Human keratinocytes utilize the integrated stress response to adapt to environmental stress

Abstract

Human skin, consisting of the outer epidermis and inner dermis, serves as a barrier that protects the body from an onslaught of environmental stresses. Keratinocytes in the stratified epidermis undergo sequential differentiation that consists of multiple layers of cells differing in structure and function. Therefore, keratinocytes must not only combat environmental stress, but need to undergo massive changes in gene expression and morphology to form a proper barrier. One mode by which cells cope with stress and differentiation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P), which causes global inhibition of protein synthesis coincident with preferential translation of select gene transcripts. Translational repression allows stressed cells to conserve energy and prioritize pro-survival processes to alleviate stress damage. Since eIF2α kinases are each activated by distinct types of stress, this pathway is referred to as the Integrated Stress Response (ISR). We sought to identify the roles of the ISR in the keratinocyte response to the stresses associated with differentiation and ultraviolet B (UVB) irradiation. In this thesis, we show that both general and gene-specific translational control in the ISR are activated following differentiation or UVB irradiation of human keratinocytes. ISR deficiency through genetic modifications or pharmacological interventions caused severe divergence from the appropriate keratinocyte response to differentiation or UVB. Differentiation genes were selectively translated by eIF2α-P, and inhibition of the ISR diminished their induction during differentiation. Furthermore, loss of the eIF2α kinase GCN2 (EIF2AK4) adversely affected the ability of keratinocytes to stratify in three dimensional cultures. Our analysis also revealed a non-canonical ISR response following UVB irradiation, in which downstream factors ATF4 (CREB2) and CHOP (DDIT3/GADD153) were poorly expressed due to repressed transcription, despite preferential translation in response to eIF2α-P. The ISR was cytoprotective during UVB and we found that eIF2α-P was required for a UVB induced G1 arrest, cell fate determination, and DNA repair via a mechanism involving translational control of human CDKN1A (p21 protein) transcript variant 4 mRNA. Collectively, this thesis describes novel roles for the ISR in keratinocyte differentiation and response to UVB, emphasizing the utility of targeting translational control in skin disease therapy.