Defective calcium uptake in keratinocyte cell cultures from vitiliginous skin

Summary ⁴⁵Ca²⁺ has been used to measure the kinetics for the uptake, efflux, and steady state of this regulatory cation in keratinocytes grown from the involved and uninvolved skin of one donor (JM) with vitiligo. Cells grown from uninvolved skin yielded a very rapid uptake and efflux of this isotope before reaching steady state. A similar profile has been found for keratinocytes from normal healthy adult controls. However, cells established from vitiliginous skin showed a slow uptake of ⁴⁵Ca²⁺ before reaching the same steady state as the controls. ⁴⁵Ca²⁺ efflux has not been observed in vitiliginous keratinocytes. Furthermore, vitiliginous keratinocytes yielded a higher concentration of extracellular bound ⁴⁵Ca²⁺ compared with keratinocytes from uninvolved skin. Since Ca²⁺ has been found to be an allosteric inhibitor of membrane-associated thioredoxin reductase, this defect in Ca²⁺ transport may explain the proposed breakdown in free radical defense in vitiligo. These findings may also shed some more light on the etiology of this disorder.     

Expression of melanoma-associated antigens in melanoma cell cultures

The efficiency of melanoma immunotherapy appears to depend on both melanoma- and immune system-specific factors. Melanoma-specific factors include melanoma-associated antigen (MAA) expression as well as HLA class I molecule expression. We investigated the expression of five MAA - Melan-A/MART-1, tyrosinase, gp100, MAGE-1 and MAGE-3 - by means of FACS analysis in 50 melanoma cell cultures and compared them to the cultures of human foreskin-derived melanocytes and melanoma cell line UKRV-Mel2. Melan-A, tyrosinase and gp100 expression was frequently reduced in melanoma cell cultures, compared to that in foreskin melanocytes, whereas MAGE-1 and MAGE-3 expression showed variable degree of upregulation, compared to that in foreskin melanocytes. The expression of all tested MAA demonstrated high interindividual variability. We further show that cell cultures derived from the same tissue sample are oligoclonal in nature, by demonstrating the presence of up to three cell populations bearing distinct MAA profile. Analysing samples derived from the same patient but each at a different time point, we show that MAA expression profile changes over time either in positive (increase) or in negative (decrease) direction. Finally, we demonstrate that brain metastasis-derived cell cultures significantly overexpress Melan-A and MAGE-3, compared to primary tumours and other metastatic sites (P-value range: 0.05-0.001). Elucidation of the MAA expression patterns and the kinetics within the same patient as well as during the course of the disease may help improve current and develop new immunotherapeutic strategies.     

Differential effects of retinoids on DNA synthesis in calcium-regulated murine epidermal keratinocyte cultures

To study the possibility that the state of proliferation of epidermal keratinocytes can influence the action of retinoids, the rate of proliferation of murine epidermal keratinocytes was manipulated by growing the cells in media containing high or low concentrations of Ca++. In contrast to what other investigators have reported, keratinocytes cultured in medium containing 1.4 mM Ca++ proliferate faster, instead of slower, than cells cultured in medium with 0.09 mM Ca++. Other experiments showed that Ca++ was stimulatory to keratinocytes in medium containing a low level of growth factors, and inhibitory in medium containing a high level of growth factors, suggesting that the discrepancy could be due to a difference in the sera used. The high Ca++ cells prominently expressed the 48kD/56kD pair of keratin, showing that they were in a hyperproliferative state. Exposure of the faster growing high Ca++ cells to all-trans retinoic acid, 13-cis retinoic acid, etretinate, etretin, and arotinoid ethyl ester caused dose-dependent inhibition of DNA synthesis. In contrast, exposure of the slower growing low Ca++ cells to these retinoids resulted in dose-dependent stimulation of DNA synthesis. In addition, all-trans retinoic acid caused dose-related increases in cell number in the low Ca++ cultures. These findings correlate with the reported differential effects of retinoids on normal and hyperproliferative epidermis, and suggest that Ca++ and low growth factor-regulated keratinocyte cultures are useful for studying the mechanism of hyperproliferation and retinoid actions.     

Sphingolipid metabolism in organotypic mouse keratinocyte cultures

Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine. These results indicate that considerable diversity of ceramide structures occurs among mammalian species and that cultured keratinocytes may only partially reproduce the in vivo complement of ceramides. Using labeled serine in keratinocyte cultures, we have also demonstrated the de novo synthesis of ceramides and the transfer of label from glucosylceramides to ceramides during terminal differentiation of lifted cultures. The covalently bound corneocyte lipid envelope, which has recently been characterized in pig and human epidermis, was also present in mouse epidermis and was reproduced by the lifted cultures. Very-long-chain omega-hydroxyceramides were the dominant bound lipid and labeling studies in culture indicated that they were derived from ceramides synthesized in the viable epidermis.     

Antiproliferative potential of zidovudine in human keratinocyte cultures

Because the beneficial effects of zidovudine in human immunodeficiency virus infection-associated psoriasis have recently been observed, this study focused on the drug's action on the rapidly proliferating human HaCaT keratinocyte line as an in vitro model for epidermal hyperproliferation. Cultures in log growth phase were exposed to zidovudine for 2 days. Zidovudine slowed proliferation in a dose-dependent fashion as evidenced by 50% inhibition concentrations of 33 mumol/L (cell number), 30 mumol/L (protein content), 0.9 mumol/L (protein synthesis), and 0.7 mumol/L (DNA synthesis). Significant (p less than 0.01) reduction of cell viability to 94.6% and 87.2%, as well as morphologic manifestations of cytotoxicity, were first evident after 2 days' exposure to maximal drug concentrations of 10 and 100 mumol/L, respectively. Control viability, assayed by trypan blue exclusion, was 98.0%. Direct cytotoxic plasma membrane injury could be ruled out by the absence of any increase in cytoplasmic lactate dehydrogenase release into supernatants at least during the 1 day of maximal dosage exposure. The drug-induced inhibition of proliferation was reversible within 7 days after a 2-day exposure to 100 mumol/L zidovudine. Two days of treatment with a 10 mumol/L dose did not alter the pattern and synthesis of keratins in vitro. Thus the known antipsoriatic efficacy of zidovudine might be explained, at least partly, by the drug's cytostatic potency.     

Expression of epidermal integrins in human organotypic keratinocyte cultures

Abstract In organotypic coculture of human epidermal keralinocyles (HEK) or follicular outer root sheath (ORS) cells with human dermal fibroblasts, a stratified epithelium develops which in many regards re-sembles inleifollicular epidermis. The epithelium growing on type I collagen gels in the absence of a preformed basement membrane itself produces only low or moderate amounts of laminin and collagen type IV, so that a well-structured basement membrane cannot be formed. This results in loose and insufficient anchoring of basal cells in the collagen gel, frequently leading to cleft formation at the junction. Because integrins are important receptors for cell-cell and cell-matrix adhesion of keratino-cytes which under certain circumstances may also influence epidermal differentiation, we studied their expression under this culture condition which provides adhesional stress but leaves epidermal differentiation largely unaltered. The localization of integrins differed markedly from that in normal epidermis or normal outer root sheath since all integrin chains were polarized to the epithelium-collagen I interface. Thus, not only the 7.6 and β4-chains showed preferential expression at the basal attachment site of keratinocytes as in normal epidermis, but also the α2-, α3-, βM-chains which in normal epidermis under “steady stale” conditions appeal-primarily involved in cell-cell interaction of keralinocytes and are preferen-tially expressed at the lateral sides of their plasma membranes. Interest-ingly, the altered expression of integrins in organotypic cultures is not accompanied by significant disturbances in terminal differentiation.     

Pemphigus antibodies fixation and keratinocyte differentiation in organ cultures

Summary The effect of some agents, influencing the cyclic adenosine 3,5-monophosphate (cAMP) content of human cells, on the ability of the keratinocytes of binding pemphigus antibodies was studied by using tissue cultures of rabbit esophagus. As demonstrated by immunofluorescence (IF) for IgG, the bound antibodies appeared markedly decreased on esophagus explants grown under standard conditions, that is without test agents, when compared to ones fixed on fresh esophagus. But the IF reaction was remarkably more intense when methylxanthines or epinephrine were added to the growth medium of the cultures. Following the addition of these agents to the cultures some histologic modifications appeared in the explants, indicating that the keratinization process had probably been stimulated. This temporal relationship of immunofluorescence and histologic findings seems to suggest the hypothesis that keratinocyte differentiation, regulation of cAMP intracellular content, and pemphigus antibodies fixation are related processes.     

Effects of high glucose on NO synthesis in human keratinocyte cell line (HaCaT)

BACKGROUND: There is a possibility that alteration of nitric oxide (NO) synthesis by high glucose leads to a variety of diabetic complications. OBJECTIVE: In this study, we examined whether NO synthesis is altered by high glucose in spontaneously immortalized human keratinocyte cell line (HaCaT) that have three isoforms of NO synthases (NOS). METHODS: We measured NO end product nitrite in the culture medium using the Griess reagent and analyzed mRNA expression of three isoforms of NOS in HaCaT cells by RT-PCR. RESULTS: High glucose enhanced constitutively produced NO production in HaCaT cells, which persisted for 10 days and was attenuated by an inhibitor of protein kinase C (PKC), without altering eNOS/nNOS mRNA levels. Cytokine stimulation induced iNOS mRNA in HaCaT cells. Pretreatment with high glucose for 24 h enhanced cytokine-induced NO production in HaCaT cells. However, when these cells were exposed to high glucose for 10 days, cytokine treatment did not induce iNOS mRNA and nitrite production. CONCLUSION: These diverse alterations in NO production by high glucose may be involved in impaired host-defense and wound healing in the skin of diabetic patients.     

Epidermolysis bullosa: preliminary observations of blister formation in keratinocyte cultures

Keratinocytes from patients with different types of epidermolysis bullosa (EB) demonstrated an abnormal tendency to blister or bleb formation after 14-22 days in culture. The changes were most marked in samples from cases of junctional EB and were not observed in control cultures of normal skin. These findings suggest the possibility of a primary abnormality of keratinocyte adhesion in different varieties of EB and that keratinocyte culture should provide a useful model for further studies on disorders of adhesion in EB.     

Modulation of collagen type synthesis in organ and cell cultures of fibroblasts

Fibroblast cultures are widely used to study abnormalities of collagen metabolism in both inborn and acquired diseases. However, there is reason to question the extent to which the experimental information obtained from in vitro culture systems in fact reflects the in vivo situation. In the present study we analyzed the proportions of collagens I and III synthesized by human and mouse skin fibroblasts maintained under various culture conditions. The amount of type III collagen extracted from skin specimens was lower than that which was newly synthesized in organ culture. Cells obtained by enzymatic disintegration of skin specimens synthesized more type III collagen than fibroblasts grown from explants. However, subcultivation of the enzymatically liberated cells resulted in a continuous decline of type III collagen production which eventually reached levels similar to those observed in explant cultures.     

Disturbed keratinocyte differentiation in transgenic mice and organotypic keratinocyte cultures as a result of spermidine/spermine N-acetyltransferase overexpression

Overexpression of the rate-limiting enzyme in polyamine catabolism spermidine/spermine N1-acetyltransferase (SSAT) in transgenic (Tg) mouse leads to accumulation of putrescine in the skin and permanent hair loss at the age of 3 wk. The hair follicles of these mice are replaced by dermal cysts and epidermal utriculi. Increased putrescine production is also seen in hyperproliferative cutaneous disorders such as in psoriasis. These disorders are characterized by delayed onset of epidermal differentiation characterized as reduced expression of terminal differentiation markers such as cytokeratins 1/10, and filaggrin and persisting expression of basal cell cytokeratins 5/14 in the suprabasal layers. The use of these markers in immunohistological analysis of SSAT Tg skin clearly showed signs of disturbed differentiation. To exclude the possibility that changes in differentiation originated from underlying connective tissue, we introduced SSAT gene into an established rat epidermal cell line. Organotypic cultures derived from the transfected cells displayed similar changes in their differentiation pattern as keratinocytes in Tg skin. The role of accumulated putrescine in cutaneous changes of SSAT Tg mice was verified by an experiment in which putrescine level was reduced by systemic putrescine biosynthesis inhibition. The putrescine reduction was sufficient to alleviate the cutaneous changes to such an extent that distinct hair regrowth could be seen. These results suggest that the cutaneous changes of SSAT Tg animals are due to disorders of the keratinocyte differentiation. Moreover, they strengthen the view that the proper regulation of polyamine metabolism plays an important role in the keratinocyte maturation.     

Effect of hydrocortisone on Trichophyton rubrum growth in human epidermal keratinocyte cultures

A large volume of evidence suggests that topical steroids intensify fungal infections. To verify this observation further, we inoculated Trichophyton rubrum into human epidermal keratinocyte cultures in the presence of hydrocortisone. Our results reveal that hydrocortisone does stimulate mycelium growth. However, it only occurs in the cultures containing human epidermal cells, and the stimulation is greater at a higher concentration of the drug (20 micrograms/ml) than a lower one (2 micrograms/ml).     

Barrier function of human keratinocyte cultures grown at the air-liquid interface

Stratum corneum (SC), the outermost and least permeable layer of skin, is the major barrier to passive transepidermal water loss. In the research described in this paper, we have used human keratinocyte cultures, grown at the air-liquid (A/L) interface, to examine the relationship between epidermal differentiation (including SC formation) and barrier function. Histologically, the A/L culture showed several markers of complete differentiation, including the presence of well-organized and defined epidermal cell layers, keratohyalin granules, and a multilayered SC. The permeability of tritiated water through epidermal cultures, which had grown for 3 weeks at the A/L interface, was measured with a microdiffusion apparatus. The results of these experiments demonstrated that: a) the human keratinocyte cultures developed a substantial barrier (i.e., a multilayered SC) to water diffusion across the entire surface. If the relative humidity of the culturing environment was lowered from 100% to around 75%, the barrier was significantly improved; b) the differentiation promoter, 1.25-dihydroxy-vitamin-D3, increased the number of SC layers and reduced water permeation through the culture; c) the nature of the keratinocyte support matrix could be altered to improve the morphology as well as the barrier function of the epidermal cultures. Overall, the observations are consistent with the relationship that is believed to exist between SC intercellular lipid content and percutaneous penetration. Confirmation of this hypothesis will further the considerable potential of human keratinocyte A/L cultures as a valuable and relevant model in which to study drug absorption and metabolism.     

Investigating human keratinocyte stem cell identity

Studies of the regulatory networks controlling intrinsic properties and fate of adult stem cells are in a large part performed in animal models. Epidermis is one of the most accessible human tissues for researchers, which is a critical parameter for conducting programs dedicated to this knowledge in human stem cell systems. Keratinocyte stem cells constitute a particularly valuable model, because of this practical aspect, but more importantly because their existence is for decades validated by the clinical demonstration of their impressive capacity for epidermis regeneration. For the fundamentalist, human keratinocyte stem cells represent a unique system to dissect the genetic and epigenetic controls of "stemness" and self-renewal. For this purpose, a highly limiting point is our current inability of obtaining a cellular material corresponding to keratinocyte stem cells with homogeneous phenotypic and functional characteristics. The search for tools suitable for the prospective selection of keratinocyte stem cells will benefit from studies conducted at the broad level of the global stem cell field, as well as from more specifically targeted approaches. Advances in that direction are tightly linked to the development of functional assays allowing reliable assessment and modeling of the different stem cell-associated functional characteristics.     

Lamellar granule extrusion and stratum corneum intercellular lamellae in murine keratinocyte cultures

Lamellar granules are specialized epidermal organelles containing stacks of membranous disks that are extruded into the intercellular spaces in the upper portion of the granular layer. The extruded disks are believed to undergo biochemical and biophysical changes to form the stratum corneum intercellular lipid sheets that constitute the epidermal permeability barrier. Little is known about this important component of epidermal differentiation, in part due to lack of a suitable in vitro model. We have demonstrated microscopically the presence of characteristic lipid membrane structures in a primary keratinocyte culture system which shows morphologic differentiation comparable to that seen in vivo. A basal cell-enriched fraction of isolated neonatal mouse keratinocytes was plated into Vitrogen-coated 30 mm Millicell (Millipore, Bedford, Massachusetts) wells, fed daily with Medium 199 containing 10% fetal bovine serum, 10 micrograms/ml each of insulin and hydrocortisone, and kept at 32 degrees C in a 5% CO2/95% air atmosphere in a humidified incubator. Three days after plating, cultures were placed on living, epidermis-free mouse dermis at the air/liquid interface. At 2 wk, histologic examination showed multiple well-organized cell layers, including a distinct granular layer and a well-developed stratum corneum. Transmission electron microscopy demonstrated numerous lamellar granules and extrusion of their contents into the intercellular space. After fixation with ruthenium tetroxide, stacked intercellular lamellae in the stratum corneum were seen. Both the presence of dermis and growth at the air/liquid interface were necessary to achieve complete differentiation. This system conclusively demonstrates the formation of complex epidermal lipid structures in vitro and should allow the mechanisms and regulation of their synthesis to be elucidated.     

Relationship between actinic damage and chronologic aging in keratinocyte cultures of human skin.

The relationship of actinically-induced "premature aging" to chronological aging was studied in paired keratinocyte cultures obtained from the habitually sun-exposed (lateral) and nonexposed (medial) aspects of the arm of 5 male donors, aged 41 to 80 yr. In all cases, the number of cell generations in vitro was greater for cultures derived from sun-exposed skin, and this discrepancy increased with donor age and the severity of clinical aging changes. Hence, chronic sun exposure does accelerate aging in human skin by at least one previously established in vitro criterion: it decreased the lifespan of cultured keratinocytes. Plating efficiency was 11- to 32-fold higher for keratinocytes from chronically sun-exposed skin than nonexposed controls, perhaps reflecting the recognized carcinogenic potential of actinic radiation. Keratinocyte cultures appear to be as amenable to gerontologic studies as the already widely used human fibroblast cultures.     

The water permeability of primary mouse keratinocyte cultures grown at the air-liquid interface

In order to study the development of the epidermal permeability barrier in vitro, tritiated water (HTO) flux was measured across murine keratinocytes cultured at the air-liquid interface. Using a micro-diffusion technique, it was shown that air-liquid cultures form areas where the water diffusion is comparable to that of intact neonatal mouse skin. When water permeability is measured over a large area of the culture surface, however, significantly higher flux is obtained. These results show that under the culture conditions used, areas of water barrier comparable to intact neonatal mouse skin coexist with regions of less complete barrier formation.     

Increased calmodulin levels in psoriasis and low Ca++ regulated mouse epidermal keratinocyte cultures

Calcium has been shown to regulate the proliferation of epidermal keratinocytes in vitro. We became interested in the role of the calcium binding protein, calmodulin, in hyperproliferative, low calcium regulated keratinocytes in vitro and in the in vivo hyperproliferative state, psoriasis. Calmodulin levels were measured by radioimmune assay in neonatal mouse keratinocytes grown in 0.02 mM calcium (hyperproliferative) and 1.2 mM calcium (normal) media, and in cells that had been grown in low calcium medium and then switched to normal calcium. On a whole culture basis the normal cells had more calmodulin than the low calcium cells. However, when low calcium monolayers were compared to the normal basal monolayer, the low calcium hyperproliferative cells had more calmodulin. Cells that were switched from 0.02 mM calcium to 1.2 mM calcium showed increasing calmodulin levels over time. Psoriatic plaques contained 2-3 times more calmodulin than the skin of normal controls when examined on a per micrograms of DNA, per micrograms of protein, and per gram of wet weight basis. Adjacent uninvolved psoriatic skin also had significantly elevated calmodulin levels in all data bases except per microgram of protein/cm2. These data suggest that increased calmodulin levels are associated with epidermal hyperproliferation and/or with the state of differentiation.     

角质形成细胞μ-阿片受体表达的研究

目的：研究不同特性的角质形成细胞μ-阿片受体的表达情况。方法：以体外培养的角质形成细胞株HaCaT细胞、人鳞状细胞癌(简称鳞癌)细胞株A431、神经母细胞瘤SK-N-SH细胞株为对象，采用逆转录(RT)-PCR方法研究细胞μ-阿片受体的表达。结果：在常规体外培养条件下，角质形成细胞株HaCaT细胞、人鳞癌细胞株A431的RT-PCR结果显示有μ-阿片受体mRNA的表达，后者的表达水平略高于前者。结论：μ-阿片受体在角质形成细胞的表达，为神经系统和皮肤通过神经肽直接发生作用提供依据。