Ann E. Collier, Ronald C. Wek, Dan F. Spandau

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Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2 Ann E. Collier, Ronald C. Wek, Dan F. Spandau Journal of Investigative Dermatology Volume 137, Issue 9, Pages 1924-1934 (September 2017) DOI: 10.1016/j.jid.2017.04.029 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Global translation initiation is repressed during keratinocyte differentiation. (a) Phase-contrast images of undifferentiated (Undiff) and differentiated (Diff) N-TERT keratinocytes generated by switching confluent N-TERT keratinocytes to a media containing 2 mM Ca2+/2% fetal bovine serum for 72 hours. (b) Polysome profiles for these conditions are shown. No CHX (blue) indicates that cycloheximide was omitted from the protocol. (c) 3D organotypic cultures were fixed at day 0 (Undiff) and 7 (Diff) after raising to the air-liquid interface and stained with hematoxylin and eosin. (d) Polysome profiles generated for these conditions compared with an undifferentiated monolayer control are shown. For all polysome profiles, the ratio of polysomes to monosomes (p/m) is indicated to the right of each sample label. Scale bars = 50 μm. CHX, cycloheximide. Journal of Investigative Dermatology 2017 137, 1924-1934DOI: (10.1016/j.jid.2017.04.029) Copyright © 2017 The Authors Terms and Conditions

Figure 2 The integrated stress response is activated in differentiated keratinocytes. (a) Undifferentiated (Undiff), differentiated (Diff), and tunicamycin (TM) treated N-TERT keratinocytes were subjected to immunoblot analysis to measure levels of the indicated proteins. (b) Levels of eIF2α-P normalized to total eIF2α as measured by densitometry are indicated. (c) Alternatively, RNA was collected from samples and total mRNA levels were measured for IVL. (d) Keratinocyte differentiation was also monitored for the indicated number of days and subjected to immunoblot analysis. (e) Full-thickness skin was stained for antibodies against eIF2α-P, ATF4, CHOP, or an IgG isotype control. The basement membrane is demarcated with a gray line. Scale bars: large image = 50 μm, inset = 25 μm. Error bars = mean ± SD. ∗P < 0.05. ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; eIF2α, eukaryotic initiation factor 2 alpha; eIF2α-P, phosphorylated eukaryotic initiation factor 2; IVL, involucrin; KRT1, keratin 1; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1924-1934DOI: (10.1016/j.jid.2017.04.029) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Gene-specific translational control during keratinocyte differentiation. RNA was isolated from sucrose fractions taken from polysome profiles in Figure 1b and used to generate cDNA. qPCR was used to measure mRNA levels of (a) ATF4, (b) CHOP, (c) IVL, and (d) eIF4E in each fraction. Levels of mRNA were normalized to a spike-in luciferase transcript, and represented as a percent total for each mRNA so as to omit changes in gene transcript levels. Arrows represent shifts toward (green) or away from (red) heavy polysomes (fractions 5–7), and the total percentage of each gene transcript that shifts during differentiation is indicated. Error bars = mean ± SD. ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; IVL, involucrin; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1924-1934DOI: (10.1016/j.jid.2017.04.029) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Inhibition of the ISR suppresses keratinocyte differentiation. (a) Polysome profiles were generated for undifferentiated (Undiff), differentiated (Diff+Vehicle), and differentiated during GADD34 overexpression (Diff+DOX) N-TERT keratinocytes grown in monolayer culture. Polysome/monosome (p/m) ratios are listed beside each sample. (b) Alternatively, lysates were subjected to immunoblot analysis to measure the indicated protein levels. (c) Measurement of eIF2α-P normalized to total eIF2α and IVL proteins is indicated. (d) p/m values were calculated for each indicated gene in differentiated cells treated with vehicle or DOX to induce GADD34 overexpression that sharply lowers eIF2α-P by dividing the percent of the gene transcript in polysome (5–7) by monosome (1–3) sucrose fractions. (e) IVL p/m values are indicated. (f) Alternatively, total RNA was isolated from cells and qPCR was performed to measure levels of IVL mRNA. *P < 0.05, error bars = mean ± SD. ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; DOX, doxycycline; eIF2α, eukaryotic initiation factor 2 alpha; eIF2α-P, phosphorylated eukaryotic initiation factor 2; GADD34, growth arrest and DNA damage protein 34; ISR, integrated stress response; IVL, involucrin; KRT1, keratin 1; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1924-1934DOI: (10.1016/j.jid.2017.04.029) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Loss of GCN2 abrogates differentiation gene expression. (a) GCN2 mRNA knockdown efficiency was measured by qPCR. (b) Undifferentiated (Undiff), differentiated (Diff), or tunicamycin (TM) treated N-TERT keratinocytes with control (shCTRL) or shGCN2 shRNA were subjected to immunoblot analysis to measure the indicated proteins. (c) Measurement of eIF2α-P normalized to total eIF2α and IVL proteins is indicated. (d) RNA was also isolated from these treatments and IVL mRNA levels were measured by qPCR. (e) An mRNA half-life assay was performed by treating shCTRL or shGCN2 cells that had been allowed to differentiate for 24 hours with 10 μg/ml actinomycin D (AD). RNA was isolated from each sample after the indicated number of hours, and qPCR was performed to measure levels of IVL mRNA. (f) N-TERTs were left untreated (NT), or treated with 2 μM TM or 100 nM halofuginone (HF) for 6 hours or were differentiated. Lysates were then subjected to immunoblot analysis to measure GCN2 activation. *P < 0.05. Error bars = mean ± SD. CA, calcium; eIF2α, eukaryotic initiation factor 2 alpha; eIF2α-P, phosphorylated eukaryotic initiation factor 2; GCN2, general control nonderepressible 2; IVL, involucrin; KRT1, keratin 1; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 1924-1934DOI: (10.1016/j.jid.2017.04.029) Copyright © 2017 The Authors Terms and Conditions

Figure 6 GCN2 is required for proper epidermal differentiation. 3D organotypic cell cultures were made using shCTRL and shGCN2 primary human keratinocytes that were seeded on a collagen-fibroblast matrix and raised to the air-liquid interface to differentiate and stratify. After 7 days the samples were fixed, sectioned, and stained with (a) hematoxylin and eosin (H&E) or antibodies against (b) IVL, (c) eIF2α-P, (d) Ki67, or (e) an IgG isotype control. (f) Relative GCN2 knockdown efficiency in primary keratinocytes was measured by qPCR. ∗P < 0.05. Error bars represent mean ± standard deviation of three separate experiments. Scale bar = 25 μm for every image. eIF2α-P, phosphorylated eukaryotic initiation factor 2; IVL, involucrin. Journal of Investigative Dermatology 2017 137, 1924-1934DOI: (10.1016/j.jid.2017.04.029) Copyright © 2017 The Authors Terms and Conditions