MiP2005: Session 4

Mitochondrial Physiology Network 10.9: 50-51 (2005) - download pdf


  • Note added after publication: Abstracts published with A Garedew as first author have to be critically re-evaluated and the corresponding data will not be published as presented in these abstracts (Erich Gnaiger).

v-Raf antagonizes impairment of mitochondrial respiratory function following growth factor removal.

A Garedew,1,2 C Doblander,1 B Haffner,1 E Gnaiger1, Jakob Troppmair,1

1Daniel-Swarovski Research Lab., Dept. General Transplant Surgery, Innsbruck Medical Univ.; 2OROBOROS INSTRUMENTS, Innsbruck, Austria. jakob.troppmair@uibk.ac.at

    The cell’s ability to respond to extrinsic stimuli depends on the provision of adequate energy. Increasing evidence suggests that cellular energy production itself is subject to regulation by extrinsic signals through signaling pathways, which control cell proliferation and survival. In tumors, where signaling components are frequently affected by mutations, an increased dependence on glycolytic energy has been recognized long ago and more recent experiments suggested direct targeting of components of the glycolytic machinery as one of the underlying mechanisms [1,2]. Indirect evidence from studies on the control of cell survival by C-Raf suggests a role for this kinase in maintaining mitochondrial integrity during apoptosis induction [3]. These experiments also suggested cooperation in this process with two other major guardians of cell survival, Bcl-2 and PKB. To test for direct effects of C-Raf on mitochondrial energy production we performed high resolution respirometry on the mouse pro-myeloid 32D cell line. These cells strictly depend on IL-3 for growth and survival. IL-3 removal results in growth arrest and subsequent apoptosis, which can be prevented through over-expression of the oncogenic form of C-Raf, v-Raf. In the comparison of the effects of growth factor withdrawal on mitochondrial respiratory function in 32D cells versus 32D cells protected by v-Raf, our experimental design focused on early time points before cells bocome irreversibly committed to cell death.

    Cells were incubated in RPMI 1640 + 10% FCS supplemented with penicillin-streptomycin and 2 mM L-glutamine without IL-3 for a period of 8 h at a cell density of 0.5×106 cells∙ml-1. Controls were cultured in the same medium supplemented with IL-3 (15 % WEHI). During the 8 h time interval, no significant difference in viability (<4 % trypan blue or annexin V staining) was observed between control and  growth factor deprived cells. The respiratory activities of intact cells were measured using the OROBOROS Oxygraph-2k for high resolution respirometry. After recording cellular routine respiration in the respective incubation media, ATP-synthase was inhibited by oligomycin, followed by a stepwise FCCP titration to achieve maximum uncoupled respiration. Respiration was then inhibited by rotenone and antimycin A. Data (means ± SD) were analysed by a paired t-test.

Activities of citrate synthase (mitochondrial matrix marker enzyme) and lactate dehydrogenase (glycolytic marker enzyme) per million cells remained unchanged, irrespective of IL-3 withdrawal, indicating that mitochondrial content and glycolytic capacity were maintained. Analysis of ERK and AKT phosphorylation, two main signaling effectors of the IL-3 receptor, revealed no measurable decline in their activities at the end of the WEHI starvation period. However, a significant decrease in cell volume (measured by CASY®) was observed in 32D (0.93 ± 0.10 pL versus 0.76 ± 0.05 pL) but not in cells protected by v-Raf. Regardless of the significant decrease in cell size, the protein content of 32D cells remained unaffected upon IL-3 withdrawal.

    Mitochondrial respiratory function of IL-3-deprived 32D cells dropped significantly in all respiratory states (Fig. 1). Routine and oligomycin-inhibited respiration of 32D-v-Raf cells deprived of IL-3 were not significantly lower compared to their controls with IL-3 (Fig. 1).

The uncoupling control ratio (UCR = Cr,u/Cr) is a sensitive indicator for the integrity of mitochondrial function in intact cells. In 32D controls, the UCR declined from 2.3 ± 0.23 to 1.23 ± 0.61 after growth factor removal. In contrast, the UCR of 32D-v-Raf cells remained unaffected by IL-3 withdrawal. The inverse of the respiratory control ratio (Cr,o/Cr,u), an index of the extent of oligomycin inhibited leak rate of respiration relative to the maximum capacity of the respiratory chain, was significantly different between controls (0.20 ± 0.005) and growth factor deprived 32D cells (0.43 ± 0.18), indicating primarily the loss of respiratory capacity, and providing indirect evidence for simultaneous partial uncoupling [4].

    Even though IL-3 withdrawal showed a significant effect on the respiratory rates of the different respiratory states of 32D cells, the oxygen kinetics in coupled intact cells was not significantly affected, with p50 values of 0.044 ± 0.001 kPa for controls and 0.039 ± 0.006 kPa for IL-3 deprived cells.

    Decline of mitochondrial respiratory capacity comprised an early event in the pathway to apoptosis after growth factor withdrawal, before the onset of inactivation of the main signaling effectors of the IL-3 receptor. This time course suggests a primary role of mitochondrial respiratory function in these cells. Our results clearly demonstrate that IL-3 withdrawal severely compromises mitochondrial respiratory function in a fashion that is almost completely suppressible by v-Raf. This for the first time suggests a direct link between the key mitogenic and survival kinase C-Raf and mitochondrial energy homeostasis.

1.  Dang CV, Semenza GL (1999) Oncogenic alterations of metabolism. Trends Biochem. Sci. 24: 68-72.

2.  Le Mellay V, Houben R, Troppmair J, Hagemann C, Mazurek S, Frey U, Beigel J, Weber C, Benz R, Eigenbrodt E, Rapp UR (2002) Regulation of glycolysis by Raf protein serine/threonine kinases. Adv. Enzyme Regul. 42: 317-332.

3.  Troppmair J, Rapp UR (2003) Raf and the road to cell survival: a tale of bad spells, ring bearers and detours. Biochem. Pharmacol. 66: 1341-1345.

4.  Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol. 286: H1633–H1641.

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Mitochondrial Physiology