle cell proliferation and collagenase release. However, although transforming growth factor-beta inhibited the transforming growth factor-alpha-stimulated proliferation, it enhanced the release and activation of collagenases and other gelatin-degrading enzymes detectable by gelatin zymography. Stimulation of collagenolytic activity depended on the three-dimensional hair follicle structure and did not occur in monolayer cultures of hair follicle cells. Comparison of hair follicle buds with more developed dermis-derived hair follicles, plated at the same cell density (based on DNA content), suggested that a greater fraction of cells in the bud-stage follicle responded to the growth factors by release of collagenases. Possibly only the cells in the advancing portion of growing hair follicles that are closest to the dermal papilla cell cluster produce the collagenases in response to growth factors. To examine the participation of dermal papilla cells in collagenase release and activation, several immortalized rat whisker dermal papilla cell lines were co-cultured with mouse hair follicle buds. Co-culture resulted in a marked enlargement of follicles as well as activation of the 92-kDa type IV collagenase, produced by hair follicle buds, that correlated with ability of the dermal papilla cells to stimulate hair formation in grafts of hair follicle buds on nude mice. Dermal papilla cells cultured alone produced the 72-kDa type IV collagenase, which was also activated during co-culture with hair follicle buds. Thus, two activities, both relevant for hair follicle development, namely, cell proliferation and release and activation of collagenases, have been stimulated in immature hair follicle buds by either growth-factor supplementation or interaction with dermal papilla cells.(ABSTRACT TRUNCATED AT 400 WORDS)

RETINOIDS INHIBIT PROMOTER‐DEPENDENT PRENEOPLASTIC PROGRESSION IN MOUSE EPIDERMAL CELL LINES

We have recently described a cell culture system for studying promoterdependent preneoplastic progression.l.2 The JB6 cell line has been derived from primary mouse epidermal cultures and found to respond to tumor-promoting but not to nonpromoting phorbol esters with an irreversible induction of tumor-cell phenotype, as measured by colony formation in 0.33% agar at 14 days. The mechanism appears to involve induction of a new phenotype rather than selection of preexisting anchorage-independent cells.3 The JB6 precursor cells are noatumorigenic while the phorbol ester-induced anchorage-independent transformants are tumorigenic (Colburn, submitted). Thus in view of its rapidity and irreversibility this anchorage-independence response to phorbol esters by JB6 cells appears to be analogous to late-stage skin tumor promotion in vivo. Inhibition of preneoplastic progression by retinoids has been shown for a number of epithelial systems.4~5 Boutwell and coworkers have reported that retinoids applied to mouse skin one hour before TPA during the promotion phase inhibited tumor yield substantially.6,7 The experiments described in this report were undertaken (1) to determine whether retinoids are active in inhibiting late-stage promotion in cell culture; (2) to use retinoid inhibition as an approach to identifying molecular and cellular events that are required in the process of tumor promotion, and (3) to investigate the mode of action of retinoids in tumor prophylaxis.