β-Adrenergic stimulation induces activation of protein kinase C and inositol 1,4,5-trisphosphate increase in epidermis

Abstract Epidermal keratinocytes express β₂-adrenergic receptors on the cell membrane. The binding of the agonists to the β₂-adrenergic receptors regulates activation of adenylate cyclase. This transmembrane signaling system has been regarded to be one of the important pathways for the functions of keratinocytes. We previously reported that β-adrenergic stimulation induced a transient increase of intracellular Ca²⁺ in normal human epidermal keratinocytes. Thus we investigated the effects of epinephrine on another transmembrane signaling system, the phosphatidylinositol signal transduction pathway in pig epidermis. Treatment of pig pure epidermis with epinephrine resulted in a transient increase in inositol 1,4,5-trisphosphate with a peak at 30 s. Epinephrine induced translocation of protein kinase C from cytosol to the membrane fraction. The activation of protein kinase C, translocation of protein kinase C from cytosol to the membrane fraction, was confirmed using the β-adrenergic agonist isoproterenol. Moreover, the effect of epinephrine on the activation of protein kinase C was inhibited by preincubation with propranolol, a β-adrenergic antagonist. The increase in inositol 1,4,5-trisphosphate and translocation of protein kinase C by epinephrine are consistent with the view that β-adrenergic stimulation induces turnover of inositol phospho-lipid in pig epidermis.     

Distribution of an estrogen receptor-related protein (P29) in normal skin and in cultured human keratinocytes

A monoclonal antibody, ERD5, which recognizes a 29Kd phosphoprotein associated with human estrogen receptor of myometrium was used to study the expression of this protein in normal skin and in cultured human keratinocytes. By indirect immunofluorescence, both in vivo and in vitro keratinocytes showed a variable cytoplasmic staining which increased with cell differentiation. SDS gel electrophoresis of soluble extracts of cultured keratinocytes and normal epidermis showed that P29 was a minor protein. Immunoblot analysis demonstrated that ERD5 strongly reacted only with a 29Kd polypeptide band without any cross-reactivity. These data suggest that keratinocytes might be estrogen sensitive like other cells in which P29 has already been located. The exact role of this protein in the keratinocyte differentiation process and its relationship with estrogen receptors remain to be elucidated.     

Dynamics of intracellular calcium in cultured human keratinocytes after localized cell damage

Recovery of cultured keratinocytes after scratch damage is considered to be a wound‐healing model. In this study, we observed changes in intracellular calcium concentration ([Ca²⁺]_i) in cultured human keratinocytes after scratch damage. Immediately after scratch damage, a wave of increased [Ca²⁺]_i radiated outward from the damaged area and then disappeared gradually. But, [Ca²⁺]_i remained elevated in a peripheral layer of cells around the damaged area for several minutes. This layer did not appear in calcium‐free medium. When the culture was switched to calcium‐free medium for 30 min immediately after scratch damage, then switched back to standard (Ca²⁺‐containing) medium, the recovery ratio after 24 h was approximately 25% lower than that of the culture in standard medium throughout. We speculate that delineation of damage sites by a layer of cells with increased [Ca²⁺]_i might be part of a signalling pathway that appropriately directs the wound‐healing process in epidermis.     

Substance P induces intracellular calcium increase and translocation of protein kinase C in epidermis

Substance P is a neuropeptide present in, and released from, peripheral C nerve endings. The presence of substance P-positive nerve fibres in the epidermis has been reported. We investigated the effect of substance P on the transmembrane signalling system of pig epidermal keratinocytes. Treatment of pig epidermis with substance P resulted in an increase in inositol 1,4,5-trisphosphate (IP3), and in intracellular free calcium. The treatment also resulted in translocation of protein kinase C from a cytosol to a membrane fraction. Substance P, however, did not affect the beta-adrenergic- or histamine (H2)- adenylate cyclase responses of the epidermis. Neither forskolin-induced, nor cholera toxin-induced cyclic AMP accumulation were affected by substance P treatment. These results consistent with the view that substance P stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis of keratinocytes, resulting in IP3-Ca2+ and diacylglycerol-protein kinase C signal activation. Although protein kinase C is known to affect the epidermal adenylate cyclase system, no evidence for such 'cross-talk regulation' was detected in keratinocytes by substance P treatment.     

Ultraviolet radiation B induces differentiation and protein kinase C in normal human epidermal keratinocytes

Mid-wave ultraviolet radiation (UVB, 280–320 nm) is highly efficient at inducing erythema, pyrimidine dimers in DNA, oncogene expression and initiation of cutaneous tumors. These UVB-induced responses of epidermal cells have been correlated with the direct effects of UVB on DNA. However, UVB has also been shown to have biologic effects at the cellular level that appear to mimic some of the membrane-associated effects produced by phorbol ester tumor promoters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA). For example, we have previously shown that both UVB irradiation and TPA treatment are followed by release of arachidonic acid and a rapid, dose-dependent inhibition of epidermal growth factor (EGF) binding. TPA generates cellular responses through activation of a phospholipid-dependent, calcium-sensitive protein kinase, protein kinase C (PKC). The primary goal of the studies described here was to compare the cellular effects of TPA with those of UVB with special regard to PKC and keratinocyte growth control, using normal human epidermal keratinocytes. The results obtained showed that both TPA and UVB radiation induced differentiation in normal human keratinocytes. UVB radiation, however, increased both cytosolic and membrane-associated levels of PKC, in contrast to TPA, which increased PKC primarily in the membrane fraction. PKC is probably not the initial chromophore or target molecule of UVB, but because activation of PKC has been shown to be essential for keratinocyte differentiation, differentiation induced by UVB may be caused by activation of PKC by UVB-induced release of diacylglycerol or arachidonic acid.     

Purification of natural antikeratin autoantibodies from normal human serum and their effect on human keratinocytes cultured in vitro

BACKGROUND: Antikeratin (AK) autoantibodies, circulating antibodies against epidermal keratins, have been detected in all normal human sera. However, direct evidence on the biological significance of AK autoantibodies is still lacking. OBJECTIVES: To purify AK autoantibodies from human serum and to make a preliminary study of their biological effects on human keratinocytes. METHODS: We first extracted keratin polypeptides from human stratum corneum and analysed their purity using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, a keratin affinity column was prepared with the extracted keratins, and AK autoantibodies were purified from pooled normal human serum. Antibodies obtained were identified with SDS-PAGE, enzyme-linked immunosorbent assay, immunoperoxidase staining, immunoelectron microscopy and Western blotting. The biological effect of AK autoantibodies on cultured human keratinocytes was studied using a DNA synthesis assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric determination and cell cycle analysis. RESULTS: On average, 1.83 +/- 0.24 mg of antibodies could be purified from 10 mL of pooled human serum. High-titre IgG (about 1 : 70) and low-titre IgM (about 1 : 30) AK autoantibodies were obtained. The DNA synthesis assay and MTT colorimetric determination demonstrated that AK autoantibodies have a significant dose-dependent inhibitory effect on cultured keratinocytes. Correlation coefficients in the two experiments were - 0.583 and - 0.797, respectively. Cell cycle analysis indicated that a small dose of AK autoantibodies leads to inhibition of proliferation of cultured keratinocytes, whereas a large dose of AK autoantibodies causes a visible hypodiploid peak, suggesting apoptosis of keratinocytes. CONCLUSIONS: The present research lays a solid foundation for further investigation into the biological significance of natural AK autoantibodies.     

Adenylate cyclase induces intracellular calcium increase in single human epidermal keratinocytes measured by fluorescence microscopy using Fura 2-AM

Intracellular calcium ([Ca2+]i) is an important second messenger of extracellular signals to induce various cellular responses. Extracellular and intracellular Ca2+ are considered to be important for cellular differentiation and proliferation of epidermal keratinocytes. Several mechanisms which increase [Ca2+]i have been demonstrated in various tissues, but in epidermal keratinocytes these mechanisms are poorly understood. In epidermal keratinocytes the adenylate cyclase-cyclic AMP response is thought to regulate cell proliferation and differentiation. However, the series of reactions which follow the cyclic AMP response remain unknown. Beta-adrenergic agonists increase [Ca2+]i in cultured epidermal keratinocytes, and we have therefore studied whether stimulation of keratinocyte adenylate cyclase could induce [Ca2+]i increase, by using fluorescence microscopy with Fura 2-AM. Adenosine and histamine, which are known to be keratinocyte adenylate cyclase receptor agonists, induced transient [Ca2+]i increase, as did epinephrine. In addition, forskolin, a direct adenylate cyclase activator, and dibutyryl-cyclic AMP also induced an increase in [Ca2+]i. In a calcium-free medium epinephrine, adenosine, histamine and dibutyryl-cyclic AMP induced an increase in [Ca2+]i. These results suggest that cyclic AMP in human epidermal keratinocytes regulates [Ca2+]i, which is released from intracellular stores.     

Aminoisobutyric acid uptake in normal and transformed human epidermal keratinocytes

α-Aminoisobutyric acid transport has been demonstrated in cultured human epidermal keratinocytes as well as in the transformed state of these cells. The concentrative uptake is sodium-dependent and may be ascribed to one Michaelis-Menten component whose maximal velocity is 6.9 nmol/min × 10⁶ cells, with an apparent affinity of 3.8 mM. These parameters may be modified, depending upon the nature of malignant transformation. In SVK14 SV40-virus-transformed cells, there is no change of affinity but the maximal velocity is 1.5 fold less than in normal cells. In the spontaneous epidermoid carcinoma-derived A431 line, this phenomenon is inverted; the maximal velocity is unmodified but the system affinity is 2.2-fold higher than in normal cells. The unresponsiveness of this transport system to insulin and epidermal growth factor (EGF) shows that it behaves differently from those of many other cell types.     

Growth and differentiation stimuli induce different and distinct increases in intracellular free calcium in human keratinocytes

The effect of growth and differentiation stimuli on intracellular free calcium ([Ca²⁺]_i) in cultured human keratinocytes was investigated using micro-spectrofluori-metric techniques and the calcium-sensitive dye FURA-2. The mean [Ca²⁺]_i of keratinocytes in 70 M calcium medium was 104 ± 3 nM (mean ± SEM), significantly lower than the transformed keratinocyte line SVK₁₄ (128 ± 2 nM). When cultured in 2.0 mM calcium medium the [Ca²⁺]_i increased in both normal and transformed keratinocytes to 135 ± 4 nM and 180 ± 4 nM, respectively. Keratinocytes grew more slowly in the absence of EGF, but [Ca²⁺]_i was unaltered. Stimulation with EGF (10 ng/ml) induced, over 4 min, a large transient rise in [Ca²⁺]_i up to 230 nm, due to an influx of extracellular calcium. Heterogeneity of keratinocytes was observed with 46% (n=13) responding, but confluent or differentiated keratinocytes did not respond. TGF- (1 ng/ml) reduced cell growth without inducing differentiation and was not associated with any change in [Ca²⁺]_i. The phorbol ester TPA (50 nM) induced irreversible growth arrest and terminal differentiation and increased the [Ca²⁺]_i from 102 ± 2 nM to 126 ± 3 nM at 2 h, an effect similar to that of 2 mM extracellular calcium. Addition of 500 nM TPA was associated with a rise in [Ca²⁺]_i, over several minutes to a plateau of 200–300 nM, due to release from internal stores and an influx of extracellular calcium. In normal human keratinocytes an increase in [Ca²⁺]_i appears to be an early event in differentiation, whether induced by calcium or TPA, but not during growth inhibition without differentiation.     

Betulinic acid induces apoptosis in skin cancer cells and differentiation in normal human keratinocytes

Betulinic acid (BA), a pentacyclic triterpene of plant origin, induces cell death in melanoma cells and other malignant cells of neuroectodermal origin. Little is known about additional biological effects in normal target cells. We show, in this study, that BA induces differentiation as well as cell death in normal human keratinocytes (NHK). Cytotoxicity profiles of BA are compared among normal human keratinocytes, HaCaT cells, IGR1 melanoma cells and normal melanocytes. As expected, BA is toxic to all cell types, normal and malignant, but varies in its cytotoxic potency and in the extent of induction of apoptotic vs. necrotic cell death in the four different skin cell types. Apoptosis is proved by annexin V and Apo2.7 binding and by DNA fragmentation. Induction of differentiation-associated antigens in keratinocytes--filaggrin and involucrin--is demonstrated, together with specific morphological changes in treated cell cultures. BA, apart from its cytotoxic activities in cellular systems, is capable of inducing differentiation of NHK into corneocytes without immediately provoking apoptotic cell death.     

Adenylate cyclase induces intracellular Ca2+ increase in single human epidermal keratinocytes of the epidermal sheet as measured by digital imaging microscopy using Fura 2-AM

Summary Intracellular Ca²⁺ ([Ca²⁺]_i) is thought to act as a second messenger of transmembrane signalling systems. However, no measurement of [Ca²⁺]_i has been made in intact epidermal keratinocytes. We have developed a method for measuring [Ca²⁺]_i in human keratinocytes from pure epidermal sheet by the application of digital imaging fluorescence microscopy with the use of Fura 2-AM. Normal human pure epidermal sheets were obtained by dispase treatment. Epinephrine and salbutamol induced transient [Ca²⁺]_i increases. Propranolol, a -antagonist, inhibited this response, while prazosin and yohimbine (alpha₁- and alpha₂-antagonists, respectively) did not affect the response. Histamine and adenosine, also receptor agonists of the epidermal adenylate cyclase system, induced a similar [Ca²⁺]_i increase, as did forskolin, a direct activator of adenylate cyclase. These data coincide with those previously presented for cultured human epidermal keratinocytes, and reveal that adenylate cyclase activation induces an increase of [Ca²⁺]_i in intact epidermal cells. This technique enables the kinetics of [Ca²⁺]_i in various skin disorders to be investigated.     

Fumaric acid derivatives evoke a transient increase in intracellular free calcium concentration and inhibit the proliferation of human keratinocytes

Systemic administration of fumaric acid (FA) derivatives was originally an empirical antipsoriatic treatment, which showed promising clinical results. In the present study, FURA-2-loaded suspensions of cultured normal keratinocytes and SV40-transformed keratinocytes (SVK-14 cells) were used to study the effects of FA derivatives on the intracellular free calcium concentration ([ca²⁺]_i). Monomethylfumarate (MMF), dimethylfumarate (DMF) and monoethylfumarate (MEF) induced a rapid, transient [ca²⁺]_i increase in both cell types. This immediate increase reached maximal values of 396 nmol/1 10 s after addition of MMF, and fell to basal values within 90–120 s (173 nmol/1 for normal keratinocytes and 68 nmol/1 for transformed keratinocytes). This increase was not affected by the prior addition of EGTA, indicating that FA derivatives released Ca²⁺ mainly from intracellular stores into the sytoplasm. Subsequently, dose-dependent inhibitory effects of FA derivatives on keratinocyte proliferation were demonstrated. The results of these experiments revealed that DMF was the most potent. MMF and MEF intermediate, and FA and malonic acid the least potent growth inhibitors. These antiproliferative effects of FA derivatives might be linked to the observed, transient [Ca²⁺]_i elevations.     

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 extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes

Abstract:  The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro , the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 m m . In NHK, addition of calcium-induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.     

Sirtuin 4 identification in normal human epidermal keratinocytes and its relation to sirtuin 3 and energy metabolism under normal conditions and UVB-induced stress

Sirtuins (SIRT) are NAD(+) -dependent deacetylases and ADP-ribosyltransferases that play a critical role in metabolism and epigenetics. SIRT3 and SIRT4 are of particular interest because they are localized in the mitochondria where energy is generated and their expression is inversely proportional to each other. Here, we report data, for the first time, demonstrating the presence of SIRT4 in normal human epidermal keratinocytes (NHEK) and confirm that its expression is inversely related to SIRT3 in these cells and that they follow a temporal cycle. Further, UVB radiation modified their expression, as well as ATP and H2 O2 levels. These deviations from the normal sirtuin cycles after UVB exposure can be an epigenetic indicator of lower metabolism levels.     

Ultraviolet B induces hyperproliferation and modification of epidermal differentiation in normal human skin grafted on to nude mice

BACKGROUND: For ethical and technical reasons, the in vivo biological effects of ultraviolet (UV) radiation on skin are difficult to study in human volunteers. The use of human skin grafted on to nude mice may circumvent this difficulty. OBJECTIVES: To investigate the effects of a single moderate UVB exposure on human skin grafted on to nude mice. METHODS: Modifications of epidermal differentiation markers and patterns of keratin expression were assessed from 24 h to 14 days after a physiological UVB irradiation characterized by the induction of sunburn cells. RESULTS: During the first 48 h postexposure, involucrin, loricrin, transglutaminase type I, filaggrin and keratin K2e expression were altered together with the formation of abnormal horny layers. Constitutive keratin K14 was increased while keratin K10 expression was delayed. Newly synthesized keratins K6, K16, K17 and K19 were induced in parallel with an increase in the epidermal proliferation rate. A progressive normalization of both keratinocyte proliferation and differentiation took place during the following days, reaching completion within 2 weeks. CONCLUSIONS: Exposure of human skin to a UVB dose corresponding to a mild sunburn reaction induces epidermal hyperproliferation and alterations of several constitutive differentiation markers, as well as a drastic modification in the pattern of epidermal keratins. Although these modifications were shown to be progressively reversed in a single exposure model, the data also suggest that subsequent UV exposures occurring during the recovery period may lead to potentially deleterious long-term consequences, such as photoageing and photocarcinogenesis. Grafted human skin appeared to be an attractive and promising model for investigating the biological consequences of UVB radiation in vivo.     

Cytochrome P450, CYP26AI, is expressed at low levels in human epidermal keratinocytes and is not retinoic acid-inducible

Retinoids, and their synthetic analogues, are well-established regulators of the squamous differentiation programme both in vivo and in vitro. Despite this, very few studies have focused on the mechanism by which retinoid action is terminated, e.g. metabolism. Recently, a new cytochrome P450 family member (CYP26AI) was cloned. CYP26AI was reported to have substrate specificity for retinoids and to be retinoid-inducible. In this study, we have examined the expression and retinoic acid (RA) inducibility of CYP26AI in human epidermis and cultured keratinocytes. We found very low levels of CYP26AI mRNA expression in both epidermis and keratinocytes. Furthermore, we found no evidence for RA inducibility of CYP26 mRNA expression. This lack of RA inducibility was not due to inactivity of the retinoids, as we show that transglutaminase was still repressed by RA in the same cultures. Despite the low levels of CYP26AI expression in the keratinocytes, the keratinocytes were still capable of significant RA metabolism. In conclusion, our study reports, for the first time, that CYP26AI is unlikely to contribute to RA metabolism in keratinocytes. These studies also indicate that as yet unknown isoforms of cytochrome P450 may be involved in RA metabolism in keratinocytes.     

Effects of calcium concentration on the SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes

BACKGROUND/PURPOSE: Cellular differentiation due to the extracellular calcium (Ca(2+)) concentration affects the level of several antioxidant enzymes in cultured human keratinocytes. Because the epidermis includes well- and un-differentiated keratinocytes, we expected that keratinocytes possess different antioxidant capacity and sensitivity to damaging effects of ultraviolet-B (UVB) depending on the differentiation. We examined the effects of Ca(2+) concentration of culture medium (DMEM (Dulbecco's modified Eagle's medium)) on the superoxide dismutase (SOD) activity and UVB-induced cytotoxicity in cultured human keratinocytes in order to investigate the relationship between cell differentiation and antioxidant defense. METHODS: Human keratinocytes (HaCaT cells) were incubated in high Ca(2+) (>1 mM) or low Ca(2+) (<0.1 mM) concentration DMEM for 24 h at 37 degrees C in 5% CO(2). Then, we measured total SOD activity and also individual Cu,Zn- and Mn-SOD activities in keratinocytes. Furthermore, after incubation in high or low Ca(2+) concentration DMEM, human keratinocytes were irradiated with 10, 20 or 30 mJ/cm(2) UVB. The quantity of lactate dehydrogenase (LDH) leaked in the supernatant from damaged keratinocytes, cell viability and TdT-mediated dUTP nick end labelings (TUNEL) positive keratinocytes were measured at 24 h after UVB irradiation. RESULTS: Total SOD activity and Cu,Zn-SOD activity in human keratinocytes cultured in low Ca(2+) were significantly lower than in keratinocytes cultured in high Ca(2+) concentration DMEM. In contrast, Mn-SOD activity was not affected. LDH leakage in the supernatant from keratinocytes cultured in low Ca(2+) concentration was significantly higher than that from keratinocytes cultured in high Ca(2+) concentration DMEM after UVB irradiation. The cell viability of keratinocytes cultured in low Ca(2+) concentration DMEM was significantly decreased compared to that of keratinocytes cultured in high Ca(2+) concentration DMEM after UVB irradiation. Furthermore, UVB-induced apoptosis was increased in keratinocytes cultured in low Ca(2+) concentration DMEM by the TUNEL method. CONCLUSIONS: These results suggest that cellular differentiation due to the change of Ca(2+) concentration of culture medium affects the Cu,Zn-SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes.