New insights into cadherin function in epidermal sheet formation and maintenance of tissue integrity

Abstract

Co-expression and gene linkage have hampered elucidating the physiological relevance of cadherins in mammalian tissues. Here, we combine conditional gene ablation and transgenic RNA interference to uncover new roles for E- and P-cadherins in epidermal sheet formation in vitro and maintenance of epidermal integrity in vivo. By devising skin-specific RNAi technology, we demonstrate that cadherin inhibition in vivo impairs junction formation and intercellular adhesion and increases apoptosis. These defects compromise epidermal barrier function and tissue integrity. In vitro, with only E-cadherin missing, epidermal sheet formation is delayed, but when both cadherins are suppressed, defects extend to adherens junctions, desmosomes, tight junctions and cortical actin dynamics. Using different rescue strategies, we show that cadherin level rather than subtype is critical. Finally, by comparing conditional loss-of-function studies of epidermal catenins and cadherins, we dissect cadherin-dependent and independent roles of adherens junction components in tissue physiology.

Fig. 1.

Generation of Pcad RNAi transgenic mice. (A) Schematic of the Pcad and Ecad chromosomal locus. (B) The K14-Pcad RNAi construct used to generate transgenic (Tg) mice. (C) Images of WT and PcadRNAi Tg littermates and WT and Pcad KO littermate adult mice. (D and F) Newborn (P0) tail skins of indicated mice were processed for indirect immunofluorescence with indicated Abs. Arrows denote downregulation of Ecad at sites of HF downgrowth in WT, but not Pcad KO or PcadRNAi Tg mice. (E) Immunoblot analyses of P0 epidermal lysates with indicated Abs; α-tub (α-tubulin). epi, epidermis; der, dermis; hf, hair follicle. Dotted lines represent junction between epidermis and dermis. (Scale bars, 10 μm.)

Fig. 2.

Inhibition of classical cadherins in mouse epidermis results in cell dissociation, blistering skin lesions, and defective epidermal barrier. PcadRNAi Tg and K14-Cre/Ecad cKO mice were bred to generate P0 mice WT or conditionally null for the Ecad allele and either negative (Ecad cKO) or positive (cKO/Tg) for the PcadRNAi transgene. Arrows in A denote blistered skin lesions, seen only in cKO/Tg mice. Barrier function analyses in B assessed by exclusion of an XGAL-containing solution. Arrows indicate areas, found only in cKO/Tg mice, where skin barrier function is disrupted and endogenous β-galactosidase converts dye to blue. Immunofluorescence in C shows absence of P-cadherin in cKO/Tg backskin epidermis (epidermal Ecad is completely absent in Ecad cKO mice) (10). H&E staining in D reveals that such regions lacking both cadherins exhibit gross perturbations in tissue integrity.

Fig. 3.

Ultrastructural abnormalities in cadherin-deficient epidermis. (A) 0.8 μm semithin sections of P0 backskins were stained with toluidine blue. Asterisks denote intercellular gaps between keratinocytes of cKO/Tg epidermis. Also note epidermal hyperthickening and marked disorganization and altered cuboidal morphology of basal cells in cKO/Tg skin. (B and C) Transmission electron microscopy. Asterisks in (B and B′) and double arrows in (B′) denote intercellular gaps. Despite gaps and signs of cellular degeneration, double membranes appear to persist between cKO/Tg keratinocytes. Note also that desmosomes (Dm), which were not reduced in number, appear to be intact even in areas where intercellular gaps occurred (B′). Region in C contrasts the normal desmosome-keratin filament network of suprabasal cells of WT epidermis versus the irregular aggregates of keratin filaments (Kf) that were frequently observed in cKO/Tg epidermis. Such alterations in keratin organization frequently reflect defects in mechanical integrity. Additional abbreviations: BL (basal layer); Sp (spinous layer); Gr (granular layer); SC (stratum corneum). Dotted lines in A and B represent junction between epidermis and dermis. (Scale bars: A, 5 μm (A); B, 2 μm; B′, 500 nm; C, 5 μm.)

Fig. 4.

Perturbations in intercellular junctions, cytoskeleton organization, and tight junction function in epidermis lacking E- and P-cad. (A–F) P0 skins from tail (D) and back (all others) were processed for fluorescence microscopy with indicated Abs or TRITC-Phalloidin (actin, red). Additional Ab abbreviations: α-cat (α-catenin); β4 (β4 integrin), hemidesmosomal component restricted to the base of the basal layer; DP (desmoplakin); K1 (keratin 1), component of suprabasal IF network; ClaI (claudin 1); Occl (occludin). Asterisk in A denotes nonspecific 2⁰Ab staining of cornified layer. Abnormalities unique to cKO/Tg skin are denoted by: Arrows in B and D, gaps between basal and suprabasal cells; arrowhead in B, absence of cortical actin network between two suprabasal keratinocytes; asterisk in B, expansion of β4 integrin localization into spinous layers (cornified cell staining is nonspecific as per above); arrowhead in D, non-uniform staining patterns of keratin in some suprabasal keratinocytes. (F) Inside-out permeability assessed by monitoring impedance to biotin flow at TJs in granular layer. Arrows indicate occludin-based TJs. Arrowheads denote biotin flow past TJs in cKO/Tg epidermis only. (G) Immunoblot analysis of total P0 epidermal lysates with indicated Abs; Pan Cad (pan cadherin); β-cat (β-catenin); p120 (p120-catenin); Afad (afadin); PG (plakoglobin). (Scale bars: 10 μm.)

Fig. 5.

Overall cadherin level governs epidermal sheet formation in vitro. (A–E) Confluent monolayers of WT and Ecad KO primary mouse keratinocytes (1⁰MKs) alone or stably expressing indicated constructs were shifted from low (0.05 mM) to high (1.5 mM) Ca²⁺ media for indicated times before processing for fluorescence microscopy with Abs or Alexa 647-phalloidin (actin, red) as indicated and immunoblot analysis of total lysates (D). Actin in A and B represents epifluorescence from K14-GFPactin (28) 1⁰MKs WT or null for Ecad. Arrows in all images indicate sites of cell-cell interactions. (A) Despite increased Pcad at puncta, Ecad KO 1⁰MKs display delayed kinetics of catenin localization and actin organization. (B) Expression of either Ecad-GFP or Pcad-GFP rescues the early delay in epidermal sheet formation in Ecad KO 1⁰MKs. Asterisks denote sites of interactions between Ecad KO cells that do not express cadherin-GFP. (C and D) Immunofluoresence and immunoblot analysis of PcadRNAi in vitro. (E) Inhibition of Ecad and Pcad blocks epidermal sheet formation in vitro and this is rescued by expression of silencing resistant Pcad-cherry fusion protein. Anti-RFP antibody was used to detect mutant Pcad protein. Asterisks specifically indicate sites of interactions between Ecad KO + PcadRNAi cells lacking rescue construct. (Scale bars: 10 μm.)

Fig. 6.

In vivo cadherin and catenin inhibition results in an increase in epidermal apoptosis, yet loss of epidermal cadherins does not perturb proliferative and inflammatory responses. (A and B) P0 backskins and E18.5 embryos, respectively, were processed for labeling of fragmented DNA via TUNEL. (C and D) Quantification of active-caspase 3 (pCasp3) immunofluorescence of indicated littermate samples. Data were collected from two independently processed sets of animals. Results are shown as percent anti-pCasp3 immunoreactive cells of the total epidermal cells counted. Asterisks denote significant difference from WT cells determined by t test: (C): P < 1 × 10⁻⁵; (D): P < 1 × 10⁻¹⁰. Error bars represent SD. (E and H) P0 backskins were processed for indirect immunofluorescence and immunohistochemistry with indicated Abs. (F) Quantification of BrdU-labeling experiments. P0 mice were injected s.c. with 50 μg/g wt BrdU and killed 2 h. later. Data were collected from two independently labeled sets of animals. Results are shown as percent BrdU-labeled cells of the total epidermal cells counted. Error bars represent SD. (G) Total P0 epidermal lysates were processed for immunoblot analysis with indicated Abs. (Scale bars: 10 μm.)