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.

Relevance of the directionality of skin elasticity to aging and sagging of the face.

BACKGROUND: Forces acting in facial skin have been suggested to show directionality. Non-invasive methods of measuring this directionality may thus provide information related to aging processes. The Reviscometer(®) RVM600 device is capable of measuring directionality of forces on the skin. This device has not been used previously in a published study to evaluate changes in directionality of forces on facial skin with aging. AIM: The first objective of this pilot study was to investigate relationships between mechanical directionality using the Reviscometer(®) RVM600, the Cutometer(®) MPA580, and aging of the facial skin in a supine position. In addition, the study investigated relationships between mechanical directionality and 'skin sagging,' which may be caused by gravity. To validate this as a new measurement of mechanical directionality, we also performed double-blinded trials on two groups of subjects, with one group using a product containing an anti-aging substance and the other group using a placebo product without an anti-aging substance. METHODS: We examined 91 healthy Japanese women with a mean age of 48.5 years (range, 20-79 years) at the three sites on the face using the Reviscometer(®) RVM600 and the Cutometer(®) MPA580, and evaluation was performed for skin sagging in September and November 2008, and January 2009. The Reviscometer(®) RVM600 was used to measure resonance-running time (RRT) every 10° from 0° to 350°. Evaluation of skin sagging was undertaken by making marks on the face and using face photographs taken in both sitting and supine positions to calculate the sagging index. Usage testing was conducted on 38 healthy Japanese women in a double-blinded study with one group, using a preparation containing Yomogi AGEs Clearing (YAC) extract and another group using the same preparation without the YAC extract from October 2008 to April 2009. Mean age of these subjects was 44.0 years (range, 30-60 years). Measurements were taken at the three sites on the face using the Reviscometer(®) RVM600 and the Cutometer(®) MPA580 and sagging index. RESULTS: A significant correlation was identified between RRT parameters and subject age at all three measurement sites. Significant correlations between sagging index and RRT values were found for 110-170° and 290-350° only at the center of the cheek. Significant differences in RRT values were noted for 110-150° and 300-350° at this site between subjects with and without the use of YAC extract. A similar trend was found in sagging index for this site alone between subjects with and without YAC extract. CONCLUSION: The use of non-invasive procedures to measure skin mechanical parameters on the face in all directions may evaluate aging and effective preventive and restorative support.

A novel agent, methylophiopogonanone B, promotes Rho activation and tubulin depolymerization.

Cytoskeletal reorganization, including reconstruction of actin fibers and microtubules, is essential for various biological processes, such as cell migration, proliferation and dendrite formation. We show here that methylophiopogonanone B (MOPB) induces cell morphological change via melanocyte dendrite retraction and stress fiber formation. Since members of the Rho family of small GTP-binding proteins act as master regulators of dendrite formation and actin cytoskeletal reorganization, and activated Rho promotes dendrite retraction and stress fiber formation, we studied the effects of MOPB on the small GTPases using normal human epidermal melanocytes and HeLa cells. In in vitro binding assay, MOPB significantly increased GTP-Rho, but not GTP-Rac or GTP-CDC42. Furthermore, a Rho inhibitor, a Rho kinase inhibitor and a small GTPase inhibitor each blocked MOPB-induced stress fiber formation. The effect of MOPB on actin reorganization was blocked in a Rho dominant negative mutant. These results suggest MOPB acts via the Rho signaling pathway, and it may directly or indirectly activate Rho. Quantitative Western blot analysis indicated that MOPB also induced microtubule destabilization and tubulin depolymerization. Thus, MOPB appears to induce Rho activation, resulting in actin cytoskeletal reorganization, including dendrite retraction and stress fiber formation.

Centaureidin promotes dendrite retraction of melanocytes by activating Rho.

Melanosomes synthesized within melanocytes are transferred to keratinocytes through dendrites, resulting in a constant supply of melanin to the epidermis, and this process determines skin pigmentation. During screening for inhibitors of melanosome transfer, we found a novel reagent, centaureidin, that induces significant morphological changes in normal human epidermal melanocytes and inhibits melanocyte dendrite elongation, resulting in a reduction of melanosome transfer in an in vitro melanocyte-keratinocyte co-culture system. Since members of the Rho family of small GTP-binding proteins act as master regulators of dendrite formation, and activated Rho promotes dendrite retraction, we studied the effects of centaureidin on the small GTPases. In in vitro binding assay, centaureidin activated Rho and furthermore, a Rho inhibitor (C. botulinum C3 exoenzyme), a Rho kinase inhibitor (Y27632) and a small GTPase inhibitor (Toxin B) blocked dendrite retraction induced by centaureidin. These results suggest centaureidin could act via the Rho signaling pathway, and it may directly or indirectly activate Rho. Thus, centaureidin appears to inhibit dendrite outgrowth from melanocytes by activating Rho, resulting in the inhibition of melanosome transfer from melanocytes to keratinocytes.