Browsing by Author "Collier, Ann E."

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    Human keratinocyte differentiation requires translational control by the eIF2α kinase GCN2
    (Elsevier, 2017) Collier, Ann E.; Wek, Ronald C.; Spandau, Dan F.; Department of Dermatology, IU School of Medicine
    Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. While 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 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 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.
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    Human keratinocytes utilize the integrated stress response to adapt to environmental stress
    (2017-06) Collier, Ann E.; Spandau, Dan F.; Wek, Ronald C.; Travers, Jeffrey B.; Turchi, John J.; Turner, Matthew J.
    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.
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    Insulin‐like growth factor‐1 receptor regulates repair of ultraviolet B‐induced DNA damage in human keratinocytes in vivo
    (Wiley, 2016-10) Loesch, Mathew M.; Collier, Ann E.; Southern, David H.; Ward, Rachel E.; Tholpady, Sunil S.; Lewis, Davina A.; Travers, Jeffrey B.; Spandau, Dan F.; Dermatology, School of Medicine
    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., IGF‐1R is necessary for optimal repair of UVB‐induced DNA damage in keratinocytes.Novel separation technique allows study specifically in basal layer keratinocytes.IGF‐1R activation enhances the expression of nucleotide excision repair genes.
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    Translational Repression Protects Human Keratinocytes from UVB-Induced Apoptosis through a Discordant eIF2 Kinase Stress Response
    (Nature Publishing Group, 2015-10) Collier, Ann E.; Wek, Ronald C.; Spandau, Dan F.; Department of Dermatology, IU School of Medicine
    This study delineates the mechanisms by which 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) that causes decreased global protein synthesis coincident with enhanced translation of selected stress-related transcripts, such as activating transcription factor 4 (ATF4). ATF4 is a transcriptional activator of the integrated stress response (ISR) that has cytoprotective functions as well as apoptotic signals through the downstream transcriptional regulator C/EBP homologous protein (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 with 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 before 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 that was alleviated by cycloheximide (CHX), indicating that translation repression through eIF2-P is central to keratinocyte survival.