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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

Differentiation-dependent Dsg1 maintains adhesion in the superficial layers of epidermal raft cultures. (A) IHC analysis of Dsg1 and Dsg3 in frozen sections prepared from 9-d-old rafts. Dsg1 was concentrated in the suprabasal layers (insets with dashed lines), whereas Dsg3 was prominent in the basal layer (insets with continuous lines). (B) Real-time PCR analysis of Dsg1–4 mRNA levels from keratinocytes maintained as submerged cultures (Sub day 0) or on rafts for 3, 6, or 9 d. The Dsg mRNA levels were normalized to cyclophilin 1 levels interpolated from a standard curve, and the mean of three independent experiments (±SEM) is represented in the graph. Dsg1 mRNA levels were tightly coordinated with raft maturation, whereas Dsg3 remained relatively constant. Maximal Dsg2 (0.0468 ± 0.0318) and Dsg4 (0.000874 ± 0.000101) mRNA transcript levels were detected in submerged and day 9 raft cultures, respectively. (C) H&E-stained sections of 9-d-old rafts incubated with or without 5 µg/ml recombinant ETA (acute ETA) for 24 h. Boxed insets show areas of cell–cell dissociation in the uppermost layers of ETA-treated rafts, whereas control cultures remained intact. (D) Western blot analysis of these raft cultures using a cytoplasmic domain (CYTO) antibody for Dsg1 demonstrated that the N-terminal portion (aa 1–381) of the adhesive ectodomain (ECTO) of the full-length protein (Dsg1 FL) was efficiently cleaved in the presence of ETA, resulting in a membrane-associated cytoplasmic fragment (Dsg1 CL; aa 382–1,049) that was retained in whole cell lysates. Dsg2–4 were not cleaved by ETA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TM, transmembrane domain. Bars: (A and C) 50 µm; (insets) 20 µm.
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fig1: Differentiation-dependent Dsg1 maintains adhesion in the superficial layers of epidermal raft cultures. (A) IHC analysis of Dsg1 and Dsg3 in frozen sections prepared from 9-d-old rafts. Dsg1 was concentrated in the suprabasal layers (insets with dashed lines), whereas Dsg3 was prominent in the basal layer (insets with continuous lines). (B) Real-time PCR analysis of Dsg1–4 mRNA levels from keratinocytes maintained as submerged cultures (Sub day 0) or on rafts for 3, 6, or 9 d. The Dsg mRNA levels were normalized to cyclophilin 1 levels interpolated from a standard curve, and the mean of three independent experiments (±SEM) is represented in the graph. Dsg1 mRNA levels were tightly coordinated with raft maturation, whereas Dsg3 remained relatively constant. Maximal Dsg2 (0.0468 ± 0.0318) and Dsg4 (0.000874 ± 0.000101) mRNA transcript levels were detected in submerged and day 9 raft cultures, respectively. (C) H&E-stained sections of 9-d-old rafts incubated with or without 5 µg/ml recombinant ETA (acute ETA) for 24 h. Boxed insets show areas of cell–cell dissociation in the uppermost layers of ETA-treated rafts, whereas control cultures remained intact. (D) Western blot analysis of these raft cultures using a cytoplasmic domain (CYTO) antibody for Dsg1 demonstrated that the N-terminal portion (aa 1–381) of the adhesive ectodomain (ECTO) of the full-length protein (Dsg1 FL) was efficiently cleaved in the presence of ETA, resulting in a membrane-associated cytoplasmic fragment (Dsg1 CL; aa 382–1,049) that was retained in whole cell lysates. Dsg2–4 were not cleaved by ETA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TM, transmembrane domain. Bars: (A and C) 50 µm; (insets) 20 µm.

Mentions: Dsg1 was present in rafts and exhibited a restricted expression pattern in differentiating keratinocytes (Fig. 1, A and B). As expected, Dsg1 was primarily found in the suprabasal layers that formed after 9 d at an air–liquid interface, whereas Dsg3 was immunolocalized between adjacent basal keratinocytes and was more abundant in the lower epidermal cell layers (Fig. 1 A). To test whether Dsg1 adhesive functions were conserved in these three-dimensional cultures, keratinocytes were matured on rafts for 9 d and then incubated for 24 h in the presence or absence of recombinant Staphylococcus aureus exfoliative toxin A (ETA; acute ETA treatment), a Ser protease which specifically cleaves after a Glu residue (381) located between EC3 and EC4 of Dsg1 but does not target other cadherins (Amagai et al., 2000; Hanakawa et al., 2004; Nagasaka et al., 2004). Similar to the phenotypic outcome in humans with ETA-induced bullous impetigo or Staphylococcal scalded skin syndrome, efficient removal of EC1–3 from Dsg1 in mature rafts resulted in prominent areas of dissociation between adjacent keratinocytes in the uppermost cell layers (Fig. 1, C and D). These regions corresponded to cell layers in which Dsg1 expression was most abundant and which further lacked Dsg3. Importantly, ETA had no effect on the protein levels or integrity of Dsg3 or the related Dsg2 and Dsg4, with the latter two found in lower abundance in raft cultures (Fig. 1, B and D). These observations helped validate the raft model for Dsg1 functional analysis.


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)

Differentiation-dependent Dsg1 maintains adhesion in the superficial layers of epidermal raft cultures. (A) IHC analysis of Dsg1 and Dsg3 in frozen sections prepared from 9-d-old rafts. Dsg1 was concentrated in the suprabasal layers (insets with dashed lines), whereas Dsg3 was prominent in the basal layer (insets with continuous lines). (B) Real-time PCR analysis of Dsg1–4 mRNA levels from keratinocytes maintained as submerged cultures (Sub day 0) or on rafts for 3, 6, or 9 d. The Dsg mRNA levels were normalized to cyclophilin 1 levels interpolated from a standard curve, and the mean of three independent experiments (±SEM) is represented in the graph. Dsg1 mRNA levels were tightly coordinated with raft maturation, whereas Dsg3 remained relatively constant. Maximal Dsg2 (0.0468 ± 0.0318) and Dsg4 (0.000874 ± 0.000101) mRNA transcript levels were detected in submerged and day 9 raft cultures, respectively. (C) H&E-stained sections of 9-d-old rafts incubated with or without 5 µg/ml recombinant ETA (acute ETA) for 24 h. Boxed insets show areas of cell–cell dissociation in the uppermost layers of ETA-treated rafts, whereas control cultures remained intact. (D) Western blot analysis of these raft cultures using a cytoplasmic domain (CYTO) antibody for Dsg1 demonstrated that the N-terminal portion (aa 1–381) of the adhesive ectodomain (ECTO) of the full-length protein (Dsg1 FL) was efficiently cleaved in the presence of ETA, resulting in a membrane-associated cytoplasmic fragment (Dsg1 CL; aa 382–1,049) that was retained in whole cell lysates. Dsg2–4 were not cleaved by ETA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TM, transmembrane domain. Bars: (A and C) 50 µm; (insets) 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712955&req=5

fig1: Differentiation-dependent Dsg1 maintains adhesion in the superficial layers of epidermal raft cultures. (A) IHC analysis of Dsg1 and Dsg3 in frozen sections prepared from 9-d-old rafts. Dsg1 was concentrated in the suprabasal layers (insets with dashed lines), whereas Dsg3 was prominent in the basal layer (insets with continuous lines). (B) Real-time PCR analysis of Dsg1–4 mRNA levels from keratinocytes maintained as submerged cultures (Sub day 0) or on rafts for 3, 6, or 9 d. The Dsg mRNA levels were normalized to cyclophilin 1 levels interpolated from a standard curve, and the mean of three independent experiments (±SEM) is represented in the graph. Dsg1 mRNA levels were tightly coordinated with raft maturation, whereas Dsg3 remained relatively constant. Maximal Dsg2 (0.0468 ± 0.0318) and Dsg4 (0.000874 ± 0.000101) mRNA transcript levels were detected in submerged and day 9 raft cultures, respectively. (C) H&E-stained sections of 9-d-old rafts incubated with or without 5 µg/ml recombinant ETA (acute ETA) for 24 h. Boxed insets show areas of cell–cell dissociation in the uppermost layers of ETA-treated rafts, whereas control cultures remained intact. (D) Western blot analysis of these raft cultures using a cytoplasmic domain (CYTO) antibody for Dsg1 demonstrated that the N-terminal portion (aa 1–381) of the adhesive ectodomain (ECTO) of the full-length protein (Dsg1 FL) was efficiently cleaved in the presence of ETA, resulting in a membrane-associated cytoplasmic fragment (Dsg1 CL; aa 382–1,049) that was retained in whole cell lysates. Dsg2–4 were not cleaved by ETA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TM, transmembrane domain. Bars: (A and C) 50 µm; (insets) 20 µm.
Mentions: Dsg1 was present in rafts and exhibited a restricted expression pattern in differentiating keratinocytes (Fig. 1, A and B). As expected, Dsg1 was primarily found in the suprabasal layers that formed after 9 d at an air–liquid interface, whereas Dsg3 was immunolocalized between adjacent basal keratinocytes and was more abundant in the lower epidermal cell layers (Fig. 1 A). To test whether Dsg1 adhesive functions were conserved in these three-dimensional cultures, keratinocytes were matured on rafts for 9 d and then incubated for 24 h in the presence or absence of recombinant Staphylococcus aureus exfoliative toxin A (ETA; acute ETA treatment), a Ser protease which specifically cleaves after a Glu residue (381) located between EC3 and EC4 of Dsg1 but does not target other cadherins (Amagai et al., 2000; Hanakawa et al., 2004; Nagasaka et al., 2004). Similar to the phenotypic outcome in humans with ETA-induced bullous impetigo or Staphylococcal scalded skin syndrome, efficient removal of EC1–3 from Dsg1 in mature rafts resulted in prominent areas of dissociation between adjacent keratinocytes in the uppermost cell layers (Fig. 1, C and D). These regions corresponded to cell layers in which Dsg1 expression was most abundant and which further lacked Dsg3. Importantly, ETA had no effect on the protein levels or integrity of Dsg3 or the related Dsg2 and Dsg4, with the latter two found in lower abundance in raft cultures (Fig. 1, B and D). These observations helped validate the raft model for Dsg1 functional analysis.

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