Differential effects of UV irradiation on nuclear retinoid receptor levels in cultured keratinocytes and melanocytes

A major risk factor for skin cancer is UV irradiation, which not only damages DNA and other photosensitive compounds like vitamin A, but may also perturb cellular signaling, e.g. via the retinoid receptor system believed to be important for cancer protection. We used cultured normal human keratinocytes and melanocytes to examine the effects of UV irradiation on the expression of the predominant retinoid receptors in the human skin (RARalpha, RARgamma and RXRalpha) and the AP-1 protein c-Jun; mRNA levels were studied by real-time PCR and protein levels by Western blot. In keratinocytes, a single dose of UVB (50 mJ/cm2) caused a rapid drop in the expression of all three receptors (mRNA levels minus 35-50% after 4 h; protein levels minus 20-45% after 8 h), which was followed over the next 40 h by a variable response, leading to full normalization for RARalpha only. In contrast, the levels of c-Jun did not change significantly after UV exposure. In melanocytes, UVB caused a similar drop of the retinoid receptor levels as in keratinocytes but this was soon followed by an increased expression leading to a complete normalization of all receptor levels within 1-3 days. The c-Jun levels in melanocytes increased 1 day after UV exposure and remained high (plus 50%) thereafter. In both cell types, a approximately 3-fold increase in apoptosis (measured by DNA fragmentation) was observed 8-48 h after UVB irradiation. In conclusion, a depletion of vitamin A and retinoid receptors by UV irradiation, together with unchanged or even increased c-Jun levels, might seriously interfere with retinoid signaling and thus promote future tumor development, especially in keratinocytes.     

Human keratinocytes release high levels of inducible heat shock protein 70 that enhances peptide uptake

Abstract: Background: The stress‐inducible chaperone heat shock protein (HSP) 70 is considered a ‘danger signal’ if released into the extracellular environment. It has been proposed to play a role in the pathogenesis of skin diseases such as psoriasis and lupus erythematosus (LE). Objectives: The aim of this study was to decipher the role of human primary keratinocytes with regard to release and reactivity to HSP70. Methods: We determined HSP70 and IFNγ in cell supernatants by ELISA. Uptake of labelled HSP70 or labelled peptide by human primary keratinocytes or macrophages was analysed by flow cytometry and fluorescent microscopy. Results: We found that living keratinocytes are an important source of HSP70 in the skin compartment. They release considerably more HSP70 than fibroblasts, macrophages or lymphocytes. Interestingly, keratinocytes also bind and internalise HSP70/HSP70–peptide complexes. TNFα, IL‐27 as well as HMGB‐1 enhanced the uptake of HSP70. No difference with regard to HSP70 release or uptake was observable between keratinocytes from healthy donors or patients with cutaneous LE. Keratinocytes pulsed with HSP70–peptide complexes significantly increased IFNγ production by autologous T cells. Conclusions: Production and uptake of inducible HSP70 by keratinocytes may critically influence the chronic course of inflammatory skin diseases.     

培养角质形成细胞构成性表达热休克蛋白72

目的 了解热休克蛋白７２（ＨＳＰ７２）在培养角质形成细胞中的表达情况。方法 无血清培养基培养原代角质形成细胞,免疫组织化学（ＳＡＢＣ）及核酸原位杂交技术检测ＨＳＰ７２的表达。结果 角质形成细胞的细胞中常见阳性染色,少数大细胞中ＨＳＰ７２表达增强,集中在细胞核周围。结论：普通条件培养的皮肤角质形成细胞即可构成性表达ＨＳＰ７２,可能对维持角质形成细胞的正常功能和活性方面个有重要作用。     

Protein kinase C agonist and antagonist effects in normal human epidermal keratinocytes

Abstract Several lines of evidence implicate protein kinase C (PKC) in the development of basal cell and squamous cell carcinomas, tumors which originate from epidermal keratinocytes. To examine PKC in a model relevant to human skin, we exposed normal human epidermal keratinocytes (NHEK) in serum-free media to a variety of PKC agonists and antagonists. NHEK PKC activity increased up to 10-fold within the 1st hour of exposure to tetradecanoyl phorbol acetate (TPA), and gradually returned to control values within 72 h. TPA-induced PKC activity was enhanced by pretreatment of cultures with protein and RNA synthesis inhibitors. TPA-induced growth arrest and differentiation was antagonized by staurosporine. Down-regulation by bryostatin pretreatment blocked TPA-stimulated differentiation. Our overall conclusion is that activation of PKC in cultured human keratinocytes is required for differentiation. These results are crucial to the analysis of compounds suspected of promoting or inhibiting epidermal tumors.     

Effects of thermal shocks on interleukin-1 levels and heat shock protein 72 (HSP72) expression in normal human keratinocytes

Interleukin-1 expression is reported to be modified under a number of cell conditions including physiological stress, injury and activation. We report the effects of the physiological stresses cold and heat shock on IL-1 levels in keratinocytes. Having observed that normal human skin obtained from plastic surgery, usually stored at 4C for a few hours, highly expressed HSP72, a constant feature of stressed human keratinocytes, we wondered whether this induction could be linked to a cold shock and to modification of IL-1 levels in keratinocytes. Cultured keratinocytes were incubated at 4, 37, 40 and 43C for 1.5, 4, 8 and 16 h in a defined medium. HSP72 expression was studied by immunohistochemistry and immunoblot and IL-1 was quantified using specific and sensitive radio-immunoassay. Our findings showed that intracellular IL-1 and IL-1 levels are not significantly modified by thermal shock. HSP72 is only induced after cell exposure to 43C and is not a cold-shock protein. These results demonstrate that thermal stress is not an inductive signal for IL-1 modification in keratinocytes.     

Induction of apoptosis in human HaCaT keratinocytes

Although cell death by apoptosis has been recognized as an important control mechanism in the maintenance of tissue homeostasis and in the elimination of cells with damaged DNA, information on the induction and characteristics of apoptosis in keratinocytes is rather scarce. Apoptotic mechanisms may play an important role in normal and disturbed homeostasis of the skin. In the present study, we therefore investigated the effects of several potential inducers of apoptosis in the human keratinocyte cell line HaCaT. Apoptosis was assessed with respect to morphological changes by light and electron microscopic examinations and to DNA integrity by a specific ELISA. UVB irradiation induced the morphology and internucleosomal DNA fragmentation characteristic of apoptosis in a dose- and time-dependent manner. Interferon-γ caused DNA cleavage at the linker regions without producing morphological features consistent with apoptotic cell death. In contrast, treatment with dithranol and NP-40 resulted in necrotic alterations in the keratinocytes. Treatment with the calcium ionophore A23187 caused morphological changes which were similar to the characteristics of ‘nonapoptotic programmed cell death’. Dexamethasone, tumor necrosis factor-α, transforming growth factor-β, TPA, retinoic acid, the podophyllin derivative etoposide, the thromboxane A ₂ analogue U46619, cycloheximide, and the nitric oxide donors sodium nitroprusside and S-nitroso-glutathione, which are all known to induce apoptosis in other cell types, did not affect HaCaT keratinocytes. These results demonstrate that apoptosis can be induced in keratinocytes in vitro but the apoptosis differs from that in other cell types, such as haematopoietic cells, with regard to the type of inducer and/or the sensitivity of the target cells. Since keratinocytes are affected by numerous external and internal stimuli, they might posses several protective mechanisms to prevent apoptosis and to ensure the structural integrity of the outermost barrier of the body. Received: 28 November 1995     

Ultraviolet irradiation induces cyclooxygenase-2 expression in keratinocytes

We examined the effect of ultraviolet (UV) irradiation on the expression of cyclooxygenases in cultured HaCaT keratinocytes and in human skin in vivo. UVB irradiation (10 and 50 mJ/cm2) and hydrogen peroxide (200 micromol/L) increased cyclooxygenase-2 mRNA expression in HaCaT keratinocytes. No clear expression of cyclooxygenase-1 mRNA was detected in either control or stimulated HaCaT cells. Genistein, a tyrosine kinase inhibitor, suppressed both the basal and stimulated expression of cyclooxygenase-2 in HaCaT cells. UVB-induced cyclooxygenase-2 mRNA expression was partly inhibited by the antioxidant N-acetylcysteine and by H-7, a non-specific inhibitor of protein kinase C. Solar-simulated irradiation (40 mJ/cm2) was found to induce in vivo both cyclooxygenase-2 mRNA and protein expression in human skin, whereas the expression of cyclooxygenase-1 mRNA remained at the basal level. Our results show that cyclooxygenase-2 expression is induced by UV irradiation and suggest that tyrosine kinases and reactive oxygen intermediates are involved in this induction of cyclooxygenase-2.     

Co-culture of human melanocytes and keratinocytes in a skin equivalent model: effect of ultraviolet radiation

Melanocytes grown in pure monolayer culture lack the three-dimensional organization and many of the cellular interactions that exist in vivo. This can be partially overcome by growing melanocytes together with other epidermal cells in skin equivalent models. In this study skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in various ratios onto de-epidermized dermis. They were cultured in DMEM/Ham's F12 (31) for 3 days and then lifted to the air-liquid interface and maintained for 11 days. Histological examination revealed a structure that closely resembled human interfollicular epidermis. Melanocytes, identified by their dendritic appearance, positive dopa reaction and positive staining with a melanocyte-specific antibody (MEL5), were located in the basal layer. Melanin was seen both in melanocytes and in neighbouring keratinocytes. Whilst the skin equivalent became more pigmented following UV irradiation (total UVB 4760 J/m² over 3 days), the quantity and distribution of melanin at the light microscopic level appeared to be unchanged. However, the number and dendricity of melanocytes increased, as did their staining with dopa and MEL5. These results indicate that melanocytes are functional and capable of responding to UV irradiation.     

Induction of the Low-Molecular-Weight Stress Protein HSP27 in Organ-Cultured Normal Human Skin

To examine the inducibility of 27-kD-molecular-weight heat shock protein (HSP27) in human skin, an indirect immunofluorescence (IF) study was performed on organ-cultured normal human skin by using a monoclonal antibody specific for HSP27. After heat treatment at 45°C for 1 h, nuclear IF was observed in the epidermal cells. When the organ-cultured skin explants were exposed to 10.0 μg/ml or 100.0 μg/ml 8-methoxypsoralen (8-MOP) for 1 h and then irradiated with UVA (320–400 nm), positive nuclear IF was also observed 6 h after UVA irradiation. Considering these results with the previous reports about HSP72, it appears that, in human skin, HSP27 as well as HSP72 plays an important role in resisting various environmental stresses.     

Differential response of basal keratinocytes in a human skin equivalent to ultraviolet irradiation

Abstract The human skin equivalent (HSE) provides a convenient model system for studying the cellular responses of basal keratinocytes to UV irradiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were raised to an air-liquid interface to promote epidermal differentiation. HSEs were exposed to ultraviolet radiation from a 500-W Hot Quartz Hanovia therapeutic sunlamp, at a total dose of 100 J/m². The HSEs were then frozen every 4 h over a 48-h period and cryosectioned. For each time period, the expression of β₁ integrin and cyclin E, p53, or Bcl-2 were quantified using dual immunolocalization. Basal cells expressing β₁ integrin were divided into two subpopulations, denoted β₁ ^high or β₁ ^low. The proportion of β₁ ^high keratinocytes expressing Bcl-2 and cyclin E increased significantly 4 and 8 h, respectively, after exposure to UV; during the subsequent 16 h, this basal cell subpopulation expressed p53. By contrast, significant numbers of β₁ ^low basal keratinocytes expressed p53, but not Bcl-2. These results suggest that β₁ ^high and β₁ ^low populations of basal epidermal cells in HSEs respond differently to UV irradiation. Received: 30 October 1997     

Ultraviolet A and B affect human melanocytes and keratinocytes differently. A study of oxidative alterations and apoptosis

Ultraviolet (UV) radiation is an etiologic agent for malignant melanoma and non-melanoma skin cancer, but the spectral range responsible for tumor induction is still to be elucidated. In this study, we compared effects of UVA and UVB irradiation on normal human melanocytes (MCs) and keratinocytes (KCs) in vitro. We demonstrate that UVA irradiation induces immediate loss of reduced glutathione (GSH) in both MCs and KCs. Exposure to UVA also causes reduced plasma membrane stability, in both cell types, as estimated by fluorescein diacetate retention and flow cytometry. Furthermore, we noted reduction in proliferation and higher apoptosis frequency 24 h after UVA irradiation. UVB irradiation of KCs caused instant reduction of reduced GSH and impaired plasma membrane stability. We also found decline in proliferation and increased apoptosis after 24 h. In MCs, on the other hand, UVB had no effect on GSH level or plasma membrane stability, although increased apoptotic cell death and reduced proliferation was detected. In summary, MCs and KCs showed similar response towards UVA, while UVB had more pronounced effects on KCs as compared to MCs. These results might have implications for the induction of malignant melanoma and non-melanoma skin cancer.     

Use of enhanced green fluorescent protein to monitor retroviral-mediated gene therapy in human keratinocytes

Keratinocytes have great promise as targets for gene therapy involving both skin as well as for systemic disorders due to their availability and potential long life span. Improvement of gene transfer into keratinocytes will be greatly facilitated by markers that will allow both rapid detection and efficient selection of transduced cells. For these purposes, a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP) was placed into a replication-deficient retroviral vector. High-titer retrovirus was used to transduce both primary cultures of neonatal foreskin-derived human keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Both cell types stably expressed the EGFP, and this marker allowed rapid purification of transduced cells by fluorescence-activated cell sorting. EGFP expression was seen in HaCaT keratinocytes for at least 40 passages, and the presence of this construct did not effect cell growth, or apoptosis in response to UVB or etoposide. Transduced populations of HK were grafted into SCID mice, resulting in a functional epidermis. EGFP expression was readily seen in vivo by exposing the xenografts to an ultraviolet light source. These studies demonstrate the feasibility of using EGFP as a convenient and rapid marker to monitor keratinocyte gene transfer both in vitro and in vivo.     

特应性皮炎皮损HSP70的表达及他克莫司对其表达的影响

目的:探讨热休克蛋白(HSP)70在特应性皮炎(AD)中的表达及外用他克莫司对HSP70表达的影响。方法:采用免疫组化二步法检测8例中至重度AD患者急性发作期炎性皮损HSP70表达及外用0.1%或0.03%他克莫司软膏治疗AD3周后HSP70表达的变化。结果:免疫组化检测显示HSP70在AD急性发作期炎性皮损角质形成细胞(KC)的胞核,胞浆及胞膜外过度表达,其中在3例大面积泛发或红皮病型急性AD皮损KC中,HSP70以弥漫性细胞核表达伴胞浆表达为特征,主要定位于基底层至颗粒层;外用他克莫司软膏治疗3周后特应性皮炎皮损KCHSP70的核表达消失,胞浆及膜(表面)表达信号减弱。结论:急性期AD皮肤KC的HSP70过度表达;他克莫司可通过直接或间接作用抑制KC的HSP70诱导性或过度表达,从而可抑制或下调与内源性HSP70危险信号有关的AD皮肤天然自身免疫炎症反应。     

Arsenite and arsenate activate extracellular signal-regulated kinases 1/2 by an epidermal growth factor receptor-mediated pathway in normal human keratinocytes

BACKGROUND: Inorganic arsenic is an environmental contaminant and is associated with the increased risk of human skin cancer. Arsenic has been reported to activate or inhibit a variety of cellular signalling pathways which has effects on cell growth, differentiation and apoptosis. However, the molecular mechanisms of these arsenic-induced biological effects are not completely understood. OBJECTIVES: To understand the molecular basis for the mode of action of arsenicals, we examined the effect of arsenite and arsenate on the activation of mitogen-activated protein kinases (MAPK) and the upstream signalling cascade in normal human epidermal keratinocytes (NHEK). METHODS: NHEK were exposed to arsenite or arsenate. Western blot analysis was performed to determine the activation of extracellular signal-regulated kinases (ERK) 1/2, c-jun N-terminal kinases (JNK), p38, and MAPK or ERK kinases (MEK) 1/2. Epidermal growth factor receptor (EGFR) tyrosine phosphorylation and recruitment of its adaptor proteins, Shc and Grb2, to EGFR were detected by immunoprecipitation and Western blot analysis. RESULTS: Both arsenicals activated ERK1/2, which are most highly activated in response to mitogenic stimulation, in addition to JNK and p38, which show greater activation in response to cellular stresses. The kinetics of ERK1/2 activation differed from those of JNK and p38 activation. Both arsenicals transiently activated ERK1/2 prior to JNK and p38 activation. MEK1/2, upstream kinases of ERK1/2, were also activated by arsenicals with similar time kinetics to that of ERK1/2 activation. To investigate a signalling pathway leading to activation of MEK1/2-ERK1/2, we examined the tyrosine phosphorylation of EGFR and Shc adapter protein. Both arsenicals stimulated tyrosine phosphorylation of EGFR and Shc. After arsenical treatment, Shc immunoprecipitates contained coprecipitated EGFR and Grb2, suggesting that both arsenicals induce the assembly of EGFR-Shc-Grb2 complexes. Both the EGFR inhibitor tyrphostin AG1478 and anti-EGFR blocking antibody markedly attenuated ERK1/2 activation induced by arsenicals, but did not affect JNK and p38 activation. CONCLUSIONS: Our data indicate that both arsenite and arsenate activate the EGFR-Shc-Grb2-MEK1/2-ERK1/2 signalling cascade in NHEK.     

Induction of the 72-kD Heat Shock Protein in Human Skin Melanoma and Squamous Cell Carcinoma Cell Lines

Heat shock proteins (HSPs) or stress proteins comprise a characteristic group of proteins synthesized in cells exposed to heat or other environmental stimuli. Of the many HSPs, the 72-kD heat shock protein (HSP72) is the most stress-inducible one. In the present study, we examined the effects of heat, chemicals (azetidine and sodium arsenite), ultraviolet (UV) light, and γ-ray irradiation on the induction of HSP72 in cultured human skin melanoma cell lines (P-39 and G-361), a human skin squamous cell carcinoma cell line (HSC-1), and an SV40-transformed human lung fibroblast cell line (WI38VA13) as a control. In these cell lines, heat treatment induced HSP72 more rapidly and intensely than did chemical exposure. Compared with the SCC cell line, the two melanoma cell lines produced less HSP72 with heat treatment. UVC irradiation (20 J/m²) induced HSP72 only in the WI38VA13 cells. After γ-ray irradiation, no HSP72 induction was detected in any of the cell lines examined. These observations suggest that, in cultured cells, inducibility of HSP72 depends not only on the inducer but also on the origin of each cell line.