Laminin 332 Is Indispensable for Homeostatic Epidermal Differentiation Programs

Abstract

The skin epidermis is attached to the underlying dermis by a laminin 332 (Lm332)–rich basement membrane. Consequently, loss of Lm332 leads to the severe blistering disorder epidermolysis bullosa junctionalis in humans and animals. Owing to the indispensable role of Lm332 in keratinocyte adhesion in vivo, the severity of the disease has limited research into other functions of the protein. We have conditionally disrupted Lm332 expression in basal keratinocytes of adult mice. Although blisters develop along the interfollicular epidermis, hair follicle basal cells provide sufficient anchorage of the epidermis to the dermis, making inducible deletion of the Lama3 gene compatible with life. Loss of Lm332 promoted the thickening of the epidermis and exaggerated desquamation. Global RNA expression analysis revealed major changes in the expression of keratins, cornified envelope proteins, and cellular stress markers. These modifications of the keratinocyte genetic program are accompanied by changes in cell shape and disorganization of the actin cytoskeleton. These data indicate that loss of Lm332-mediated progenitor cell adhesion alters cell fate and disturbs epidermal homeostasis. The skin epidermis is attached to the underlying dermis by a laminin 332 (Lm332)–rich basement membrane. Consequently, loss of Lm332 leads to the severe blistering disorder epidermolysis bullosa junctionalis in humans and animals. Owing to the indispensable role of Lm332 in keratinocyte adhesion in vivo, the severity of the disease has limited research into other functions of the protein. We have conditionally disrupted Lm332 expression in basal keratinocytes of adult mice. Although blisters develop along the interfollicular epidermis, hair follicle basal cells provide sufficient anchorage of the epidermis to the dermis, making inducible deletion of the Lama3 gene compatible with life. Loss of Lm332 promoted the thickening of the epidermis and exaggerated desquamation. Global RNA expression analysis revealed major changes in the expression of keratins, cornified envelope proteins, and cellular stress markers. These modifications of the keratinocyte genetic program are accompanied by changes in cell shape and disorganization of the actin cytoskeleton. These data indicate that loss of Lm332-mediated progenitor cell adhesion alters cell fate and disturbs epidermal homeostasis. IntroductionLaminin 332 (Lm332) is the central component in a multiprotein network that constitutes the basement membrane attaching the skin epidermis to the dermis. Laminins are glycoproteins endowed with structural, adhesive, signaling, and mechanical functions through which they control the homeostasis of cells and tissues (Aumailley, 2013Aumailley M. The laminin family.Cell Adh Migr. 2013; 7: 48-55Crossref PubMed Scopus (202) Google Scholar). Alterations in laminin expression or processing and mutations in laminin-coding genes are associated with pathologies such as cancer, autoimmune diseases, or inborn disorders (Durbeej, 2015Durbeej M. Laminin-α2 chain-deficient congenital muscular dystrophy: pathophysiology and development of treatment.Curr Top Membr. 2015; 76: 31-60Crossref PubMed Scopus (43) Google Scholar; Funk et al., 2018Funk S.D. Lin M.H. Miner J.H. Alport syndrome and Pierson syndrome: diseases of the glomerular basement membrane.Matrix Biol. 2018; 71–72: 250-261Crossref PubMed Scopus (55) Google Scholar; Qin et al., 2017Qin Y. Rodin S. Simonson O.E. Hollande F. Laminins and cancer stem cells: partners in crime?.Semin Cancer Biol. 2017; 45: 3-12Crossref PubMed Scopus (38) Google Scholar; Simon and Bromberg, 2017Simon T. Bromberg J.S. Regulation of the immune system by laminins.Trends Immunol. 2017; 38: 858-871Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In particular, mutations in the genes coding for any one of the α3, β3, and γ2 chains of Lm332 lead to absence or strong reduction of the protein, causing the severe skin-blistering disorder epidermolysis bullosa junctionalis (Aumailley et al., 2006Aumailley M. Has C. Tunggal L. Bruckner-Tuderman L. Molecular basis of inherited skin-blistering disorders, and therapeutic implications.Expert Rev Mol Med. 2006; 8: 1-21Crossref PubMed Scopus (66) Google Scholar; Kiritsi et al., 2013Kiritsi D. Has C. Bruckner-Tuderman L. Laminin 332 in junctional epidermolysis bullosa.Cell Adh Migr. 2013; 7: 135-141Crossref PubMed Scopus (67) Google Scholar; Uitto et al., 1994Uitto J. Pulkkinen L. Christiano A.M. Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes.J Invest Dermatol. 1994; 103: 39S-46SAbstract Full Text PDF PubMed Scopus (121) Google Scholar). The condition is often associated with early death, thus restricting studies on the role of Lm332 in vivo.Using an inducible conditional transgenic mouse model in which the disruption of the Lama3 gene in the basal keratinocytes (KCs) of adult mice results in a progressive loss of Lm332, we unequivocally showed that Lm332 is crucial to maintain the physical attachment of the epidermis to the dermis (Pesch et al., 2017Pesch M. König S. Aumailley M. Targeted disruption of the Lama3 gene in adult mice is sufficient to induce skin inflammation and fibrosis.J Invest Dermatol. 2017; 137: 332-340Abstract Full Text Full Text PDF PubMed Google Scholar). These transgenic mice develop blisters at the interfollicular epidermis (IFE) and epidermal thickening, but the epidermis remains anchored to the dermis within the hair follicles (Pesch et al., 2017Pesch M. König S. Aumailley M. Targeted disruption of the Lama3 gene in adult mice is sufficient to induce skin inflammation and fibrosis.J Invest Dermatol. 2017; 137: 332-340Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, the model is compatible with life in contrast to the lethality associated with the constitutive deletion of Lm332, allowing us to study the longer-term consequences of the loss of Lm332 in vivo. It is particularly well-suited for determining which cellular activities shown to be under the control of Lm332 in vitro are also under its control in adult skin in vivo. Except for the essential role of Lm332 for stable adhesion of KCs, firmly established by clinical, genetics, and biological studies of junctional epidermolysis bullosa, in vivo studies supporting the role of Lm332 for the control of other cellular activities are very scarce and are limited to newborns or mouse embryos.To uncover how the loss of Lm332 leads to morphological changes in the epidermis, especially the thickening, we performed RNA sequencing (RNA-seq) of the epidermal cells from transgenic and control mice. Of the 1,961 genes that were significantly regulated in the epidermis of Lm332-depleted mice, genes coding for keratins, cornified envelope proteins, and cell stress markers were strongly upregulated. In addition, there were profound changes in the cell shape and disorganization of the actin cytoskeleton, pointing to substantial perturbation of epidermal homeostasis in the absence of Lm332.ResultsBlistering and epidermal thickening during an inducible loss of Lama3The loss of Lama3 and consequently of Lm332 in the adult epidermis was induced by putting Lama3flox/flox/K14-CreERT (Lama3e knockout [Lama3e KO]) mice on a chow diet containing tamoxifen for 8–12 weeks. Whereas the Lm332 in the skin of Lama3e KO mice is reduced to 5–30% that of the controls, blisters develop along the IFE (Pesch et al., 2017Pesch M. König S. Aumailley M. Targeted disruption of the Lama3 gene in adult mice is sufficient to induce skin inflammation and fibrosis.J Invest Dermatol. 2017; 137: 332-340Abstract Full Text Full Text PDF PubMed Google Scholar) of skin revealed epidermal thickening the blisters of Lama3e KO mice with that of the with the of blistering at of the of the epidermal the blisters was to that of of blisters or that of to as Lama3e KO mice with [Lama3e in the of blistering of the in Lama3e KO mice, the epidermal was to that of the to as Lama3e KO mice epidermal to blistering but to Lm332 in the skin that thickening is a of the of an of the cornified the blisters of Lama3e mice These results indicate that the absence of epidermal anchorage to the dermis through Lm332 leads to an of genes coding for cornified and during the loss of how Lm332 epidermal from the skin of control and Lama3e mice in which at of the IFE blisters were to the of the of mice revealed that a of 1,961 genes were significantly by at of which were and were in the Lama3e The of the and genes in the are shown in in the biological of the of genes and to the consequences of Lama3 in the epidermis, we to the et al., and analysis of gene PubMed Scopus Google Scholar). analysis that the significantly biological in the Lama3e mice were associated with epidermal cell skin and cell the were the and the of cornified envelope that are a of et al., M. M. D. The epidermal cornified envelope and the Dermatol. PubMed Scopus Google Scholar; and C. The in and Invest Dermatol. 2017; 137: Full Text Full Text PDF PubMed Scopus Google Scholar). These genes to the of proteins, and of genes coding for the cornified envelope and for genes coding for markers of such as or were also In genes and cornified envelope were and analysis of genes in keratinocytes of Lama3e mice. Top for the biological associated with genes in skin keratinocytes of Lama3e mice with that of the genes to the biological shown in gene are to the the The to the genes coding for or in the of the epidermal The to the genes coding for of the keratinocyte or associated with gene Lama3e Lama3e Lama3e Lama3e knockout mouse with gene in the of the is the gene the major of the epidermal that is crucial for and et al., C. in the role in skin and PubMed Scopus Google Scholar). of the skin of Lama3e mice the of expression at the with a of the in the and an in the of cells within the of the epidermis is a of epidermal the cells to they the of the et al., C. in the role in skin and PubMed Scopus Google Scholar). Thus, the of at the RNA and is with the of in the epidermis of Lama3e KO mice and of and of in the of the epidermis of Lama3e mice. of Lama3e and mice skin were for were with the thickening of the and an in the of cells in the epidermis of Lm332-depleted skin with that in the and cells in were on for mice for shown are The Lama3e mice, and the the of skin of Lama3e and mice. the of at the of the epidermis a in a Lama3e Lama3e Lama3e Lama3e Lama3e knockout mouse with results that of basal to loss of Lm332 in the basement membrane strongly with an exaggerated expression of genes coding for to and for epidermal of epidermal expression during the loss of the of the and genes were genes coding for In particular, the was the gene in the epidermis of Lama3e mice and was at a in control IFE and was on IFE such as during and in diseases et al., M. T. M. et in through a containing and Full Text Full Text PDF PubMed Scopus Google Scholar; et al., T. M. et mouse are in the hair and PubMed Scopus (66) Google Scholar; et al., F. S. keratinocyte by and Biol. 2018; PubMed Scopus Google Scholar). of the genes was by with RNA from the epidermis of Lama3e mice with blisters but from Lama3e KO mice with or very blisters for and its were also and as as other of which are in or and are in hair follicles In of which are hair follicles were strongly for with of the we an at the was genes were of Lm332 a in the expression of in the epidermis of Lama3e mice. The in the for and hair and hair genes along the of was by The results for Lama3e KO mouse were to that of the The the results for Lama3e mice blisters and Lama3e mice with blisters at the they were of and on of skin of Lama3e KO and mice. is in the of the Lama3e Lama3e Lama3e Lama3e knockout mouse with Lama3e Lama3e knockout mouse Lm332, laminin RNA Lama3e KO mice in the expression of and and by basal and KCs, the epidermal thickening was to an the by and the by was performed markers. analysis revealed a strong expression of also in the of the epidermis of Lama3e mice, of an of the In strong was in the of the epidermis of mice, which also was in of the of with that of the was accompanied by cell we performed of the was in the of cells in the epidermis of Lama3e KO and control mice, but cells were in the epidermal It that the blisters induced during the loss of Lm332 the in epidermal and in epidermal and that the loss of Lm332 in the basement membrane underlying basal and the blisters expression and epidermal of on loss of Lm332 and of the revealed substantial of genes coding for the and a and in Lama3e mice with that in the control mice. of genes was by analysis on RNA from epidermal of Lama3e mice of strong and the was in the epidermis of Lama3e mice with blisters but in Lama3e KO mice with or blisters at the they were and a of or associated with which are essential to and et al., M. M. S. et in skin role of the and to induce 2018; PubMed Scopus Google Scholar). These are at a in the epidermis, expression is in et al., A.M. D. during epidermal and PubMed Scopus Google Scholar; et al., J. J. M. T. et and are by genes.J Full Text Full Text PDF PubMed Scopus Google Scholar; et al., L. L. L. D. et skin disease and by inducible epidermal deletion of in PubMed Scopus Google Scholar). In of in the the blisters in Lm332-depleted skin showed strongly cells in the epidermal of Lama3e mice These data the in epidermal homeostasis on of Lm332 and to the of inflammation in the of the of and analysis of and data the results of Lama3e KO mice with of its The to mice with blisters (Lama3e and the to mice blisters (Lama3e for of on the skin of and Lama3e mice. keratinocytes are in the of the epidermis of Lama3e mice. Lama3e Lama3e Lama3e Lama3e knockout mouse with Lama3e Lama3e knockout mouse of adhesion to Lm332 a in the shape of basal network is a major of shape and et al., the of epidermal Rev Mol Biol. PubMed Scopus Google Scholar). the expression of is strongly in the epidermis of Lama3e KO mice we an on the of and the the were for to and cell of showed that were in the epidermis of Lama3e KO and control mice but that the shape of epidermal cells In the epidermis of control mice, the basal were along the and the were In basal and be by in the epidermis of Lama3e KO mice of and actin in the epidermis of Lama3e mice. of mice and mice with (Lama3e and (Lama3e blisters were for and collagen were for actin and collagen VII in the and Lama3e mice. the with and blisters as is with other or shown as The actin network in epidermis has in Lama3e mice. analysis of is shown for Lama3e and Lama3e mouse with that of the of and collagen VII in and Lama3e KO mice. Lama3e Lama3e Lama3e Lama3e knockout mouse with Lama3e Lama3e knockout mouse shape is on the to a and and a central in cell shape and PubMed Scopus Google Scholar). the of the cytoskeleton, actin was by In the control the of basal were by a of In for Lama3e mice, the network was in the epidermal cells in a has or in epidermal cells in the of the the gene coding for actin was in the analysis the expression of RNA from epidermal of Lama3e mice showed expression of actin in epidermal cells the of the blisters in Lama3e mice, the was in the basal of the control mice the results that the and adhesion is during the loss of epidermal we that of the Lama3 gene in adult mouse causing loss of Lm332 and blistering along the the thickening of the epidermis and alters the essential role of Lm332 for of the epidermis to the dermis has firmly established by the of epidermolysis bullosa in humans or the constitutive deletion of coding genes in mice et al., M.H. L. in the gene results in junctional epidermolysis 8: PubMed Scopus Google Scholar; et al., T. et of laminin gene junctional epidermolysis Invest Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar; et al., Y. Targeted disruption of the gene in mice in and of Biol. PubMed Scopus Google Scholar; Uitto et al., 1994Uitto J. Pulkkinen L. Christiano A.M. Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes.J Invest Dermatol. 1994; 103: 39S-46SAbstract Full Text PDF PubMed Scopus (121) Google Scholar). the is by a Lm332 and by basal et al., F. et is a for laminin and 1994; PubMed Scopus Google Scholar; and Aumailley M. is laminin in adhesion of keratinocytes and other cells and has a for Biol. 1994; PubMed Scopus Google Scholar). In in vivo studies a role of Lm332 in other cellular activities such as or gene expression in vitro and by the of Lm332 with cellular the and et al., functions for in and in a stable of to Biol. PubMed Scopus Google Scholar; et al., F. C. A.M. M. et of to by keratinocyte J. PubMed Scopus Google Scholar; et al., L. et keratinocyte through and of PubMed Scopus Google Scholar; et al., F. et is a for laminin and 1994; PubMed Scopus Google Scholar; et al., L. L. L. T. et Biol. PubMed Scopus Google Scholar; et al., T. C. T. et of cells on Biol. PubMed Scopus Google Scholar). is in mice with disruption of genes Lm332 or its and et al., and for are essential for epidermal and homeostasis during skin PubMed Google Scholar; et al., J. is for cell adhesion and cell Biol. PubMed Scopus Google Scholar; et al., L. M. of leads to epidermolysis bullosa and in PubMed Scopus Google Scholar; et al., M.H. L. in the gene results in junctional epidermolysis 8: PubMed Scopus Google Scholar; et al., T. et of laminin gene junctional epidermolysis Invest Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar; et al., M. J. and in conditional knockout PubMed Scopus Google Scholar; et al., Y. Targeted disruption of the gene in mice in and of Biol. PubMed Scopus Google Scholar; et al., J. to absence of in PubMed Scopus Google Scholar). in and was in mouse with deletion of the et al., S. C. Loss of in keratinocytes leads to an in and and in expression of genes.J PubMed Scopus Google and a in and was for mice of the gene et al., T. et of laminin gene junctional epidermolysis Invest Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, Lm332 and its through are for skin in with the that the epidermis and Lm332 to in the mouse et al., D. C. expression of adhesion 1994; PubMed Scopus Google Scholar) and in humans et al., of γ2 and its in and J. PubMed Scopus Google Scholar; et al., C. C. et and for the of the of the of laminin and during PubMed Scopus Google results that Lm332 is essential for epidermis anchorage in adult skin but also for epidermal homeostasis. loss leads to epidermal thickening, in and of Lm332 with its collagen which in collagen in the dermis and T. Aumailley M. The of the with Dermatol. PubMed Scopus Google Scholar). there is for the loss of Lm332-mediated cell Although other with the as Lm332 are basal (Aumailley and M. Laminins of the Biol. PubMed Scopus Google Scholar; et al., M. of laminin in the of the epidermal basement 2006; PubMed Scopus Google Scholar; et al., J. S. T. C. Aumailley M. the of epidermal stem 2015; PubMed Scopus Google has the of to collagen which the of Thus, Lm332 to be in the multiprotein the network to the collagen network of the homeostasis results from a cell in the basal and cell in the of the epidermis, to desquamation. in the epidermis of Lama3e KO mice, especially the We an of of KCs, the of was strongly is of the epidermal are from to and of and to of the envelope during of the epidermal Biol. 76: PubMed Scopus Google Scholar). We the thickening of the epidermis with a of basal deletion of the which adhesion to Lm332 et al., C. T. L. et is for epidermal and hair follicle Biol. PubMed Scopus Google Scholar). thickening associated with and but with a major in cell is also in a mouse model of congenital et al., J. The and epidermal thickening in a mouse model for Invest Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). In epidermal thickening was by by an in cell be for epidermis thickening in Lama3e KO the through the the of cell and in the skin et al., in the in and Invest Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar; et al., Y. D. of in Biol. 2018; PubMed Scopus Google Scholar). In the epidermis, attached to and the from the and the cell the actin and the associated the to the and T. as mechanical in Biol. 2015; PubMed Scopus Google Scholar; et al., D. T. M. et and cell and to epidermal Biol. 2018; PubMed Scopus Google Scholar; and S. the and of cell Biol. PubMed Scopus Google Scholar). Consequently, the cellular and shape but also and et al., gene and in epidermal development and PubMed Scopus Google Scholar). In Lama3e KO mice, we that the of the and induced by the loss of Lm332 and loss of adhesion of the epidermal cells in the of the blisters studies on mouse epidermal within the genes of the epidermal a of the to changes in et al., et analysis of the and in skin 2017; PubMed Scopus Google Scholar). is a for the of of the genes coding for the epidermal in mouse has that mechanical results in epidermal thickening in skin et al., Y. Y. on skin the epidermal and the basement 2015; PubMed Scopus Google Scholar) and in skin in vivo et al., M. M. Y. J. S. et of skin at PubMed Scopus Google Scholar; and M. for stem cells the PubMed Scopus Google Scholar). In the of homeostasis of the adult epidermis on the of basal through Lm332, cellular stress and the of from the in the dermis and the of the basal in the and are in the and Lama3flox/flox/K14-CreERT (Lama3e and mice were (Pesch et al., 2017Pesch M. König S. Aumailley M. Targeted disruption of the Lama3 gene in adult mice is sufficient to induce skin inflammation and fibrosis.J Invest Dermatol. 2017; 137: 332-340Abstract Full Text Full Text PDF PubMed Google Scholar). were and in with the for the of animals. were by the as in the study by et al., 2017Pesch M. König S. Aumailley M. Targeted disruption of the Lama3 gene in adult mice is sufficient to induce skin inflammation and fibrosis.J Invest Dermatol. 2017; 137: 332-340Abstract Full Text Full Text PDF PubMed Google of skin the of the on the with as and as blisters was with The of blistering was as a epidermal were performed along the for control mice and in the blisters for Lama3e KO and sequencing and from Lama3e KO and control mice were in in for at to the of the epidermis from the dermis. cells were by by and for and sequencing of biological regulated and with an were for analysis with the et al., and analysis of gene PubMed Scopus Google Scholar). data have to for was performed to sequencing data have to for M. of is an of The of IntroductionLaminin 332 (Lm332) is the central component in a multiprotein network that constitutes the basement membrane attaching the skin epidermis to the dermis. Laminins are glycoproteins endowed with structural, adhesive, signaling, and mechanical functions through which they control the homeostasis of cells and tissues (Aumailley, 2013Aumailley M. The laminin family.Cell Adh Migr. 2013; 7: 48-55Crossref PubMed Scopus (202) Google Scholar). Alterations in laminin expression or processing and mutations in laminin-coding genes are associated with pathologies such as cancer, autoimmune diseases, or inborn disorders (Durbeej, 2015Durbeej M. Laminin-α2 chain-deficient congenital muscular dystrophy: pathophysiology and development of treatment.Curr Top Membr. 2015; 76: 31-60Crossref PubMed Scopus (43) Google Scholar; Funk et al., 2018Funk S.D. Lin M.H. Miner J.H. Alport syndrome and Pierson syndrome: diseases of the glomerular basement membrane.Matrix Biol. 2018; 71–72: 250-261Crossref PubMed Scopus (55) Google Scholar; Qin et al., 2017Qin Y. Rodin S. Simonson O.E. Hollande F. Laminins and cancer stem cells: partners in crime?.Semin Cancer Biol. 2017; 45: 3-12Crossref PubMed Scopus (38) Google Scholar; Simon and Bromberg, 2017Simon T. Bromberg J.S. Regulation of the immune system by laminins.Trends Immunol. 2017; 38: 858-871Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In particular, mutations in the genes coding for any one of the α3, β3, and γ2 chains of Lm332 lead to absence or strong reduction of the protein, causing the severe skin-blistering disorder epidermolysis bullosa junctionalis (Aumailley et al., 2006Aumailley M. Has C. Tunggal L. Bruckner-Tuderman L. Molecular basis of inherited skin-blistering disorders, and therapeutic implications.Expert Rev Mol Med. 2006; 8: 1-21Crossref PubMed Scopus (66) Google Scholar; Kiritsi et al., 2013Kiritsi D. Has C. Bruckner-Tuderman L. Laminin 332 in junctional epidermolysis bullosa.Cell Adh Migr. 2013; 7: 135-141Crossref PubMed Scopus (67) Google Scholar; Uitto et al., 1994Uitto J. Pulkkinen L. Christiano A.M. Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes.J Invest Dermatol. 1994; 103: 39S-46SAbstract Full Text PDF PubMed Scopus (121) Google Scholar). The condition is often associated with early death, thus restricting studies on the role of Lm332 in vivo.Using an inducible conditional transgenic mouse model in which the disruption of the Lama3 gene in the basal keratinocytes (KCs) of adult mice results in a progressive loss of Lm332, we unequivocally showed that Lm332 is crucial to maintain the physical attachment of the epidermis to the dermis (Pesch et al., 2017Pesch M. König S. Aumailley M. Targeted disruption of the Lama3 gene in adult mice is sufficient to induce skin inflammation and fibrosis.J Invest Dermatol. 2017; 137: 332-340Abstract Full Text Full Text PDF PubMed Google Scholar). These transgenic mice develop blisters at the interfollicular epidermis (IFE) and epidermal thickening, but the epidermis remains anchored to the dermis within the hair follicles (Pesch et al., 2017Pesch M. König S. Aumailley M. Targeted disruption of the Lama3 gene in adult mice is sufficient to induce skin inflammation and fibrosis.J Invest Dermatol. 2017; 137: 332-340Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, the model is compatible with life in contrast to the lethality associated with the constitutive deletion of Lm332, allowing us to study the longer-term consequences of the loss of Lm332 in vivo. It is particularly well-suited for determining which cellular activities shown to be under the control of Lm332 in vitro are also under its control in adult skin in vivo. Except for the essential role of Lm332 for stable adhesion of KCs, firmly established by clinical, genetics, and biological studies of junctional epidermolysis bullosa, in vivo studies supporting the role of Lm332 for the control of other cellular activities are very scarce and are limited to newborns or mouse embryos.To uncover how the loss of Lm332 leads to morphological changes in the epidermis, especially the thickening, we performed RNA sequencing (RNA-seq) of the epidermal cells from transgenic and control mice. Of the 1,961 genes that were significantly regulated in the epidermis of Lm332-depleted mice, genes coding for keratins, cornified envelope proteins, and cell stress markers were strongly upregulated. In addition, there were profound changes in the cell shape and disorganization of the actin cytoskeleton, pointing to substantial perturbation of epidermal homeostasis in the absence of

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