The human skin forms an essential barrier, which not only protects the body from the environment but also from transepidermal water loss. Various detrimental factors such as UV radiation, mechanical tension, different humidity levels, air pollution and irritating chemical substances can have tremendous impact on the skin as they contribute to barrier disruption and inflammation, dry and fragile skin as well as premature skin ageing. Hence it is of major interest to develop cosmetic treatment approaches that support the formation of a healthy skin barrier. Here we present a novel, targeted approach based on modulating the TRPV4 ion channel by small-molecule bioactive compounds to improve epidermal barrier formation.
The TRPV4 (transient receptor potential vanilloid 4) ion channel belongs to the family of temperature-sensitive Ca²⁺-permeable channels. While it was recently shown that TRPV4 plays a role in epidermal barrier integrity in other tissues, its function in the human skin is not well understood. We therefore extensively characterized TRPV4 and its function in human keratinocytes using different techniques such as CRISPR/Cas9 genome editing, electrophysiological patch clamp analysis and live cell Ca²⁺ imaging. We determined the contribution of TRPV4 to epidermal development by analyzing Ca²⁺-induced in vitro 2D differentiation and 3D barrier function assays using reconstituted epidermal equivalents. In addition, we created a novel spheroid-based 3D epidermal model, which permits both the study of epidermal development and the study of real-time confocal Ca²⁺ live cell imaging with cellular resolution in a 3D organization.
We discovered that expression of TRPV4 changes during keratinocyte differentiation and that absence of TRPV4 in keratinocyte knockout cells led to increased expression of differentiation marker genes. TRPV4 knockout cells completely lacked agonist-induced Ca²⁺ fluxes as measured by electrophysiological patch clamping and by live cell Ca²⁺ imaging. By applying our novel 3D epidermal spheroid model, we achieved to visualize the dynamic Ca²⁺ fluxes occurring within the different epidermal layers of the spheroids after specific TRPV4 activation. We revealed that TRPV4-dependet Ca²⁺ uptake was restricted to the outer differentiated layers whereas the inner proliferating core of the spheroid was not responding. In agreement with this finding, we observed increased cytokeratin 10 and involucrin staining in the outer cellular layers when TRPV4 was absent. Knockout of TRPV4 also enhanced the penetration of lucifer yellow dye in reconstituted 3D epidermal equivalents, which is indicative for a disrupted barrier.
We next aimed at identifying bioactive compounds that are capable of modulating TRPV4 ion channel activity. We therefore combined our cell-based functional TRPV4 assays with our proprietary cherry-picked library composed of natural compounds and performed a pilot screening campaign. We uncovered natural small-molecule activators and inhibitors of TRPV4, which enhanced or inhibited Ca²⁺ uptake in keratinocytes, respectively. Strikingly, the TRPV4 modulators were also capable of enhancing or suppressing keratinocyte in vitro differentiation and barrier function depending on their mode of modulation highlighting the great potential of TRPV4 as a new molecular target for improving epidermal barrier function.
In summary, our study suggests a crucial role of TRPV4 for mediating Ca²⁺ fluxes in the human epidermis. TRPV4 seems to contribute to late epidermal differentiation and maturation and is required for intercellular barrier formation. Moreover, we identified a first set of small-molecule compounds that not only modulate TRPV4 activity but also modulate in vitro epidermal development and barrier function. These BioActives serve as a promising starting point for the development of novel cosmetic ingredients for skin care products to improve the barrier function of the skin.