102 Loss of volume-regulated anion channel LRRC8 interferes with cell volume regulation and epidermal homeostasis

Abstract

The volume-regulated anion channel LRRC8 contributes to cell volume regulation upon hypoosmotic stress in keratinocytes. In a hypotonic environment water enters the cell and leads to an increase in cell volume, which is counteracted by a chloride efflux mediated by LRRC8. We found that the essential LRRC8 subunit namely LRRC8A is mainly expressed in the basal epidermal layers with highest expression in transient amplifying cells. Thus, we hypothesize that it potentially plays an important role during the switch between proliferation and differentiation in these cells. To analyze the function of LRRC8A in human keratinocytes, we established a siRNA-mediated knockdown in primary keratinocytes. These cells displayed aberrant expression of involucrin and keratin 10 in differentiating cells, while filaggrin and keratin 14 were unchanged. To overcome experimental variations and get a better understanding of the underlying processes, we generated an LRRC8A- knockout cell line from immortalized keratinocytes using CRISPR/Cas9. The knockout was validated on DNA and protein levels and further verified by manual patch clamping, which showed complete disappearance of VRAC-mediated ionic currents. LRRC8A-/- cells showed a reduced proliferation rate and disturbed epidermal maturation in 2D differentiation assays. Interestingly, LRRC8A-/- cells were unable to reconstitute a 3D epidermis in vitro, indicating that LRRC8 is potentially involved in differentiation initiation. Taken together, our findings suggest that LRRC8 ion channels control cell volume changes in the epidermis, which could represent an essential mechanism, when keratinocytes leave the proliferative, basal compartment and initiate differentiation. Further work will indicate via which molecular processes LRRC8 regulates epidermal renewal and contributes to skin homeostasis.