Role of mitogen-activated protein kinases and c-Jun/AP-1 trans-activating activity in the regulation of protease mRNAs and the malignant phenotype inNIH 3T3 fibroblasts

Ras activates a multitude of downstream activities with roles in cellular p roliferation, invasion and metastasis, differentiation, and programmed cell death. In this work we have evaluated the requirement of extracellular sig nal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase kinase (JNKK) , and c-Jun/AP-1 activities in transformation and extracellular matrix inva sion of ras oncogene expressing NIH 3T3 fibroblasts by expressing stable mu tant genes that constitutively inhibit these activities. Whereas the inhibi tion of ERK activity reverts the transformed and invasive phenotype, the in hibition of the JNK pathway and AP-1 transactivating activities by JNKK[K12 9R] and c-Jun(TAM67) had no effect on the ability of the ras oncogene-expre ssing cells to grow in soft agar or invade Matrigel basement membrane. Thus an elevated JNK activity and/or c-Jun/AP-1 trans-activating activity are n ot absolute requirements for ras transformation or invasion through basemen t membrane, and the dependence on AP-1 activity for transformation is cell- specific. However, inhibition of JNH kinase (JNKK) in ras-transformed cells with normally elevated JNK activity switches the protease-dependent invasi ve phenotype from a urokinase plasminogen activator (uPA)-dependent to a ca thepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-t ransformed cells of low constitutive JNK activity with the JNH stimulator, anisomycin, converts the protease mRNA levels from those characteristic of a CL-dependent to a uPA-dependent phenotype, These protease phenotypes can be duplicated in untransformed NIH 3T3 cells that express platelet-derived growth factor receptors and mi muscarinic receptors that selectively stimul ate the ERK or JNK pathways, respectively. It is concluded that high ERK ac tivity is required for both protease phenotypes, whereas the JNK pathway an d c-Jun/AP-1 activity are not required for transformation but regulate a sw itch between uPA and CL protease phenotypes in both transformed and untrans formed cells. In ras-transformed NIH 3T3 fibroblasts, the uPA- and CL-depen dent protease phenotypes are redundant in their ability to invade through b asement membrane.