Importance of transient receptor potential vanilloid 4 (TRPV4) in epidermal barrier function in human skin keratinocytes.

The state of the skin changes drastically depending on the ambient temperature. Skin epidermal keratinocytes express thermosensitive transient receptor potential vanilloid (TRPV) cation channels, TRPV3 and TRPV4. These multimodal receptors are activated by various kinds of chemical and physical stimuli, including warm temperatures (>30°C). It has been suggested that TRPV4 is involved in cell-cell junction maturation; however, the effect of temperature fluctuations on TRPV4-dependent barrier homeostasis is unclear. In the present study, we demonstrated that activation of TRPV4 was crucial for barrier formation and recovery, both of which were critical for the prevention of excess dehydration of human skin keratinocytes. TRPV4 activation by physiological skin temperature (33°C), GSK1016790A or 4α-PDD allowed influx of Ca(2+) from extracellular spaces which promoted cell-cell junction development. These changes resulted in augmentation of intercellular barrier integrity in vitro and ex vivo. TRPV4 disruption reduced the increase in trans-epidermal resistance and increased intercellular permeation after a Ca(2+) switch. Furthermore, barrier recovery after the disruption of the stratum corneum was accelerated by the activation of TRPV4 either by warm temperature or a chemical activator. Our results suggest that physiological skin temperatures play important roles in cell-cell junction and skin barrier homeostasis through TRPV4 activation.

Tight junction regulates epidermal calcium ion gradient and differentiation.

It is well known that calcium ions (Ca(2+)) induce keratinocyte differentiation. Ca(2+) distributes to form a vertical gradient that peaks at the stratum granulosum. It is thought that the stratum corneum (SC) forms the Ca(2+) gradient since it is considered the only permeability barrier in the skin. However, the epidermal tight junction (TJ) in the granulosum has recently been suggested to restrict molecular movement to assist the SC as a secondary barrier. The objective of this study was to clarify the contribution of the TJ to Ca(2+) gradient and epidermal differentiation in reconstructed human epidermis. When the epidermal TJ barrier was disrupted by sodium caprate treatment, Ca(2+) flux increased and the gradient changed in ion-capture cytochemistry images. Alterations of ultrastructures and proliferation/differentiation markers revealed that both hyperproliferation and precocious differentiation occurred regionally in the epidermis. These results suggest that the TJ plays a crucial role in maintaining epidermal homeostasis by controlling the Ca(2+) gradient.

Perturbation of lamellar granule secretion by sodium caprate implicates epidermal tight junctions in lamellar granule function.

BACKGROUND: Polarized secretion of lamellar granules (LGs) delivers various lipids, proteases, and protease inhibitors into the stratum corneum (SC) of the epithelium. Disruption of LGs is associated with severe cutaneous diseases, but the mechanism of their polarized secretion is not known. On the other hand, recent study shows epidermal tight junctions (TJs) localize in stratum granulosum (SG), and TJs are involved in polarized molecule secretion. Thus, we hypothesized epidermal TJs relate to polarized LGs secretion. OBJECTIVE: To assess the possibility that epidermal TJs are involved in polarized LGs secretion. METHODS: In order to examine LGs secretion, we used fluorescent ceramide (BODIPY FL C(5)-ceramide) and a natural LG cargo, lympho-epithelial Kazal-type-related inhibitor (LEKTI), in cultured normal human epidermal keratinocytes and a reconstructed human epidermis. We investigated their alteration using the medium-chain fatty acid sodium caprate (C10), TJs inhibitor. In addition, LG distribution was observed by electron microscopy. RESULTS: C10 significantly inhibited secretion of both fluorescent ceramide and LEKTI in cultured normal human epidermal keratinocytes and a reconstructed human epidermis. C10 also disturbed the polarized localization of fluorescent ceramide and LEKTI in the reconstructed epidermis. Electron microscopy revealed that a large number of LGs remained in corneocytes in the C10-treated epidermis, rather than being secreted. CONCLUSION: Our data indicate that C10 perturbs the polarized secretion of LGs. Our study therefore suggests that epidermal TJs are possibly involved in polarized LG secretion and provides new insights into potential of treatments for skin diseases caused by abnormal LG secretion.

Regulation of tight junction permeability by sodium caprate in human keratinocytes and reconstructed epidermis.

Tight junctions (TJs) restrict paracellular flux of water and solutes in epithelia and endothelia. In epidermis, the physiological role of TJs is not fully understood. In this study, sodium caprate (C10), which dilates intestinal TJs, was applied to cultured human epidermal keratinocytes and reconstructed human epidermis to investigate the effects of C10 on epidermal TJs. C10 treatment decreased transepithelial electrical resistance and increased paracellular permeability, although Western blots showed that the expression of TJ-related transmembrane proteins was not decreased. The effects of C10 were reversible. Immunofluorescence microscopy and immuno-replica electron microscopy showed that the localization of TJ strands were disintegrated, concomitant with the dispersion and/or disappearance of TJ-related molecules from the cell surface. These findings suggest that C10 impairs barrier function by physically disrupting TJ conformation in the epidermis. Furthermore, these results also show that proper localization of the molecules on the cellular membrane is important for TJ barrier function.

Relationships between changes in mechanical properties of the skin, wrinkling, and destruction of dermal collagen fiber bundles caused by photoaging.

BACKGROUND/PURPOSE: Long-term exposure to ultraviolet (UV) radiation induces various cutaneous changes that differ from those because of physiological aging, including structural destruction of dermal collagen fiber bundles (DCFBs), which comprise the major component of the dermis. Wrinkling, a representative change in skin surface associated with photoaging, is often seen at the corners of the eyes and in the space between the eyebrows. These are locations where the skin contracts repeatedly and routinely. Lowered resiliency to skin contraction induced by marked structural changes in DCFBs may represent one cause of photo-induced wrinkles. Using animal models of photoaging, changes in mechanical properties of the skin caused by UV irradiation were measured, and relationships between UV-induced changes were analyzed. METHODS: Animal models of photoaging were prepared by irradiating hairless mice with UVB light. Dorsal skin surface replicas of animals were taken using silicon rubber, and volume of wrinkles was calculated using an image analyzer. Stress of the skin against horizontal contraction was measured using a new device called the Resiliometer. Three-dimensional organization of dermal collagen structures in skin samples collected from the back of each animal was observed under scanning electron microscopy, and compactness of DCFBs was assessed from electron micrography. RESULTS: With time and therefore increasing UV dose, deep wrinkles formed on the backs of mice. Volume of wrinkles peaked at 8 weeks. All parameters obtained from Resiliometer measurements were increased by irradiation. DCFB structure was degraded in a radiation dose-dependent manner. DCFB grading was significantly correlated with each Resiliometer parameter. Significant correlations were also observed between each Resiliometer parameter and volume of wrinkles. CONCLUSION: Stress of the skin against horizontal contraction obtained using the Resiliometer changes following UV irradiation, correlating with photo-induced wrinkling and destruction of DCFBs. These results support the hypothesis that changed force of restitution to skin contraction induced by marked structural changes in DCFBs represents one cause of photo-induced wrinkles. The resiliometric parameter may offer a good indicator for monitoring the condition of DCFB structure, as changes in these would induce failure in restitution to skin contraction, leading to wrinkling.