Keratinocytes in Research: Epidermal Differentiation and Senescence

Function and Differentiation of Keratinocytes

Keratinocytes make up the majority of cells in the epidermis, the outer layer of the skin. The progenitor cells that give rise to keratinocytes are found in the basal epidermal layer. As these cells proliferate and differentiate into keratinocytes, they move to the upper layers of the epidermis. Once they reach the stratum corneum (outermost epidermal layer), they become post-mitotic, dead cells that are referred to as corneocytes. These corneocytes form the tough, protective outer layer of skin and are continually shed as new corneocytes are made.

Lifeline® Keratinocytes in Epidermal Differentiation and Senescence Research

The process of differentiation from a progenitor cell to mature differentiated cell involves the coordinated efforts of chromatin modifiers like Polycomb group proteins (PcG; epigenetic silencers) and Trithorax group proteins (trxG; epigenetic activators). Previous research has shown that transcription repression by PcG maintains epidermal progenitor cells and release of this repression stimulates the differentiation program. However, the roles of activating trxG in this process are less well studied. In a study from 2012, Hopkin et al. set out to define how trxG-mediated gene regulation is involved in epidermal differentiation. In the epidermis, the Grainyhead transcription factor (GRHL3) is an important regulator of differentiation.

To determine how GRHL3 mediates gene regulation, the authors used Lifeline® human neonatal epidermal keratinocytes (NHEKs) and differentiated them in vitro using calcium. The authors first confirmed that the enzyme Transglutaminase 1 (TGM1), which is involved in the formation of the cornified cell envelope, was a direct target of GRHL3. Additionally, increased occupancy of the TGM1 promoter by GRHL3 was associated with increased levels of H3K4me3 marks (indicating an active promoter), which were mediated by GRHL3 expression. Using siRNA knockdown of epidermal trxG members, the authors found that MLL2 regulates TGM1 methylation in a GRHL3-dependent manner.

To more fully understand how GRHL3 and MLL2 globally regulate epidermal differentiation, the researchers performed microarray analysis following loss of GRHL3 or MLL2 in NHEKs. The largest overlap was in terminal differentiation genes, suggesting that GRHL3 and MLL2 acts during terminal differentiation. Using their microarray data and co-immunoprecipitation assays to confirm binding, the authors identified a set of genes that are targets of GRHL3-mediated MLL2 binding. Next, to examine whether GRHL3 could bind trxG complex proteins, the authors used co-immunoprecipitation to confirm that GRHL3 bound WDR5, an important member of the trxG complex.

Using chromatin immunoprecipitation and sequencing (ChIP-seq), the group then examined GRHL3-WDR5 occupancy in differentiated NHEK cells on a genome-wide level, and found that the majority of genes with a GRHL3 signal also had a WDR5 signal, suggesting that GRHL3 recruitment of WDR5 facilitates epidermal differentiation.
Finally, to understand how trxG-mediated regulation of epidermal differentiation works along with PcG-mediated regulation, the authors performed ChIP assays at different time points following induction of differentiation to examine H3K4me3 and H3K27me3 (a repressive chromatin mark) on GRHL3-MLL2 targets. Interestingly, they identified two sets of genes: those activated by PcG-trxG co-regulation and those activated by trxG only.
Together, the results of this study demonstrate that along with PcG proteins, trxG proteins are also key regulators of epidermal differentiation. The authors suggest that since changes in differentiation underly the development of multiple skin diseases like psoriasis, interrogation of these differentiation pathways may lead to advances in the development of new therapies.

Calmodulin-like skin protein (CLSP) expression is a key modulator of keratinocyte differentiation. In a study from this year, Takahara and colleagues investigated whether CLSP plays a role in keratinocyte senescence. Using Lifeline® NHEKs, which differentiate and undergo senescence over the course of 6-14 days in culture, the authors demonstrated that CLSP expression increases over time as NHEKs become senescent. The authors next induced senescence by treating NHEKs with H2O2 or UV-A/UV-B light and found CLSP expression also increased under these senescence-inducing conditions. To determine the role of CLSP during senescence, the authors treated cells with recombinant CLSP after inducing senescence with H2O2 or UV-B light. Interestingly, CLSP treatment decreased senescence under both conditions, suggesting that CLSP is induced by cells to combat senescence. Although the authors were unable to determine the mechanism behind CLSP-induced reduction in cellular senescence, they hypothesized it was through the heterotrimeric humanin receptor.

Check out the Lifeline® catalog for our keratinocyte cell models, which are optimized for growth in our DermaLife® medium:

• Oral keratinocytes
• Neonatal epidermal keratinocytes
• Adult epidermal keratinocytes
• Epidermal keratinocytes (10-donor pool)

Let us know how you are using Lifeline® keratinocytes to answer your research questions and your published study could be featured on our next blog!