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Desmoglein 1-dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis.

Getsios S, Simpson CL, Kojima S, Harmon R, Sheu LJ, Dusek RL, Cornwell M, Green KJ - J. Cell Biol. (2009)

Bottom Line: Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1).Instead, Dsg1 was required for suppression of epidermal growth factor receptor-Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program.In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

ABSTRACT
Dsg1 (desmoglein 1) is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor-Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.

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Dsg1 promotes differentiation in the absence of robust PG binding. (A) To test which domains of Dsg1 would be sufficient to drive differentiation, we generated three Flag-tagged Dsg1 cDNA constructs: WT Dsg1 (Dsg1WT), a triple point mutant harboring three Ala substitutions (Dsg1AAA) within the predicted binding region for PG, or a truncation mutant lacking the ectodomain (ECTO) and transmembrane (TM) region (ΔN-Dsg1). CYTO, cytoplasmic domain. (B) The subcellular localization of Dsg1WT, Dsg1AAA, or ΔN-Dsg1 was determined in keratinocytes immunostained using a rabbit polyclonal antibody directed against Flag and a chicken polyclonal antibody against PG after exposing cells to high Ca2+ for 4 h to induce junction assembly. Both Dsg1WT and Dsg1AAA were efficiently recruited to areas of cell–cell contact; however, ΔN-Dsg1 was diffusely distributed throughout the cytoplasm. PG staining highlighted the intercellular borders; its localization at junctions was largely unaffected by any of the Dsg1 constructs. (C) Western blot analysis of keratinocytes transduced with these Dsg1 constructs and induced to differentiate for 2 d as submerged cultures. Although Dsg1WT and Dsg1AAA were sufficient to increase Dsc1/K10/loricrin, ΔN-Dsg1 did not affect these markers of differentiation compared with EGFP-transduced (Dsg1−) control cultures. FL, full length; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bar, 20 µm.
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fig6: Dsg1 promotes differentiation in the absence of robust PG binding. (A) To test which domains of Dsg1 would be sufficient to drive differentiation, we generated three Flag-tagged Dsg1 cDNA constructs: WT Dsg1 (Dsg1WT), a triple point mutant harboring three Ala substitutions (Dsg1AAA) within the predicted binding region for PG, or a truncation mutant lacking the ectodomain (ECTO) and transmembrane (TM) region (ΔN-Dsg1). CYTO, cytoplasmic domain. (B) The subcellular localization of Dsg1WT, Dsg1AAA, or ΔN-Dsg1 was determined in keratinocytes immunostained using a rabbit polyclonal antibody directed against Flag and a chicken polyclonal antibody against PG after exposing cells to high Ca2+ for 4 h to induce junction assembly. Both Dsg1WT and Dsg1AAA were efficiently recruited to areas of cell–cell contact; however, ΔN-Dsg1 was diffusely distributed throughout the cytoplasm. PG staining highlighted the intercellular borders; its localization at junctions was largely unaffected by any of the Dsg1 constructs. (C) Western blot analysis of keratinocytes transduced with these Dsg1 constructs and induced to differentiate for 2 d as submerged cultures. Although Dsg1WT and Dsg1AAA were sufficient to increase Dsc1/K10/loricrin, ΔN-Dsg1 did not affect these markers of differentiation compared with EGFP-transduced (Dsg1−) control cultures. FL, full length; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bar, 20 µm.

Mentions: Previous experiments showed that the misexpression of Dsc3 in the suprabasal epidermis increased β-catenin stability and signaling (Hardman et al., 2005). However, Dsg1-deficient rafts exhibited a variable decrease in Dsc3 levels but no changes in β-catenin distribution or activation (Fig. S1). Moreover, ectopic expression of Dsg1 was capable of inducing differentiation without altering levels of Dsc3 or β-catenin (unpublished data). The desmosomal counterpart to β-catenin, PG, is a major binding partner for the Dsg1 cytoplasmic domain and has been shown to play a key role in nuclear signaling and keratinocyte differentiation (Chitaev et al., 1998; Teuliere et al., 2004; Williamson et al., 2006). Thus, this armadillo family member served as a logical candidate for mediating Dsg1-dependent differentiation. Therefore, we mutated three hydrophobic residues important for PG binding within the Dsg1 cytoplasmic domain to assess its contribution to Dsg1-mediated differentiation (Dsg1AAA; Fig. 6 A; Chitaev et al., 1998). Although Dsg1AAA still concentrated at areas of cell–cell contact (Fig. 6 B), its association with PG was severely limited as compared with the WT protein (Fig. S5). Despite this profound deficiency in PG binding, Dsg1AAA and WT Dsg1 were equally capable of restoring the levels of Dsc1, K10, and loricrin in Dsg1-deficient rafts (Fig. 4 A). Moreover, ectopic expression of either of these Dsg1 constructs increased differentiation markers (Fig. 6 C). These findings demonstrate that introduction of Dsg1 into keratinocytes is sufficient to accelerate a program of terminal differentiation and emphasize that recruitment of additional PG by this ectopic desmosomal cadherin is not a critical step in this cascade of differentiation-promoting events.


Desmoglein 1-dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis.

Getsios S, Simpson CL, Kojima S, Harmon R, Sheu LJ, Dusek RL, Cornwell M, Green KJ - J. Cell Biol. (2009)

Dsg1 promotes differentiation in the absence of robust PG binding. (A) To test which domains of Dsg1 would be sufficient to drive differentiation, we generated three Flag-tagged Dsg1 cDNA constructs: WT Dsg1 (Dsg1WT), a triple point mutant harboring three Ala substitutions (Dsg1AAA) within the predicted binding region for PG, or a truncation mutant lacking the ectodomain (ECTO) and transmembrane (TM) region (ΔN-Dsg1). CYTO, cytoplasmic domain. (B) The subcellular localization of Dsg1WT, Dsg1AAA, or ΔN-Dsg1 was determined in keratinocytes immunostained using a rabbit polyclonal antibody directed against Flag and a chicken polyclonal antibody against PG after exposing cells to high Ca2+ for 4 h to induce junction assembly. Both Dsg1WT and Dsg1AAA were efficiently recruited to areas of cell–cell contact; however, ΔN-Dsg1 was diffusely distributed throughout the cytoplasm. PG staining highlighted the intercellular borders; its localization at junctions was largely unaffected by any of the Dsg1 constructs. (C) Western blot analysis of keratinocytes transduced with these Dsg1 constructs and induced to differentiate for 2 d as submerged cultures. Although Dsg1WT and Dsg1AAA were sufficient to increase Dsc1/K10/loricrin, ΔN-Dsg1 did not affect these markers of differentiation compared with EGFP-transduced (Dsg1−) control cultures. FL, full length; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bar, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2712955&req=5

fig6: Dsg1 promotes differentiation in the absence of robust PG binding. (A) To test which domains of Dsg1 would be sufficient to drive differentiation, we generated three Flag-tagged Dsg1 cDNA constructs: WT Dsg1 (Dsg1WT), a triple point mutant harboring three Ala substitutions (Dsg1AAA) within the predicted binding region for PG, or a truncation mutant lacking the ectodomain (ECTO) and transmembrane (TM) region (ΔN-Dsg1). CYTO, cytoplasmic domain. (B) The subcellular localization of Dsg1WT, Dsg1AAA, or ΔN-Dsg1 was determined in keratinocytes immunostained using a rabbit polyclonal antibody directed against Flag and a chicken polyclonal antibody against PG after exposing cells to high Ca2+ for 4 h to induce junction assembly. Both Dsg1WT and Dsg1AAA were efficiently recruited to areas of cell–cell contact; however, ΔN-Dsg1 was diffusely distributed throughout the cytoplasm. PG staining highlighted the intercellular borders; its localization at junctions was largely unaffected by any of the Dsg1 constructs. (C) Western blot analysis of keratinocytes transduced with these Dsg1 constructs and induced to differentiate for 2 d as submerged cultures. Although Dsg1WT and Dsg1AAA were sufficient to increase Dsc1/K10/loricrin, ΔN-Dsg1 did not affect these markers of differentiation compared with EGFP-transduced (Dsg1−) control cultures. FL, full length; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bar, 20 µm.
Mentions: Previous experiments showed that the misexpression of Dsc3 in the suprabasal epidermis increased β-catenin stability and signaling (Hardman et al., 2005). However, Dsg1-deficient rafts exhibited a variable decrease in Dsc3 levels but no changes in β-catenin distribution or activation (Fig. S1). Moreover, ectopic expression of Dsg1 was capable of inducing differentiation without altering levels of Dsc3 or β-catenin (unpublished data). The desmosomal counterpart to β-catenin, PG, is a major binding partner for the Dsg1 cytoplasmic domain and has been shown to play a key role in nuclear signaling and keratinocyte differentiation (Chitaev et al., 1998; Teuliere et al., 2004; Williamson et al., 2006). Thus, this armadillo family member served as a logical candidate for mediating Dsg1-dependent differentiation. Therefore, we mutated three hydrophobic residues important for PG binding within the Dsg1 cytoplasmic domain to assess its contribution to Dsg1-mediated differentiation (Dsg1AAA; Fig. 6 A; Chitaev et al., 1998). Although Dsg1AAA still concentrated at areas of cell–cell contact (Fig. 6 B), its association with PG was severely limited as compared with the WT protein (Fig. S5). Despite this profound deficiency in PG binding, Dsg1AAA and WT Dsg1 were equally capable of restoring the levels of Dsc1, K10, and loricrin in Dsg1-deficient rafts (Fig. 4 A). Moreover, ectopic expression of either of these Dsg1 constructs increased differentiation markers (Fig. 6 C). These findings demonstrate that introduction of Dsg1 into keratinocytes is sufficient to accelerate a program of terminal differentiation and emphasize that recruitment of additional PG by this ectopic desmosomal cadherin is not a critical step in this cascade of differentiation-promoting events.

Bottom Line: Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1).Instead, Dsg1 was required for suppression of epidermal growth factor receptor-Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program.In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

ABSTRACT
Dsg1 (desmoglein 1) is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor-Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.

Show MeSH
Related in: MedlinePlus