Ann E. Collier

Translational Repression Protects Human Keratinocytes from UVB-Induced Apoptosis through a Discordant eIF2 Kinase Stress Response

This study delineates the mechanisms by which ultraviolet B (UVB) regulates protein synthesis in human keratinocytes and the importance of translational control in cell survival. Translation initiation is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2~P), which causes decreased global protein synthesis coincident with enhanced translation of selected stress-related transcripts, such as ATF4. ATF4 is a transcriptional activator of the Integrated Stress Response (ISR), which has cytoprotective functions as well as apoptotic signals through the downstream transcriptional regulator CHOP (GADD153/DDIT3). We determined that UVB irradiation is a potent inducer of eIF2~P in keratinocytes, leading to decreased levels of translation initiation. However, expression of ATF4 or CHOP was not induced by UVB as compared to traditional ISR activators. The rationale for this discordant response is that ATF4 mRNA is reduced by UVB, and despite its ability to be preferentially translated there are diminished levels of available transcript. Forced expression of ATF4 and CHOP protein prior to UVB irradiation significantly enhanced apoptosis, suggesting that this portion of the ISR is deleterious in keratinocytes following UVB. Inhibition of eIF2~P and translational control reduced viability following UVB, which was alleviated by cycloheximide, indicating that translation repression through eIF2~P is central to keratinocyte survival.

Insulin-like growth factor-1 receptor regulates repair of ultraviolet B-induced DNA damage in human keratinocytes in vivo

The activation status of the insulin‐like growth factor‐1 receptor (IGF‐1R) regulates the cellular response of keratinocytes to ultraviolet B (UVB) exposure, both in vitro and in vivo. Geriatric skin is deficient in IGF‐1 expression resulting in an aberrant IGF‐1R‐dependent UVB response which contributes to the development of aging‐associated squamous cell carcinoma. Furthermore, our lab and others have reported that geriatric keratinocytes repair UVB‐induced DNA damage less efficiently than young adult keratinocytes. Here, we show that IGF‐1R activation influences DNA damage repair in UVB‐irradiated keratinocytes. Specifically, in the absence of IGF‐1R activation, the rate of DNA damage repair following UVB‐irradiation was significantly slowed (using immortalized human keratinocytes) or inhibited (using primary human keratinocytes). Furthermore, inhibition of IGF‐1R activity in human skin, using either ex vivo explant cultures or in vivo xenograft models, suppressed DNA damage repair. Primary keratinocytes with an inactivated IGF‐1R also exhibited lower steady‐state levels of nucleotide excision repair mRNAs. These results suggest that deficient UVB‐induced DNA repair in geriatric keratinocytes is due in part to silenced IGF‐1R activation in geriatric skin and provide a mechanism for how the IGF‐1 pathway plays a role in the initiation of squamous cell carcinoma in geriatric patients.

Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2

Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. Although transcriptional regulation has proven to be important for keratinocyte differentiation, little is known about the role of translational control. A key mechanism for modulating translation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2). A family of different eIF2α kinases function in the integrative stress response to inhibit general protein synthesis coincident with preferential translation of select mRNAs that participate in stress alleviation. Here we demonstrate that translational control through eIF2α phosphorylation is required for normal keratinocyte differentiation. Analyses of polysome profiles revealed that key differentiation genes, including involucrin, are bound to heavy polysomes during differentiation, despite decreased general protein synthesis. Induced eIF2α phosphorylation by the general control nonderepressible 2 (GCN2) protein kinase facilitated translational control and differentiation-specific protein expression during keratinocyte differentiation. Furthermore, loss of GCN2 thwarted translational control, normal epidermal differentiation, and differentiation gene expression in organotypic skin culture. These findings underscore a previously unknown function for GCN2 phosphorylation of eIF2α and translational control in the formation of an intact human epidermis.

Translational control of a human CDKN1A mRNA splice variant regulates the fate of UVB-irradiated human keratinocytes

Phosphorylation of eIF2 and the integrated stress response in response to UVB are cytoprotective by a mechanism featuring preferential translation of a specific splice variant of CDKN1A that facilitates G1 arrest and subsequent DNA repair.