Fibroblast Growth Factor-2 Suppression of Tumor Necrosis Factor α-Mediated Apoptosis Requires Ras and the Activation of Mitogen-activated Protein Kinase

Abstract

Treatment of L929 cells with tumor necrosis factor alpha (TNFalpha) activates a programmed cell death pathway resulting in apoptosis. We investigated the intracellular signaling pathways activated in L929 cells by TNFalpha. TNFalpha robustly activates Jun kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family. In addition, p42(MAPK) is activated, but a 10-fold greater concentration of TNFalpha was required for substantial MAPK activation than was needed for maximal JNK stimulation. Simultaneous treatment of L929 cells with fibroblast growth factor (FGF-2) significantly reduced the apoptotic response to TNFalpha. FGF-2 substantially activated the Raf/MEK/MAPK (where MEK is mitogen-activated protein kinase kinase) pathway but did not affect TNFalpha activation of JNK. These results indicate that although JNK may play an important role in transmitting the TNFalpha signal from the cell surface to the nucleus, activation of the JNK pathway is not sufficient to induce apoptosis. Expression of dominant-negative Asn-17 Ras in L929 cells diminished the FGF-2 stimulation of p42(MAPK) and eliminated the protective effect of FGF-2. Asn-17 Ras expression did not affect JNK activity and had no effect on TNFalpha activation of JNK. Pharmacological inhibition of MEK-1 activity by incubation of cells with the compound PD 098059 blocked p42(MAPK) activation and FGF-2 protection against apoptosis. Interestingly, activated Val-12 Ras expression substantially enhanced TNFalpha-mediated apoptosis in L929 cells, but Val-12 Ras did not constitutively activate MAPK in L929 cells and FGF-2 partially protected Val-12 Ras-expressing cells from TNFalpha-mediated apoptosis. Our data indicate that activation of the MAPK pathway mediates an FGF-2 protective effect against apoptosis and highlights the important role that integration of multiple intracellular signaling pathways plays in the regulation of cell growth and death.

Regulation of BAD phosphorylation at serine 112 by the Ras-mitogen-activated protein kinase pathway

“…The Ras-MAPK pathway has been shown to promote cell survival in many di erent systems (Xia et al, 1995;Gardner and Johnson, 1996;Parrizas et al, 1997;Kurada and White, 1998;Bergmann et al, 1998). There has been a lack of functional connection between the Ras-MAPK pathway and a pro-apoptotic molecule or death antagonist of the BCL-2 family although the Ras-MAPK pathway has been shown to down-regulate the proapoptotic activity of the gene head involution defective (hid) (Kurada and White, 1998;Bergmann et al, 1998).…”

“…The Ras-MAPK signaling pathway is essential for cell proliferation and differentiation (Cowley et al, 1994). It also plays an important role in the regulation of cell survival (Xia et al, 1995;Gardner and Johnson, 1996;Parrizas et al, 1997;Bergmann et al, 1998). MAPK activity was examined in HEK293 cells stimulated with EGF, IGF-I or insulin using a phospho-speci®c antibody and by means of gel-mobility shift assays.…”

Potentiation of apoptosis by low dose stress stimuli in cells expressing activated MEK kinase 1

“…L929 and Swiss 3T3 cells were transfected using CaPO 4 (LassignalJohnson et al, 1996;Gardner and Johnson, 1996) with the vector 3'SS (Stratagene, La Jolla, CA) expressing the LACI repressor. Stable clones were selected in 200 mg/ml hygromycin and screened for LACI expression using indirect immuno¯uorescence with a rabbit antisera to LACI.…”

“…Blots were blocked in 5% powdered milk in Tris-HCl, pH 7.5, bu ered saline. The 37 kDa DMEKK1 was detected using a rabbit antisera raised against a peptide encoding the last 13 amino acids of MEKK1 (Lange-Carter et al, 1993 Gardner and Johnson, 1996). Cells were then incubated in the presence or absence of 5 mM IPTG and allowed to grow for an additional 8 h. The cells were then treated with di erent stimuli, ®xed in 3% paraformaldehyde and stained with 1 mg/ml propidium iodide.…”

“…An emerging theme is that the pathways signaling apoptosis overlap with those that mediate growth and di erentiation (LassignalJohnson et al, 1996;Fanidi et al, 1992;Gardner et al, 1993;Evan et al, 1992). In the context of signal transduction pathways, integration of signals initiated by growth factor and cytokine receptors, therefore, can commit a cell to either proliferation or apoptosis (Xia et al, 1995;Heasley et al, 1996;Gardner and Johnson, 1996). For example, we recently demonstrated that FGF-2 protects L929 cells from TNFa-stimulated apoptosis .…”