Keratinocytes In Terms Research Articles

Different impact of vitamin D on mitochondrial activity and morphology in normal and malignant keratinocytes, the role of genomic pathway

Deregulation of mitochondria activity is one of the hallmarks of cancerogenesis and an important target for cancer therapy. Therefore, we compared the impact of an active form of vitamin D3 (1,25(OH)2D3) on mitochondrial morphology and bioenergetics in human squamous cell carcinoma (A431) and immortalized HaCaT keratinocytes. It was shown that mitochondria of cancerous A431 cells differ from that observed in HaCaT keratinocytes in terms of network, morphology, bioenergetics, glycolysis, and mitochondrial DNA copy number, while treatment of A431 with 1,25(OH)2D3 partially eliminates these differences. Furthermore, mitochondrial membrane potential, basal respiration, and mitochondrial reactive oxygen species production were decreased in A431 cells treated with 1,25(OH)2D3. Additionally, the expression and protein level of mitophagy marker PINK1 was significantly increased in A431 1,25(OH)2D3 treated cells, but not observed in treated HaCaT cells. Knockout of VDR (vitamin D receptor) or RXRA (binding partner retinoid X receptor) partially altered mitochondrial morphology and function as well as mitochondrial response to 1,25(OH)2D3. Transcriptomic analysis on A431 cells treated with 1,25(OH)2D3 revealed modulation of expression of several mitochondrial-related genes involved in mitochondrial depolarization, mitochondrial protein translation (i.e. LYRM9, MARS2), and fusion-fission (OPA1, FIS1, MFN1 and 2), however, none of the genes coded by mitochondrial DNA was affected. Interestingly, in silico analyses of nuclear-encoded mitochondrial genes revealed that they are rather activated by the secondary genomic response to 1,25(OH)2D3. Taken together, 1,25(OH)2D3 remodels mitochondrial architecture and bioenergetics through VDR-dependent and only partially RXRA-dependent activation of the genomic pathway, thus outlining a new perspective for anticancer properties of vitamin D3 in relation to mitochondria in squamous cell carcinoma.

Ionizing Radiation-Induced DNA Damage Response in Primary Melanocytes and Keratinocytes of Human Skin

Currently, our knowledge of how different cell types in a tissue microenvironment respond to low and high linear energy transfer (LET) radiation is highly restricted. In this study, a comparative analysis was performed on γ-ray-induced DNA damage and repair in primary human melanocytes and keratinocytes isolated from 3 donors. Our study demonstrates a modest interindividual variability in both melanocytes and keratinocytes in terms of both spontaneous and ionizing radiation (IR)-induced 53BP1 foci formation and persistence. Melanocytes, in general, showed a slightly elevated (1.66–2.79 folds more) 53BP1 foci induction relative to keratinocytes after exposure to different doses of γ-rays (0.1–2.5 Gy) radiation. To verify the influence of ATM kinase on IR-induced 53BP1 foci formation, melanocytes and keratinocytes were treated with a specific ATM kinase inhibitor (KU55993, 10 μM) for 1 h prior to radiation. ATM kinase inhibition resulted in the reduction of both spontaneous and IR-induced 53BP1 foci by 17–42% in both melanocytes and keratinocytes of all the 3 donors. Increased persistence of IR-induced 53BP1 foci number was observed in ATM-inhibited melanocytes and keratinocytes after different post exposure times (6 h and 24 h). Taken together, our study suggests that interindividual variations exist in the induction and repair of DNA double-strand breaks (DSBs) in melanocytes and keratinocytes and that ATM is crucial for an optimal DSB repair efficiency in both human skin cell types.

Random/aligned electrospun PCL fibrous matrices with modified surface textures: Characterization and interactions with dermal fibroblasts and keratinocytes

Micro- or nano-surface topography of a biomaterial can improve various cellular activities for obtaining functional tissues. Electrospun fibers can gain further functionality when introduced topographic details to their surfaces. In this regard, we produced random and aligned polycaprolactone (PCL) micron/submicron fibers by the electrospinning method. Simultaneously, the surface structure of the fibers was altered by applying phase separation processes including non-solvent-induced phase separation (NIPS) and vapor-induced phase separation (VIPS) mechanisms. As a result, PCL fibers with porous, wrinkled, grooved, and crater-like morphology were obtained. Human dermal fibroblasts (BJ cells) and human keratinocytes (HS2) were cultured onto the fiber surfaces and the data were evaluated in terms of cell-material interactions. Results showed that not only the orientation of fibers but also fiber topography affected both cell-fiber and cell-cell interactions in different manners. It was observed that the wrinkled topography is the most suitable for both dermal fibroblasts and keratinocytes in terms of cell attachment and proliferation. We also concluded that cellular behavior was varied according to the morphology of the cells used. Morphological observations showed that HS2 cells proliferated more intensively on all surfaces compared to BJ cells. All these findings can be evaluated in terms of the design of tissue scaffolds, especially in skin tissue engineering.

Advances in our understanding of canine atopic dermatitis.

Canine atopic dermatitis (cAD) is a genetically inherited clinical syndrome that encompasses a diversity of mechanisms and can have a variety of triggers. Development of clinical disease is the result of genetic factors and environmental conditions, which shape the resulting immunological response. Clinical disease becomes evident once a threshold of inflammatory response is achieved. Skin barrier impairment plays a role in promoting cutaneous dysbiosis and increased allergen penetration. Keratinocytes shape the response of dendritic cells and subsequent lymphocytic response. Thymic stromal lymphopoietin is one of the links between the damaged skin barrier and the modulation of a T-helper (Th)2 response. It is still unclear whether mutations in skin barrier genes exist in atopic dogs, as they do in humans, or whether the observed alterations are purely secondary to inflammation. A dysregulated immune response with increased Th2, Th17 and CD4+ CD25+ regulatory T cells has been reported. A variety of cytokines [interleukin(IL)-31, IL-34, Macrophage migration inhibitory factor] are proposed as potential biomarkers and treatment targets because they are increased in the serum of atopic dogs when compared to controls, although a correlation between serum levels of these factors and severity of disease is not always present. The main issue with many published studies is that atopic dogs are always only compared to normal controls. Thus, it is unclear whether the changes that we find are truly a signature of cAD or merely a manifestation of nonspecific broad inflammatory responses. Studies considering comparison with other inflammatory diseases different from cAD are urgently needed to correctly identify what is specific to this complicated syndrome.

Interfollicular epidermal stem-like cells for the recreation of the hair follicle epithelial compartment

BackgroundHair follicle (HF) development and growth are dependent on epithelial-mesenchymal interactions (EMIs). Dermal papilla (DP) cells are recognized as the key inductive mesenchymal player, but the ideal source of receptive keratinocytes for human HF regeneration is yet to be defined. We herein investigated whether human interfollicular epidermal keratinocytes with stem-like features (EpSlKCs), characterized by a α6bri/CD71dim expression, can replace human hair follicular keratinocytes (HHFKCs) for the recreation of the HF epithelium and respective EMIs.MethodsThe α6bri/CD71dim cellular fraction was selected from the whole interfollicular keratinocyte population through fluorescence-activated cell sorting and directly compared with follicular keratinocytes in terms of their proliferative capacity and phenotype. The crosstalk with DP cells was studied in an indirect co-culture system, and EpSlKC hair forming capacity tested in a hair reconstitution assay when combined with DP cells.ResultsEpSlKCs exhibited a phenotypic profile similar to follicular keratinocytes and were capable of increasing DP cell proliferation and, for short co-culture times, the number of alkaline phosphatase-active cells, suggesting an improvement of their inductivity. Moreover, the recreation of immature HFs and sebaceous glands was observed after EpSlKC and DP cell co-grafting in nude mice.ConclusionsOur results suggest that EpSlKCs are akin to follicular keratinocytes and can crosstalk with DP cells, contributing to HF morphogenesis in vivo, thus representing an attractive epithelial cell source for hair regeneration strategies.

990 0.005% hypochlorite reduces serine protease in cultured human keratinocytes and alleviates itch of atopic skin

Proper serine protease (SP) regulation is essential for the homeostasis of epidermal permeability barrier. The imbalance between the activities of SPs and SP inhibitors contributes to disease flares in atopic dermatitis (AD). Bleach bath (0.005% hypochlorite (HOCl) has been included in the armamentarium for treating AD, but the mechanism involved in this treatment strategy remains unclear. This study aimed to determine the direct effect of 0.005% HOCl on cultured human keratinocytes in terms of SP expressions and to document the clinical effects of bleach bath on atopic skin. Stimulated cultured human keratinocytes were pretreated with 0.005% HOCl, and alterations in SP and SP inhibitor expressions as well as SP activities were evaluated. The potential involved molecular pathway was investigated. Additionally, the itch scores and skin physiologic profiles were documented before and within one day after bleach bath in patients with moderate to severe AD. Our results showed that the expression of SP kallikrein-related peptidase 5 (KLK5) and KLK7 in cultured keratinocytes were significantly upregulated by IL-1β. Pretreatment with 0.005% HOCl abrogated this phenomenon. Attenuation of nuclear factor kappa B (NFκB) signaling was found to involve in this process. The itch scores and skin pH were remarkably reduced within one day after bleach bath in AD patients while no significant change was noted in terms of stratum corneum hydration and epidermal water loss profiles. In summary, 0.005% HOCl has direct impact on cultured human keratinocytes in terms of SP expression and activity via modulation of NFκB pathway. As SP plays an important role maintaining epidermal integrity and modulating itching sensation, the direct effects of HOCl on epidermal keratinocytes contributed to the favorable therapeutic effect of bleach bath on atopic skin.

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

Surface air plasma-induced cell death and cytokine release of human keratinocytes in the context of psoriasis

Cold atmospheric plasma (CAP) has shown promise for wound healing, although little is understood of the underpinning mechanisms. Little has been reported so far of its potential use in the treatment of immune-mediated diseases such as psoriasis. To study CAP-induced cell death and cytokine release in human keratinocytes as a first assessment of possible CAP use for psoriasis. Using a CAP generator free of energetic ions, we observed its effects on keratinocytes in terms of morphology, cell viability and apoptosis, intracellular and mitochondrial reactive oxygen species (ROS), lysosomal integrity and mitochondrial membrane potential; and on secretion and expression of eight cytokines at protein and gene levels. CAP-induced reduced cell viability, apoptotic death and production of intracellular and mitochondrial ROS in dose-dependent manner. Mitochondrial dysfunction and lysosomal leakage were found in CAP-treated cells. It also induced release of interleukin (IL)-6, IL-8, tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF), and enhanced the mRNA expression of IL-1β, IL-6, IL-8, IL-10, TNF-α, interferon-γ and VEGF. By contrast, IL-12 declined monotonically. The results suggest that with appropriate control of its dose, physical plasma could induce cell death via apoptotic pathways and enable simultaneous reduction in IL-12. These effects may be used to suppress keratinocyte hyperproliferation and to target T-cell activation to control amplification of inflammation. This provides an initial basis for further studies of CAP as a potential therapeutic option for inflammatory and immune-related diseases in dermatology, including psoriasis.

Non-thermal atmospheric-pressure plasma possible application in wound healing.

Non-thermal atmospheric-pressure plasma, also named cold plasma, is defined as a partly ionized gas. Therefore, it cannot be equated with plasma from blood; it is not biological in nature. Non-thermal atmospheric-pressure plasma is a new innovative approach in medicine not only for the treatment of wounds, but with a wide-range of other applications, as e.g. topical treatment of other skin diseases with microbial involvement or treatment of cancer diseases. This review emphasizes plasma effects on wound healing. Non-thermal atmospheric-pressure plasma can support wound healing by its antiseptic effects, by stimulation of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect. We summarize the effects of plasma on eukaryotic cells, especially on keratinocytes in terms of viability, proliferation, DNA, adhesion molecules and angiogenesis together with the role of reactive oxygen species and other components of plasma. The outcome of first clinical trials regarding wound healing is pointed out.

Keratinocytes in the treatment of severe burn injury: an update.

Burns are among the most life-threatening physical injuries, in which fast wound closure is crucial. The surgical burn care has evolved considerably throughout the past decennia resulting in a shift of therapeutic goals. Therapies aiming to provide coverage of the burn have been replaced by treatments that have both functional as aesthetic outcomes. The standard in treating severe burns is still early excision followed by skin grafting. The use of cultured keratinocytes to cover extensive burn wounds appeared very promising at first, but the technique still has several limitations of which the long time to culture, the major costs, the risk of infection and the need for an adequate dermal layer limit clinical application. The introduction of dermal substitutes, composite grafts, tissue engineering based on stem cell application have been advocated. The aim of this review is to assess the use of cultured keratinocytes in terms of technical aspects, clinical application, limitations and future perspectives. Cultured keratinocytes are expected to keep playing a role in wound healing, especially in the field of chronic wounds. In severe burns, despite its limitations, keratinocytes can be beneficial if implemented as one of the elements in a broader wound management.

Differential regulation of nuclear factor-kappa B subunits on epidermal keratinocytes by ultraviolet B and tacrolimus

Modulation of nuclear factor-kappa B (NF-κB) expression has important clinical implications including anti-inflammation. Recently, we have shown that direct regulation of NF-κB/p65 subunit may account for tacrolimus ointment’s remarkable clinical efficacy on treating inflammatory dermatoses. However, NF-κB is a dimeric transcription factor formed by hetero- or homodimeration of the five Rel family proteins. The complete operational scheme of different NF-κB subunits remains obscure. It has been shown that homodimers consist of NF-κB/p50 may serve an inhibitory role in suppressing inflammation while dimers consisting of NF-κB/p65 activate inflammatory pathway. Our current study aimed to explore the effects of ultraviolet B (UVB) on epidermal keratinocytes in terms of specific NF-κB subunits NF-κB/p50 and NF-κB/p65. Additionally, the effects of tacrolimus on differential regulation of NF-κB subunits of UVB irradiated keratinocytes were also investigated. Our result showed that UVB sequentially regulated the activities of different subunits of NF-κB: the activity of NF-κB/p50 was downregulated in the early stage (6 hours), followed by upregulation of NF-κB/p65 in the later stage (12 hours). The results from immunofluorescence, immunocytochemical, and immunohistochemical analyses indicated that the nuclear expression of NF-κB/p50 could be seen constitutively while the nuclear expression of NF-κB/p65 could only be seen after UVB irradiation. Furthermore, treatment with tacrolimus didn’t affect the nuclear activation and translocation of NF-κB/p50, while the UVB induced NF-κB/p65 nuclear expression was suppressed by tacrolimus. In summary, we have shown that UVB irradiation sequentially regulated different NF-κB subunits. The clinical efficacy of tacrolimus may be attributed to its specific regulatory effect on NF-κB/p65 but not NF-κB/p50 of the NF-κB pathway.

Bi‐layered collagen nano‐structured membrane prototype (collagen matrix 10826®) for oral soft tissue regeneration: an “in vitro” study

To evaluate fundamental cell functions, such as adhesion, IL-6 production and proliferation of human gingival keratinocytes cultured on a newly engineered collagen matrix (CM-10826) and to assess the degree of specific biocompatibility of this new device. Primary cultures of human keratinocytes were derived "in vitro" from biopsies of independent donors. Their true epithelial origin was ensured by the expression of cytokeratin 14. Adhesion, proliferation and production of IL-6 cytokine was then measured in the presence or absence of CM-10826 activity or of its relevant components. Functional tests revealed that keratinocytes adhered to CM-10826 and up-regulated their basal IL-6 production. The type of keratinocytes used expressed cytokeratin 14. Proliferation experiments demonstrated that the best cellular response was observed in the presence of Collagen I, the main component of CM-10826. No undesired effects were observed as for keratinocyte viability, morphology or differentiation. Our results demonstrate that CM-10826 has a favourable biological effect on the "in vitro" response of gingival keratinocytes in terms of IL-6 production, cell growth and adhesion. These findings may encourage a possible use of this collagen membrane as a tissue which, alone, may substitute for autologous gingival grafts thereby overcoming the limitations of autologous tissue.

Hyperglycaemic conditions decrease cultured keratinocyte mobility: implications for impaired wound healing in patients with diabetes

Elevated blood glucose in patients with diabetes mellitus (DM) leads to complications including poor wound healing. Proper keratinocyte migration and proliferation are the crucial steps during re-epithelialization. We hypothesize that the impaired wound healing in patients with DM is due to the disruption of proper re-epithelialization. We aimed to explore the effects of high glucose on keratinocytes in terms of cell migration and proliferation. Keratinocytes were cultivated in normal and high glucose conditions. Their viability was evaluated by MTS assay. Transwell migration and in vitro scratch assays were used to evaluate their mobility. The mRNA expressions and activities of matrix metalloproteinase (MMP)-2 and MMP-9 were determined. The mRNA of their respective physiological inhibitors, tissue inhibitor of MMP (TIMP)-1 and TIMP-2, was also evaluated. Immunofluorescent staining and Western blotting were used to examine the expression of phosphorylated focal adhesion kinase (pp125(FAK)). The impacts of high glucose on keratinocyte proliferation were assessed by 5-bromo-2'-deoxyuridine incorporation assay. High glucose treatment did not affect keratinocyte viability up to 3 days. In contrast, the mobility of keratinocytes, the activities and gene expressions of MMP-2 and MMP-9, the expression of pp125(FAK), and the cell proliferation after 5 days were significantly downregulated after hyperglycaemic treatments while the mRNA expression of TIMP-1 increased. Under hyperglycaemic conditions, keratinocytes demonstrate reduced migration and decreased proliferation capacities. These impairments of keratinocyte functions are likely to result in inadequate re-epithelialization. These defective physiological events provide a reasonable explanation for the poor wound healing commonly observed in patients with DM.

The neonatal Fc receptor (FcRn) expression in the human skin

The neonatal Fc receptor, FcRn, has a wide expression within the human body, including adult cells; however, it is not ubiquitously expressed (reviewed in [2]). The receptor is involved both in IgG transcytosis as well as recycling, being able to bind both free and antigen complexed IgG molecules (reviewed in [4]). Cauza et al. [1] have shown that the neonatal receptor is present in the human epidermal keratinocytes, using both biopsies and cultivated keratinocytes. Furthermore, they were able to demonstrate that most of the FcRn molecules are expressed intracellularly, in acidified vesicles [1], and that the receptor is functional within these cells. However, the FcRn role in keratinocytes in terms of recycling, transcytosis, or perhaps both remains speculative. In this paper, we show by immunohistochemistry that FcRn expression in human skin is not restricted only to keratinocytes but has a wider expression. Tissues were obtained from five human cadavers (harvested in an interval of 24 h from the time of death), and a surgical biopsy was performed for diagnostic purposes (nevus). Immunohistochemistry was done on consecutive skin sections as previously described [2]. The epithelial cells were evidenced with an antihuman cytokeratins antibody (clone MNF116, Dako). As expected, the antibody stained the epithelial cells of the hair follicles and sebaceous glands and the keratinocytes (data not shown). The FcRn-staining pattern proved to be more complex. The epithelial structures, evidenced by the cytokeratins targeting, proved to be positive as well for the Fc neonatal receptor (Fig. 1). Furthermore, we were able to show that the cytokeratinnegative, S-100-positive melanocytes (data not shown) [3] express also this receptor (Fig. 2). Histiocytes and dendritic cells were also shown to be FcRn positive by others [1, 5]. The characterization of the exact nature of the cells scattered throughout the investigated tissues was beyond the goals of this study. However, we can speculate that the isolated FcRn-positive cells in our skin sections are histiocytes and dendritic cells. The presence of FcRn in endothelial cells was investigated by labeling sequential sections with anti-FcRn and respectively anti-CD34 II (clone QBEnd, Dako) antibodies, as well as double labeling with HRP/DAB (brown) for FcRn, and Alkaline Phosphatase/FastRed (Dako, Denmark) (red) for CD34 II. Consistent with the results obtained on human mammary gland sections [2], blood vessels are either negative for the neonatal Fc receptor or express this molecule at a level that is undetectable by the immunohistochemical method (Fig. 3). IgG is the main antibody isotype in the extravascular body spaces. Hence, the wide expression of the neonatal Fc receptor throughout the organism, including in various cell types of the skin, does not come as a surprise. Although the receptor was proven to be functional in keratinocytes [1], the FcRn function (recycling, transcytosis, or both) in the skin structures remains to be elucidated. On the other hand, Virchows Arch (2007) 451:859–860 DOI 10.1007/s00428-007-0467-7

Calcipotriol Affects Keratinocyte Proliferation by Decreasing Expression of Early Growth Response-1 and Polo-like Kinase-2

Calcipotriol is a potent drug for topical treatment of psoriasis because it manages to inhibit keratinocyte proliferation. In the present study we investigated the effects of calcipotriol on gene expression in human keratinocytes in terms of mechanism of how calcipotriol decreases proliferation. Cell proliferation was analyzed by MTT assay. The differential display approach together with qPCR was used to assess the gene expression after treatment. In addition, Western immunoblotting revealed differences on the protein level. Finally, transfection of the KCs with specific small interfering RNA determined the genes necessary to inhibit proliferation. KCs proliferation was decreased in a concentration-dependent manner. Moreover, calcipotriol dowregulated the expression of two proliferation factors: early growth response-1 (EGR1) and polo-like kinase-2 (PLK2). The protein levels of EGR1 and PLK2 were also decreased. Specific siRNA against EGR1 and PLK2 in KCs resulted in marked reduction of EGR1 and PLK2 expression. In both cases, the reduction resolved in the decreased proliferation of KCs. This study provides a new insight into how calcipotriol affects proliferation of keratinocytes by decreasing the expression of EGR1 and PLK2. Furthermore, the results offer groundwork for developing novel compounds for the treatment of hyperproliferative skin disorders like psoriasis.

FK506 inhibits tumour necrosis factor-alpha secretion in human keratinocytes via regulation of nuclear factor-kappaB

Tacrolimus (FK506) ointment has been used for treatment of inflammatory dermatoses with remarkable success. Our previous studies have indicated that direct modulation of tacrolimus on keratinocytes (KCs) may have an impact on its therapeutic effect. The use of monoclonal antibody specific for tumour necrosis factor (TNF)-alpha has shown efficacy in treating both psoriasis and Crohn disease. Topical tacrolimus has also been shown to be effective for treating cutaneous manifestations of both diseases. To explore the effects of FK506 on human KCs in terms of TNF-alpha secretion and to investigate the regulatory pathway involved. Ultraviolet (UV) B-irradiated cultured KCs were treated with various concentrations of FK506. At indicated time points after UVB irradiation we determined: (i) the TNF-alpha concentrations present in the culture supernatants; (ii) the activation and translocation of nuclear factor (NF)-kappaB in the cell nucleus; and (iii) the protein expressions of IkappaB kinase (IKK) and IkappaB in the cell lysates. In addition, a mouse model was used to corroborate our in vitro findings in vivo. More specifically, topical tacrolimus was applied on to mouse skin unilaterally after UVB irradiation. The effects of FK506 on nuclear NF-kappaB expression of UVB-irradiated mouse skin were determined. Our results showed that FK506 dose-dependently downregulated the secretion of TNF-alpha from UVB-irradiated KCs. The activation and translocation of NF-kappaB in UVB-irradiated KCs were also dose-dependently suppressed by FK506. The degradation of IkappaB induced by UVB was also inhibited by FK506, while no change in IKK expression was noted regardless of UVB and FK506 treatment. Murine skin biopsies showed that nuclear NF-kappaB expression induced by UVB was inhibited by topical tacrolimus treatment. Our results indicate that FK506 inhibits TNF-alpha secretion in human KCs via direct regulation of NF-kappaB. This modulatory effect of FK506 on KCs offers a possible mechanism for how topical tacrolimus regulates cutaneous inflammatory conditions.

FK506 independently upregulates transforming growth factor beta and downregulates inducible nitric oxide synthase in cultured human keratinocytes: possible mechanisms of how tacrolimus ointment interacts with atopic skin.

Tacrolimus ointment (FK506) has been used in recent years for the treatment of atopic dermatitis (AD), with favourable results. Most of the therapeutic efficacy of FK506 in AD has been attributed to its immunomodulatory effects on different immune cell types, but its effects on keratinocytes (KCs) have rarely been discussed. Studies have shown that low expression of transforming growth factor (TGF)-beta and high expression of nitric oxide synthase (NOS) are implicated in the pathogenesis of AD. To investigate the direct effects of FK506 on KCs in terms of TGF-beta and inducible NOS (iNOS), and to explore the interactions between TGF-beta and iNOS in the KC system. Cultured human KCs treated with different concentrations of FK506 were used for investigation. The changes in the KC system induced by FK506 were documented in terms of TGF-beta and iNOS using enzyme-linked immunosorbent assay and Western blotting techniques, respectively. The gene expression of both TGF-beta and iNOS was also determined. A certain amount of tumour necrosis factor (TNF)-alpha was introduced to mimic atopic skin in vivo. Our results showed that the release of TGF-beta was upregulated in FK506-treated KCs, particularly in the presence of TNF-alpha, while the expression of iNOS was downregulated. The gene expression of iNOS was also downregulated, as shown by reverse transcriptase-polymerase chain reaction analysis. However, the addition of TNF-alpha did not further downregulate the expression of iNOS protein, suggesting that FK506 may regulate TGF-beta and iNOS through different pathways. Our findings indicate that the direct effects of FK506 on KCs probably contribute to its therapeutic efficacy in the treatment of AD.

Coenzyme Q10, a cutaneous antioxidant and energizer.

The processes of aging and photoaging are associated with an increase in cellular oxidation. This may be in part due to a decline in the levels of the endogenous cellular antioxidant coenzyme Q10 (ubiquinone, CoQ10). Therefore, we have investigated whether topical application of CoQ10 has the beneficial effect of preventing photoaging. We were able to demonstrate that CoQ10 penetrated into the viable layers of the epidermis and reduce the level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was also shown. CoQ10 was determined to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, activation of specific phosphotyrosine kinases and prevention of oxidative DNA damage. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging.

Incomplete Differentiation of Fetal Keratinocytes in the Skin Equivalent Leads to the Default Pathway of Apoptosis

The program of epidermal morphogenesis and differentiation changes dramatically during development of human fetal skin. Keratinocytes derived from fetal basal cells at early stages undergo only an incomplete keratinization and are eventually replaced by newly formed fetal keratinocytes that complete the terminal differentiation program and form the first stratum granulosum and stratum corneum. Once established, this program is reiterated throughout life, as the epidermis continually renews. To test the developmental potential of early fetal keratinocytes, we have cultured them in the physiologic skin equivalent (SE) system in the presence of varied retinoic acid (RA) concentrations and have compared them to neonatal keratinocytes cultured under the same conditions. The responses of fetal and neonatal SEs have been characterized by analysis of epidermal morphology, the presence and distribution of RA-responsive and differentiation-specific keratins and filaggrin, proliferation, and apoptosis. Our study shows that fetal basal keratinocytes already are programmed to form the granular layer and incomplete stratum corneum, even when isolated from a stage prior to formation of these layers. Fetal keratinocytes respond differently than neonatal keratinocytes to RA in terms of modulation of both epidermal morphology and expression of differentiation markers. Modulation of RA-responsive K1 and K19 occurs in both fetal SE and neonatal SE but the fetal keratinocyte responds at lower RA concentrations in the medium. In contrast, fetal keratinocytes appear to be less responsive than neonatal keratinocytes in terms of filaggrin expression and stratum corneum formation. These differences in the differentiation and RA response in vitromay be related to inherent stage-specific differences between fetal and neonatal keratinocytes in RA-signaling pathways including expression of the retinoic acid receptor, RARβ. Furthermore, high rates of apoptosis in the fetal SE suggest that apoptosis is the default pathway that is taken in the absence of complete keratinocyte differentiation.

L-Serine Potentiates the Mitogenic Effects of Growth Factors on Cultured Human Keratinocytes

L-Serine increased the growth of passaged human foreskin keratinocytes in terms of DNA and protein per dish of cells, and potentiated the mitogenic effects of serum and epidermal growth factor, and also insulin, keratinocyte growth factor, and a bovine pituitary extract. The effects of serine were apparent with as little as 0.05 mM. The stimulatory effects of these various growth factors were additive, without synergism. Exposure to L-[3H]serine resulted in labeling of the phospholipids phosphatidyl (Ptd) serine, Ptd ethanolamine, sphingomyelin, and, to a slight extent, Ptd choline. The time course of labeling suggested synthesis of Ptd serine was initiated; Ptd serine was converted to Ptd ethanolamine; little methylation of Ptd ethanolamine to form Ptd choline took place. Separation of nonradioactive phospholipids and phosphorus assay of individual lipids showed that serine-supplemented cells had only slightly increased content of Ptd serine and Ptd ethanolamine. The significance of these for activation of protein kinase C is discussed.