RAF and antioxidants prevent cell death induction after growth factor abrogation through regulation of Bcl-2 proteins.

We have shown previously that mitochondrial ROS production is essential to turn growth factor (GF) removal into cell death. Activated RAF, AKT, Bcl-2 and antioxidants protected equally well against ROS accumulation and subsequent death. Here we investigated whether protection by survival signaling and antioxidants utilizes shared or distinct targets. Using serum deprivation from NIH 3T3 fibroblasts and IL-3 withdrawal from promyeloid 32D cells, we showed that pro-survival signaling by activated RAF but not AKT prevented the decline in Mcl-1 following GF abrogation. GF starvation increased levels of Bim in both model systems, which was prevented by RAF in 32D cells but not in NIH 3T3 fibroblasts. RAF and AKT suppressed activation and mitochondrial translocation of BAX. Also, antioxidant treatment efficiently prevented BAX activation and death of 32D cells but showed little effect on its mitochondrial translocation. No significant impact of antioxidant treatment on Bim or Mcl-1 expression was observed. ROS produced during GF abrogation also did not alter the activity of intracellular signaling pathways, which have been implicated previously in cell killing by pro-oxidants. Together these data suggest Bcl-2 family proteins as convergence point for RAF and ROS in life and death decisions.

Cytoplasmic signaling in the control of mitochondrial uproar?

The concept of a pre-emptive strike as a good means to prevent greater harm may be frequently over-stressed in daily life. However, biological systems in a homeostatic balance are prepared to withstand a certain degree of hostile fire by rather passive means. This also applies to the maintenance of cell survival, where a plethora of protective proteins provide safeguard against erroneous activation of death pathways. Apart from these mechanisms active processes are also essential for the maintenance of cellular homeostasis, commonly referred to as survival signaling. Frequently their targets may be mitochondrial, assuring organelle integrity, which is essential for continued energy production and survival. Transient or permanent failures in these cellular defense strategies result in pathophysiological conditions, which manifest themselves e.g. as cancer or ischemia/reperfusion-associated organ damage.

Survival signaling by C-RAF: mitochondrial reactive oxygen species and Ca2+ are critical targets.

Survival signaling by RAF occurs through largely unknown mechanisms. Here we provide evidence for the first time that RAF controls cell survival by maintaining permissive levels of mitochondrial reactive oxygen species (ROS) and Ca(2+). Interleukin-3 (IL-3) withdrawal from 32D cells resulted in ROS production, which was suppressed by activated C-RAF. Oncogenic C-RAF decreased the percentage of apoptotic cells following treatment with staurosporine or the oxidative stress-inducing agent tert-butyl hydroperoxide. However, it was also the case that in parental 32D cells growing in the presence of IL-3, inhibition of RAF signaling resulted in elevated mitochondrial ROS and Ca(2+) levels. Cell death is preceded by a ROS-dependent increase in mitochondrial Ca(2+), which was absent from cells expressing transforming C-RAF. Prevention of mitochondrial Ca(2+) overload after IL-3 deprivation increased cell viability. MEK was essential for the mitochondrial effects of RAF. In summary, our data show that survival control by C-RAF involves controlling ROS production, which otherwise perturbs mitochondrial Ca(2+) homeostasis.