148 Disruption of nucleolar functions variably affect epidermal differentiation

Nucleoli are multifunctional organelles and the site of ribosome synthesis, which is critical for protein translation. A hallmark of epidermal differentiation is the expression of a defined set of proteins in suprabasal keratinocytes where each epidermal layer has a unique identity and protein profile. However, the link between nucleolar function and cell differentiation remains unexplored. To determine whether nucleoli play a role in the process of epidermal differentiation, we examined nucleoli number and area in different epidermal layers of mature 3D human skin equivalents (3D HSE). As keratinocytes became more differentiated, nucleolar number (fibrillarin punctae) and area (relative to the nucleus) decreased (P ≤ 0.1). Blocking differentiation by silencing EPHA2 receptor tyrosine kinase reduced the number of nucleoli in the basal layer and increased their area (P < 0.0001). These results demonstrate the dynamics of nucleoli during keratinocyte differentiation. To address whether nucleolar structure and/or ribosome synthesis are important for differentiation, we knocked down POLR1A (segregating nucleolar structure) or UTP4 (disrupting processing of rRNA without segregating nucleoli) in 3D HSE. Knockdown of either protein reduces ribosome synthesis. Assessment of tissue morphology revealed both knockdowns reduced epidermal stratification. However, there were contrasting effects on the extent of differentiation. POLR1A knockdown accelerated differentiation with increased deposition of keratohyalin granules and enhanced cornification. In contrast, loss of UTP4 impaired these aspects of terminal differentiation. Further, filaggrin expression was enhanced in siPOLR1A 3D HSE tissue sections, but reduced in UTP4 knockdown samples. The changes in nucleolar characteristics and the variable effects of nucleolar segregation on keratinocyte stratification and protein expression suggest a functional role for nucleoli in the process of epidermal differentiation. Future studies will tease out the mechanisms of nucleoli in keratinocyte differentiation.