Figure 5.

Squamous cell differentiation is promoted by synergistic activation of Wnt/β-catenin and JNK/SAPK. Mammary epithelial cells C57MG (A–E) and C57MGWnt1 (F–J), a derivative of C57MG constitutively expressing Wnt1, were immunochemically stained with specific antibodies for the expression of PJun63 (A, F), PJun73 (B, G), and HMW-K (E, J). The immunochemistry (brown).stained cells were counterstained with hematoxylin (blue). Immunostaining of C57MG and C57MGWnt1 cells with an antibody recognizing only activated β-catenin (Act. β-cat; C, H) was counterstained by propidium iodide (D, I). Note that the C57MG cell line contains certain levels of JNK/SAPK activity, leading to the phosphorylation of c-Jun at Ser63 and Ser73 (A–B and F–G). However, no squamous differentiation was evident because HMW-K is negative (E). Constitutive expression of Wnt1 induced β-catenin signaling as it accumulated in the nucleus (H), which, together with the existing JNK/SAPK signaling, promotes squamous differentiation (J). Synergistic activation of Wnt/β-catenin and JNK/SAPK signaling by transient expression of a β-catenin-LEF1 fusion protein (catC-Lef1) and a dominant MEKK1 (ΔMEKK1), respectively, strongly promotes squamous differentiation in mammary epithelial cells (N). The control transfected with vehicle only showed no expression of squamous cell marker HMW-K (K). Expression of either ΔMEKK1 (L) or catC-Lef1 (M) slightly enhances the HMW-K expression. (O, P) The presence of JNK inhibitor has no effect on the expression of HMW-K in C57MGWnt1 cells. In C57MG cells, the induction of HMW-K by transfection of catC-Lef1 and ΔMEKK1 is prohibited by JNK inhibitor (Q, R). Scale bar, 100 µm (A–R).