The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids

Abstract

Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds. Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds. Although healthy individuals heal cutaneous wounds without complication, there are many patients whose wounds do not resolve and require long-term care. In fact, chronic cutaneous wounds are an epidemic that are frequently associated with diabetes, aging, and poor nutrition (1Sen C.K. Human wounds and its burden: An updated compendium of estimates.Adv. Wound Care (New Rochelle). 2019; 8: 39-48Crossref PubMed Scopus (200) Google Scholar, 2Frykberg R.G. Banks J. Challenges in the treatment of chronic wounds.Adv. Wound Care (New Rochelle). 2015; 4: 560-582Crossref PubMed Google Scholar, 3Han G. Ceilley R. Chronic wound healing: A review of current management and treatments.Adv. Ther. 2017; 34: 599-610Crossref PubMed Scopus (563) Google Scholar). Wound healing occurs in four sequential phases: hemostasis, inflammation, proliferation, and maturation. An important process during the proliferation phase is re-epithelialization that involves keratinocyte collective cell migration (KCCM) where sheets of connected cells migrate in concert to cover the wound bed (4Reinke J.M. Sorg H. Wound repair and regeneration.Eur. Surg. Res. 2012; 49: 35-43Crossref PubMed Scopus (816) Google Scholar). If these phases are dysregulated or do not occur in an orderly fashion, a wound will not properly heal. A chronic wound is defined as one that does not heal within 3 months. Chronic wounds are most often arrested in the inflammation phase, stalling keratinocyte migration and the subsequent sealing of the wound bed (5Rousselle P. Braye F. Dayan G. Re-epithelialization of adult skin wounds: Cellular mechanisms and therapeutic strategies.Adv. Drug Deliv. Rev. 2019; 146: 244-365Crossref Scopus (112) Google Scholar). Because re-epithelialization is impaired in many types of chronic wounds, further research is warranted to find novel therapeutic strategies to restore and enhance epithelial barrier formation (6Pastar I. Stojadinovic O. Yin N.C. Ramirez H. Nusbaum A.G. Sawaya A. Patel S.B. Khalid L. Isseroff R.R. Tomic-Canic M. Epithelialization in wound healing: A comprehensive review.Adv. Wound Care (New Rochelle). 2014; 3: 445-464Crossref PubMed Google Scholar). To study keratinocyte epithelial migration during wound healing, wounds are often modeled by “scratching” or removing sections of confluent sheets of differentiated keratinocytes and monitoring KCCM in two-dimensional cell culture (7Poujade M. Grasland-Mongrain E. Hertzog A. Jouanneau J. Chavrier P. Ladoux B. Buguin A. Silberzan P. Collective migration of an epithelial monolayer in response to a model wound.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 15988-15993Crossref PubMed Scopus (578) Google Scholar, 8Friedl P. Gilmour D. Collective cell migration in morphogenesis, regeneration and cancer.Nat. Rev. Mol. PubMed Scopus Google Scholar, O. P. of collective cell migration a Sci. PubMed Scopus Google Scholar). KCCM to occur and a cells that intact during migration and the of the to a to KCCM requires of the to a the collective migrate P. Gilmour D. Collective cell migration in morphogenesis, regeneration and cancer.Nat. Rev. Mol. PubMed Scopus Google Scholar). These and to types of cells in the and cells E. R. Collective cell migration of epithelial and Mol. Sci. PubMed Scopus Google Scholar, E. B. E. S. A. R. cell collective PubMed Scopus Google Scholar). is that of reactive oxygen species (ROS) in keratinocytes the of the wound is critical for cell migration of the to lamellipodia and A. A. F. reactive oxygen the and in cell 2014; PubMed Scopus Google Scholar, the PubMed Scopus Google Scholar). of during wound formation a within the An important for cell of involves phosphorylation of the of eukaryotic initiation factor 2 to amino and and of in the with eIF2 and 34: PubMed Scopus Google Scholar, of in and to cellular PubMed Scopus Google Scholar). of to and of gene expression to the in of that are critical for as the factor of gene and of gene expression to Because to to the is to as the response H. I. M. B. R. J.M. D. An response amino and to PubMed Scopus Google Scholar). is important to that is for of gene expression in the in response to many there are as in keratinocytes of a human the of human PubMed Scopus Google Scholar, human keratinocytes from a eIF2 kinase 2015; PubMed Scopus Google where the of the occur of GCN2 in the is to with to to with activation of the and of and the are by or of in and to cellular PubMed Scopus Google Scholar, D. signaling in Rev. PubMed Scopus Google Scholar). is to is by the a that to and restore of the important for GCN2 in skin and the of in we the role that GCN2 and the in KCCM during of cutaneous wounds. a of biochemical, and cellular in cell culture and model we that GCN2 and its attendant gene expression are important for appropriate management of amino acids, generation, lamellipodia formation, and focal adhesion dynamics that are for KCCM and wound healing. of human keratinocytes is a model to study skin and KCCM of a human the of human PubMed Scopus Google Scholar, human keratinocytes from a eIF2 kinase 2015; PubMed Scopus Google Scholar, Human keratinocyte requires by the kinase 2017; PubMed Scopus Google Scholar, in human keratinocytes requires a and PubMed Scopus Google Scholar, G. S. D. P. G. S. wound healing in the skin by 2017; PubMed Scopus Google Scholar). KCCM during wound healing using an of the cells with a sequential to that the differentiated keratinocytes the as a of cells A and These results that model KCCM A. M. F. of collective cell migration using the wound healing 2017; PubMed Scopus Google Scholar). that of the wound occur within this that KCCM a of migration and not proliferation keratinocytes a of to in vitro S. D. D. S. L. and of human keratinocyte cells in long-term PubMed Scopus Google Scholar). We the role of GCN2 in KCCM by using and the of or a the critical in the kinase the GCN2 a of keratinocytes GCN2 expression cells in wound healing we a in wound of the cells with the We these results using human keratinocytes that from from J. of the the of human keratinocytes following J. PubMed Scopus Google Scholar). to GCN2 in from individuals and of wound in cells delayed We that GCN2 KCCM in during wounding. To the of GCN2 in the initiation of KCCM following we a the of keratinocytes in to the wound using to a to wounds culture the the well in and a of wounds the of cells to the initiation of that of the 2 and we cell from to to the and of the and cells differentiated epithelial sheets for to analyses of that to activation of as by GCN2 within to the cells in the cells with and these in the cells the in the cells and of wounding. These results indicate that GCN2 during is necessary to and is critical for a role in the of the during many expression following of the of GCN2 is important for induction of there are mechanisms in and and significantly to and these A. L. cellular amino with for of 2017; PubMed Scopus Google Scholar, I. E. H. L. R. F. a with the PubMed Scopus Google Scholar, M. L. A. D. role of and in of cells with Res. PubMed Scopus Google Scholar, M. H. H. J. E. D. G. of the by the 2014; PubMed Scopus Google Scholar, J. J. D. to is by of and and PubMed Scopus Google Scholar, H. B. S. A. P. O. response by Res. 2012; PubMed Scopus Google Scholar, inhibition of gene expression and the PubMed Scopus Google Scholar). role of in the wound will is in by expression of and in and expression following in and These results that of GCN2 with and to in in and cells to to using a in in cells with and in there to a in in keratinocytes to of with These results the model that GCN2 and associated to following wounding. are frequently as of the is a of GCN2 A. S. D. J. O. A. R. A. of GCN2 cells to by the amino Natl. Acad. Sci. U. S. A. PubMed Scopus Google and with of in activation P. D. A. inhibition of a of PubMed Scopus Google Scholar). and cells in the wound healing and with or to and during the impaired wound of for the of GCN2 and KCCM in cells of cells with not wound not treatment of cells with not KCCM following not These results further the model that by GCN2 is critical for wound closure. is that expression in and cells as we that activation of GCN2 in keratinocytes does not to expression of a human the of human PubMed Scopus Google Scholar, human keratinocytes from a eIF2 kinase 2015; PubMed Scopus Google Scholar). We to an of as by in cells to a there in the cells this of the cell and cells in the wound healing in wound and cells However, treatment of the cells with delayed wound that the cells the for These results are with the results that expression in cells is of GCN2 and for wound in To gene expression in we an and cells to or and and by to S. of and for with 2014; PubMed Scopus Google Scholar). with of and of for further of significantly and significantly deletion of GCN2 in with cells gene a in with In gene a in to gene a significantly that of GCN2 the of expression These results that in the in keratinocytes and that of GCN2 significantly expression in and To by we using from in the keratinocytes gene expression in cellular and inflammation there activation for these in in the cells in of we the to with deletion of GCN2 activation for by the factor and activation for by the factor associated with signaling and activation in the is that associated with and formation activation in cells These results that GCN2 is critical for appropriate of cell inflammation, and We in gene expression in and keratinocytes that the for wound healing to In the there expression of in the response with cells the the in cells to and in a cellular with and of amino in the of and in the cells an an that free cysteine, in the of and maintenance of free cellular is critical for and management P. L. B. the of and of PubMed Scopus Google Scholar). We in of GCN2 for gene in and In a there in the of gene as well as many of and and with deletion of as of in is for necessary for formation of in management in the of and significantly an response However, to to generation, and in These results that GCN2 is critical for appropriate management of critical in These are to by the of these cells to phases in wound healing. the role for GCN2 as a of amino initiation factor 2 phosphorylation and in 2012; 3: PubMed Scopus Google we amino with activation of GCN2 following wounding. to amino during we that free in cells with and further in cells is not and by the of in and cells following wounding. of in cells to with in and cells and These results that enhance of that GCN2. We the that GCN2 plays in appropriate in as there is in that that of appropriate to for free amino as in the analyses expression of and of a in cells and in cells of However, of in cells of and in in the cells These results that GCN2 to enhance of and appropriate of of GCN2 the of these gene to the wound a of the that the a to the keratinocytes and are the keratinocytes in a as a (5Rousselle P. Braye F. Dayan G. Re-epithelialization of adult skin wounds: Cellular mechanisms and therapeutic strategies.Adv. Drug Deliv. Rev. 2019; 146: 244-365Crossref Scopus (112) Google is from KCCM of the cells is the of KCCM in the keratinocytes with to keratinocytes and lamellipodia in the keratinocytes of these in that of the is a in the in KCCM A. A. F. reactive oxygen the and in cell 2014; PubMed Scopus Google Scholar, the PubMed Scopus Google Scholar, M. PubMed Scopus Google and expression of in and we and keratinocytes with in the of H. of in by the Natl. Acad. Sci. U. S. A. 2015; PubMed Scopus Google Scholar). to cells within the of cells However, in the cells not in cells the of the wound there in cells the keratinocyte pharmacological inhibition of GCN2 using in cells to a KCCM with impaired activation of the of cells to critical for M. PubMed Scopus Google and that signaling in keratinocytes and To inhibition of activation or inhibition of the and the of GCN2 in this signaling keratinocyte monolayers with activation or and with of of these impaired the of the to in the pharmacological inhibition of and the wound of cells to with of GCN2 Cellular of the and of as by in the these and further of GCN2 and in in or expression and the in the of with of GCN2 to enhance the of is to and for lamellipodia the M. L. H. O. P. A. G. of during cell PubMed Scopus Google Scholar, P. J. cell migration activation of the PubMed Scopus Google Scholar, signaling in cell migration and PubMed Scopus Google Scholar). To of GCN2 activation of and we an that of analyses of or from from or cells that GCN2 is to in an monolayer of keratinocytes and during and in with further wounding. in of wounding. in the of and or and these results an important role for GCN2 in induction of the activation of or activation to the subsequent of the to a of from using two-dimensional cell is important to the results using skin in we the wound healing of skin using mice and mice a deletion of GCN2 A wound model to of the wound A. S. M. S. C.K. S. of inflammation and cutaneous wound healing in PubMed Scopus Google Scholar, A. R. S. C.K. S. expression in human chronic wound kinase 2012; PubMed Scopus Google Scholar). is necessary to the human of wound healing that involves and formation of of the wound by of human wound Mol. PubMed Scopus Google Scholar). of the wound and that wound significantly impaired in with the A and the skin and the determined in the the wound bed that with an to and with to cellular and by and These experiments that there in wound re-epithelialization in the skin with These results an role for GCN2 to wound in the of skin. we the that GCN2 kinase is critical for KCCM and wound closure. is in differentiated and activation of GCN2 is for deletion of GCN2 led to of and by and in the model in GCN2 and attendant gene expression that are critical for of there is a of free amino and is to an of GCN2. GCN2 for maintenance of are important for appropriate and of critical for of is to critical for activation of GCN2 that are GCN2 is important for with the and of in the the in these in cells in the of keratinocytes in the is critical for in the and cells the the or pharmacological of GCN2 led to and the of GCN2 in healing using wound model in for GCN2 a impaired in the of wound these results indicate that GCN2 to KCCM by and the of GCN2 in keratinocytes many that are critical for wound healing that an expression of occurs in response to wound healing, this process of GCN2 we that is for the KCCM and wound healing These results that the in wound healing involves that to critical and in the We that the of in response to and in keratinocytes of a human the of human PubMed Scopus Google Scholar, human keratinocytes from a eIF2 kinase 2015; PubMed Scopus Google Scholar, Human keratinocyte requires by the kinase 2017; PubMed Scopus Google Scholar). wound healing, and analyses that GCN2 to activation of and a process that of expression significantly in These results that GCN2 to of enhance the to for and that to the in KCCM and GCN2 the expression of and the that the kinase a critical in in is that the in and are in cells and These results that by GCN2 plays a critical role in mechanisms of the are to to for these without these are to and kinase the of the and with eIF2 and 34: PubMed Scopus Google Scholar, of in and to cellular PubMed Scopus Google Scholar, P. D. to PubMed Scopus Google is important of the that is for KCCM P. Gilmour D. Collective cell migration in morphogenesis, regeneration and cancer.Nat. Rev. Mol. PubMed Scopus Google Scholar). that deletion of in keratinocytes and keratinocyte These results that in the in the maintenance of the of skin. Chronic wounds are associated with diabetes, aging, and poor nutrition (1Sen C.K. Human wounds and its burden: An updated compendium of estimates.Adv. Wound Care (New Rochelle). 2019; 8: 39-48Crossref PubMed Scopus (200) Google Scholar, 2Frykberg R.G. Banks J. Challenges in the treatment of chronic wounds.Adv. Wound Care (New Rochelle). 2015; 4: 560-582Crossref PubMed Google Scholar, 3Han G. Ceilley R. Chronic wound healing: A review of current management and treatments.Adv. Ther. 2017; 34: 599-610Crossref PubMed Scopus (563) Google and long-term care. is associated with of However, with chronic and the from its to one that of in and to cellular PubMed Scopus Google Scholar, D. signaling in Rev. PubMed Scopus Google Scholar, I. M. A. PubMed Scopus Google Scholar). the important of GCN2 in the KCCM and wound healing, is to that of the is a to the to chronic wounds. of the to chronic wounds the and poor nutrition and In these the the from its to one that is and in GCN2 to and appropriate and the the and a critical therapeutic to wound healing are a of that to or inhibition of the and of in and to cellular PubMed Scopus Google Scholar, A. S. D. J. O. A. R. A. of GCN2 cells to by the amino Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, L. J. S. M. by a chronic 2019; PubMed Scopus Google and the of these and for treatment of chronic wounds. using an human keratinocyte cell that to keratinocyte Human keratinocytes that and a that and PubMed Scopus Google or human keratinocytes that from as J. of the the of human keratinocytes following J. PubMed Scopus Google and with by the of and cells and in with human keratinocyte and Human keratinocytes are in a by the of and in If and are to 2 and the cells by an appropriate of P. E. G. of in vitro keratinocytes requires PubMed Scopus Google Scholar). We that of GCN2 keratinocyte by this process is Human keratinocyte requires by the kinase 2017; PubMed Scopus Google Scholar). with as in during a monolayer or during the and and To a of with the to the kinase of GCN2 in 2 or in cells or in To and in to and an in and the and in to a of and by for and to to by the with and for with 2 or by using one keratinocytes using the with the Human and gene by using the and by of GCN2 and to KCCM during the re-epithelialization process of healing. a and cell wound from of a a and of the to a to wounds in a differentiated keratinocyte monolayer keratinocytes well in in with and to and the keratinocytes with with and 2 to P. E. G. of in vitro keratinocytes requires PubMed Scopus Google Scholar). an process that the cells a monolayer and a critical to re-epithelialization of and not to and the for (5Rousselle P. Braye F. Dayan G. Re-epithelialization of adult skin wounds: Cellular mechanisms and therapeutic strategies.Adv. Drug Deliv. Rev. 2019; 146: 244-365Crossref Scopus (112) Google Scholar). treatment with and the from the cells and with to the and to a and cells with and and for using the that to KCCM and wound healing the of keratinocytes to enhance of the and that collective cell the one keratinocytes in a and to and the keratinocytes differentiated by in with and 2 wounds in the with that and the of the and and the to a a of the from the and the cells in and for or by removing the and the cells in culture in and in a and and or in a in and for and for by a by determined by the of the from to of and with and for a of for and in to with in the by of and for the the of the in for 2 to to with the in an and for with with the indicated with in and To four for to in by with the for further four for to in a and the with and a for by the of cellular as A. of in using 2017; PubMed Scopus Google Scholar). with differentiated keratinocyte monolayers and for and with for to or as cells with for to cells with and cells with for as An to of cellular using to the with and in the with with a and and to using of in a with gene expression from GCN2 a using defined and for to in expression a gene and of Human keratinocyte and cells and or to in of from and and GCN2 cells using to the to where and A of to for and and GCN2 and for with expression from with and with is in the the for and with poor using M. B. A for 2014; PubMed Scopus Google Scholar). to human using a A. F. J. S. P. M. PubMed Scopus Google Scholar). of to the human of to the of using a from the An for to 2014; PubMed Scopus Google Scholar). the to S. of and for with 2014; PubMed Scopus Google Scholar). we and GCN2 for for and GCN2 the and and GCN2 with and GCN2 with of and of for further These for using from this study are in the with the for To significantly in of gene for by a the activation is is to the of a in an To in treatment the by a in the to the of to the an activation for that gene A. J. J. S. in 2014; PubMed Scopus Google Scholar). from with a with a and the for of intracellular free amino An of and the a of A using a amino amino to in in the A by the in from to in the for amino to a S. A. M. for and PubMed Scopus Google to the the for amino to amino within the for from and by in to in and as an to cell and to a and to in or to or of in with and of the for and the and the from the the If the of of the to of in the of the amino Cellular as J. S. G. B. L. M. H. R. J. J. of the gene is for to 2019; PubMed Scopus Google Scholar). from cells using with or in in for by with with and to using a in for to the using An to the for using for of with the following human human to by the from A with and for phase and using a and for is a for in is and does not in a environment. is the and with monolayers as and to for collective migration for to the culture a of and with the cells for and cells with with using the using the To confluent monolayers of or cells and in and with with and and mice J. L. A. H. D. P. GCN2 kinase to amino in PubMed Scopus Google and for of wound healing using the wound model to of the wound and the human of wound healing that involves and formation of of wound by A. S. M. S. C.K. S. of inflammation and cutaneous wound healing in PubMed Scopus Google Scholar, A. R. S. C.K. S. expression in human chronic wound kinase 2012; PubMed Scopus Google Scholar, of human wound Mol. PubMed Scopus Google Scholar). and to that wounds, and the in with a A. S. M. S. C.K. S. of inflammation and cutaneous wound healing in PubMed Scopus Google Scholar, A. R. S. C.K. S. expression in human chronic wound kinase 2012; PubMed Scopus Google Scholar). the from the the skin and the wound in a not connected to wound with Wound by and for to and wound determined using of of wound using as with with and with by subsequent with by with Wound re-epithelialization from sections using by the of the wound and the of the wound that as S. S. S. G. J. C.K. of a model of chronic PubMed Scopus Google Scholar). experiments by the Care and with the for the and of by the to and as appropriate for the using or If in the using or A defined as in as the from the or of in for or and and defined with using the following of are as in and in this study are within the and and from the from this study are in the R. as a to that of with the of this We the of the and and G. for R. R. S. D. F. and R. R. R. M. B. J. S. and D. F. S. R. R. P. H. M. B. J. E. A. M. D. S. D. F. and R. D. F. S. and R. R. R. P. H. M. B. J. E. A. M. D. S. D. F. and R. R. R. P. H. M. B. J. A. M. D. S. and D. F. S. D. F. S. and R. R. S. D. F. and R. R. R. P. H. J. E. A. M. D. S. and D. F. S. R. R. P. H. M. B. D. F. and R. R. R. P. H. M. B. J. E. A. M. D. S. D. F. and R. R. R. P. H. D. F. and R. review and study by of and and of F. is the of the and does not the of the of

Topics & Concepts