Role of Mitogen-activated Protein Kinases and c-Jun/AP-1 trans-Activating Activity in the Regulation of Protease mRNAs and the Malignant Phenotype in NIH 3T3 Fibroblasts

Abstract

Ras activates a multitude of downstream activities with roles in cellular proliferation, invasion and metastasis, differentiation, and programmed cell death. In this work we have evaluated the requirement of extracellular signal-regulated protein kinase (ERK), c-Jun NH 2 -terminal kinase kinase (JNKK), and c-Jun/AP-1 activities in transformation and extracellular matrix invasion of ras oncogene expressing NIH 3T3 fibroblasts by expressing stable mutant genes that constitutively inhibit these activities. Whereas the inhibition of ERK activity reverts the transformed and invasive phenotype, the inhibition of the JNK pathway and AP-1 transactivating activities by JNKK[K129R] and c-Jun(TAM67) had no effect on the ability of the ras oncogene-expressing 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 not absolute requirements for ras transformation or invasion through basement membrane, and the dependence on AP-1 activity for transformation is cell-specific. However, inhibition of JNK kinase (JNKK) in ras-transformed cells with normally elevated JNK activity switches the proteasedependent invasive phenotype from a urokinase plasminogen activator (uPA)-dependent to a cathepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-transformed cells of low constitutive JNK activity with the JNK 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 m1 muscarinic receptors that selectively stimulate the ERK or JNK pathways, respectively. It is concluded that high ERK activity is required for both protease phenotypes, whereas the JNK pathway and c-Jun/AP-1 activity are not required for transformation but regulate a switch between uPA and CL protease phenotypes in both transformed and untransformed cells. In ras-transformed NIH 3T3 fibroblasts, the uPAand CL-dependent protease phenotypes are redundant in their ability to invade through basement membrane.The Ras protein is at the center of a web of multiple downstream pathways that are differentially regulated to produce signals that control processes as disparate as cell division, cell differentiation, and programmed cell death (reviewed in Refs. 1-3). Two of the better characterized pathways downstream from Ras are from Ras to ERK-1 1 and -2 through the sequential activation of Raf and MEK1, and the pathway from Ras leading to the activation of JNK1 through the activation of Cdc42 and Rac 1 which then sequentially activate PAK, MEK kinase-2 and/or -3, JNKK/SEK/MKK4, and JNK1. The ERKs and JNK1 are mitogen-activated protein kinases that translocate to the nucleus and activate transcription factors involved in Rasregulated processes. The protein c-Jun is a nuclear substrate of JNK1 (4 -7), and c-Jun is a prominent component of many AP-1 transcription factors that ...

Promotion of Cancer Cell Migration

“…JNK inhibition did not affect IGFBP-6-dependent migration in RD cells, consistent with other reports that this pathway is not involved in cell migration/invasion (49,58).…”

Phenotype-assisted transcriptome analysis identifies FOXM1 downstream from Ras–MKK3–p38 to regulate in vitro cellular invasion

“…Cells carrying this mutation colonize the lungs of athymic nude mice after tail vein injection (Bradley et al, 1986;Zhang and Schultz, 1992). Moreover, it has been demonstrated that both ERK1/2 and JNK are active in Ha-Ras EJ stably transfected NIH3T3 cells, but only ERK was required for an invasive phenotype (Janulis et al, 1999). We have previously demonstrated that p38 is also required for in vitro invasion of v-Ha-Ras EJ stably transfected NIH3T3 cells (Behren et al, 2005).…”

The small-GTPase RalA activates transcription of the urokinase plasminogen activator receptor (uPAR) gene via an AP1-dependent mechanism

“…We focused on the activation of uPAR transcription by Ras as it is a point of convergence for diverse extracellular signal-stimulated pathways and frequently su ers activating mutations in a wide range of cancers. In addition, the introduction of oncogenic Ras into cells increases extracellular protease activity in general and the expression of uPAR in particular (Westermark and KaÈ haÈ ri, 1999; Lengyel et al, 1995;Jankun et al, 1991;Allgayer et al, 1999;Janulis et al, 1999;Muller et al, 2000). The work of White et al (1995) established that the activation of multiple-ras e ector pathways is necessary to induce cell transformation.…”