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doi: 10.1242/10.1242/jcs.00141

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ß-Catenin is not required for proliferation and differentiation of epidermal mouse keratinocytes

¹ Institute of Animal Pathology, University of Bern, Länggassstr. 122, 3012 Bern, Switzerland
² Max Planck Institute for Immunobiology, PO Box 1169, Stübeweg 51, 7800 Freiburg Zähringen, Germany
³ Max Planck Institute for Developmental Biology, Spemannstr.35, 72076 Tübingen, Germany

View larger version (27K): [in a new window]   Fig. 1. Geno- and phenotyping of selected keratinocyte cultures before and after conditional ß-catenin gene ablation by Adenovirus-encoded Cre-recombinase. (A) PCR performed on genomic DNA of cultured mouse keratinocytes depicts complete recombination of ß-cat+loxP into ß-cat- alleles in cultures 1-3. (B) Western blot analysis of whole cell lysates was used to confirm lack of ß-catenin expression in conditional null mutant keratinocytes. Plakoglobin was assessed on the same blot and used as a loading control. ß-, deleted ß-catenin allele; ß+loxP, transgenic allele carrying two loxP sites; ß+, wild-type allele; PG, plakoglobin.

View larger version (70K): [in a new window]   Fig. 3. Establishment of intercellular adhesion in homozygous, heterozygous and ß-catenin-null mutant keratinocytes after incubation in medium containing 1.2 mM CaCl2. (A) Cytoplasmic, TritonX-100-soluble and -insoluble protein fractions were assessed by western blot analysis using the indicated antibodies. Compared are cell equivalents at the ratio of 2:1:1 in the respective fractions. E-cad, PG and ß-cat expression were assessed on the same blot, whereas Pph3, Pph1, -cat and Dsg3 expression were investigated on a second blot. Anti-tubulin and anti-keratin 14 antibodies were used as loading controls on all blots of the cytoplasmic or Triton-insoluble fraction, respectively (here shown for one blot). (B) Double-immunofluorescence staining of cultured mouse keratinocytes from the indicated genotype show linear membrane localization of adherens junction proteins, E-cadherin, -catenin, plakoglobin and insertion of actin filaments in adherens junctions. Similar results were obtained for desmosomal proteins. Experimental procedures and photographic exposures were held constant to obtain semi-quantitative results. (C) Adherens junctions and desmosomes demonstrate identical ultrastructure in wild-type and ß-catenin-null mutant keratinocytes (Scale bars, 435 nm). (D) Intercellular adhesiveness was quantified. Values are means of n=4; bars indicate standard deviations. Note that the differences were not significant. Dsg3, desmoglein3; E-cad, E-cadherin; -cat, -catenin; ß-cat, ß-catenin; PG, plakoglobin; Pph, plakophilin.

View larger version (24K): [in a new window]   Fig. 2. Proliferation capacity and transcriptional transactivation from Tfc/Lef1 promoters in homozygous, heterozygous and ß-catenin-null mutant keratinocytes. (A) Proliferation curves were established under low Ca2+ conditions for the indicated keratinocyte cultures. Error bars represent standard deviation of the mean of triplicate samples within one out of two experiments. (B) During the proliferative phase (in A, 96 hours), cyclin D1 expression was simultaneously assessed by western blot analysis. Plakoglobin expression examined on the same blot served as a control for equal loading. (C) Keratinocytes were transfected with pTOP-flash (gray bars) or the same amount of pFOP-flash (open bars) reporter along with Lef1 or ß-catenin as indicated. Relative light units (firefly luciferase over renilla luciferase) are indicated. Numbers are the ratios of normalized TOP-flash over FOP-flash activity. Bars represent minimum and maximum values of duplicate samples of one experiment. This experiment was repeated three times. (D) Expression of Tcf/Lef family members assessed by RT-PCR. Total RNA was isolated from confluent cell cultures grown under low (1) or high (h) Ca2+ conditions. Plasmids, full-length cDNA as positive control; DNA, genomic DNA used as negative control; H2O, PCR performed without template. For Tcf-3 and GAPDH, the number of cycles was reduced to 25 and 15, respectively, to allow semi-1uantitative analysis.

View larger version (39K): [in a new window]   Fig. 4. Substitution of ß-catenin by plakoglobin in ß-catenin-null mutant keratinocytes in the TritonX-100-soluble fraction (same as in Fig. 3). Co-precipitation was done with E-cadherin, plakoglobin or desmoglein3 antibodies. The same western blots were sequentially developed with the indicated antibodies. Note that clearly more plakoglobin associated with E-cadherin in ß-catenin-/- cells than in ß-catenin+/- or ß-catenin+/+ cells. Dsg3, desmoglein3; E-cad, E-cadherin; -cat, -catenin; ß-cat, ß-catenin; PG, plakoglobin; Pph, plakophilin.

View larger version (71K): [in a new window]   Fig. 5. Expression of early and late terminal differentiation markers in homozygous, heterozygous and ß-catenin-null mutant keratinocytes. Western blot analysis of whole cell lysates of cultures 130 hours in KSFM high Ca2+ were developed with the indicated antibodies. The arrow indicates the partially crosslinked form of loricrin. Dsg1/2: desmoglein1/2.