Role of Piezo2 in Schwann Cell Volume Regulation and Its Impact on Neurotrophic Release Regulation.

BACKGROUND/AIMS: Tactile perception relies on mechanoreceptors and nerve fibers, including c-fibers, Aß-fibers and Aδ-fibers. Schwann cells (SCs) play a crucial role in supporting nerve fibers, with non-myelinating SCs enwrapping c-fibers and myelinating SCs ensheathing Aß and Aδ fibers. Recent research has unveiled new functions for cutaneous sensory SCs, highlighting the involvement of nociceptive SCs in pain perception and Meissner corpuscle SCs in tactile sensation. Furthermore, Piezo2, previously associated with Merkel cell tactile sensitivity, has been identified in SCs. The goal of this study was to investigate the channels implicated in SC mechanosensitivity and the release process of neurotrophic factor secretion. METHODS: Immortalized IFRS1 SCs and human primary SCs generated two distinct subtypes of SCs: undifferentiated and differentiated SCs. Quantitative PCR was employed to evaluate the expression of differentiation markers and mechanosensitive channels, including TRP channels (TRPV4, TRPM7 and TRPA1) and Piezo channels (Piezo1 and Piezo2). To validate the functionality of specific mechanosensitive channels, Ca2+ imaging and electronic cell sizing experiments were conducted under hypotonic conditions, and inhibitors and siRNAs were used. Protein expression was assessed by Western blotting and immunostaining. Additionally, secretome analysis was performed to evaluate the release of neurotrophic factors in response to hypotonic stimulation, with BDNF, a representative trophic factor, quantified using ELISA. RESULTS: Induction of differentiation increased Piezo2 mRNA expression levels both in IFRS1 and in human primary SCs. Both cell types were responsive to hypotonic solutions, with differentiated SCs displaying a more pronounced response. Gd3+ and FM1-43 effectively inhibited hypotonicity-induced Ca2+ transients in differentiated SCs, implicating Piezo2 channels. Conversely, inhibitors of Piezo1 and TRPM7 (Dooku1 and NS8593, respectively) had no discernible impact. Moreover, Piezo2 in differentiated SCs appeared to participate in regulatory volume decreases (RVD) after cell swelling induced by hypotonic stimulation. A Piezo2 deficiency correlated with reduced RVD and prolonged cell swelling, leading to heightened release of the neurotrophic factor BDNF by upregulating the function of endogenously expressed Ca2+-permeable TRPV4. CONCLUSION: Our study unveils the mechanosensitivity of SCs and implicates Piezo2 channels in the release of neurotrophic factors from SCs. These results suggest that Piezo2 may contribute to RVD, thereby maintaining cellular homeostasis, and may also serve as a negative regulator of neurotrophic factor release. These findings underscore the need for further investigation into the role of Piezo2 in SC function and neurotrophic regulation.

Preliminary evidence that Merkel cells exert chemosensory functions in human epidermis.

The mechanotransduction of light-touch sensory stimuli is considered to be the main physiological function of epidermal Merkel cells (MCs). Recently, however, MCs have been demonstrated to be also thermo-sensitive, suggesting that their role in skin physiologically extends well beyond mechanosensation. Here, we demonstrate that in healthy human skin epidermal MCs express functional olfactory receptors, namely OR2AT4, just like neighbouring keratinocytes. Selective stimulation of OR2AT4 by topical application of the synthetic odorant, Sandalore®, significantly increased Piccolo protein expression in MCs, as assessed by quantitative immunohistomorphometry, indicating increased vesicle trafficking and recycling, and significantly reduced nerve growth factor (NGF) immunoreactivity within MCs, possibly indicating increased neurotrophin release upon OR2AT4 activation. Live-cell imaging showed that Sandalore® rapidly induces a loss of FFN206-dependent fluorescence in MCs, suggesting OR2AT4-dependent MC depolarization and subsequent vesicle secretion. Yet, in contrast to keratinocytes, OR2AT4 stimulation by Sandalore® altered neither the number nor the proliferation status of MCs. These preliminary ex vivo findings demonstrate that epidermal MCs also exert OR-dependent chemosensory functions in human skin, and invite one to explore whether these newly identified properties are dysregulated in selected skin disorders, for example, in pruritic dermatoses, and if these novel MC functions can be therapeutically targeted to maintain/promote skin health.

Higher oxytocin concentrations occur in subjects who build affiliative relationships with companion robots.

Building affiliative relationships with others is important for mental health. Recently, robots have been expected to play a role in improving mental health, but there is little scientific evidence as to whether they can build affiliative relationships with humans. To investigate that, we conducted studies combining behavior, physiology and questionnaires for companion robot Owners and Non-Owners. The results reveal that the steady-state concentration of oxytocin, a hormone related to affiliative relationships, was significantly higher in Owners than in Non-Owners. In addition, the Owners showed more behaviors indicative of intimacy than the Non-Owners. These results suggest that humans can build affiliative relationships with robots. Fifteen minutes of contact with the robot decreased the concentration of cortisol in both groups, suggesting that even a brief contact can contribute to improving mental health. Therefore, relationships between humans and robots may be one option to improve mental health and enhance well-being.

Abnormal Morphology of Blood Vessels in Erythematous Skin From Atopic Dermatitis Patients.

Previous studies suggest that altered peripheral blood circulation might be associated with erythema or inflammation in atopic dermatitis (AD) patients. However, the overall structure of blood vessels and capillaries in AD skin is poorly understood because most studies have involved light-microscopic observation of thin skin sections. In the present study, we compared the 3-dimensional structures of peripheral blood vessels of healthy subjects and AD patients in detail by means of 2-photon microscopy. In skin from healthy subjects, superficial vascular plexus and capillaries originating from flexous blood vessels were observed. However, skin from AD patients contained thickened, flexuous blood vessels, which might be associated with increased blood flow, in both erythematous and nonlesional areas. However, patients with lichenification did not display these morphological changes. Bifurcation of vessels was not observed in either erythematous or lichenification lesions. These results might be helpful for developing new clinical strategies to treat erythema in AD patients.

Japanese Cedar (Cryptomeria japonica) pollen allergen induces elevation of intracellular calcium in human keratinocytes and impairs epidermal barrier function of human skin ex vivo.

Cry j1 is the major peptide allergen of Japanese cedar (Sugi), Cryptomeria japonica. Since some allergens disrupt epidermal permeability barrier homeostasis, we hypothesized that Cry j1 might have a similar effect. Intracellular calcium level in cultured human keratinocytes was measured with a ratiometric fluorescent probe, Fura-2 AM. Application of Cry j1 significantly increased the intracellular calcium level of keratinocytes, and this increase was inhibited by trypsin inhibitor or a protease-activated receptor 2 (PAR-2) antagonist. We found that Cry j1 itself did not show protease activity, but application of Cry j1 to cultured keratinocytes induced a rapid (within 30 s) and transient increase of protease activity in the medium. This transient increase was blocked by trypsin inhibitor or PAR-2 antagonist. The effect of Cry j1 on transepidermal water loss (TEWL) of cultured human skin was measured in the presence and absence of a trypsin inhibitor and PAR-2 antagonist. Cry j1 significantly impaired the barrier function of human skin ex vivo, and this action was blocked by co-application of trypsin inhibitor or PAR-2 antagonist. Our results suggested that interaction of Cry j1 with epidermal keratinocytes leads to the activation of PAR-2, which induces elevation of intracellular calcium and disruption of barrier function. Blocking the interaction of Cry j1 with epidermal keratinocytes might ameliorate allergic reaction and prevent disruption of epidermal permeability barrier homeostasis.

Possible role of epidermal keratinocytes in the construction of acupuncture meridians.

Acupuncture meridians consist of a network of acupuncture points on the skin, stimulation of which is well established to have a variety of physiological effects. We have previously demonstrated that epidermal keratinocytes contain multiple sensory systems for temperature, mechanical stimuli, electric potentials and other stimuli. These sensory systems generate changes in the calcium-ion concentration in the epidermis, so epidermal keratinocytes can generate spatially-localized electro-physiological patterns in the skin. We have previously demonstrated signaling between epidermal keratinocytes and peripheral nerve systems. Therefore, stimuli sensed by epidermal keratinocytes might be transferred to the unmyelinated nerve fibers that are known to exist in the epidermis and, thence, to the spinal cord and brain. We propose that epidermal keratinocytes form an information-gathering network in the skin and that this network plays a key role in whole-body homeostasis in response to the changing environment. We also hypothesize that this network corresponds to the acupuncture meridians. As supporting examples, we present some striking calcium propagation patterns observed in cultured human keratinocytes after adenosine-triphosphate (ATP) stimulation. These results support the ideas that keratinocytes can generate spatially-restricted signaling patterns after environmental stimulation and that the cultures might be in-vitro models of meridians as an information-gathering network in skin.

Mathematical modeling of calcium waves induced by mechanical stimulation in keratinocytes.

Recent studies have shown that the behavior of calcium in the epidermis is closely related to the conditions of the skin, especially the differentiation of the epidermal keratinocytes and the permeability barrier function, and therefore a correct understanding of the calcium dynamics is important in explaining epidermal homeostasis. Here we report on experimental observations of in vitro calcium waves in keratinocytes induced by mechanical stimulation, and present a mathematical model that can describe the experimentally observed wave behavior that includes finite-range wave propagation and a ring-shaped pattern. A mechanism of the ring formation hypothesized by our model may be related to similar calcium propagation patterns observed during the wound healing process in the epidermis. We discuss a possible extension of our model that may serve as a tool for investigating the mechanisms of various skin diseases.

Frontiers in epidermal barrier homeostasis--an approach to mathematical modelling of epidermal calcium dynamics.

Intact epidermal barrier function is crucial for survival and is associated with the presence of gradients of both calcium ion concentration and electric potential. Although many molecules, including ion channels and pumps, are known to contribute to maintenance of these gradients, the mechanisms involved in epidermal calcium ion dynamics have not been clarified. We have established that a variety of neurotransmitters and their receptors, originally found in the brain, are expressed in keratinocytes and are also associated with barrier homeostasis. Moreover, keratinocytes and neurons show some similarities of electrochemical behaviour. As mathematical modelling and computer simulation have been employed to understand electrochemical phenomena in brain science, we considered that a similar approach might be applicable to describe the dynamics of epidermal electrochemical phenomena associated with barrier homeostasis. Such methodology would also be potentially useful to address a number of difficult problems in clinical dermatology, such as ageing and itching. Although this work is at a very early stage, in this essay, we discuss the background to our approach and we present some preliminary results of simulation of barrier recovery.

Coculture system of keratinocytes and dorsal-root-ganglion-derived cells for screening neurotrophic factors involved in guidance of neuronal axon growth in the skin.

The density of peripheral nerve fibres is increased in atopic dermatitis. Moreover, reduction in the fibres in a mouse model of atopic dermatitis reduces scratching behaviour. Thus, regulation of nerve fibre extension could be an effective strategy to reduce itching in pruritus dermatosis. In this study, we established a new coculture system of keratinocytes and dorsal-root-ganglion-derived cells using an apparatus, AXIS(™) , which consists of two different channels connected via a set of microgrooves, through which signalling molecules and axons, but not living cells, can pass. When we seeded keratinocytes in one chamber, extension of nerve fibres was observed from dorsal root ganglion cells seeded in the other chamber. Addition of anti-BDNF antibody in the keratinocyte-seeded chamber significantly reduced the extension. Application of Semaphorin 3A also reduced the extension by approximately 50%. We suggest that this coculture system may be useful for screening of anti-itching drugs.

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 ([Ca(2+) ]i ) in cultured human keratinocytes after scratch damage. Immediately after scratch damage, a wave of increased [Ca(2+) ]i radiated outward from the damaged area and then disappeared gradually. But, [Ca(2+) ]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 (Ca(2+) -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 [Ca(2+) ]i might be part of a signalling pathway that appropriately directs the wound-healing process in epidermis.

In vitro formation of organized structure between keratinocytes and dorsal-root-ganglion cells.

We recently found that the morphology in a co-culture system of keratinocytes and dorsal-root ganglion-derived cells depended on the timing of seeding of the two cell types. In skin, epidermis is formed first, followed by construction of peripheral nerve structure. Therefore, we hypothesized that formation of peripheral nerve structure in the epidermis might be driven by interaction between keratinocytes and nerve cells. In the present study, we tested this idea by incubating keratinocytes and dorsal-root ganglion cells in a spatially separated manner and observing the morphological changes in the co-culture system. Extension of nerve fibre-like structures from the ganglion cells was observed, and within 3 days after seeding, many nerve fibre-like extensions penetrated into the keratinocyte cluster, subsequently forming a network that appeared to resemble the cutaneous peripheral nervous system. Our present model may be useful for studying the formation of peripheral nerve structure in the skin.

Oxytocin is expressed in epidermal keratinocytes and released upon stimulation with adenosine 5'-[γ-thio]triphosphate in vitro.

Oxytocin is a neuropeptide produced primarily in the hypothalamus and is best known for its roles in parturition and lactation. It also influences behaviour, memory and mental state. Recent studies have suggested a variety of roles for oxytocin in peripheral tissues, including skin. Here we show that oxytocin is expressed in human skin. Immunohistochemical studies showed that oxytocin and its carrier protein, neurophysin I, are predominantly localized in epidermis. RT-PCR confirmed the expression of oxytocin in both skin and cultured epidermal keratinocytes. We also show that oxytocin is released from keratinocytes after application of adenosine 5'-[γ-thio]triphosphate (ATPγS, a stable analogue of ATP) in a dose-dependent manner. The ATPγS-induced oxytocin release was inhibited by removal of extracellular calcium, or by the P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). These results suggest that oxytocin is produced in human epidermal keratinocytes and is released in response to calcium influx via P2X receptors.

Ryanodine receptors are expressed in epidermal keratinocytes and associated with keratinocyte differentiation and epidermal permeability barrier homeostasis.

Ryanodine receptors (RyRs) have an important role as calcium channels in the regulation of intracellular calcium levels in the nervous system and muscle. In the present study, we investigated the expression of RyR in human epidermis. Immunohistochemical studies and reverse transcription-PCR indicated the expression of RyR type 1, 2, and 3 proteins in epidermal keratinocytes. The expression level of each RyR subtype was higher in differentiating keratinocytes than in proliferative cells. We also demonstrated the functional expression of RyR by calcium imaging. In cultured human keratinocytes, application of the RyR agonist 4-chloro-m-cresol (CMC) induced elevation of the intracellular calcium concentration, and co-application of the RyR antagonist 1,1'-diheptyl-4,4'-bipyridinium dibromide (DHBP) blocked the elevation. Application of CMC accelerated keratinocyte differentiation in vitro. On the other hand, topical application of CMC after tape-stripping of hairless mouse skin delayed barrier recovery, whereas application of an RyR antagonist, dantrolene or DHBP, accelerated the barrier recovery. These results suggest that RyR expressed in epidermal keratinocytes is associated with both differentiation of keratinocytes and epidermal barrier homeostasis.

Intracellular calcium response to high temperature is similar in undifferentiated and differentiated cultured human keratinocytes.

A series of thermo-sensitive proteins is expressed in epidermal keratinocytes, where they function as cation channels that are activated at temperatures in the range of 28-42°C. Here, we examined the influence of high temperature on intracellular calcium level in undifferentiated and differentiated cultured human keratinocytes, which are considered to be representative of deeper and upper epidermal layers, respectively. We found that the percentages of responsive keratinocytes among undifferentiated and differentiated cells exposed to temperatures in the warm-hot range (30-50°C) were essentially the same. In contrast, we previously demonstrated that undifferentiated keratinocytes were more sensitive than differentiated cells to low temperature (<22°C). Thus, the deeper layer of the epidermis is more sensitive to cold than the upper layer, but the response to warm-hot temperatures appears to be homogeneous throughout the epidermis.

Morphological and functional differences in coculture system of keratinocytes and dorsal-root-ganglion-derived cells depending on time of seeding.

Previous study indicated that in a coculture system of keratinocytes and dorsal-root-ganglion-derived (DRG) cells, mechanical stimulation of keratinocytes induced ATP-mediated calcium propagation and excitation of DRG cells. Here, we examined two different coculture systems of keratinocytes and DRG cells. In one, we seeded keratinocytes first and then seeded DRG cells on the keratinocytes. In this system, nerve fibres from DRG cells passed between keratinocytes. Mechanical stimulation of keratinocytes did not induce excitation of DRG cells. In the other, we seeded both cell types together. At first, each cell type grew separately, forming cell aggregates. Then, nerve fibres grew out from the DRG cell aggregates to keratinocyte aggregates and penetrated into them. In this system, mechanical stimulation of keratinocytes induced excitation of the nerve fibres, but the excitation was not completely blocked by apyrase, an ATP-degrading enzyme. These results suggest that coculture of keratinocytes and DRG can generate a variety of structures, depending on the seeding conditions.

Phosphodiesterase inhibitors block the acceleration of skin permeability barrier repair by red light.

We previously demonstrated that exposure to red light (550-670 nm) accelerates epidermal permeability barrier recovery after barrier disruption. Furthermore, we showed that photosensitive proteins, originally found in retina, are also expressed in epidermis. In retina, transducin and phosphodiesterase 6 play key roles in signal transmission. In this study, we evaluate the role of phosphodiesterese 6 in the acceleration by red light of epidermal permeability barrier recovery. Immunohistochemical study and reverse transcription-PCR assays confirmed the expression of both transducin and phosphodiesterase 6 in epidermal keratinocytes. Topical application of 3-isobutyl-1-methylxanthine, a non-specific phosphodiesterase inhibitor, blocked the acceleration of the barrier recovery by red light. Topical application of zaprinast, a specific inhibitor of phosphodiesterases 5 and 6, also blocked the acceleration, whereas T0156, a specific inhibitor of phosphodiesterase 5, had no effect. Red light exposure reduced the epidermal hyperplasia induced by barrier disruption under low humidity, and the effect was blocked by pretreatment with zaprinast. Our results indicate phosphodiesterase 6 is involved in the recovery-accelerating effect of red light on the disrupted epidermal permeability barrier.

Roles of transient receptor potential proteins (TRPs) in epidermal keratinocytes.

Epidermal keratinocytes are the epithelial cells of mammalian skin. At the basal layer of the epidermis, these cells proliferate strongly, and as they move towards the skin surface, differentiation proceeds. At the uppermost layer of the epidermis, keratinocytes undergo apoptosis and die, forming a thin, water-impermeable layer called the stratum corneum. Peripheral blood vessels do not reach the epidermis, but peripheral nerve fibers do penetrate into it. Until recently, it was considered that the main role of epidermal keratinocytes was to construct and maintain the water-impermeable barrier function. However, since the functional existence of TRPV1, which is activated by heat and low pH, in epidermal keratinocytes was identified, our understanding of the role of keratinocytes has changed enormously. It has been found that many TRP channels are expressed in epidermal keratinocytes, and play important roles in differentiation, proliferation and barrier homeostasis. Moreover, because TRP channels expressed in keratinocytes have the ability to sense a variety of environmental factors, such as temperature, mechanical stress, osmotic stress and chemical stimuli, epidermal keratinocytes might form a key part of the sensory system of the skin. The present review deals with the potential roles of TRP channels expressed in epidermal keratinocytes and focuses on the concept of the epidermis as an active interface between the body and the environment.

Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold-sensitive TRP receptors in epidermal permeability barrier homoeostasis.

TRPA1 and TRPM8 receptors are activated at low temperature (A1: below 17 degrees C and M8: below 22 degrees C). Recently, we observed that low temperature (below 22 degrees C) induced elevation of intracellular calcium in keratinocytes. Moreover, we demonstrated that topical application of TRPA1 agonists accelerated the recovery of epidermal permeability barrier function after disruption. In this study, we examined the effect of topical application of TRPM8 modulators on epidermal permeability barrier homoeostasis. Immunohistochemical study and RT-PCR confirmed the expression of TRPM8 or TRPM8-like protein in epidermal keratinocytes. Topical application of TRPM8 agonists, menthol and WS 12 accelerated barrier recovery after tape stripping. The effect of WS12 was blocked by a non-selective TRP antagonist, Ruthenium Red, and a TRPM8-specific antagonist, BTCT. Topical application of WS12 also reduced epidermal proliferation associated with barrier disruption under low humidity, and this effect was blocked by BTCT. Our results indicate that TRPM8 or a closely related protein in epidermal keratinocytes plays a role in epidermal permeability barrier homoeostasis and epidermal proliferation after barrier insult.