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First published online 6 February 2007
doi: 10.1242/jcs.03392

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Suprabasal Dsg2 expression in transgenic mouse skin confers a hyperproliferative and apoptosis-resistant phenotype to keratinocytes

Donna Brennan¹, Ying Hu¹, Sohaila Joubeh¹, Yoo Won Choi¹, Diana Whitaker-Menezes², Thomas O'Brien^1,3, Jouni Uitto¹, Ulrich Rodeck¹ and M G. Mahoney^1,*

¹ Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA
² Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA
³ Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA

View larger version (35K): [in this window] [in a new window]   Fig. 1. Targeting construct and Dsg2-Flag expression in transgenic mice. (A) Schematic diagram of the mDsg2-Flag transgenic construct inserted at NotI restriction sites downstream of the involucrin promoter in the pH2700-pL2 vector mDsg2, mouse desmoglein 2 cDNA; Flag, nucleotide sequence encoding for the Flag octapeptide; polyA, polyadenylation signal. (B) Immunofluorescent staining of wild-type (upper panels) and Inv-Dsg2 transgenic (lower panels) adult mouse skin with Flag (green, left panels) and Dsg2 MP6 (red, middle panels) antibodies showing expression of Dsg2-Flag in the transgenic, but not control epidermis. Co-staining (merge, right panels) for Dsg2 and Flag in the Inv-Dsg2 transgenic but not control wild-type skin. Nuclei were stained with DAPI (blue). (C) Immunoblot analysis of adult control and transgenic mouse skin extracts using Flag and MP6 antibodies showing Dsg2 and Flag expression in the transgenic but not wild-type skin. Blots were reprobed with actin antibody as a loading control. In all panels: Tg, transgenic; WT, wild type.

View larger version (88K): [in this window] [in a new window]   Fig. 2. Inv-Dsg2 mice develop epidermal hyperplasia. Hematoxylin and Eosin staining of newborn back skin (A,B) and adult (C-J) back skin (C,D,I,J), ear (E,F) and tongue (G,H) of wild-type control (A,C,E,G,I) and Inv-Dsg2 transgenic (B,D,F,H,J) littermate mice. Newborn and adult mice were 2 days and 3 months old, respectively. Histological analysis shows varying degrees of epidermal thickening in all Inv-Dsg2 transgenic tissues examined. Minor compaction (B,D; asterisk) and the presence of nuclei (B,D; arrows) was observed in the outermost granular and horny layer interface. A more dramatic difference in epidermal thickness was observed in the adult transgenic mice compared that of control littermates (C,D; brackets) and to newborn transgenic skin. Pink translucent flattened eosinophilic keratinocytes were apparent in the stratum corneum (F; open arrow). Cells and nuclei in the spinous layers of transgenic epithelia appear slightly enlarged relative to those in wild-type epithelia. Bars, 25 µm.

View larger version (103K): [in this window] [in a new window]   Fig. 3. Transmission electron microscopy of adult control wild-type (A) and Inv-Dsg2 transgenic (B) epidermis. The granular cells appeared more compact and interconnected and corneocytes were thicker in the axial direction of the Inv-Dsg2 transgenic epidermis (brackets). Detached desmosomes with intercellular gaps were detected in the stratum corneum (*). Arrows, desmosomes at the granular and stratum corneum interface; arrowheads, desmosome-like electron dense intercellular structures; d, desmosome; k, keratohyalin granules. Bars, 1 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 4. Changes in expression patterns of cornified envelope and keratin proteins in Inv-Dsg2 mice. (A) Immunofluorescent staining of 3-month old adult wild-type and Inv-Dsg2 transgenic mouse skin for cytokeratin (CK14, CK10 and CK6), involucrin (Inv), filaggrin (Fil), and loricrin (Lor). CK14 was observed only in the basal layer of the control but extended into the suprabasal epidermal layers of Inv-Dsg2 skin. Increased expression of CK10 was observed in the Inv-Dsg2 epidermis. CK6 expression was observed in the interfollicular epidermis in the Inv-Dsg2 mice. Suprabasal Dsg2 expression was associated with expression of involucrin, filaggrin, and loricrin. Insets are higher resolution and increased exposure images. Nuclei were stained with DAPI (blue). (B) Immunoblotting analysis of adult control wild-type and Inv-Dsg2 transgenic mouse skin with antibodies against Flag, Dsg1 (antibody 27B2; recognizes mouse Dsg1-, Dsg1- and Dsg1-), Dsg3 (AP904), PG, CK6, CK10, CK14, Lor, Fil and actin (loading control).

View larger version (76K): [in this window] [in a new window]   Fig. 5. Inv-Dsg2 transgenic mice show hyperproliferative epidermis and increase in mitotic activity. Paraffin embedded sections of adult wild-type (A,C) and Inv-Dsg2 transgenic (B,D) skin were stained with antibodies to detect either PCNA expression (A,B) or BrdU incorporation (C,D; green). Insets show higher resolution images. Also shown in red in C and D is immunostaining for CK14.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Characterization of the signaling events in Inv-Dsg2 epidermis. Total protein lysates of wild-type and Inv-Dsg2 transgenic adult skin were analyzed by western analysis to assess activation (phosphorylation) states of signaling intermediates. Immunoblotting for actin, pan cytokeratin (CK) and -tubulin expression showed equal loading of lanes.

View larger version (59K): [in this window] [in a new window]   Fig. 7. Establishment of cultured primary keratinocytes from Inv-Dsg2 transgenic and wild-type newborn mouse skin. (A) Immunoblotting analysis for the Flag tag of wild-type and transgenic cell lines grown in low (80 nM; –) or high (1 mM; +) calcium for 5 days. Dsg2-Flag (160 kDa) expression was detected in the calcium-treated transgenic but not the untreated transgenic or the wild-type control keratinocytes. Differentiation was confirmed by increased involucrin expression (*) in response to calcium. Upregulation of Dsg2 enhanced P-STAT3 level but not total STAT3 in transgenic compared with wild-type skin. Actin was used as a loading control. (B) Wild-type and transgenic keratinocytes were grown to confluency in low-calcium-containing CnT medium and then 1 mM calcium was added for 24 hours. Cells were fixed and stained with Flag and DG3.10 antibodies. Transgenic but not wild-type keratinocytes expressed the Dsg2-Flag protein. DG3.10 recognized the endogenous Dsg2 in the wild-type and as well as the Dsg2-Flag in the transgenic cells. (C) Wild-type and Inv-Dsg2 transgenic cells were trypsinized and put in suspension culture for up to 72 hours. Cells were collected at the time points indicated. Dsg2-Flag was detected by immunoblot analysis in Inv-Dsg2 cells within 24-72 hours in suspension culture. Immunoblotting analysis for actin expression showed equal loading. Similar results were observed with three independent wild-type and transgenic cell lines.

View larger version (54K): [in this window] [in a new window]   Fig. 8. Enhanced survival of Inv-Dsg2 keratinocytes in forced suspension culture is dependent on NF-B activation. (A) Clonal growth of control and Inv-Dsg2 cell lines after 0, 24, 48 and 72 hours in forced suspension culture was assessed by replating cells on tissue culture plastic and allowing cell proliferation for 7-9 days after replating. Inv-Dsg2 transgenic cells showed dramatically increased survival and re-growth compared to wild-type cells, in this setting. (B) Inv-Dsg2 transgenic cells were subjected to cell suspension culture for 48 hours with EGF and/or Bay11-7082, an IB-alpha phosphorylation inhibitor. Cell survival was assessed by replating cells on tissue cultured-treated plastic for 7 days. Activation of the EGF receptor with exogenous EGF further enhanced survival of Inv-Dsg2 transgenic cells in forced suspension cultures. Bay11-7082 inhibition of NF-B completely abolished cell survival in suspension culture. EGF-R activation was unable to counteract the effect of Bay11-7082. (C) Immunoblot analysis of NF-B p65 expression in wild-type and transgenic skin. Actin was used as a loading control. (D) Immunoblot results were confirmed by immunofluorescent staining, which showed the presence of NF-B p65 in nuclei of transgenic but not wild-type skin. Dotted lines demarcate dermal-epidermal junction. Bars, 50 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 9. Increased tumor formation in Inv-Dsg2 transgenic mice. (A,B) Histological analysis of epidermal hyperplasia and early forms of papillomas in Inv-Dsg2 transgenic but not control wild-type mice at 6 weeks (A) and 3 months (B) (10x magnification). Arrows indicate hyperproliferation; box, hyperkeratosis; asterisk, encapsulated stratum corneum. (C) After 25 weeks of DMBA and TPA treatment, the Inv-Dsg2 transgenic mice developed more tumors than control wild-type mice. (D,E) The frequency and average number of papillomas in wild-type and transgenic mice after 25 weeks of tumor promotion. For tumors greater than 2 mm, *P=0.0063. (F) Histopathological appearance of carcinogen-induced tumors from wild-type and transgenic mice showing differentiated and hyperkeratotic papillomas.

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