Abstract
The Wnts can be classified into two classes based on their ability to transform cells. The Wnt5a class can antagonize the effects of transforming Wnts partly through effects on cell migration. To understand the mechanisms of regulation of Wnt5a, we investigated its expression in human normal and breast cancer cell lines. Elevation of Wnt5a in HB2, a normal breast epithelial cell line, was linearly correlated with cell density, but this did not occur in cancer cell lines. We examined intracellular events responsible for the regulation of Wnt5a by cell to cell contacts, using various metabolic agents known to affect signal transduction pathways. Agents that selectively blocked protein kinase C (calphostin C) or protein tyrosine kinases (genistein) reduced the level of Wnt5a expression markedly. Protein kinase C activation by phorbol 12-myristate 13-acetate up-regulated Wnt5a partly through prolongation of Wnt5a mRNA half-life. Cytoskeleton reorganization following cytochalasin D treatment caused an induction of Wnt5a, which was associated with changes in cell morphology. Calphostin C did not block these effects, showing that protein kinase C is acting upstream of cytoskeletal modulation. However, the cancer cell lines treated with cytochalasin D showed no changes in cell morphology or Wnt5a induction, suggesting disruption of this regulatory pathway in cancer.
Highlights
We have investigated the signalling pathways involved in the regulation of Wnt5a expression in cell density and cell to cell contact
We have shown that protein kinase C (PKC), tyrosine kinase activities and cytoskeleton rearrangement are involved in the regulation of Wnt5a at cell confluence
Results from RNase protection assays showed a linear correlation between increased cell density and Wnt5a message level, i.e. lowest expression at early subconfluence compared with the highest message level in confluent cells (Figure 1)
Summary
Mycoplasma-free HB2 cells [a subclone of the MTSVI-7 line (Bartek et al, 1991)] obtained from Dr Joyce Taylor-Papadimitriou. Exposing cells to different drugs may modulate GAPDH expression, thereby questioning the validity of GAPDH as loading control To circumvent this problem, we spiked all samples with 1 jig of total RNA from K562 cells (which strongly overexpress x1-globin) and included a [a-32P]CTP labelled antisense cx-globin mRNA to the hybridization solutions. We spiked all samples with 1 jig of total RNA from K562 cells (which strongly overexpress x1-globin) and included a [a-32P]CTP labelled antisense cx-globin mRNA to the hybridization solutions This hybridizes to the aglobin mRNA in the spike (Frith and Ratcliffe, 1992), thereby providing an external loading control. To study the effect of E-cadherin extracellular domain blocking on Wnt5a expression, confluent cells were incubated with 50 mg ml-' HECD- 1, mouse anti E-cadherin monoclonal antibody (Shimoyama et al, 1989) or isotype-matched control mouse IgG for 16 h. The level of E-cadherin protein in confluent and subconfluent HB2 cells was measured using immunoblotting as described above
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