Various sensors that respond to physical or chemical environmental factors have been identified in the peripheral nervous system. Some of them, which respond to mechanical stress, osmotic pressure, temperature and chemical stimuli (such as pH), are also expressed in epidermal keratinocytes. Neurotransmitters and their receptors, as well as receptors that regulate the neuroendocrine system of the skin, are also present in keratinocytes. Thus, broadly speaking, epidermal keratinocytes appear to be equipped with sensing systems similar to those of the peripheral and central nervous systems. It had long been considered that only nerve C-terminals in the epidermis play a role in skin surface perception. However, building on earlier work on skin receptors and new findings introduced here, we present in this review a novel hypothesis of skin sensory perception, i.e. first, keratinocytes recognize various environmental factors, and then the information is processed and conveyed to the nervous system. Sensory receptors in keratinocytesThe traditional view of the skin surface sensory system for environmental factors, such as temperature, humidity, mechanical stress and chemical stimuli, has been that the key sensors are the C fibres, which penetrate into the epidermis (1). However, the terminals of the nerve fibres are quite sparse. For example, the pressure points, which detect mechanical stimuli, are localized at distances of millimetres from each other (2). The skin can detect pattern on a much smaller scale (3-6) than would be expected on the basis of sampling theory if the nerve terminals were the only sensors (7). We previously suggested that epidermal keratinocytes might be at the forefront of skin surface perception (8). This idea has been supported by the recent cloning of a series of receptors, which are activated by temperature, mechanical stress, osmotic pressure and chemical stimuli. Many of these receptors are expressed in epidermal keratinocytes.One of the most interesting receptor families is called transient receptor potential (TRP). The TRP receptor family members have been reported to act as sensors of temperature or other physical or chemical factors (9), and TRPV1, TRPV3 and TRPV4 were shown to be present in epidermal keratinocytes (Fig. 1a) (10-12). TRPV1 seems to be most strongly expressed in the upper and basal layers of the epidermis. High expression of the receptor at the surface of the epidermis would be consistent with a role in detecting external temperature. The reason for the high expression at the basal layer is not clear, but TRPV1 may also have some role in epidermal-dermal interaction. TPRV1 is activated by heat (>43°C), acidic conditions (pH < 6.6) and capsaicin (13). TRPV3 is activated by heat (>35°C), mechanical stress, camphor and 2-aminoethoxydiphenyl borate (2APB) (14,15). TRPV4 is activated by osmotic pressure (9). Previous reports suggest the existence of a water flux sensor in the epidermis (16,17). TRPV4 might play an important role as a sensor of water flux from the ...

Calcium dynamics in the epidermis play a crucial role in barrier homeostasis and keratinocyte differentiation. We have recently suggested that the electro-physiological responses of the keratinocyte represent the frontier of the skin sensory system for environmental stimuli. In the present study, we have evaluated the responses of proliferating and differentiated human keratinocytes to mechanical stress by measuring the intracellular calcium level. Before differentiation, mechanical stress induces a calcium wave over a limited area; this is completely blocked by apyrase, which degrades ATP. In the case of differentiated keratinocytes, the calcium wave propagates over a larger area. Application of apyrase does not completely inhibit this wave. Thus, in differentiated cells, the induction of calcium waves might involve not only ATP, but also another factor. Immunohistochemical studies indicate that connexins 26 and 43, both components of gap junctions, are expressed in the cell membrane of differentiated keratinocytes. Application of octanol or carbenxolone, which block gap junctions, significantly reduces calcium wave propagation in differentiated keratinocytes. Thus, signaling via gap junctions might be involved in the induction of calcium waves in response to mechanical stress at the upper layer of the epidermis.

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons. Keywords: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, development, GluR1 subunit, post-synaptic density-95/ Dlg/ ZO-1 domain, Shank3, synapse. Transmission at excitatory synapses is primarily mediated by glutamate acting on three classes of ligand-gated ion channels, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate and NMDA receptors (Wisden and Seeburg 1993;Hollmann and Heinemann 1994). In addition to their role in synaptic transmission, these glutamate receptors (GluRs) have been thought to play a crucial role in many brain functions, including activity-dependent synaptogenesis during development and synaptic plasticity (McDonald and Johnston 1990;Bliss and Collingridge 1993).Many excitatory synapses in young developing neurons have been found to express only NMDA receptors, which are continuously blocked by magnesium at resting membrane potentials. As no evoked transmission is observed even when glutamate is present, these synapses are referred to as 'silent synapses'. During later development, AMPA receptors are delivered and clustered on the synaptic membrane in an activity-dependent manner, and the synapses subsequently become functionally active (Durand et al. 1996;Wu et al. 1996;Pickard et al. 2000;Liao et al. 2001;Isaac 2003). Thus, the clustering of AMPA receptors on the synaptic membrane is an essential event during synaptogenesis. Address correspondence and reprint requests to S. Kohsaka, Department of Neurochemistry, National Institute of Neu...

Previous reports have suggested the existence of photoreceptors for visible radiation at the surface of the human body. Rhodopsin is a well-known photosensitive protein found in the rod cells of the retina and detects light ⁄ dark contrast. Cone opsins are also photosensitive receptors in the cone cells of the retina and detect colour. Here, we describe immunochemical studies using anti-rhodopsin and anti-opsin antibodies on human skin. Both mouse retina and human epidermis showed clear immunoreactivity with each antibody. Interestingly, immunoreactivity against longer-wavelength opsin antibody was observed in the basal layer of the epidermis, while immunoreactivity against rhodopsin and shorter-wavelength opsin was observed in the upper layer. PCR analysis confirmed the expression of rhodopsin-like and opsin-like genes in human retina and the skin. These results suggest that a series of proteins, which play a crucial role in visual perception, are expressed in human epidermis.

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.