Activation of NF-kappaB is required for PDGF-B chain to transform NIH3T3 cells.

Elucidating the secondary signaling molecules that are necessary for platelet-derived growth factor (PDGF) to stimulate tumor development will be crucial to the understanding and treatment of a variety of cancers. Several lines of evidence have indicated that the transcription factor NF-kappaB plays a central role in transformation induced by Ha-ras and Bcr-abl, but nothing is known concerning its role in transformation by PDGF. Here we demonstrate that transcription from a promoter containing NF-kappaB binding sequences as well as the DNA binding activity of NF-kappaB were increased in PDGF-B-chain-transformed mouse fibroblast cells. Focus formation of PDGF-B-chain-transformed mouse fibroblasts was suppressed by treatment with acetylsalicylic acid (ASA) and salicylic acid, which are known inhibitors of NF-kappaB activation, but other nonsteroidal anti-inflammatory drugs that do not have an effect on NF-kappaB activity did not affect focus formation in these cells. Furthermore, expression of a dominant negative mutant of IkappaBalpha, pMEIkappaBalpha67CJ, and a dominant negative mutant of p65, p65DeltaC, resulted in decreased focus formation and NF-kappaB activity. Therefore, the transcription factor NF-kappaB plays a vital role in PDGF-B chain transformation of mouse fibroblast cells, and the NF-kappaB activity is sensitive to treatment with ASA.

Transactivation of Fra-1 and consequent activation of AP-1 occur extracellular signal-regulated kinase dependently.

Mitogen-activated protein (MAP) kinase, extracellular-signal-regulated kinases (ERKs) play an important role in activating AP-1-dependent transcription. Studies using the JB6 mouse epidermal model and a transgenic mouse model have established a requirement for AP-1-dependent transcription in tumor promotion. Tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor induce activator protein 1 (AP-1) activity and neoplastic transformation in JB6 transformation-sensitive (P(+)) cells, but not in transformation-resistant (P(-)) variants. The resistance in one of the P(-) variants can be attributed to the low levels of the MAP kinases, ERKs 1 and 2, and consequent nonresponsiveness to AP-1 activation. The resistant variant is not deficient in c-fos transcription. The purpose of these studies was to define the targets of activated ERK that lead to AP-1 transactivation. The results establish that the transactivation domain of Fra-1 can be activated, that activation of Fra-1 is ERK dependent, and that a putative ERK phosphorylation site must be intact for activation to occur. Fra-1 was activated by TPA in ERK-sufficient P(+) cells but not in ERK-deficient P(-) cells. A similar activation pattern was seen for c-Fos but not for Fra-2. Gel shift analysis identified Fra-1 as distinguishing mitogen-activated (P(+)) from nonactivated (P(-)) AP-1 complexes. A second AP-1-nonresponsive P(-) variant that underexpresses Fra-1 gained AP-1 response upon introduction of a Fra-1 expression construct. These observations suggest that ERK-dependent activation of Fra-1 is required for AP-1 transactivation in JB6 cells.