DNA breakdown in epidermal keratinocytes

The outermost layer of the skin, the stratum corneum, functions as an efficient barrier against entry of chemicals and microbes from the environment and protects the body from dehydration. The stratum corneum is formed by terminal differentiation of keratinocytes, the predominant cell type in the epidermis. The final differentiation step of epidermal keratinocytes is the conversion of living cells into corneocytes, i.e. rigid cell remnants devoid of all intracellular organelles including the nucleus. This special mode of programmed cell death involves the complete breakdown of nuclear DNA and thereby terminates the genetic control of the cellular metabolism. Aberrant retention of nuclear DNA in corneocytes is characteristic of diseases such as psoriasis and certain forms of ichthyosis. Although the breakdown of the nucleus is known to be a key step in stratum corneum formation, the molecular mechanisms underlying this process are poorly understood at present. In a previous study we have identified DNase1-like 2 (DNase1L2) as a keratinocyte-specific endonuclease. In vitro DNase1L2 is essential for the breakdown of nuclear DNA during keratinocyte differentiation in a human skin equivalent model and in vivo it is strongly expressed in the course of all keratinocyte differentiation processes that result in cell death, namely the formation of stratum corneum, sebum, hair, and nail. An additional role of DNase1L2 in the antimicrobial defence of the skin is suggested by our finding that DNase1L2 is present in its catalytically active form in the stratum corneum and that it suppresses the establishment of bacterial biofilms in a standard in vitro assay. The aims of this project are (1) to determine the role of DNase1L2 in DNA degradation during keratinocyte differentiation in vivo, (2) to characterize the consequences of defective nuclear breakdown on stratum corneum formation and function, and (3) to assess the molecular regulation of DNase1L2 activity in keratinocytes. DNase1L2 expression will be ablated by gene knockout in the mouse and by siRNA-mediated knockdown in a human skin equivalent model. The effects of DNase1L2 deficiency will be analyzed at the molecular, cellular, and histological levels. This study will define the mechanism and physiological role of DNA breakdown in the course of the formation of the stratum corneum. The results will have implications for the prevention and treatment of skin diseases associated with epidermal barrier defects.

The stratum corneum of the skin protects the body against desiccation and prevents the entry of toxic substances and microbes from the environment. Like hair and nails, the stratum corneum is produced by the main cell type of the epidermis, i.e. the keratinocytes, in a process that involves cell death and the degradation of DNA in the nucleus. The aim of our research is to uncover the molecular mechanisms of DNA degradation in epidermal keratinocytes. In this project the DNA-degrading enzyme DNase1l2, which is expressed specifically in the epidermis, was deleted in a gene knockout mouse model. Histological and biochemical studies showed that the absence of DNase1L2 leads to incomplete degradation of DNA in hairs and nails whereas the stratum corneum appeared normal, at least under non-stressed conditions. Investigations of human and murine skin revealed the existence of several DNases which, besides DNase1L2, are active in the stratum corneum. The generation of the DNase1L2 knockout mouse and the preliminary characterization of further epidermal DNases in this project provides the basis for a follow-up project which is also funded by the Austrian Science Fund. The impact of non-degraded DNA on physiologically relevant properties of hair and claws will be investigated in the DNase1L2 knockout mouse. Furthermore, the mouse model will be used for studies on the role of DNase1L2 in stressed skin, and the characterization and identification of epidermal DNases will be continued. Together, these projects will help to explain the role of DNA breakdown in the formation of hair, nails and stratum corneum and to understand the molecular processes that underlie the disturbances in DNA degradation in diseases such as psoriasis.