The skin barrier function resides in the epidermis, particularly in the SC, the outermost cornified layer. SC is composed of corneocytes, which is the end product of keratinocytes terminal differentiation and SC intercellular lipid, the lamellar bilayers composed of ceramides, cholesterol, and free fatty acid. Lamellar bodies (LBs), the specialized organelles in the skin play a central role in the formation of the SC lipid, protein and anti-microbial barrier via delivering pro-barrier lipids and enzymes crucial for lipid processing along with proteases and anti-microbial peptides to the SC16. Prior studies demonstrated that the rate of LBs secretion, lipid synthesis and permeability barrier homeostasis are regulated by the changes in extracellular calcium concentration of the upper epidermis, which is triggered by permeability barrier disruption567. Acute barrier disruption by topical solvent application or tape-stripping induces an immediate depletion of both extracellular calcium ions in the epidermis, especially in the upper granular layers, and results in the loss of normal epidermal calcium gradient61718. The calcium levels in the upper epidermis then progressively restored over 6~24 hours in parallel with barrier recovery6717. Inhibition of extracellular calcium loss in the upper epidermis by immersion of barrier disrupted skin in the high calcium-containing solutions or occlusion with a vapor-impermeable membrane impairs the barrier recovery71920. These findings indicate that acute loss of calcium concentration in the stratum granulosm following barrier disruption is an important regulatory signal, initiating the immediate release of pre-stored LBs contents to the SC interstices, accelerated synthesis of new LBs and epidermal lipid synthesis leading to barrier repair. In addition to the extracellular calcium contents, calcium influx into keratinocytes also regulates barrier recovery. Lee et al.45 demonstrated that L-type Ca²⁺ channel blocker, verapamil reverses the high extracellular Ca²⁺-induced inhibition of barrier recovery. Both extracellular calcium and calcium influx via calcium channels regulates epidermal permeability barrier homeostasis. Later, Choi et al.21 demonstrated that high frequency sonophoresis or iontophoresis at energies that do not cause alterations in skin barrier function can trigger LBs secretion and cytokines expression to an extent comparable with barrier disruption via triggering the change in the epidermal calcium gradient. These observations suggest that alteration of calcium levels in the outer epidermis without influencing skin barrier function can be a strategy to enhance permeability barrier function.

Calcium ion is a major regulator in keratinocyte differentiation and proliferation82223. The skin is characterized by the vertical differentiation from basal layer to SC. The basal layer contains proliferating cells. As differentiation proceeds, keratinocytes progress upwards through the different epidermal layers along with the stage-specific changes in expression of numerous differentiation markers, and finally become the terminally differentiated corneocytes in the cornified layer of the SC. Calcium plays critical roles in the all processes of keratinocyte differentiation from the commitment to differentiation in the basal and spinous layer to the terminal differentiation in the stratum granulosum. Calcium regulates the transcription of all genes encoding keratinocyte differentiation- specific proteins. Activator protein-1 (AP-1) transcription factors are present in many keratinocyte-specific genes, including transglutaminase, loricrin, involucrin, profilaggrin, and other keratins to control the transcription of various differentiation markers24. Ng et al.25 revealed that gene encoding involucrin has AP-1 responsive element in the promoter region and the AP-1 site in the involucrin gene is essential for the calcium response, suggesting that nuclear Ca²⁺ regulates synthesis of differentiation specific proteins.

The calcium contents required for the stage-specific expression of differentiation-related proteins is different between each layers of the epidermis. For example, the extracellular calcium content required for expression of profilaggrin, the late differentiation marker, is higher than that required for the keratin 1 and keratin 10 expression8. These findings suggest that the epidermal calcium gradient is essential for proper epidermal differentiation and barrier formation. Calcium is also important in posttranslational processing of profilaggrin to filaggrin. Profilaggrin has N-terminal domain containing Ca²⁺-binding motifs, which share similarity with the EF-hands of the S100 Ca²⁺-binding protein family. Calcium, via binding to the head domain of profilaggrin, induces conformational changes, thereby exposing the crucial cleavage sites of profilaggrin to initiate the processing pathway26. During the terminal differentiation, many envelope precursor proteins, including involucrin, loricrin, elafin, small proline-rich proteins, filaggrin, and keratin are covalently cross-linked to form the cornified envelope by transglutaminase 1 and the transglutaminases mediate crosslinking of cornified envelope precursors in a calcium dependent-manner27. Therefore calcium modulates cornified envelope formation during terminal differentiation.

In addition to extracellular calcium ions, calcium sensitive proteins are also known to induce keratinocyte differentiation. Protein kinase C (PKC), which is activated by the rise in diacylglycerol and intracellular calcium induces differentiation markers of granular keratinocytes, including loricrin, filaggrin, and transglutaminases282930. Among the isozymes, PKCalpha and delta are activated by calcium ions in human and murine epidermis and regulate the extracellular calcium-induced transcription of these differentiated genes2930.

Moreover, intracellular calcium dynamics play a role in keratinocyte migration and wound healing47. It is well known that asymmetries in the distribution of the intracellular Ca²⁺ concentration regulate cellular polarity, guidance, and migration via providing spatial and temporal information to control cellular extension and migration4849. Previous study has observed the asymmetric distributions of lamellipodial Ca²⁺ sparks in frequency during keratinocyte migration47. They also demonstrated that Ca²⁺-permeable channels within these cells are mechanically activated and among the mechanosensitive TRP channels, TRPV1 was revealed to be involved in the Ca²⁺ influx in response to the membrane tension during keratinocyte migration. These findings suggest that modulations of calcium and mechanosensitive TRP channels can be the potential strategies for wound healing, especially during the proliferation phase.

Recently, it is recognized that epidermal hyaluronan regulates epidermal differentiation and lipid synthesis/secretion, which in turn influence permeability barrier homeostasis through the interaction with its receptor, CD4450. Previously, our group demonstrated that permeability barrier perturbation induces the expression of hyaluronan and CD44 in murine epidermis and the epidermal calcium gradient change hyaluronan is the important signal for the barrier disruption-induced hyaluronan synthesis in keratinocytes51. In addition, the authors demonstrated that high frequency sonophoresis at the intensity that do not cause alterations in barrier function also induces epidermal hyaluronan and CD44 expression via triggering the change in the epidermal calcium gradient.

Keratinocytes express five TRPV subfamilies including four nonselective cation channels (TRPV1, TRPV2, TRPV3, and TRPV4) and one highly Ca²⁺ selective channel (TRPV6)5859. Among the TRPV channels, TRPV1, TRPV3, and TRPV6 were demonstrated to regulate keratinocyte differentiation/proliferation. TRPV1 is activated by heat (>43℃), capsaicin and low pH and showed stronger expression in the stratum basale compared to upper layers of the skin606162. TRPV1 was demonstrated to be required for the endocannabinoid-mediated suppression of keratinocyte proliferation and induction of apoptosis63. Among TRP channels, TRPV3 is primarily expressed in the skin, especially in epidermal and follicular keratinocytes and activated by innocuous warm temperatures (>33℃), chemicals, and inflammatory mediators such as arachidonic acids64. The role of TRPV3 in keratinocyte differentiation, proliferation, and skin barrier function has been unraveled from TRPV3 knockout mice model and human disease caused by the mutations in TRPV3, the so-called “TRPV3 channelopathy”65666768697071. TRPV3-deficient mice show dry skin phenotype with defective barrier formation and altered late terminal differentiation along with abnormal hair morphogenesis68. Cheng et al.68 demonstrated that TRPV3 forms a signaling complex with transforming growth factor-alpha/epidermal growth factor receptor, two growth factors that regulate keratinocyte proliferation in basal layer and differentiation in suprabasal layers, to modulate the activity of transglutaminases to induce terminal differentiation and cornified envelope formation. In addition, transgenic mice with the gain-of-function mutation of the TRPV3 gene (TRPV3Gly573Ser) and patients with Olmsted syndrome (OMIM 607066) caused by the identical or other ‘gain-of-function’ mutation of TRPV3 showed similar clinical features characterized by a pruritic and hyperkeratotic skin inflammation with massive acanthosis and hyperkeratosis in histopathological examination697071. These findings indicate that hyperactive TRPV3 in keratinocytes disrupts the balance of keratinocyte proliferation and differentiation and emphasize its relevance in inflammation and pruritus. TRPV6, a highly Ca²⁺-selective channel, has been shown to be expressed in keratinocytes and play a crucial role in Ca²⁺/1,25-dihydroxyvitamin D3-induced differentiation of keratinocytes72. Knockdown of TRPV6 in human keratinocytes has been shown to impair the Ca²⁺-induced differentiated phenotype with inhibited expression of differentiation markers as involucrin, transglutaminase-1, and cytokeratin-10. 1,25-Dihydroxyvitamin D₃ increases the expression of TRPV6 in human keratinocytes, which in turn mediates, at least in part, the pro-differentiating effects of 1,25-dihydroxyvitamin D3 by increasing Ca²⁺ entry, thereby promoting differentiation73.

Store-operated channels (SOC)-related Ca²⁺ entry is a Ca²⁺ entry pathway that is activated in response to depletion of Ca²⁺ stores within the ER, and contributes to the control of various cellular functions. Among the TRP channels, TRPC subfamily has been suggested to participate in SOC-related Ca²⁺ entry7475. TRPC1, TRPC4, and TRPC6 have been also implicated in the CaSR triggered elevation of [Ca²⁺]_i and keratinocytes differentiation76777879. Knockdown of TRPC1 and TRPC4 in human keratinocytes has been shown to prevent the induction of Ca²⁺-induced differentiation76. It was also demonstrated that activation of TRPC6 with hyperforin induces full differentiation and inhibits proliferation similar to high [Ca²⁺]_ex79. Previous studies demonstrated a defective SOC-related Ca²⁺ entry and reduced expression of TRPC1, TRPC4, and TRPC6 in psoriatic keraitnocytes. TRPC6 activation was observed to partly restore the disturbed differentiation and proliferation in psoriatic keratinocytes80. Furthermore, an up-regulation of TRPC1 was observed in keratinocytes of SERCa2+/− mice and Darier's disease patients and this upregulated TRPC1 was thought to augments cell proliferation and restrict apoptosis. These findings indicate an important role of TRPC channels-induced calcium influx in keratinocyte differentiation and proliferation81. However, it has also been demonstrated that ER Ca²⁺ release itself can promote keratinocyte differentiation, suggesting that keratinocyte differentiation is regulated by increased [Ca²⁺]_i via both Ca²⁺ release from intracellular stores, such as the ER and Ca²⁺ influx mechanisms.

Denda et al.82 has demonstrated that several TRP channels such as TRPV1, TRPV4, and TRPA1 are involved in the regulation of epidermal permeability barrier homeostasis. They found that thermal (at 42℃) or pharmacological activation of TRPV1 (capsaicin) delayed barrier recovery, whereas thermal pharmacological activation of TRPV4 (4α-Phorbol 12,13-didecanone) accelerated barrier recovery, suggesting that TRPV1 and TRPV4 play important roles in skin permeability barrier homeostasis. A later study reported that a TRPV1 inhibitor compound, PAC-14028 improved epidermal barrier function in Dermatophagoides farina-and hapten-induced atopic dermatitis murine models83. TRPV4 that is activated by moderate heat (>30℃), hypo-osmolarity, and inflammatory metabolites has been demonstrated to regulate skin barrier formation. TRPV4-deficient mice showed the impaired epidermal barrier and it was demonstrated that TRPV4 is functionally co-expressed and interacts with β-catenin and E-cadherin, the crucial components linking adherens junctions and the actin cytoskeleton, thereby enhancing the formation of the epidermal TJ barrier848586. Further, pharmacological activation of TRPV4 has shown to strengthen the epidermal tightjunction barrier87. Other TRP channels, TRPA1 (below 17℃) and TRPM8 (below 22℃), which are expressed in keratinocytes and activated by low temperature have been demonstrated to play a role in epidermal barrier homeostasis. Denda et al.88 have demonstrated that brief exposure to cold (10℃ to 15℃) or pharmacological activation of TRPA1 (allyl isothiocyanate or cinnamaldehyde) accelerated barrier recovery. They later found that exposure to low temperature (<22℃) induced elevation of intracellular calcium in cultured human keratinocytes and topical application of TRPM8 agonists (menthol and WS 12) accelerated barrier recovery in vivo89. These findings indicate that modulation of TRP channels can be a therapeutic approach for skin diseases with barrier impairment such as atopic dermatitis and psoriasis.

Although many TRP channels in sensory nerve contribute to itch, TRPV3 and TRPV4 which are mainly expressed in keratinocytes, at much higher levels than those seen in neurons, have been suggested to be involved in non-histaminergic itch. The characteristic severe itch found in mice and humans with TRPV3 gain-of-function mutations strongly suggest the involvement of TRPV3 in keratinocytes in the production and transduction of itch signal possibly through the release of itch mediators to activate neurons in dorsal root ganglia. A number of candidate mediators such as prostaglandin E₂ (PGE₂), ATP, nerve growth factor, and thymic stromal lymphopoietin (TSLP) have been demonstrated to be released by TRPV3 activation on keratinocytes90919293. TRPV4 is an osmoreceptor in the skin and has been shown to be functionally required to generate dry skin–associated itch in mice. It was also found that TRPV4 mediate serotonin-evoked itch and 5-hydroxytryptamine signal is required for TRPV4- dependent chronic itch conditions94. These findings suggest a role of TRPV3 and TRPV4 in the link between keratinocyte calcium signal, epidermal barrier, and itch.