Epidermal regeneration relies on a balance between keratinocyte proliferation and differentiation. Both processes are accompanied by cell volume changes. The anion channel LRRC8 mediates cell volume regulation upon hypotonic stress in keratinocytes and its essential subunit LRRC8A is mainly expressed in the basal, transient amplifying cell population of the human epidermis. In vitro differentiation experiments of primary keratinocytes confirmed these findings, as LRRC8A is upregulated during the onset of differentiation leading to a bell-shaped expression pattern. Therefore, we asked whether LRRC8A is also involved in the process of cell volume changes during differentiation of keratinocytes. In order to understand the role of LRRC8A we performed different loss-of-function strategies. First, we used CRISPR/Cas9 to edit LRRC8A in HaCaT cells and in the immortalized keratinocyte cell line NHEK/SV-TERT. In contrast to HaCaT cells, NHEK-SV/TERT can differentiate after Ca2+ switch in 2D and show stratification in epidermal equivalents. In both cell lines, we found aberrant differentiation in 2D and 3D experiments. Next, we used siRNA to reduce LRRC8A levels in primary keratinocytes, which can better simulate in vivo differentiation. This significantly reduced RNA and protein levels leading to a reduced ion channel activity. Surprisingly, LRRC8A knockdown did not interfere with differentiation. As LRRC8A has a short window of high expression during differentiation initiation, we specifically targeted transient amplifying cells with siRNA. Strikingly, knockdown of LRRC8A in transient amplifying cells indeed leads to reduced differentiation capability. Our work identifies LRRC8A as an essential molecule during keratinocyte differentiation, with its main function in transient amplifying cells, where LRRC8A protein is needed for the switch from proliferation to differentiation.

Epidermal regeneration relies on a balance between keratinocyte proliferation and differentiation. Both processes are accompanied by cell volume changes. The anion channel LRRC8 mediates cell volume regulation upon hypotonic stress in keratinocytes and its essential subunit LRRC8A is mainly expressed in the basal, transient amplifying cell population of the human epidermis. _In vitro_ differentiation experiments of primary keratinocytes confirmed these findings, as LRRC8A is upregulated during the onset of differentiation leading to a bell-shaped expression pattern. Therefore, we asked whether LRRC8A is also involved in the process of cell volume changes during differentiation of keratinocytes. In order to understand the role of LRRC8A we performed different loss-of-function strategies. First, we used CRISPR/Cas9 to edit LRRC8A in HaCaT cells and in the immortalized keratinocyte cell line NHEK/SV-TERT. In contrast to HaCaT cells, NHEK-SV/TERT can differentiate after Ca^2+^ switch in 2D and show stratification in epidermal equivalents. In both cell lines, we found aberrant differentiation in 2D and 3D experiments. Next, we used siRNA to reduce LRRC8A levels in primary keratinocytes, which can better simulate _in vivo_ differentiation. This significantly reduced RNA and protein levels leading to a reduced ion channel activity. Surprisingly, LRRC8A knockdown did not interfere with differentiation. As LRRC8A has a short window of high expression during differentiation initiation, we specifically targeted transient amplifying cells with siRNA. Strikingly, knockdown of LRRC8A in transient amplifying cells indeed leads to reduced differentiation capability. Our work identifies LRRC8A as an essential molecule during keratinocyte differentiation, with its main function in transient amplifying cells, where LRRC8A protein is needed for the switch from proliferation to differentiation.

ESDR202 - The importance of LRRC8 ion channel in keratinocyte differentiation

The scientific meeting of the European Society for Dermatological Research (ESDR) is held yearly, usually in September. With an international attendance of more than 1000 skin scientists from around the globe, the four-day event includes short oral presentations from peer-reviewed abstracts, keynote lectures from international experts, sponsored symposia, satellite programs, educational events as well as an important focus on posters, networking and building collaborations amongst scientists. For more information about this year’s meeting visit our website.