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• W2616687321 abstract "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. 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. In human skin, differentiation of keratinocytes is required to form a stress-resistant, impermeable barrier that protects against infection, water loss, UV damage, and other environmental stresses (Bikle et al., 2012Bikle D.D. Xie Z. Tu C.L. Calcium regulation of keratinocyte differentiation.Expert Rev Endocrinol Metab. 2012; 7: 461-472Crossref PubMed Scopus (173) Google Scholar, Fuchs, 2007Fuchs E. Scratching the surface of skin development.Nature. 2007; 445: 834-842Crossref PubMed Scopus (646) Google Scholar). The process of keratinocyte differentiation involves reprogramming of gene expression and cell morphology (Bikle et al., 2012Bikle D.D. Xie Z. Tu C.L. Calcium regulation of keratinocyte differentiation.Expert Rev Endocrinol Metab. 2012; 7: 461-472Crossref PubMed Scopus (173) Google Scholar, Green et al., 1982Green H. Fuchs E. Watt F. Differentiated structural components of the keratinocyte.In: Cold Spring Harbor Symp Quant Biol. 1982; 46: 293-301Crossref PubMed Google Scholar). Undifferentiated epidermal keratinocytes are attached to the cutaneous basement membrane that separates the epidermis and the underlying dermis. These basal layer keratinocytes actively divide until select progeny receive a signal to exit the cell cycle, detach from the basement membrane, and begin to migrate to the upper layers of the epidermis. During this process, cells begin to synthesize differentiation-specific proteins, including involucrin (IVL), loricrin, filaggrin, and various keratins (KRT1, KRT10) that are essential for changes in cell morphology and function (Abhishek and Palamadai Krishnan, 2016Abhishek S. Palamadai Krishnan S. Epidermal differentiation complex: a review on its epigenetic regulation and potential drug targets.Cell J. 2016; 18: 1-6PubMed Google Scholar, Moll et al., 1982Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4532) Google Scholar, Steven et al., 1990Steven A.C. Bisher M.E. Roop D.R. Steinert P.M. Biosynthetic pathways of filaggrin and loricrin—two major proteins expressed by terminally differentiated epidermal keratinocytes.J Struct Biol. 1990; 104: 150-162Crossref PubMed Scopus (172) Google Scholar, Warhol et al., 1985Warhol M.J. Roth J. Lucocq J.M. Pinkus G.S. Rice R.H. Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes.J Histochem Cytochem. 1985; 33: 141-149Crossref PubMed Scopus (55) Google Scholar). Disruption of normal keratinocyte differentiation results in a diminished capacity of the epidermis to maintain barrier function, a hallmark of skin diseases such as psoriasis, atopic dermatitis, and nonmelanoma skin cancers (Bouwstra and Ponec, 2006Bouwstra J.A. Ponec M. The skin barrier in healthy and diseased state.Biochim Biophys Acta. 2006; 1758: 2080-2095Crossref PubMed Scopus (472) Google Scholar, Menon et al., 1994Menon G.K. Elias P.M. Feingold K.R. Integrity of the permeability barrier is crucial for maintenance of the epidermal calcium gradient.Br J Dermatol. 1994; 130: 139-147Crossref PubMed Scopus (105) Google Scholar). Although changes in transcriptional and epigenetic networks during keratinocyte differentiation are well documented (Botchkarev, 2015Botchkarev V.A. Integration of the transcription factor-regulated and epigenetic mechanisms in the control of keratinocyte differentiation.J Investig Dermatol Symp Proc. 2015; 17: 30-32Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), little is known about the contributions of translational control. Recent studies have suggested that posttranscriptional regulation has a role in skin development. Changes in amino acid incorporation into proteins were reported between the layers of the epidermis, and a recent genome-wide analysis of psoriatic tissue suggested increased expression of the translational machinery (Swindell et al., 2015Swindell W.R. Remmer H.A. Sarkar M.K. Xing X. Barnes D.H. Wolterink L. et al.Proteogenomic analysis of psoriasis reveals discordant and concordant changes in mRNA and protein abundance.Genome Med. 2015; 7: 86Crossref PubMed Scopus (60) Google Scholar, Zhao et al., 2005Zhao K.N. Gu W. Fang N.X. Saunders N.A. Frazer I.H. Gene codon composition determines differentiation-dependent expression of a viral capsid gene in keratinocytes in vitro and in vivo.Mol Cell Biol. 2005; 25: 8643-8655Crossref PubMed Scopus (52) Google Scholar). Furthermore, markers of the unfolded protein response (UPR), which is an adaptive response to endoplasmic reticulum (ER) stress that features translational control, were suggested to be increased in the upper layers of normal epidermis and decreased in psoriasis and squamous cell carcinoma tissues (Sugiura et al., 2009Sugiura K. Muro Y. Futamura K. Matsumoto K. Hashimoto N. Nishizawa Y. et al.The unfolded protein response is activated in differentiating epidermal keratinocytes.J Invest Dermatol. 2009; 129: 2126-2135Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Therefore, we hypothesized that translational control would occur during normal keratinocyte differentiation. Signaling pathways that modify translation have been shown to participate in cellular differentiation processes (Masciarelli et al., 2010Masciarelli S. Fra A.M. Pengo N. Bertolotti M. Cenci S. Fagioli C. et al.CHOP-independent apoptosis and pathway-selective induction of the UPR in developing plasma cells.Mol Immunol. 2010; 47: 1356-1365Crossref PubMed Scopus (53) Google Scholar, Yang et al., 2016Yang S.Y. Wei F.L. Hu L.H. Wang C.L. PERK-eIF2alpha-ATF4 pathway mediated by endoplasmic reticulum stress response is involved in osteodifferentiation of human periodontal ligament cells under cyclic mechanical force.Cell Signal. 2016; 28: 880-886Crossref PubMed Scopus (52) Google Scholar), and regulation of translation is an important means by which eukaryotic cells adapt to a variety of environmental stresses (Baird and Wek, 2012Baird T.D. Wek R.C. Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism.Adv Nutr. 2012; 3: 307-321Crossref PubMed Scopus (321) Google Scholar, Schwanhausser et al., 2011Schwanhausser B. Busse D. Li N. Dittmar G. Schuchhardt J. Wolf J. et al.Global quantification of mammalian gene expression control.Nature. 2011; 473: 337-342Crossref PubMed Scopus (4095) Google Scholar, Sonenberg and Hinnebusch, 2009Sonenberg N. Hinnebusch A.G. Regulation of translation initiation in eukaryotes: mechanisms and biological targets.Cell. 2009; 136: 731-745Abstract Full Text Full Text PDF PubMed Scopus (2248) Google Scholar). Cells repress global protein synthesis to conserve resources, coincident with preferential translation of mRNA transcripts that confer stress resistance. An important mechanism directing translational control during stress features phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P). eIF2α-P decreases initiation of global translation through a reduced ability of eIF2α to combine with guanosine triphosphate and transport initiator Met-tRNAiMet to ribosomes for mRNA translation (Baird and Wek, 2012Baird T.D. Wek R.C. Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism.Adv Nutr. 2012; 3: 307-321Crossref PubMed Scopus (321) Google Scholar, Wek et al., 2006Wek R.C. Jiang H.Y. Anthony T.G. Coping with stress: eIF2 kinases and translational control.Biochem Soc Trans. 2006; 34: 7-11Crossref PubMed Scopus (1019) Google Scholar). Four mammalian protein kinases phosphorylate serine-51 of eIF2α, each activated by distinct types of stress. Because a variety of stresses regulate eIF2α-P, this pathway is referred to as the integrated stress response (ISR) (Harding et al., 2003Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. et al.An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2378) Google Scholar). Key eIF2α kinases include general control nonderepressible 2 (GCN2/EIF2AK4), which is activated by amino starvation and UV irradiation, and PKR-like endoplasmic reticulum kinase (PERK/EIF2AK3/PEK) that responds to ER stress and participates in the UPR. In addition to repressing global translation, eIF2α-P enhances translation of a subset of cytoprotective gene transcripts, such as activating transcription factor 4 (ATF4/CREB2) and its downstream target C/EBP homologous protein (CHOP/GADD153/DDIT3) by mechanisms involving upstream open reading frames in the 5′-leaders of these mRNAs (Harding et al., 2000Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. et al.Regulated translation initiation controls stress-induced gene expression in mammalian cells.Mol Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2412) Google Scholar, Lee et al., 2009Lee Y.Y. Cevallos R.C. Jan E. An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.J Biol Chem. 2009; 284: 6661-6673Crossref PubMed Scopus (163) Google Scholar, Palam et al., 2011Palam L.R. Baird T.D. Wek R.C. Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation.J Biol Chem. 2011; 286: 10939-10949Crossref PubMed Scopus (273) Google Scholar, Vattem and Wek, 2004Vattem K.M. Wek R.C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.Proc Natl Acad Sci USA. 2004; 101: 11269-11274Crossref PubMed Scopus (1131) Google Scholar, Young et al., 2016Young S.K. Palam L.R. Wu C. Sachs M.S. Wek R.C. Ribosome elongation stall directs gene-specific translation in the integrated stress response.J Biol Chem. 2016; 291: 6546-6558Crossref PubMed Scopus (42) Google Scholar, Young and Wek, 2016Young S.K. Wek R.C. Upstream open reading frames differentially regulate gene-specific translation in the integrated stress response.J Biol Chem. 2016; 291: 16927-16935Crossref PubMed Scopus (188) Google Scholar). Additionally, eIF2α-P induces the transcriptional and translational expression of growth arrest and DNA damage-inducible protein 34 (GADD34/PPP1R15A), which facilitates the dephosphorylation of eIF2α-P and resumption of translation (Connor et al., 2001Connor J.H. Weiser D.C. Li S. Hallenbeck J.M. Shenolikar S. Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1.Mol Cell Biol. 2001; 21: 6841-6850Crossref PubMed Scopus (223) Google Scholar, Novoa et al., 2001Novoa I. Zeng H. Harding H.P. Ron D. Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha.J Cell Biol. 2001; 153: 1011-1022Crossref PubMed Scopus (1038) Google Scholar, Young et al., 2015Young S.K. Willy J.A. Wu C. Sachs M.S. Wek R.C. Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.J Biol Chem. 2015; 290: 28257-28271Crossref PubMed Scopus (48) Google Scholar). Here we demonstrate the importance of translational control mediated through eIF2α-P in the differentiation of human keratinocytes. We show that repression of translation initiation occurs during keratinocyte differentiation, and differentiation-specific genes such as IVL are resistant to translation inhibition by eIF2α-P. Strikingly, the eIF2α kinase GCN2 is activated and is required for proper formation of the human epidermis, as visualized by a three-dimensional (3D) in vitro organotypic epidermal model. These results demonstrate that translational control by the ISR is required for proper keratinocyte differentiation during the formation of human skin. Keratinocytes in vitro can be induced to differentiate by growing cells to confluence and switching to a growth media containing 2 mM Ca2+ and 2% fetal bovine serum for 72 hours (Borowiec et al., 2013Borowiec A.S. Delcourt P. Dewailly E. Bidaux G. Optimal differentiation of in vitro keratinocytes requires multifactorial external control.PloS One. 2013; 8: e77507Crossref PubMed Scopus (68) Google Scholar) (Figure 1a). This calcium switch protocol is widely accepted as a means to initiate keratinocyte differentiation in vitro (Micallef et al., 2009Micallef L. Belaubre F. Pinon A. Jayat-Vignoles C. Delage C. Charveron M. et al.Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes.Exp Dermatol. 2009; 18: 143-151Crossref PubMed Scopus (111) Google Scholar, Pillai et al., 1990Pillai S. Bikle D.D. Mancianti M.L. Cline P. Hincenbergs M. Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium.J Cell Physiol. 1990; 143: 294-302Crossref PubMed Scopus (210) Google Scholar, Poumay and Pittelkow, 1995Poumay Y. Pittelkow M.R. Cell density and culture factors regulate keratinocyte commitment to differentiation and expression of suprabasal K1/K10 keratins.J Invest Dermatol. 1995; 104: 271-276Abstract Full Text PDF PubMed Scopus (220) Google Scholar). Differentiated keratinocytes were compared with subconfluent, proliferating cultures of keratinocytes (defined here as undifferentiated). To determine the dynamics of translation initiation during keratinocyte differentiation, lysates were prepared from undifferentiated and differentiated keratinocytes and subjected to sucrose gradient ultracentrifugation. This method measures the levels of protein synthesis as judged by polysome profiling, which determines the amount of ribosomal loading onto mRNAs at a fixed point in time. Keratinocyte differentiation substantially decreased the level of cellular mRNAs bound to heavy polysomes coincident with an increase in mRNAs associated with 80S monosomes, indicating repression of translation initiation (Figure 1b). Translational efficiency can be quantified by calculating the ratio of mRNAs bound to polysomes and monosomes (p/m); larger p/m values correspond to increased translation. The p/m of differentiated keratinocytes was decreased by sixfold compared with undifferentiated controls. To determine if translational control also impacted the elongation phase of protein synthesis, polysome profiling analyses were performed without the addition of cycloheximide. If differentiation also lowered the elongation phase of translation, omission of cycloheximide should not significantly change the levels of measured polysomes. However, in the absence of cycloheximide, differentiated keratinocytes showed a further decrease in polysomes accompanied by increased levels of monosomes (Figure 1b, blue line), verifying that the repression of translation occurs predominantly at the initiation stage. Although the use of in vitro two-dimensional cell culture is a powerful tool to study keratinocytes, this culture condition may not fully represent how intact 3D skin undergoes differentiation. Therefore, 3D organotypic skin equivalents were constructed using primary keratinocytes (Figure 1c) and analyzed by polysome profiling. A monolayer of undifferentiated primary keratinocytes seeded on collagen/fibroblast matrix, the initial step in constructing a skin equivalent, displayed levels of translation similar to that of a keratinocyte monolayer grown on a plastic dish (Figure 1d). However, after seven days of growth at the air-liquid interface, fully stratified skin equivalents revealed sharply lowered levels of transcripts bound to heavy polysomes coincident with increased numbers of mRNAs associated with 80S monosomes, indicating a repression of translation initiation similar to keratinocytes differentiated in monolayers. Collectively, these results indicate that keratinocyte differentiation is concomitant with lowered translation initiation in a 3D tissue. To determine whether the ISR is induced in differentiating keratinocytes, eIF2α-P was measured in both undifferentiated and differentiated keratinocytes. Levels of eIF2α-P normalized to total eIF2α were nearly ninefold higher in differentiated keratinocytes as compared with undifferentiated cells (Figure 2a, 2b). Of importance, there were increased amounts of the differentiation-specific proteins IVL and keratin 1 (KRT1) (Figure 2a). As a control, keratinocytes were also treated with tunicamycin (TM), a potent inducer of ER stress and the eIF2α kinase PERK. Although eIF2α-P was increased after treatment with TM, there were no detectable IVL and KRT1 proteins, indicating that eIF2α-P alone does not induce keratinocyte differentiation. As expected, IVL mRNA was significantly elevated with keratinocyte differentiation but not with exposure to TM (Figure 2c). Importantly, eIF2α-P occurred early during differentiation (within 24 hours), was detected concurrently with IVL, and was sustained over the course of the experiment (Figure 2d). To address whether eIF2α-P occurs during keratinocyte differentiation in vivo, full-thickness human skin was obtained from surgical abdominoplasty procedures. The tissue was fixed, paraffin embedded, sectioned, and stained with antibodies to measure eIF2α-P, ATF4, and CHOP (Figure 2e), which are subject to preferential translation in the ISR. Fluorescence marking the increased presence of each of these ISR markers was increased specifically in the suprabasal layers of the epidermis, which contain differentiated keratinocytes. By comparison, these protein markers were not visible in the single layer of basal keratinocytes. Staining with an IgG isotype control confirms that the fluorescence is not a result of high background. These results indicate that eIF2α-P and translational control are induced in differentiated keratinocytes, in vivo and in vitro. In addition to global translation repression, eIF2α-P leads to enhanced translation of specific mRNA transcripts, such as ATF4 and CHOP. To investigate if gene-specific translational control occurs during keratinocyte differentiation, fractions were eluted and collected from polysome profiles and levels of specific mRNAs were measured by qPCR. The percent of total ATF4 and CHOP mRNAs bound to heavy polysomes (fractions 5–7) was increased by 18% and 27%, respectively, during differentiation of keratinocytes in vitro (Figure 3a, 3b), indicative of preferential translation during eIF2α-P. Average polysome (fractions 5–7) over monosome (fractions 1–3) values are indicated for each gene to further illustrate changes in polysome association during differentiation. Importantly, IVL transcripts also shifted 27% toward heavy polysomes during differentiation (Figure 3c). By comparison, eIF4E mRNA led to a 12% shift away from heavy polysomes toward monosomes (Figure 4d), which is representative of the large number of genes that are subject to translation repression in the ISR. These results show that individual mRNAs including canonical ISR markers and keratinocyte differentiation-specific transcripts are bound to heavy polysomes despite global repression of translation that occurs during keratinocyte differentiation (Figure 1b).Figure 4Inhibition 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether eIF2α-P plays a critical role in keratinocyte differentiation, we utilized a doxycycline-inducible system to overexpress GADD34 in N-TERT keratinocytes (Collier et al., 2015Collier A.E. Wek R.C. Spandau D.F. Translational repression protects human keratinocytes from UVB-induced apoptosis through a discordant eIF2 kinase stress response.J Invest Dermatol. 2015; 135: 2502-2511Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Elevated levels of GADD34 lead to robust dephosphorylation of eIF2α, which will halt the ISR and relieve global translational repression. When GADD34 was overexpressed in differentiated keratinocytes, polysome profiling analyses revealed a shift to heavy polysomes alongside a decrease in monosome association (Figure 4a), consistent with GADD34 relieving translation repression in response to keratinocyte differentiation. Importantly, GADD34-induced dephosphorylation of eIF2α reduced the amount of IVL protein over twofold in keratinocytes compared with control keratinocytes (Figure 4b, 4c). GADD34 overexpression also decreased the levels of KRT1 protein (Figure 4b), indicating a widespread effect on differentiation gene expression. Elevated levels of GADD34 lowered the polysome association and resulting p/m ratio for ATF4 and CHOP mRNAs (Figure 4d), and IVL transcript (Figure 4e) compared with differentiation in the absence of doxycycline. By contrast, the p/m value for eIF4E was significantly increased on GADD34 overexpression (Figure 4d). Of note, doxycycline-reduced levels of eIF2α-P also led to a twofold reduction in IVL mRNA levels during keratinocyte differentiation, suggesting that translational control also contributes directly or indirectly to the increase in IVL transcript (Figure 4f). These results indicate that differentiation-specific protein expression is dependent on eIF2α-P and the induction of the ISR. eIF2α kinases are activated in response to distinct stress signals (Baird and Wek, 2012Baird T.D. Wek R.C. Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism.Adv Nutr. 2012; 3: 307-321Crossref PubMed Scopus (321) Google Scholar). In the case of PERK, accumulating levels of unfolded protein in the ER activates this eIF2 kinase, which is a transmembrane protein situated in this organelle. PERK functions in the UPR in conjunction with other sensory proteins, such as inositol requiring enzyme 1, which directs transcriptional gene expression through cytosolic splicing of x-box binding protein 1(XBP1) mRNA that leads to the expression of an active XBP1(s) transcription factor. To determine if there is activation of the UPR and inferred ER stress during keratinocyte differentiation, levels of mRNA encoding XBP1(s) or the ER chaperone, binding immunoglobulin protein (BiP/GRP78/HSPA5), were measured by qPCR. Keratinocyte differentiation led to lower amounts of both XBP1(s) and GRP78 mRNAs, suggesting that there is minimal activation of the UPR (Supplementary Figure S1a, S1b online). In contrast, both UPR markers were robustly induced in keratinocytes treated with TM. Although TM also induced robust PERK expression in keratinocytes, there was no increase in PERK protein levels during keratinocyte differentiation, suggesting that PERK is not activated by this type of stress (Supplementary Figure S1d). Because the UPR was not appreciably induced during keratinocyte differentiation, we next tested whether PERK or GCN2 was responsible for differentiation-induced eIF2α-P. Knockdown N-TERT keratinocytes were created using shRNA against either GCN2 or PERK (Supplementary Figure S1c, Figure 5a). PERK knockdown had no effect on differentiation-induced eIF2α-P, IVL, or KRT1 protein expression (Supplementary Figure S1d, S1e). By comparison, depletion of GCN2 caused a decrease in differentiation-induced eIF2α-P compared with control (shCTRL) (Figure 5b, 5c). This was confirmed by the analysis of two independent shRNA knockdowns of GCN2 targeting either the coding sequence or 3′ UTR. Loss of GCN2 also caused a sharp decrease (fivefold) in IVL and KRT1 protein induced on differentiation (Figure 5b, 5c). Of interest, knockdown of GCN2 also caused a decrease in differentiation-induced IVL mRNA expression compared with control (shCTRL) (Figure 5d), similar to that seen with GADD34 overexpression. To test whether lowered levels of IVL mRNA in differentiated GCN2-depleted keratinocytes were due to an increase in IVL transcript decay, we assayed the stability of IVL mRNA. Differentiated keratinocytes were treated with actinomycin D, an inhibitor of transcript synthesis, and harvested at the indicated times after the addition of the drug (Figure 5e). There was no significant difference between IVL mRNA decay in shCTRL compared with shGCN2 keratinocytes on differentiation, suggesting that the decrease in IVL mRNA levels in shGCN2 cells is due to lowered transcription of the IVL gene. To examine whether GCN2 is directly activated by keratinocyte differentiation, levels of GCN2 phosphorylated on threonine 899 (GCN2-P) were measured by immunoblot. Activation of GCN2 leads to autophosphorylation on this residue, releasing autoinhibitory molecular interactions that enhance GCN2 phosphorylation of eIF2α (Castilho et al., 2014Castilho B.A. Shanmugam R. Silva R.C. Ramesh R. Himme B.M. Sattlegger E. Keeping the eIF2 alpha kinase Gcn2 in check.Biochim Biophys Acta. 2014; 1843: 1948-1968Crossref PubMed Scopus (191) Google Scholar). Keratinocyte differentiation caused an increase in GCN2-P similar to that seen with halofuginone, a known GCN2 activator that inhibits charging of tRNAPro (Figure 5f). Importantly, the ER stress inducer TM did not induce GCN2-P. These results indicate that GCN2 is activated during keratinocyte differentiation and is required for eIF2α-P and translational control as well as expression of differentiation proteins. Given the adverse effect of GCN2 knockdown on differentiation in monolayer keratinocytes, we next addressed the impact of GCN2 loss on epidermal differentiation and formation of an intact, stratified tissue. 3D organotypic cultures were constructed using primary keratinocytes expressing shGCN2 or shCTRL. After seven days o" @default.
• W2616687321 created "2017-05-26" @default.
• W2616687321 creator A5062376824 @default.
• W2616687321 creator A5082229689 @default.
• W2616687321 creator A5089003346 @default.
• W2616687321 date "2017-09-01" @default.
• W2616687321 modified "2023-09-26" @default.
• W2616687321 title "Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2" @default.
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• W2616687321 doi "https://doi.org/10.1016/j.jid.2017.04.029" @default.
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