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Calcium gradient in cultured slice of rat hindpaw skin. (a) High calcium concentration was observed in the uppermost area of the epidermis. (b) This shifted to the deeper area, 2 hours after removal of about half of the stratum corneum with cyanoacrylate. White particles were observed at the basal layer (arrows). (c) Fluorescence image of the skin obtained with CellTracker. Fluorescence was observed from the bottom to the upper region, i.e., the cells were viable throughout. Bar=20 M.
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Ionotropic receptors, originally found in the brain, were recently also identified in epidermal keratinocytes. Moreover, concentration gradients and movement of calcium are crucial in epidermal homeostasis. Thus, imaging of calcium in the living epidermis is expected to provide insight into epidermal physiology and pathology. Here we describe the i...
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... next examined the localization of intracellular calcium in cultured skin slices (Figure 2). A steep calcium gradient was observed in the uppermost area of epidermis, as has previously been found in fixed tissue (a) ( Mauro et al., 1998;Denda et al., 2000). ...
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... cultured cells, glutathione and glutathione transferase are ubiquitous, so the probe indicates cell viability ( Lantz et al., 2001). As shown in Figure 2c, fluorescence was observed throughout the epidermis, that is, cells in the slice were alive during the observation. ...
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... this work, we have succeeded in imaging the calcium gradient in cultured skin slices of rat (Figure 2a), demonstrating the presence of higher basal levels of calcium in the deeper layers (Figure 2b). Previous studies have shown that a steep calcium gradient exists in the uppermost epidermal layer, and this gradient disappears after barrier disruption ( Mauro et al., 1998;Denda et al., 2000). ...
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... this work, we have succeeded in imaging the calcium gradient in cultured skin slices of rat (Figure 2a), demonstrating the presence of higher basal levels of calcium in the deeper layers (Figure 2b). Previous studies have shown that a steep calcium gradient exists in the uppermost epidermal layer, and this gradient disappears after barrier disruption ( Mauro et al., 1998;Denda et al., 2000). ...
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... we found that the levels of intracellular calcium in individual keratinocytes at the bottom of the epidermis were not homogeneous, though it is possible that this might be an artifact due to cell damage during the process of skin sectioning. After the partial removal of the stratum corneum, white particles were consistently observed in the basal layer (Figure 2b, arrows). Rhod-2, the calcium indicator that we used, can penetrate into the endoplasmic reticulum, so these particles might represent sites of elevated calcium in endoplasmic reticulum, but again we cannot rule out an artifact. ...
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... pain receptors, such as TRPV1 or P2X3, are expressed at the surface of the epidermis (Denda et al., 2001b). TRPV1 is activated by heat and chemical stimuli ( Dhaka et al., 2006), and we suggested that TRPV1 expressed in keratinocytes may be involved in the sensory system of skin (Denda et al., 2007a) www.jidonline.org 587 cytes to stress induced both elevation of intracellular calcium and ATP release, and nerve cells were excited by the ATP released from keratinocytes ( Koizumi et al., 2004). ...
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... next examined the localization of intracellular calcium in cultured skin slices (Figure 2). A steep calcium gradient was observed in the uppermost area of epidermis, as has previously been found in fixed tissue (a) ( Mauro et al., 1998;Denda et al., 2000). ...
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... cultured cells, glutathione and glutathione transferase are ubiquitous, so the probe indicates cell viability ( Lantz et al., 2001). As shown in Figure 2c, fluorescence was observed throughout the epidermis, that is, cells in the slice were alive during the observation. ...
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... this work, we have succeeded in imaging the calcium gradient in cultured skin slices of rat (Figure 2a), demonstrat- ing the presence of higher basal levels of calcium in the deeper layers (Figure 2b). Previous studies have shown that a steep calcium gradient exists in the uppermost epidermal layer, and this gradient disappears after barrier disruption ( Mauro et al., 1998;Denda et al., 2000). ...
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... this work, we have succeeded in imaging the calcium gradient in cultured skin slices of rat (Figure 2a), demonstrat- ing the presence of higher basal levels of calcium in the deeper layers (Figure 2b). Previous studies have shown that a steep calcium gradient exists in the uppermost epidermal layer, and this gradient disappears after barrier disruption ( Mauro et al., 1998;Denda et al., 2000). ...
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... we found that the levels of intracellular calcium in individual keratinocytes at the bottom of the epidermis were not homogeneous, though it is possible that this might be an artifact due to cell damage during the process of skin sectioning. After the partial removal of the stratum corneum, white particles were consistently observed in the basal layer (Figure 2b, arrows). Rhod-2, the calcium indicator that we used, can penetrate into the endoplasmic reticulum, so these particles might represent sites of elevated calcium in endoplasmic reticulum, but again we cannot rule out an artifact. ...
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... pain receptors, such as TRPV1 or P2X3, are expressed at the surface of the epidermis (Denda et al., 2001b). TRPV1 is activated by heat and chemical stimuli ( Dhaka et al., 2006), and we suggested that TRPV1 expressed in keratinocytes may be involved in the sensory system of skin (Denda et al., 2007a) www.jidonline.org 587 cytes to stress induced both elevation of intracellular calcium and ATP release, and nerve cells were excited by the ATP released from keratinocytes ( Koizumi et al., 2004). ...
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Citations
... In combination with the moisture absorption by the skin brought about by the osmotic effect, a low calcium concentration was initially provided so moisture could penetrate the deeper layers of the skin. Towards the end of the test, the concentration of the calcium ion was sufficiently high that an effective influence of the epidermal calcium gradient can occur [20] [21] [22]. In order to reproduce the conditions of young skin, there should only be a low-level influence of the calcium concentration in the deeper layers, whereas the calcium concentration should be significantly increased close to the surface [21] [22] [23]. ...
... This gradient facilitates the regulation of various cellular processes in keratinocytes, including cell survival, proliferation, motility, apoptosis, and differentiation [36]. Calcium ion levels are highly dynamic, as ions are released from intracellular stores or enter from extracellular sources, driving Ca 2+ signaling in cells [37][38][39]. Ca 2+ homeostasis is associated with the control of cell cycle progression and, inevitably, proliferation [40]. Thus, we sought to determine whether ATRA treatment alters the intracellular Ca 2+ levels in HaCaT cells as a mechanism underlying its antiproliferative effect. ...
Olfactory receptors (ORs), which belong to the G-protein-coupled receptor family, have been widely studied as ectopically expressed receptors in various human tissues, including the skin. However, the physiological functions of only a few OR types have been elucidated in skin cells. All-trans retinoic acid (ATRA) is a well-known medication for various skin diseases. However, many studies have shown that ATRA can have adverse effects, resulting from the suppression of cell proliferation. Here, we investigated the involvement of OR7A17 in the ATRA-induced suppression of human keratinocyte (HaCaT) proliferation. We demonstrated that OR7A17 is expressed in HaCaT keratinocytes, and its expression was downregulated by ATRA. The ATRA-induced downregulation of OR7A17 was attenuated via RAR α or RAR γ antagonist treatment, indicating that the effects of ATRA on OR7A17 expression were mediated through nuclear retinoic acid receptor signaling. Moreover, we found that the overexpression of OR7A17 induced the proliferation of HaCaT cells while counteracting the antiproliferative effect of ATRA. Mechanistically, OR7A17 overexpression reversed the ATRA-induced attenuation of Ca2+ entry. Our findings indicated that ATRA suppresses cell proliferation through the downregulation of OR7A17 via RAR α- and γ-mediated retinoid signaling. Taken together, OR7A17 is a potential therapeutic target for ameliorating the anti-proliferative effects of ATRA.
... In the case of keratinocytes, even in a monolayer culture system, propagation and oscillation of calcium waves have been observed. 30,168,169 The ability to produce such patterns might be critical for information processing. ...
It was long considered that the role of epidermal keratinocytes is solely to construct a water-impermeable protective membrane, the stratum corneum, at the uppermost layer of the skin. However, in the last two decades, it has been found that keratinocytes contain multiple sensory systems that detect environmental changes, including mechanical stimuli, sound, visible radiation, electric fields, magnetic fields, temperature and chemical stimuli, and also a variety of receptor molecules associated with olfactory or taste sensation. Moreover, neurotransmitters and their receptors that play crucial roles in the brain are functionally expressed in keratinocytes. Recent studies have demonstrated that excitation of keratinocytes can induce sensory perception in the brain. Here, we review the sensory and information processing capabilities of keratinocytes. We discuss the possibility that epidermal keratinocytes might represent the earliest stage in the development of the brain during the evolution of vertebrates.
... Gelation is the most important feature and advantage of hydrogels. 25 Figure 1 shows that after the addition of two valence ions (calcium ions), the mixture of sodium alginate (SA) and benlysta rapidly gelled and adhered to the container wall, suggesting excellent gelation property of the benlysta-loaded SA hydrogel. At the skin wound, there are enough divalent ions, such as Ca 2+ , Mg 2+ , Fe 2+ , Zn 2+ , etc., 26,27 which can promote the rapid formation of the gelation. ...
Benlysta is a new drug approved by the US Food and Drug Administration (US FDA) in 2019 for the treatment of systemic lupus erythematosus. In this study, we loaded the benlysta in the traditional sodium alginate (SA) hydrogel to investigate the potential application of the drug-loaded hydrogel for skin dressing or hypodermic drug. Live/dead staining images and the CCK-8 results showed that the benlysta-loaded hydrogel could promote the growth of human epidermal cells (HaCat), fibroblasts (L929), and endothelial cells while inhibiting the aggregation of inflammatory cells (macrophages). In addition, the hydrogel degradation and drug release are slow and controllable, and the gel time of drug-loaded hydrogel can be adjusted by adding sodium alginate ratios according to the requirement. In summary, we prepared a time-dependent drug-loaded hydrogel for potential application in the treatment of skin injury that may be caused by other diseases.
... Keratinocytes are reported to release ATP in a critical gradientdependent manner reflecting the differentiation status of the cells, with CX signalling reported to play a role in the upper stratified layers, where CX26 tends to be spatially expressed (Tsutsumi, Denda, Inoue, Ikeyama, & Denda, 2009 Gomez-Hernandez, & Barrio, 2006). The fact that following proinflammatory challenge CX26 expression is so dramatically upregulated suggests that the CX26 hemichannels produce an excessive release of ATP, which activates excitatory purinergic receptors of neighbouring cells. ...
Dysregulation of Connexin (CX) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and nonhealing wounds. To mimic a proinflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/ml peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 min to 24 hr in the presence or absence of CX blockers and/or following CX26, CX43, PANX1 and TLR2 small interfering RNA (siRNA) knockdown (KD). Expression levels of IL‐6, IL‐8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by quantitative real‐time polymerase chain reaction, western blot analysis and/or immunocytochemistry. Nuclear factor kappa β (NF‐κβ) was blocked with BAY 11‐7082, CX‐channel function was determined by adenosine 5′‐triphosphate (ATP) release assays. Enzyme‐linked immunosorbent assay monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of CX or purinergic signalling. Exposure to PGN induced IL‐6, IL‐8, CX26 and TLR2 gene expression but it did not influence CX43, PANX1 or Ki67 messenger RNA expression levels. CX43 protein levels were reduced following 24 hr PGN exposure. PGN‐induced CX26 and IL‐6 expression were also aborted by TLR2‐KD and inhibition of NF‐κβ. ATP and IL‐6 release were stimulated following 15 min and 1–24 hr challenge with PGN, respectively. Release of both agents was inhibited by coincubation with CX‐channel blockers, CX26‐, CX43‐ and TLR2‐KD. The IL‐6 response was also reduced by purinergic blockers. CX‐signalling plays a role in the innate immune response in the epidermis. PGN is detected by TLR2, which via NF‐κβ, directly activates CX26 and IL‐6 expression. CX43 and CX26 maintain proinflammatory signalling by permitting ATP release, however, PANX1 does not participate.
... As opposed to TRPV5, TRPV6 is found expressed in a broader variety of tissues. In skin, a natural Ca 2+ gradient exists which spans from the stratum granulosum (high Ca 2+ ) to the basal layer (low Ca 2+ ) of the epidermis (Tsutsumi et al. 2009). This gradient is essential to prevent premature differentiation of keratinocytes (Hennings et al. 1980). ...
Tight control of basal cytosolic Ca2+ concentration is essential for cell survival and to fine-tune Ca2+-dependent cell functions. A way to control this basal cytosolic Ca2+ concentration is to regulate membrane Ca2+ channels including store-operated Ca2+ channels and secondary messenger-operated channels linked to G-protein-coupled or tyrosine kinase receptor activation. Orai, with or without its reticular STIM partner and Transient Receptor Potential (TRP) proteins, were considered to be the main Ca2+ channels involved. It is well accepted that, in response to cell stimulation, opening of these Ca2+ channels contributes to Ca2+ entry and the transient increase in cytosolic Ca2+ concentration involved in intracellular signaling. However, in various experimental conditions, Ca2+ entry and/or Ca2+ currents can be recorded at rest, without application of any experimental stimulation. This led to the proposition that some plasma membrane Ca2+ channels are already open/activated in basal condition, contributing therefore to constitutive Ca2+ entry. This article focuses on direct and indirect observations supporting constitutive activity of channels belonging to the Orai and TRP families and on the mechanisms underlying their basal/constitutive activities.
... Koizumi et al. reported that signal transduction from kerati- nocytes to neurons is mediated by ATP released from mechanically stimulated keratinocytes [11]. We previously reported that the increase of intracellular calcium in response to ATP varied in each layer of epidermis, and was greater in stratum basale than in the uppermost layer of rat skin slices [22]. Here, we also perfused human skin slices with ATP (Fig. 3), and observed that the elevation of calcium of cells after ATP application was greater in cells in the lower epidermal layers. ...
Background:
Changes of epidermal calcium ion concentration are involved in regulation of barrier homeostasis and keratinocyte differentiation. Moreover, intracellular calcium dynamics might play a role in skin sensation. But, although calcium dynamics of cultured keratinocytes in response to mechanical stresses has been well studied, calcium propagation in stimulated human epidermis is still poorly understood.
Objective:
The aim of this study was to demonstrate a novel method for real-time measurement of calcium dynamics in response to point stimulation of human epidermis at the single-cell level.
Methods:
We examined calcium propagation in cross-sectional samples of living human epidermis ex vivo, as well as in cultured human keratinocytes, by means of two-photon microscopy after stimulating cells in stratum granulosum with the emission laser of a two-photon microscope.
Results:
Cells in different epidermal layers showed different responses, and those in stratum basale showed the greatest elevation of intracellular calcium. Calcium propagation in epidermis was inhibited in the presence of apyrase (which degrades adenosine triphosphate; ATP) or gap-junction blockers. In cultured keratinocytes, on the other hand, calcium propagated in a simple concentric wave-like manner from the stimulation site, and propagation was strongly suppressed by apyrase.
Conclusion:
Our results suggested that ATP and gap junctions play important roles in calcium propagation induced by point laser stimulation of the uppermost layer of epidermis. Our method should be broadly useful to study calcium dynamics, epidermal physiological mechanisms, and mechanisms of skin sensation at the single-cell level.
... Several experimental (Menon et al., 1992;Mauro et al., 1998;Tsutsumi et al., 2009;Celli et al., 2010;Jungman et al., 2012) and computational approaches (Cornelissen et al., Adams et al., 2012;Denda et al., 2014;Adams et al., 2015) have been used to model or measure Ca 2+ fluxes in epidermis. While initial studies using fixed and sectioned tissue (Menon et al., 1992;Mauro et al., 1998) showed that following acute barrier perturbation, Ca 2+ drops in the viable layers and then recovers as the barrier recovers, a more recent study (Behne et al., 2011) reported a moderate increase in intracellular and extracellular calcium concentration as soon as 30 minutes after barrier perturbation by acetone lipid extraction in hairless mice. ...
Ca2+ fluxes direct keratinocyte differentiation, cell-to-cell adhesion, migration and epidermal barrier homeostasis. We previously showed that intracellular Ca2+ stores constitute a major portion of the calcium gradient especially in the stratum granulosum. Loss of the calcium gradient triggers epidermal barrier homeostatic responses. In this report, using unfixed ex vivo epidermis and Human Epidermal Equivalents (HEE) we show that endoplasmic reticulum (ER) Ca2+ is released in response to barrier perturbation, and that this release constitutes the major shift in epidermal Ca2+ seen after barrier perturbation. We find that ER Ca2+ release correlates with a transient increase in extracellular Ca2+. Lastly, we show that ER calcium release resulting from barrier perturbation triggers transient desmosomal remodeling, seen as an increase in extracellular space and a loss of the desmosomal intercellular midline. Topical application of thapsigargin (TG), which inhibits the ER Ca2+ ATPase activity without compromising barrier integrity, also leads to desmosomal remodeling and loss of the midline structure. These experiments establish the ER Ca2+ store as a master regulator of the Ca2+ gradient response to epidermal barrier perturbation, and suggest that ER Ca2+ homeostasis also modulates normal desmosomal reorganization, both at rest and after acute barrier perturbation.
... The velocity that was recorded in this study is similar to that of waves that have been detected in slime mold, in which they may be used for cellular integration [14]. Interestingly, Ca 2+ waves can be initiated by physical damage (see Figure 4), which is reminiscent of those that have been induced in damaged fish skin [47], cultured rat skin [48], and damaged brain tissue [49]. It is therefore possible that damage-associated Ca 2+ waves play a role in producing ectopic eyespots. ...
Butterfly wing color patterns emerge as the result of a regular arrangement of scales produced by epithelial scale cells at the pupal stage. These color patterns and scale arrangements are coordinated throughout the wing. However, the mechanism by which the development of scale cells is controlled across the entire wing remains elusive. In the present study, we used pupal wings of the blue pansy butterfly, Junonia orithya, which has distinct eyespots, to examine the possible involvement of Ca(2+) waves in wing development.
Here, we demonstrate that the developing pupal wing tissue of the blue pansy butterfly displayed spontaneous low-frequency Ca(2+) waves in vivo that propagated slowly over long distances. Some waves appeared to be released from the immediate peripheries of the prospective eyespot and discal spot, though it was often difficult to identify the specific origins of these waves. Physical damage, which is known to induce ectopic eyespots, led to the radiation of Ca(2+) waves from the immediate periphery of the damaged site. Thapsigargin, which is a specific inhibitor of Ca(2+)-ATPases in the endoplasmic reticulum, induced an acute increase in cytoplasmic Ca(2+) levels and halted the spontaneous Ca(2+) waves. Additionally, thapsigargin-treated wings showed incomplete scale development as well as other scale and color pattern abnormalities.
We identified a novel form of Ca(2+) waves, spontaneous low-frequency slow waves, which travel over exceptionally long distances. Our results suggest that spontaneous Ca(2+) waves play a critical role in the coordinated development of scale arrangements and possibly in color pattern formation in butterflies.
... Calcium contained in the cytosol and intracellular organelles of cells are considered together simply as intracellular calcium. Experimentally, intracellular calcium waves are known to propagate between adjacent keratinocytes [213], but these waves have insignificant magnitude (≈ 120 nM [214], which corresponds to ≈ 0.005 mg/kg) compared to the calcium profiles we investigate (Figures 2.4(a) and 2.4(b)). Hence we confine intracellular calcium to individual keratinocytes, with its velocity equal to the local keratinocyte velocity. ...
This project investigated the calcium distributions of the skin, and the growth patterns of skin substitutes grown in the laboratory, using mathematical models. The research found that the calcium distribution in the upper layer of the skin is controlled by three different mechanisms, not one as previously thought. The research also suggests that tight junctions, which are adhesions between neighbouring skin cells, cannot be solely responsible for the differences in the growth patterns of skin substitutes and normal skin.
