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.     

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.     

Ganglioside-induced terminal differentiation of human keratinocytes: early biochemical events in signal transduction

Previous studies have indicated that GQ_1b, a tetrasialoganglioside containing two disialosyl residues, may be an important regulator of cellular differentiation in murine keratinocytes. In the present study, we examined the effect of gangliosides on the differentiation of human keratinocytes. Current evidence indicates that GQ_1b induces cornified envelope formation and enhancement of transglutaminase (TGase) activity, which are characteristic parameters of terminal differentiation in human cultured keratinocytes, while the other gangliosides, GT_1b and GM₁, are much less effective. The mass contents of inositol 1,4,5-trisphosphate (1,4,5-IP₃) and the intracellular calcium concentration ([Ca⁺]_i) were also measured in keratinocytes exposed to gangliosides. A rapid increase in 1,4,5-IP₃ occurred at 30 s following stimulation, but no significant difference at the maximum level was observed among the three gangliosides in contrast to the finding in murine keratinocytes. In addition, [Ca⁺] increases occurred concurrently with the 1,4,5-IP₃ generation by the three gangliosides. On the other hand, [Ca⁺] transients were unaffected by chelating extracellular Ca⁺ with EGTA. It is thus considered that the mobilization by 1,4,5-IP₃ from internal stores plays a crucial role. These [Ca⁺]_i profiles were also indistinguishable between the gangliosides. Taken together, in human keratinocytes, gangliosides differentially affect some other as yet unidentified site(s) in the post-calcium transmission pathway(s) which leads to TGase activation.     

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.     

Regulation of transmembrane signalling system curing senescence of human epidermal keratinocytes

Cultured normal human epidermal keratinocytes show an increase in doubling time with increasing passage number and finally the cells show growth arrest. This is known as senescence of cultured cells. However, the mechanisms governing senescence of cells remain to be clarified. Stimulation from outside the cell and the response to the stimulation are important essential initial events for cell function. Alteration of intracellular Ca²⁺ is one of the essential responses of cells to stimulation from outside. Thus we studied whether cultured normal human epidermal keratinocytes show a modulation of the alteration of intracellular Ca²⁺ during senescence. Epinephrine and histamine induced transient increases in intracellular Ca²⁺ in the third to eighth passages in normal human epidermal keratinocytes. With increase in the passage number the responsiveness (the number of responding cells per examined cells) decreased particularly beyond the sixth passage. The attenuation of the responses was more obvious with epinephrine than with histamine. All-trans-retinoic acid and 1,25-dihydroxyvitamin D₃ did not augment the responsiveness to epinephrine of normal human epidermal keratinocytes. We expected that such an essential and immediate reaction would be confined to living cells, and that during senescence cells would show reduced responsiveness. Some of the changes during senescence of cultured keratinocytes may be due to the attenuation of the responsiveness to stimulation through the cell membrane.     

Thrombin and melittin activate phospholipase C in human HaCaT keratinocytes

Abstract Following the activation of specific receptors, phospholipase C has been shown to cleave the membrane phospholipid phosphatidylinositol bisphosphate into the 2nd messengers inositol 1,4,5-trisphosphate and di-acylgiycerol. Both 2nd messengers contribute to the regulation of cellular proliferation. The receptor for bradykinin is coupled to this pathway in keratinocytes, but knowledge about other activators of phospholipase C is limited. Additional mediators and agents were therefore examined regarding their ability to activate phospholipase C in HaCaT keratinocytes. Analysis for ³H-inositol phosphates was performed by anion-exchange HPLC. Thrombin and melittin induced a time- and dose-dependent release of inositol 1,4,5-trisphosphate. Several other mediators examined such as angiotension II, neurotensin, C3a, pituitary adenylate cyclase activating peptide, phenylephrin, and prostaglandin E₂, did not induce the formation of inositol phosphates. In view of the mitogenic activity and the increased formation of thrombin after tissue injury, the coupling of the thrombin receptor to phospholipase C in HaCaT keratinocytes suggests a rôle of this protease in epidermal wound healing.     

Dynamic expression of pemphigus and desmosomal antigens by cultured keratinocytes

Dynamic expression of pemphigus antigens by cultured human and mouse keratinocytes was compared with that of desmosome-associated molecules and cellular markers relating to epidermal differentiation. Plakoglobin was detected in localized areas of keratinocyte sheets in low Ca²⁺ (0·15mM) KGM medium. In minimum essential medium (MEM) containing 1·8 mM Ca²⁺. plakoglobin was expressed in the intercellular spaces (ICS) throughout the keratinocyte sheet. Desmoplakin I and II. which were present in the cytoplasm of keratinocytes in the low Ca²⁺ medium, moved to the cell surface after the medium was changed to MEM. Desmoglein 1 and pemphigus vulgaris (PV) antigens were observed in the ICS of both the monolayers and stratilied areas in the MEM. Pemphigus foliaceus (PF) antigens, frequently together with desmoglein 1, involucrin and keratins specific for the upper layer of the epidermis, were expressed by stratified keratinocytes but not the cells in the monolayers. The Western blotting study of the cultured keratinocyte extract showed 160- and 130-kDa bands positive for desmoglein 1 antigens and a 1 30-kDa band stained with PV sera. These findings suggest that although desmoglein 1 molecules bear PF antigenic sites, their expression pattern by cultured keratinocytes is closely related to that of PV rather than PF antigens. The PF antigenic sites may be formed on desmoglein 1 during epidermal differentiation.     

H2 histamine receptor-mediated increase in intracellular Ca2+ in cultured human keratinocytes.

Histamine is present in the epidermis in intracellular and extracellular area and is released from mast cells and keratinocytes in the early stage of inflammation of the skin. Such release may contribute to common itching or intensify the inflammatory responses. Histamine binds to its receptors and participate in regulation of the inflammatory responses by acting on endothelial cells, nerve endings, lymphocytes, monocytes, and leukocytes. Histamine has direct effects on keratinocytes as well. Histamine modulates the proliferation of keratinocytes. The binding of histamine to the receptor on keratinocyte membrane induces activation of adenylate cyclase and phospholipase C through GTP binding protein. We previously reported that histamine induces transient increase in intracellular Ca2+ in cultured normal human epidermal keratinocytes (NHEK) and normal epidermis. H1 and H2 histamine receptors are widely distributed in many tissues and cells. In this study, we investigated which types of histamine receptors are related to the increase in intracellular Ca2+ by histamine stimulation in cultured human epidermal keratinocytes. NHEK were cultured in serum-free KGM medium. With H1 antihistamines, mepyramine and diphenhydramine, histamine responses were moderately but not statistically significantly inhibited. With H2 antihistamine, cimetidine, histamine response was significantly inhibited. Epinephrine response was not affected by these antihistamines. Thus, it is considered that H2 antihistamines specifically block histamine-mediated increase in intracellular Ca2+ of cultured normal human keratinocytes.     

Cis-urocanic acid attenuates histamine receptor-mediated activation of adenylate cyclase and increase in intracellular Ca2+

Abstract UVB irradiation causes suppression of delayed hypersensitivity. Various photoreceptors and mediators of these changes have been proposed, one of which is cis-urocanic acid formed from the naturally occurring trans-urocanic acid in the epidermis on exposure to UV irradiation. The mechanism by which cis-urocanic acid alters the immune system is not fully clarified, but it acts through different mechanisms, perhaps via histamine or histamine-like receptors. Histamine stimulation of keratinocytes induces activation of adenylate cyclase leading to an accumulation of cyclic AMP and an increase in intracellular Ca²⁺. Thus we investigated the effects of cis-urocanic acid on these transmembrane signaling systems in keratinocytes. Normal human keratinocytes were cultured in serum-free KGM medium. Cyclic AMP was measured by radioimmunoassay. Alterations in intracellular Ca²⁺ in single living keratinocytes were measured using an inverted fluorescence microscope and an ARGUS-200/CA digital imaging system. Cis-urocanic acid itself did not induce adenylate cyclase activation in cultured normal human keratinocytes. Cis-urocanic acid inhibited histamine-induced cyclic AMP accumulation. It did not affect keratinocyte growth, and did not induce an increase in intracellular Ca²⁺, but did attenuate the histamine-induced increase in intracellular Ca²⁺ but not that induced by epinephrine. Cis-urocanic acid acts on keratinocytes via modulation of the effects of histamine. Received: 20 May 1997     

E‐cadherin mediates ultraviolet radiation‐ and calcium‐induced melanin transfer in human skin cells

Skin pigmentation is directed by epidermal melanin units, characterized by long‐lived and dendritic epidermal melanocytes (MC) that interact with viable keratinocytes (KC) to contribute melanin to the epidermis. Previously, we reported that MC:KC contact is required for melanosome transfer that can be enhanced by filopodi, and by UVR/UVA irradiation, which can upregulate melanosome transfer via Myosin X‐mediated control of MC filopodia. Both MC and KC express Ca²⁺‐dependent E‐cadherins. These homophilic adhesion contacts induce transient increases in intra‐KC Ca²⁺, while ultraviolet radiation (UVR) raises intra‐MC Ca²⁺ via calcium‐selective ORAI1 ion channels; both are associated with regulating melanogenesis. However, how Ca²⁺ triggers melanin transfer remains unclear. Here we evaluated the role of E‐cadherin in UVR‐mediated melanin transfer in human skin cells. MC and KC in human epidermis variably express filopodia‐associated E‐cadherin, Cdc42, VASP and β‐catenin, all of which were upregulated by UVR in human MC in vitro. Knockdown of E‐cadherin revealed that this cadherin is essential for UVR‐induced MC filopodia formation and melanin transfer. Moreover, Ca²⁺ induced a dose‐dependent increase in filopodia formation and melanin transfer, as well as increased β‐catenin, Cdc42, Myosin X and E‐cadherin expression in these skin cells. Together, these data suggest that filopodial proteins and E‐cadherin, which are upregulated by intracellular (UVR‐stimulated) and extracellular Ca²⁺ availability, are required for filopodia formation and melanin transfer. This may open new avenues to explore how Ca²⁺ signalling influences human pigmentation.     

Synthesis of epidermolysis bullosa acquisita antigen by simian virus 40-transformed human keratinocytes

Summary The synthesis of epidermolysis bullosa acquisita (EBA) antigen in simian virus 40 (SV40)-transformed human epidermal keratinocytes was studied. Indirect immunofluorescent staining of SV40-transformed keratinocytes employing a serum sample from an EBA patient as a source of antibodies decorated EBA antigen as a perinuclear granular fluorescence. This staining pattern was similar to that of nontransformed epidermal keratinocytes grown in a low Ca²⁺ medium. In contrast, stratified primary cultures of keratinocytes stained after growth in a high Ca²⁺ medium showed only small amounts of the antigen localized in the substrate-attached basal cells. To demonstrate biosynthesis of the EBA antigen by SB40-transformed keratinocytes, cells were metabolically labeled with ¹⁴C-amino acids and the cell lysates were immunoprecipitated with EBA antiserum. Analysis of immunoprecipitates by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography revealed that the EBA serum precipitated a protein with an apparent molecular weight of 290 kD from extracts of these cells. These results indicate that SV40 induces the synthesis of the 290 kD EBA antigen. Expression of this antigen may be a general feature of nonstratifying, proliferating epidermal cells.     

The parathyroid hormone family member TIP39 interacts with sarco/endoplasmic reticulum Ca2+‐ ATPase activity by influencing calcium homoeostasis

Darier disease (DD) is a genetic skin disease that is associated with mutations in the ATP2A2 gene encoding the type 2 sarco/endoplasmic reticulum (ER) Ca²⁺‐ ATPase (SERCA2). Mutations of this gene result in alterations of calcium homoeostasis, abnormal epidermal adhesion and dyskeratosis. Silencing of ATP2A2 in monolayer cell culture of keratinocytes reduces desmoplakin expression at the borders of cells and impacts cell adhesion. Here, we report establishment of a three‐dimensional (3D) epidermal model of DD and use this model to evaluate peptide therapy with tuberoinfundibular peptide of 39 residues (TIP39) to normalize calcium transport. Gene silencing of ATP2A2 in keratinocytes grown in a 3D model resulted in dyskeratosis, partial parakeratosis and suprabasal clefts that resembled the histological changes seen in skin biopsies from patients with DD. TIP39, a peptide recently identified as a regulator of keratinocyte calcium transport, was then applied to this ATP2A2‐silenced 3D epidermal model. In normal keratinocytes, TIP39 increased [Ca²⁺]_i through the inositol trisphosphate (IP3) receptor pathway and stimulated differentiation. In monolayer ATP2A2‐silenced keratinocytes, although TIP39 increased cytosolic calcium from the ER, the response was incomplete compared with its control. TIP39 was observed to reduce intercellular clefts of the gene‐silenced epidermal model but did not significantly upregulate keratinocyte differentiation genes such as keratin 10 and filaggrin. These findings indicate that TIP39 is a modulator of ER calcium signalling and may be used as a potential strategy for improving aspects of DD.     

Localisation of bullous pemphigoid antigen 180 (BP180) in cultured human keratinocytes: functionally relevant modification by calcium

The expression of BP180 has previously been demonstrated to be influenced by both calcium (Ca²⁺) concentration and binding of anti-BP180-antibodies in cultured keratinocytes of the skin squamous cell carcinoma line DJM-1. Here, BP180 expression was studied in cultured normal human epidermal keratinocytes by confocal laser scanning microscopy. We exploited an experimental system, in which BP180 was previously shown to mediate, upon incubation with anti-BP180 antibodies, a specific signal-transducing event that leads to the release of inflammatory mediators, such as IL-8 from cultured normal human epidermal keratinocytes (NHEK). We found that without addition of BP180-specific IgG, BP180 is predominantly expressed on the cell surface irrespective of the Ca²⁺ concentration. In contrast, cell surface BP180 was greatly reduced in NHEK kept in high Ca²⁺ medium after incubation with BP180-specific IgG for 12 h compared to low Ca²⁺ medium. This effect was seen with antibodies to both N- and C-terminal fragments of the BP180 ectodomain, respectively. In addition, a slightly higher BP180 expression was found in NHEK cultured in low compared to high Ca²⁺ medium by Western blotting. Interestingly, in contrast to NHEK kept under low Ca²⁺ conditions, in NHEK grown in high Ca²⁺ medium, no elevated levels of IL-8 were released after treatment of cells with anti-BP180 IgG compared to normal IgG. Our data indicate that the Ca²⁺-modulated expression of BP180 is functionally relevant. This finding sheds further light on the complex pathomechanism in blister formation of BP180-related autoimmune blistering skin diseases.     

Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis

Calcium controls an array of key events in keratinocytes and epidermis: localized changes in Ca²⁺ concentrations and their regulation are therefore especially important to assess when observing epidermal barrier homeostasis and repair, neonatal barrier establishment, in differentiation, signaling, cell adhesion, and in various pathological states. Yet, tissue- and cellular Ca²⁺ concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the Ca²⁺ distribution in skin were based on Ca²⁺ precipitation followed by electron microscopy, or proton-induced X-ray emission. Neither cellular and/or subcellular localization could be determined through these approaches. In cells in vitro, fluorescent dyes have been used extensively for ratiometric measurements of static and dynamic Ca²⁺ concentrations, also assessing organelle Ca²⁺ concentrations. For lack of better methods, these findings together build the basis for the current view of the role of Ca²⁺ in epidermis, their limitations notwithstanding. Here we report a method using Calcium Green 5N as the calcium sensor and the phasor-plot approach to separate raw lifetime components. Thus, fluorescence lifetime imaging (FLIM) enables us to quantitatively assess and visualize dynamic changes of Ca²⁺ at light-microscopic resolution in ex vivo biopsies of unfixed epidermis, in close to in vivo conditions. Comparing undisturbed epidermis with epidermis following a barrier insult revealed major shifts, and more importantly, a mobilization of high amounts of Ca²⁺ shortly following barrier disruption, from intracellular stores. These results partially contradict the conventional view, where barrier insults abrogate a Ca²⁺ gradient towards the stratum granulosum. Ca²⁺ FLIM overcomes prior limitations in the observation of epidermal Ca²⁺ dynamics, and will allow further insights into basic epidermal physiology.     

all-trans-Retinoic acid preserves viability of fibroblasts and keratinocytes in full-thickness human skin and fibroblasts in isolated dermis in organ culture

Human dermal fibroblast and human epidermal keratinocyte survival was examined under various conditions in organ culture. Using cell recovery from organ-cultured tissue as the criterion, it was observed that no keratinocytes and few fibroblasts survived incubation for 10–12 days in serum-free basal medium containing a low level (0.15 mM) of extracellular Ca²⁺. Increasing the extracellular Ca²⁺ concentration to 1.4 mM or treating the tissue with 3 M retinoic acid (RA) under low Ca²⁺ conditions resulted in increased keratinocyte and fibroblast survival; the two treatments together were more effective than either treatment alone. The same treatments preserved fibroblast survival when pieces of isolated dermal tissue were incubated in organ culture and also supported fibroblast survival in monolayer culture. These findings indicate that recovery of keratinocytes and fibroblasts from skin after maintenance in organ culture provides a simple but definitive measure of the viability of the major cellular elements present in the tissue. These findings suggest that RA treatment enhances survival of both fibroblasts and keratinocytes and that these effects of RA can be seen at physiological Ca²⁺ concentrations as well as at suboptimal levels of extracellular Ca²⁺. Finally, these results indicate that the dermis is a direct target of RA.     

Inositol phosphate formation and release of intracellular free calcium by bradykinin in HaCaT keratinocytes

Phospholipase C-mediated release of inositol trisposphate, followed by an increase in free intracellular calcium, is an important signal transduction pathway for several membrane receptors. In the present investigation, the coupling of various receptors to phospholipase C was studied in the human keratinocyte line HaCaT. Inositol trisphosphate formation was determined by anion-exchange chromatography, and the release of intracellular calcium was analysed with the fluorescence probe Fura-2 AM. Activation of HaCaT keratinocytes with bradykinin resulted in a time- and dose-dependent release of inositol trisphosphate and intracellular calcium, with an EC₅₀ value of 50 nM for bradykinin-induced inositol trisphosphate formation. The mediators and cytokines IL-1, IL-4, IL-6, IL-8, EGF and TGF, as well as bombesin, prolactin, carbachol, substance P and retinoic acid, did not activate this pathway. The inability of the mediators examined to activate phospholipase C may be due to lack of the respective cognate receptors or to the use of other signal transduction pathways.     

What about physical contacts between epidermal keratinocytes and sensory neurons?

Recent studies have demonstrated that keratinocytes closely participate in sensory transduction, and therefore, intra‐epidermal free nerve endings are not exclusive transducers of pain. This discovery implies the existence of close afferent communication from keratinocytes to sensory neurons. Although reciprocal interactions between keratinocytes and intra‐epidermal free nerve endings via soluble mediators are well established, little attention has been paid to physical contacts between keratinocytes and intra‐epidermal free nerve endings. This review proposes to consider the ultrastructural and functional knowledge of these contacts, in both human skin biopsies and keratinocyte‐sensory neuron cocultures to speculate on the possible existence of synaptic contacts.