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Repression of Wnt-5a impairs DDR1 phosphorylation and modifies adhesion and migration of mammary cells

Division of Experimental Pathology, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden

View larger version (46K): [in a new window]   Fig. 1. Expression of Wnt-5a protein in various cell populations. (A) Wnt-5a-HA fusion protein was detected in the lysates of Wnt-5a-HA-overexpressing cells, using an anti-HA antibody (lane 1), but was not found in the lysates of Wnt-5a antisense (lane 2) or control (lane 3) cells. (B) The content of Wnt-5a protein (Wnt-5a-HA) in Wnt-5a-overexpressing cells detected by an anti-Wnt-5a antibody, which recognises both endogenous and overexpressed Wnt-5a. (C) Examples of Wnt-5a repression in antisense transfectants: lane 1 shows the absence of Wnt-5a protein in a lysate of NIH 3T3 cells (negative control); lane 2, endogenous expression of Wnt-5a protein in a lysate of control cells; lanes 3–7, repression of endogenous Wnt-5a protein in five antisense transfectants (detected with the anti-Wnt-5a antibody), as compared to endogenous levels in control lane 2. (D) Expression of Wnt-5a-HA in lysates of the MCF-7 cells transfected with Wnt-5a (lane 1) and control MCF-7 cells (lane 2). All membranes were subsequently stripped and reprobed with an anti-actin antibody to confirm protein integrity and equal loading.

View larger version (158K): [in a new window]   Fig. 2. Formation of morphogenetic structures by various HB2 cell populations. The structures were designated ball-like, cyst shaped, and branching, as described in Materials and Methods. The micrographs show the structures formed by control cells (A), Wnt-5a-overexpressing cells (B) and Wnt-5a antisense cells (C). One hundred morphogenetic structures of each cell population were examined and classified on each of the triplicate plates. Mean values were calculated and plotted on the graph (D). The values represent the mean and standard deviation of determinations and are expressed as percent of the scored structures. Bar, 80 µm.

View larger version (153K): [in a new window]   Fig. 3. Formation of morphogenetic structures by various HB2 cell populations in the presence of 10 ng/ml human recombinant HGF. The micrographs show the structures produced by control cells (A), Wnt-5a-overexpressing cells (B) and Wnt-5a antisense cells (C). (D) The morphogenetic structures were quantified as described in Fig. 2D. Values represent the mean and standard deviation of determinations made on triplicate plates and are expressed as percent of the scored structures. Bar, 80 µm.

View larger version (114K): [in a new window]   Fig. 4. Morphological characteristics of Wnt-5a antisense cells grown on collagen type I gels. (A) Morphology of control cells grown on collagen matrices. (B) The scattered morphology of antisense cells grown under identical conditions to control cells. The antisense cells shown in panel B were from transfectant 1; transfectants 9 and 12 showed identical morphology.

View larger version (24K): [in a new window]   Fig. 5. The cell-to-collagen binding ability of various cell populations. (A) The data represent the average number of cells that adhered to a matrix containing various concentrations of collagen type I during 2 hours incubation. The values are given as the mean and standard deviation of determinations done on triplicate wells assayed in parallel. (B) Tyrosine phosphorylation of FAK in cells with different Wnt-5a protein levels. Upper panel: level of FAK expression in Wnt-5a-overexpressing cells (lane 1), in antisense cells (lane 2), and in control cells (lane 3). Lower panel: level of FAK phosphorylation in various cell populations as indicated in B. The molecular weight of FAK was estimated to 125 kDa by comparing with a molecular standard weight.

View larger version (35K): [in a new window]   Fig. 6. DDR1 expression by various HB2 cell populations. (A) DDR1 protein level detected in lysates of the following: non-transfected parental HB2 cells (lane 1), Wnt-5a-overexpressing cells (lane 2), Wnt-5a antisense cells (lane 3), and control cells (lane 4). (B) The tyrosine phosphorylation status of DDR1 receptors in various cell populations grown on collagen. Sample loading was done in the same order as in A. (C) DDR1 expression in MCF-7 control (lane 1), Wnt-5a-expressing MCF-7 (lane 2), and untransfected parental MCF-7 (lane 3) cells. (D) The tyrosine phosphorylation status of DDR1 receptors in various MCF-7 cell populations; sample loading was done in the same order as in 6C. (E) Tyrosine phosphorylation status of DDR1 receptors in cells grown on uncoated tissue culture plates; sample loading was done in the same order as in A. The molecular weight of DDR1 was estimated to 125 kDa by comparing with a molecular standard weight. The anti-DDR antibody reacts with isoform b/c (upper band) and with isoform a (lower band) of the DDR1 protein, but only isoform b/c is tyrosine phosphorylated.

View larger version (110K): [in a new window]   Fig. 7. Expression of Wnt-5a improves the morphology of MCF-7 breast cancer cells to a more normal phenotype. (A) Morphology of control MCF-7 cells transfected with the vector alone. (B) Morphology of MCF-7 cells expressing Wnt-5a protein. The Wnt-5a-expressing MCF-7 cells grow as islands containing tightly associated cells and therefore they appear to have a higher density than that of control cells, despite the fact that an equal number of both cell populations were plated and grown for 16 hours.

View larger version (16K): [in a new window]   Fig. 8. Kinetic analysis of phosphorylation of DDR1 receptors. (A) Control cells grown on collagen for various periods of time. The cells were lysed and immunoprecipitated with an anti-DDR1 antibody, and the tyrosine phosphorylation status of the DDR1 receptors was determined by immunoblotting with an anti-tyrosine antibody (4G10). (B) DDR1 phosphorylation during the indicated periods of time. The values represent the mean and standard deviation of determinations done on triplicate wells assayed in parallel.

View larger version (42K): [in a new window]   Fig. 9. Effects of ß-catenin accumulation on DDR1 phosphorylation. (A) The presence of secreted Wg protein in medium conditioned by S2HS-Wg compared to that conditioned by S2 control cells. (B) Extracts from various cell populations, grown on collagen for 30 minutes in the presence of Wg protein, were prepared and analysed for phosphorylation of DDR1 receptors. Double extracts were also prepared in parallel to detect induction of ß-catenin protein. (C) Accumulation of ß-catenin in response to incubation with concentrated Wg-conditioned medium, ensured by exposing cells to medium conditioned for 30 minutes by control S2 cells (lanes 1-3) or Wg-producing cells (lanes 4-6).