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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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Related in: MedlinePlus

Dsg1 is required for the suppression of EGFR–Erk1/2 signaling during epidermal differentiation. (A) IHC analysis of Dsg1 (left) and EGFR phosphorylated at Y1068 (p-EGFR; right) revealed a basally restricted p-EGFR staining pattern largely absent from Dsg1-positive suprabasal layers in control (miR Lmn) cultures, whereas Dsg1-deficient cultures (miR DG1) demonstrated a gross disorganization of p-EGFR, which was present throughout the basal and suprabasal layers. The dotted lines indicate the boundary between keratinocytes and the collagen matrix. (B) Western blot analysis of keratinocytes transduced with miR Lmn or miR DG1 and induced to undergo differentiation for 2 d as submerged cultures in the presence or absence of 10 µM of the EGFR–erbB2 inhibitor PKI166 (PKI). A significant increase in p-EGFR, p-erbB2, and p-Erk1/2 levels was detectable in keratinocytes upon Dsg1 silencing (Dsg1 KD) along with decreased K10/Dsc1. Blocking EGFR signaling using PKI166 suppressed p-EGFR, p-erbB2, and p-Erk levels in addition to restoring the capacity of Dsg1-deficient keratinocytes to differentiate. Ratios of p-EGFR to total EGFR or p-Erk1/2 to total Erk1/2 are indicated above the blots. Tx, treatment. (C) Dual-label indirect immunofluorescence and Apotome optical sectioning of Dsg1-Flag and EGFR after 4 h in high Ca2+ revealed areas of overlap in discrete regions of cell–cell contact (zoom) but not throughout the cell. The boxed area is magnified in the right panel. (D) Keratinocytes were transduced with EGFP, WT Dsg1-Flag (DG1-WT), or a soluble, truncated Dsg1 mutant (ΔN-DG1) and switched into high Ca2+ before being harvested in RIPA buffer. The insoluble pellet was resuspended in buffered SDS and run in parallel with equal protein amounts from the RIPA-soluble fraction. The ratio of insoluble/total erbB2, EGFR, E-cadherin (E-cad), and K14 is presented in the bar graph and is representative of three experiments. Full-length but not the truncated Dsg1 recruits EGFR–erbB2 into a more insoluble fraction where keratins are concentrated. Under these detergent conditions, the vast majority of E-cadherin remains in the soluble fraction. (E) Activation of EGFR-Ras-Raf-Erk signaling effectors as well as the EGFR–Erk1/2 gene target, EphA2, were elevated by Dsg1 knockdown but not chronic ETA treatment in rafts. Ratios of p-Erk to total Erk are indicated above the blots. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bars: (A) 50 µm; (C) 20 µm.
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fig7: Dsg1 is required for the suppression of EGFR–Erk1/2 signaling during epidermal differentiation. (A) IHC analysis of Dsg1 (left) and EGFR phosphorylated at Y1068 (p-EGFR; right) revealed a basally restricted p-EGFR staining pattern largely absent from Dsg1-positive suprabasal layers in control (miR Lmn) cultures, whereas Dsg1-deficient cultures (miR DG1) demonstrated a gross disorganization of p-EGFR, which was present throughout the basal and suprabasal layers. The dotted lines indicate the boundary between keratinocytes and the collagen matrix. (B) Western blot analysis of keratinocytes transduced with miR Lmn or miR DG1 and induced to undergo differentiation for 2 d as submerged cultures in the presence or absence of 10 µM of the EGFR–erbB2 inhibitor PKI166 (PKI). A significant increase in p-EGFR, p-erbB2, and p-Erk1/2 levels was detectable in keratinocytes upon Dsg1 silencing (Dsg1 KD) along with decreased K10/Dsc1. Blocking EGFR signaling using PKI166 suppressed p-EGFR, p-erbB2, and p-Erk levels in addition to restoring the capacity of Dsg1-deficient keratinocytes to differentiate. Ratios of p-EGFR to total EGFR or p-Erk1/2 to total Erk1/2 are indicated above the blots. Tx, treatment. (C) Dual-label indirect immunofluorescence and Apotome optical sectioning of Dsg1-Flag and EGFR after 4 h in high Ca2+ revealed areas of overlap in discrete regions of cell–cell contact (zoom) but not throughout the cell. The boxed area is magnified in the right panel. (D) Keratinocytes were transduced with EGFP, WT Dsg1-Flag (DG1-WT), or a soluble, truncated Dsg1 mutant (ΔN-DG1) and switched into high Ca2+ before being harvested in RIPA buffer. The insoluble pellet was resuspended in buffered SDS and run in parallel with equal protein amounts from the RIPA-soluble fraction. The ratio of insoluble/total erbB2, EGFR, E-cadherin (E-cad), and K14 is presented in the bar graph and is representative of three experiments. Full-length but not the truncated Dsg1 recruits EGFR–erbB2 into a more insoluble fraction where keratins are concentrated. Under these detergent conditions, the vast majority of E-cadherin remains in the soluble fraction. (E) Activation of EGFR-Ras-Raf-Erk signaling effectors as well as the EGFR–Erk1/2 gene target, EphA2, were elevated by Dsg1 knockdown but not chronic ETA treatment in rafts. Ratios of p-Erk to total Erk are indicated above the blots. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bars: (A) 50 µm; (C) 20 µm.

Mentions: EGFR is concentrated and active in the basal layer where it maintains proliferating keratinocytes in an undifferentiated state (Stoll et al., 2001; Lacouture, 2006). Although this receptor Tyr kinase continues to be expressed in the suprabasal epidermis, which is coordinated with the induction of Dsg1, it is no longer active upon stratification, allowing these cells to adopt a more differentiated phenotype. The inability of a soluble Dsg1 tail to regulate keratinocyte differentiation suggests that localization is important. This observation led us to speculate that Dsg1 might be required to suppress EGFR signaling in the suprabasal layers and allow for normal progression through the epidermal differentiation program. In support of this possibility, phosphorylated EGFR was largely restricted to the basal layer of control rafts, but sustained activation and expansion of this activated receptor into the suprabasal layers were found after Dsg1 knockdown (Fig. 7, A and B). The reciprocal relationship between EGFR activity and Dsg1 was examined in more detail by ectopic expression of this desmosomal cadherin in submerged keratinocytes. These two transmembrane proteins did not overlap extensively in the cytoplasm but were colocalized within discrete regions of cell–cell contact (Fig. 7 C, zoom). Moreover, Dsg1 was capable of reducing the levels of activated EGFR in a manner that did not require Dsc1 (Fig. 5 D). Finally, the ectopic expression of Dsg1 but not the soluble Dsg1 tail was capable of recruiting EGFR and the related erbB2 receptor into a more insoluble pool (Fig. 7 D).


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 is required for the suppression of EGFR–Erk1/2 signaling during epidermal differentiation. (A) IHC analysis of Dsg1 (left) and EGFR phosphorylated at Y1068 (p-EGFR; right) revealed a basally restricted p-EGFR staining pattern largely absent from Dsg1-positive suprabasal layers in control (miR Lmn) cultures, whereas Dsg1-deficient cultures (miR DG1) demonstrated a gross disorganization of p-EGFR, which was present throughout the basal and suprabasal layers. The dotted lines indicate the boundary between keratinocytes and the collagen matrix. (B) Western blot analysis of keratinocytes transduced with miR Lmn or miR DG1 and induced to undergo differentiation for 2 d as submerged cultures in the presence or absence of 10 µM of the EGFR–erbB2 inhibitor PKI166 (PKI). A significant increase in p-EGFR, p-erbB2, and p-Erk1/2 levels was detectable in keratinocytes upon Dsg1 silencing (Dsg1 KD) along with decreased K10/Dsc1. Blocking EGFR signaling using PKI166 suppressed p-EGFR, p-erbB2, and p-Erk levels in addition to restoring the capacity of Dsg1-deficient keratinocytes to differentiate. Ratios of p-EGFR to total EGFR or p-Erk1/2 to total Erk1/2 are indicated above the blots. Tx, treatment. (C) Dual-label indirect immunofluorescence and Apotome optical sectioning of Dsg1-Flag and EGFR after 4 h in high Ca2+ revealed areas of overlap in discrete regions of cell–cell contact (zoom) but not throughout the cell. The boxed area is magnified in the right panel. (D) Keratinocytes were transduced with EGFP, WT Dsg1-Flag (DG1-WT), or a soluble, truncated Dsg1 mutant (ΔN-DG1) and switched into high Ca2+ before being harvested in RIPA buffer. The insoluble pellet was resuspended in buffered SDS and run in parallel with equal protein amounts from the RIPA-soluble fraction. The ratio of insoluble/total erbB2, EGFR, E-cadherin (E-cad), and K14 is presented in the bar graph and is representative of three experiments. Full-length but not the truncated Dsg1 recruits EGFR–erbB2 into a more insoluble fraction where keratins are concentrated. Under these detergent conditions, the vast majority of E-cadherin remains in the soluble fraction. (E) Activation of EGFR-Ras-Raf-Erk signaling effectors as well as the EGFR–Erk1/2 gene target, EphA2, were elevated by Dsg1 knockdown but not chronic ETA treatment in rafts. Ratios of p-Erk to total Erk are indicated above the blots. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bars: (A) 50 µm; (C) 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Dsg1 is required for the suppression of EGFR–Erk1/2 signaling during epidermal differentiation. (A) IHC analysis of Dsg1 (left) and EGFR phosphorylated at Y1068 (p-EGFR; right) revealed a basally restricted p-EGFR staining pattern largely absent from Dsg1-positive suprabasal layers in control (miR Lmn) cultures, whereas Dsg1-deficient cultures (miR DG1) demonstrated a gross disorganization of p-EGFR, which was present throughout the basal and suprabasal layers. The dotted lines indicate the boundary between keratinocytes and the collagen matrix. (B) Western blot analysis of keratinocytes transduced with miR Lmn or miR DG1 and induced to undergo differentiation for 2 d as submerged cultures in the presence or absence of 10 µM of the EGFR–erbB2 inhibitor PKI166 (PKI). A significant increase in p-EGFR, p-erbB2, and p-Erk1/2 levels was detectable in keratinocytes upon Dsg1 silencing (Dsg1 KD) along with decreased K10/Dsc1. Blocking EGFR signaling using PKI166 suppressed p-EGFR, p-erbB2, and p-Erk levels in addition to restoring the capacity of Dsg1-deficient keratinocytes to differentiate. Ratios of p-EGFR to total EGFR or p-Erk1/2 to total Erk1/2 are indicated above the blots. Tx, treatment. (C) Dual-label indirect immunofluorescence and Apotome optical sectioning of Dsg1-Flag and EGFR after 4 h in high Ca2+ revealed areas of overlap in discrete regions of cell–cell contact (zoom) but not throughout the cell. The boxed area is magnified in the right panel. (D) Keratinocytes were transduced with EGFP, WT Dsg1-Flag (DG1-WT), or a soluble, truncated Dsg1 mutant (ΔN-DG1) and switched into high Ca2+ before being harvested in RIPA buffer. The insoluble pellet was resuspended in buffered SDS and run in parallel with equal protein amounts from the RIPA-soluble fraction. The ratio of insoluble/total erbB2, EGFR, E-cadherin (E-cad), and K14 is presented in the bar graph and is representative of three experiments. Full-length but not the truncated Dsg1 recruits EGFR–erbB2 into a more insoluble fraction where keratins are concentrated. Under these detergent conditions, the vast majority of E-cadherin remains in the soluble fraction. (E) Activation of EGFR-Ras-Raf-Erk signaling effectors as well as the EGFR–Erk1/2 gene target, EphA2, were elevated by Dsg1 knockdown but not chronic ETA treatment in rafts. Ratios of p-Erk to total Erk are indicated above the blots. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bars: (A) 50 µm; (C) 20 µm.
Mentions: EGFR is concentrated and active in the basal layer where it maintains proliferating keratinocytes in an undifferentiated state (Stoll et al., 2001; Lacouture, 2006). Although this receptor Tyr kinase continues to be expressed in the suprabasal epidermis, which is coordinated with the induction of Dsg1, it is no longer active upon stratification, allowing these cells to adopt a more differentiated phenotype. The inability of a soluble Dsg1 tail to regulate keratinocyte differentiation suggests that localization is important. This observation led us to speculate that Dsg1 might be required to suppress EGFR signaling in the suprabasal layers and allow for normal progression through the epidermal differentiation program. In support of this possibility, phosphorylated EGFR was largely restricted to the basal layer of control rafts, but sustained activation and expansion of this activated receptor into the suprabasal layers were found after Dsg1 knockdown (Fig. 7, A and B). The reciprocal relationship between EGFR activity and Dsg1 was examined in more detail by ectopic expression of this desmosomal cadherin in submerged keratinocytes. These two transmembrane proteins did not overlap extensively in the cytoplasm but were colocalized within discrete regions of cell–cell contact (Fig. 7 C, zoom). Moreover, Dsg1 was capable of reducing the levels of activated EGFR in a manner that did not require Dsc1 (Fig. 5 D). Finally, the ectopic expression of Dsg1 but not the soluble Dsg1 tail was capable of recruiting EGFR and the related erbB2 receptor into a more insoluble pool (Fig. 7 D).

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