Effect of a new retinoid, arotinoid (Ro 13-6298), on in vitro keratinocyte proliferation and differentiation

In preliminary clinical trials, a new retinoid, Arotinoid Ro 13-6298, was found highly effective in hyperproliferation and keratotic disorders. The aim of this investigation was to study the effect of Arotinoid on in vitro neonatal mouse keratinocyte proliferation and differentiation. Five doses of Arotinoid were tested. [3H]-Tdr labeling techniques and autoradiography were used to quantitate proliferation. Differentiation was assessed using one-dimensional polyacrylamide gel electrophoresis and autoradiography of six specific protein fractions extracted from the cells using a series of buffers. Our results indicate that Arotinoid is an extremely potent inhibitor of keratinocyte proliferation. Arotinoid significantly decreases the amount and synthesis of non-covalently and disulfide cross-linked keratins and cell envelopes while increasing the quantity of keratohyalin granule-associated proteins. No qualitative change in keratinocyte proteins was observed.     

Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts

The immortal human keratinocyte line HaCaT is frequently used as a paradigm for skin keratinocytes in vitro because of its highly preserved differentiation capacity. HaCaT cells form a nearly regular epidermal architecture when transplanted onto subcutaneous tissue of athymic mice. In order to analyze further their differentiation capacity in vitro, HaCaT cells were studied in organotypic cocultures on top of collagen gels containing human dermal fibroblasts. Within 1 wk HaCaT cells formed a still dysplastic epithelium, the thickness of which correlated with the number of fibroblasts in the collagen gel. With further culture time of up to 3 wk a remarkably well structured and differentiated squamous epithelium developed. After 1 wk, keratins 10 and 16, involucrin, and transglutaminase I were expressed in suprabasal layers, whereas filaggrin, keratin 2e, and loricrin appeared after 2-3 wk. Within this time, a nearly complete basement membrane had formed including hemidesmosomes and anchoring fibrils. Epithelial cell proliferation became restricted to the basal layer after 2 and 3 wk. Using the TdT-mediated dUTP nick end labeling assay, fragmentation of DNA was detectable in nuclei of the parakeratotic stratum corneum. Ultrastructurally, many features of keratinization accumulated after 2 and 3 wk, though an orthokeratotic keratinization was not achieved, in contrast to HaCaT transplants. This differentiation deficiency - as compared with normal keratinocytes -- might be due to a lack of paracrine factors important for keratinocyte differentiation or to a reduced sensitivity of these cells. Nevertheless, this high degree of differentiation under organotypic conditions qualifies this cell line as an appropriate model for elucidation of the molecular mechanisms regulating keratinocyte growth and differentiation and for use in pharmacotoxicology.     

Increased DNA synthesis of uninvolved psoriatic epidermis is maintained in vitro

Clinically uninvolved psoriatic epidermis shows increased DNA synthesis in vivo. We have studied the DNA synthesis of cultured keratinocytes from uninvolved psoriatic skin. Trypsinized epidermal cells were plated on plastic dishes pre-coated with bovine collagen type I. In initial studies, normal human serum was found to be superior to fetal bovine in supporting the growth of human epidermal keratinocytes. Furthermore, keratinocyte cultures established in the presence of normal human serum produced large keratin proteins (68,000 daltons) indicating that the terminal steps in cell differentiation can occur in vitro. In subsequent experiments keratinocyte cultures were grown in medium supplemented with 10% normal human serum. Confluent cultures of keratinocytes from uninvolved psoriatic epidermis had an increased DNA synthesis determined both as the incorporation of [3H]thymidine and as the autoradiographic labelling index. The DNA synthesis of both normal and psoriatic keratinocyte cultures increased in response to incubation in medium with 10% psoriatic serum. The ability of keratinocytes from uninvolved psoriatic epidermis to maintain an increased DNA synthesis suggests the presence of an inherent defect within the population of epidermal keratinocytes in psoriasis. Such a culture system can be used as an in vitro model for the study of psoriasis.     

重组人干扰素-γ对角朊细胞体外增殖分化影响的研究

以人转化型角朊细胞株 Co L o- 16细胞为模型 ,通过活细胞计数、3H- Td R掺入法、电镜及免疫组化方法研究了重组人干扰素 -γ( rh IFN-γ)对角朊细胞体外增殖分化的影响。结果表明 IFN-γ能以一种剂量依赖方式抑制人角朊细胞株 Co L o- 16细胞的体外增殖和 DNA合成 ,对角朊细胞核有直接损伤作用 ,并抑制角朊细胞角蛋白表达。提示 IFN-γ能改变角朊细胞的体外增殖分化行为     

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.     

Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation

Skin equivalents formed by keratinocytes cocultured with fibroblasts embedded in collagen lattices represent promising tools for mechanistic studies of skin physiology, for pharmacotoxicologic testing, and for the use as skin substitutes in wound treatment. Such cultures would be superior in defined media to avoid interference with components of serum or tissue extracts. Here we demonstrate that a defined medium (supplemented keratinocyte defined medium) supports epidermal morphogenesis in organotypic cocultures equally well as serum-containing medium (mixture of Ham's F12 and Dulbecco's modified Eagle's medium), as documented by hallmarks of the epidermal phenotype studied by immunofluorescence and electron microscopy. In both cases regularly structured, orthokeratinized epithelia evolved with similar kinetics. Morphology in mixture of Ham's F12 and Dulbecco's modified Eagle's medium was slightly hyperplastic, and keratins 1 and 10 synthesis less co-ordinated than in supplemented keratinocyte defined medium, but a consistently inverted sequence of expression of keratins 1 and 10 was found in either medium. The late differentiation markers filaggrin, involucrin, keratin 2e, and transglutaminase 1 corresponded in their typical distribution in upper suprabasal layers. Keratin 16 persisted under both conditions indicating the activated epidermal state. Keratinocyte proliferation was comparable in both media, whereas fibroblast multiplication and proliferation was delayed and reduced in supplemented keratinocyte defined medium. In both media, ultrastructural features of epidermal differentiation as well as reconstitution of a basement membrane occurred similarly. Immature lamellar bodies and cytoplasmatic vacuoles, however, indicated an impaired lipid metabolism in supplemented keratinocyte defined medium. Nevertheless, these defined organotypic cocultures provide a suitable basis for in vitro skin models to study molecular mechanisms of tissue homeostasis and for use in pharmacotoxicologic testing.     

砷对角质形成细胞DNA合成及E2F1表达的影响

目的探讨砷剂对角质形成细胞DNA合成和相关转录因子E2F1表达的影响。方法以体外培养的角质形成细胞株为对象,不同浓度砷剂处理后,采用3H-TdR实验检测角质形成细胞DNA合成,并用RT-PCR方法研究细胞E2F1表达情况。结果在0.5～16 nmol/L药物浓度范围内,DNA合成加速,促进细胞增殖。但随着砷剂浓度继续升高,3H-TdR掺入量降低,逐渐回到基线水平;RT-PCR结果显示:DNA合成加速的HaCaT细胞组,伴有E2F1mRNA水平的上调。结论一定浓度范围内的砷剂可促进角质形成细胞株HaCaT细胞的DNA合成加速,增强转录因子E2F1的表达,可能是砷相关皮肤病的病理机制之一。     

Low-energy helium neon laser irradiation does not alter human keratinocyte differentiation.

There are reports that low-energy HeNe irradiation can enhance wound healing in vivo. We have previously demonstrated that HeNe irradiation increases the motility of human epidermally derived keratinocytes in vitro. Here we investigate whether HeNe irradiation alters normal keratinocyte differentiation, which is essential for the formation of a normal, functioning epidermis. Subconfluent keratinocyte cultures were irradiated three times within 24 h with either 0, 0.8, 3, or 7.2 J/cm2. After cultures reached post-confluence, parameters of growth and differentiation, such as cell number, cornified envelope (CE) formation, and transglutaminase activity were measured. No significant differences were found between the control (0 J) and irradiated cultures in these assays. We also examined the pattern of newly synthesized keratins in cultures irradiated with 7.2 J/cm2 three times within a 24-h period. Both control and irradiated cultures exhibited similar keratin patterns. These results provide evidence that HeNe irradiations of up to 7.2 J/cm2 have no direct deleterious effect on normal keratinocyte differentiation needed for the formation of a functional epidermis. Hence, it is anticipated that the clinical use of the HeNe laser irradiance that enhances keratinocyte migration in vitro (0.8 J/cm2) to promote wound healing in vivo will not alter the ultimate integrity or differentiated function of the epidermis that migrates to cover the wounded area.     

Collateral occurrence of deimination of keratins with differentiation of an immortalized newborn rat keratinocyte cell line cultured at air–liquid interface

We devised a simple method to maintain an immortalized newbown rat keratinocyte cell line at the air–liquid interface using a tissue culture insert fitted with a microporous membrane. The cells formed stratified layers of flattened and anucleated cells resembling stratum corneum of the epidermis. Deiminated proteins, which are localized in the cornified layer of epidermis as the reaction products of peptidylarginine deiminase were detected immunohistochemically in the differentiated cells. Western blot analyses revealed that major deiminated proteins were type I keratins K10 and K14. Deiminated products of type II keratin K5 were found as minor components. Our observations show that deimination of keratins might be correlated with terminal differentiation of the immortalized keratinocyte cell line.     

Trans retinoic acid enhances the growth response of epidermal keratinocytes to epidermal growth factor and transforming growth factor beta

Retinoids have been shown to either stimulate or inhibit epidermal keratinocyte proliferation. We have observed that in serum and growth factor free medium (basal medium), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) stimulated DNA synthesis in mouse epidermal keratinocyte cultures (mKC) in a time- and dose-dependent manner. Incubation with all-trans retinoic acid (RA) greatly enhanced the stimulatory effect of EGF. Transforming growth factor beta (TGF beta) inhibited the EGF-induced DNA synthesis in a dose-dependent manner, and the inhibition was greatly enhanced by a low dose of RA. Treatment of growth-factor deprived human keratinocyte cultures (hKC) with RA before incubation in basal medium containing EGF or a mixture of EGF, bovine pituitary extract (BPE), and insulin caused a dose-related increase in DNA synthesis and cell growth (cell number), respectively. A low concentration of RA also enhanced the inhibitory effect of TGF beta on growth-factor-induced DNA synthesis and cell growth in hKC. These findings suggest that the differential effects of retinoids on epidermal keratinocyte proliferation are in part due to an enhancement of the response of keratinocytes to positive and negative peptide growth factors.     

银屑病患者淋巴细胞对角朊细胞增殖影响的研究

用体外细胞培养技术，＾３Ｈ－ＴｄＲ标记检测培养细胞ＤＮＡ的合成，证明银屑病患者淋巴细胞对体外培养角朊细胞增殖有促进作用，与正常人淋巴细胞作用比较，Ｐ〈０．０１差异有显著性。并用地塞米松阻断银屑病患者淋巴细胞刺激表皮角朊细胞增殖，有效地建立了以银屑病患者淋巴细胞刺激表皮角朊细胞增殖的实验模型，为今后研究其他治疗药物提供了实验基础。     

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.     

Control of epidermal differentiation by a retinoid analogue unable to bind to cytosolic retinoic acid-binding proteins (CRABP).

The role played by cytosolic retinoic acid-binding proteins (CRABP) in the control of differentiation and morphogenesis by retinoids remains unclear, which contrasts with the presence of these binding proteins in tissues known to be targets for retinoic acid effects. Human epidermis represents a good system to address this question because 1) the effect of retinoids on keratinocyte differentiation is well documented; 2) epidermis contains CRABP, and the amount of these proteins is modulated both by keratinization and retinoids; 3) the architecture of epidermis obtained in vitro by growing adult human keratinocytes on a dermal substrate can be modulated by retinoids added to the culture medium in a dose-dependent manner; and 4) most markers of epidermal differentiation are also modulated by retinoids in a dose-dependent manner. In this study, we compared, in dose-response experiments, the biologic activities of retinoic acid and CD271, a substance unable to bind to CRABP, but able to bind to nuclear retinoic acid receptors (RAR). Our results show that retinoic acid and CD271 exert similar controls on epidermal morphogenesis and keratinocyte differentiation, as shown by the inhibition of the synthesis of suprabasal keratins, filaggrin, and transglutaminase. Therefore, we exclude a qualitative role for CRABP in the control exerted by retinoids on the differentiation and morphogenesis of cultured human keratinocytes. Instead of being involved in the pathway via which retinoids control epidermal gene expression, CRABP might regulate the amount of intracellular-active retinoic acid and thus control quantitatively the intensity of biologic effects.     

Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.

Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes.     

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.     

Epidermal homeostasis in long-term scaffold-enforced skin equivalents

Epidermal homeostasis is understood as the maintenance of epidermal tissue structure and function by a fine tuned regulatory mechanism balancing proliferation and cell loss by desquamation and apoptosis. The lack of appropriate experimental models has largely prevented a better understanding of the regulatory mechanisms controlling epidermal tissue homeostasis in human skin. Keratinocyte culture studies had revealed a strict dependency of regular epidermal differentiation on dermal interactions only accomplishable in three-dimensional skin models. As major drawbacks, conventional models, employing collagen hydrogels as dermal equivalents (DEs) exhibit a rather poor stability and limited lifespan. Here, we present an improved stabilized in vitro-model for long-term growth and differentiation of keratinocytes providing the basis for tissue homeostasis. Keratinocytes were grown on DEs reinforced by modified hyaluronic acid fibers (Hyalograft-3D) and colonized with skin fibroblasts, producing genuine dermis-type matrix. These skin equivalents (SEs) develop superior epidermal architecture with regular differentiation and ultrastructure. Critical aspects of differentiation, still unbalanced in early stages, are renormalized, most strikingly the coexpression of keratins K1/K10, downregulation of regeneration-associated keratins (K16), and restriction of K15 to the basal layer. The strict localization of integrins to basal cells underlining restored tissue polarity, the drop of keratinocyte growth rates towards physiological levels and the rapid formation of a mature basement membrane with abundant anchoring fibrils are altogether features fulfilling the criteria of tissue homeostasis. Therefore, these scaffold-based SEs not only allow for studying homeostasis control but also for the first time provide proper experimental conditions for establishing a stem cell niche in vitro.