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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01689

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Keratinocytes display normal proliferation, survival and differentiation in conditional ß4-integrin knockout mice

Karine Raymond^*, Maaike Kreft^*, Hans Janssen, Jero Calafat and Arnoud Sonnenberg

Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

View larger version (29K): [in a new window]   Fig. 1. Targeting strategy and molecular analysis of recombinant ES cells and conditional ß4 knockout mice. (A) Restriction map of the 5' end of the ß4 locus, the targeting construct and different ß4 mutant alleles. Exons are indicated as numbered black boxes and loxP sites as triangles. Position of the unique SacII and KpnI sites used for insertion of the single loxP and the floxed neo-tk cassette are indicated, as are the primers used for PCR analyses to detect the different mutant alleles of the ß4 gene. Restriction sites are: H, HindIII; RI, EcoRI; X, XhoI; RV, EcoRV. (B) Southern blot and PCR analyses of recombinant ES clones. DNA from two independently targeted ES clones (lanes 1, 3) and two wild-type ES clones (lanes 2, 4) were digested with HindIII (left panel) or EcoRV (right panel), subjected to agarose gel electrophoresis, and transferred to nitrocellulose. Wild-type and mutant alleles were detected by hybridization of the filters with radiolabeled mouse ß4 genomic probes corresponding to exons 2-7 (5' probe) or exons 7-9 (3' probe) (left and right panels, respectively). The presence of the first loxP site in intron 1a of the targeted ES clones was confirmed by PCR using primers P1 and P2 (bottom panel). (C) PCR analysis on genomic DNA of conditional ß4 knockout mice before and after their crossing with K14-Cre mice. The conditional allele of the ß4 gene was detected by PCR analysis on tail DNA using primers P3 and P4 (Before and After K14-Cre, left and top right panel), and the K14-Cre transgene using the primers K14-cre3 and K14-cre5 (After K14-Cre, middle panel). The removal of exons 1b-5 by Cre-mediated recombination, thereby generating a ß4 null allele, was detected by PCR analysis using primers P1 and P4 (After K14-Cre, bottom panel). PCR fragments were resolved by agarose gel electrophoresis and visualized by ethidium bromide. Bands corresponding to the wt, floxed and null alleles of the ß4 gene as well as to the K14-Cre transgene are indicated (wt, flox, null, K14). Sizes of molecular weight markers are indicated in bp.

View larger version (114K): [in a new window]   Fig. 2. Histological and ultrastructural abnormalities in ß4 null skin. (A) Inflamed ear of a 4-month-old mouse of the genotype ß4flox/flox; K14-Cre. (B) Expression pattern of the Cre-recombinase in the skin of a 15.5-day-old embryo of the genotype ROSA26-pGK-neo-pA-LacZ-pA; K14-Cre, as assessed by X-gal staining. The non-stochastic expression of the Cre-recombinase at that stage has been described previously (Jonkers et al., 2001). The efficiency of K14-Cre-mediated recombination towards the ß4flox alleles is such that expression of the ß4 gene remained largely mosaic even after birth. (C) Cryosection of skin from a newborn ß4flox/flox; K14-Cre mouse stained with hematoxylin and eosin showing the presence of a small blister. The double-headed arrow denotes separation of the epidermis from the dermis. (D) Electron microscopy of the skin of a ß4flox/flox; K14-Cre mouse shows a blistered area (double-headed arrow) infiltrated by leukocytes (L). The keratinocytes that had detached from the dermis lacked discernible HDs. (E) Immuno-electron microscopy of ß4flox/flox; K14-Cre skin with primary antibodies against ß4 demonstrates gold particles associated with HDs at the base of a non-recombined keratinocyte. The neighboring cell to the right has probably degenerated as a result of an inflammatory reaction. The dotted line demarcates the boundary of the cell. BM, basement membrane; Nu, nucleus; HD, hemidesmosome; D, desmosome; L, leukocyte.

View larger version (71K): [in a new window]   Fig. 3. Expression and distribution pattern of different cell-adhesion receptors, BM components and hemidesmosomal proteins in regions of skin lacking ß4. (A-I) Cryosections of skin from a 2-day-old ß4flox/flox; K14-Cre mouse were processed for indirect immunofluorescence and visualized by confocal microscopy. Primary antibodies are against proteins indicated in the left lower corner of each image, and colors are coded according to FITC or Texas Red secondary antibodies. In all panels, the epidermis is at the top and the dermis at the bottom. Notice that only the distribution of the hemidesmosomal proteins plectin, BP180 and BP230 is affected by the absence of 6ß4.

View larger version (93K): [in a new window]   Fig. 4. The BM is disorganized in the inflamed ear of a 4-month-old ß4flox/flox; K14-Cre mouse. (A-F) Cryosections of inflamed ear processed for indirect immunofluorescence and visualized by confocal microscopy. Primary antibodies are against proteins that are indicated in the left lower corner of each image and colors are coded according to FITC or Texas Red secondary antibodies. (A,B) Images show that the removal of ß4 is associated with keratinocyte activation as visualized by keratin-6 staining (A) and the deposition of fibronectin into the BM (B). (C-F) Higher magnifications illustrating the presence of blisters when 6ß4 is absent (D, arrows), the alteration of the BM components collagen IV and Ln-5 in inflamed regions of the skin showing lamination (C,F, arrows) and the deposition of fibronectin into the BM (E, arrow).

View larger version (142K): [in a new window]   Fig. 5. Establishment of a mouse keratinocyte cell line, carrying a conditional allele of the ß4 gene. (A) Phase-contrast image of subconfluent NMK-1(+) and (–) keratinocytes showing their morphology. (B) Immunoblot analysis of the expression levels of p53 and different epithelial and mesenchymal markers in the NMK-1(+) and (–) cell lines. RAC-11P cells (Sonnenberg et al., 1993) and mouse embryonic endothelial cells (MEEC) (Larsson et al., 2001) are used as control cell lines. (C) FACS analysis of the levels of 6, ß4, ß1, 2, 5 and v integrin subunits expressed by NMK-1(+) and (–) cells. (D) Immunoprecipitation analysis of surface-labeled components from NMK-1(+) and (–) cells with antibodies specific for a range of integrin subunits. (E) NMK-1(+) and (–) keratinocytes adhered equally well to Ln-5 and fibronectin. (F,G) Detachment assays based on resistance to trypsin treatment (F) and centrifugal force (G) demonstrate the importance of 6ß4 in strengthening the adhesion. (H) Indirect immunofluorescence analysis of hemidesmosomal protein localization in NMK-1 cells visualized by confocal microscopy. Primary antibodies are against proteins specified in the left lower corner of each image and colors are coded according to FITC or Texas Red secondary antibodies. The colocalization of the different hemidesmosomal components indicates that these cells form type I HDs in culture. (I) Ultrastructural analysis of NMK-1(+) and (–) cells (upper and lower panels, respectively) further confirms the presence of HDs in NMK-1(+) cells whereas they are lost in NMK-1(–) population. (J) Wound healing assay shows that the motility of NMK-1(–) cells is increased compared with those of NMK-1(+). In the bar graph, results are expressed as the unadjusted means ± s.d. of four separate experiments with six replicates each (P<0.001).

View larger version (42K): [in a new window]   Fig. 6. Loss of the 6ß4 integrin does not affect stratification and differentiation of keratinocytes. (A-F) Cryosections of skin from two-day-old ß4flox/flox; K14-Cre mice processed for indirect immunofluorescence and visualized by confocal microscopy. Primary antibodies are against proteins indicated in the left lower corner of each image, and colors are coded according to FITC or Texas Red secondary antibodies. In all panels, the epidermis is at the top and the dermis at the bottom. (G) Semi-quantitative RT-PCR showing that there were no significant changes in the level of transcripts for a range of basal (6 integrin, Dsg-2 and -3 and the 2 chain of Ln-5) and suprabasal (Dsg-1 and envoplakin) cell markers in NMK-1(+) and (–) cells.

View larger version (53K): [in a new window]   Fig. 7. Loss of 6ß4 integrin does not affect proliferation/survival of basal keratinocytes when cells are adhered to the BM. (A-F) Cryosections of skin from newborn ß4flox/flox; K14-Cre mice, injected intraperitoneally with BrdU for 1 hour (A-C) or assayed by TUNEL for the presence of apoptotic cells (D,F) that were subsequently processed for indirect immunofluorescence and visualized by confocal microscopy. Primary antibodies are against proteins indicated, and colors are coded according to FITC or Texas Red secondary antibodies. In all panels, epidermis is at the top and dermis at the bottom. (G) Quantitative analysis of cell proliferation in the interfollicular regions of epidermis that lack or contain the ß4 integrin. (H) The NMK-1(+) and (–) cell populations do not form colonies in semi-solid medium (left panels) and show the same growth capacities at low density (right panel). (I) Growth curves indicate that the NMK-1(+) and (–) cell populations have the same growth characteristics under the various conditions tested. (J) Representative DNA histograms of exponentially growing NMK-1(+) and (–) cells. (K) Immunofluorescent detection and quantification of BrdU incorporation in NMK-1(+) and (–) cells. (L) Erk1/2 phosphorylation in growth-factor-stimulated NMK-1(+) and (–) cells. Cells were cultured for 16 hours in keratinocyte serum-free medium after which EGF and pituitary gland extract were added for the time periods indicated. Lysates were blotted and probed with antibodies against ß4, phospho-Erk and total Erk as control.

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