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First published online April 18, 2006
Journal of Experimental Biology 209, 1765-1776 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02152
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Regulation of the mitogen-activated protein kinase p44 ERK activity during anoxia/recovery in rainbow trout hypodermal fibroblasts

Carlo G. Ossum*, Tune Wulff and Else K. Hoffmann

Institute of Molecular Biology and Physiology, Department of Biochemistry, The August Krogh Building, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark


Figure 1
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  		Fig. 1. Effect of serum stimulation on ERK proteins in serum-starved RTHDF cells. (A) Top: western blot analysis of phosphorylation of p44ERK and p38ERK during serum stimulation of serum starved cells. The antibody recognises amino acids Thr202 and Tyr204. Bottom: western blot analysis of total ERK protein. (B) Quantification of the phospho-specific immunoreactions representing active p44ERK in RTHDF cells. (C) Quantification of the phospho-specific immunoreactions representing active p38ERK in RTHDF cells (N=4, *P<0.05).

 

Figure 2
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  		Fig. 2. Dose–response of p44ERK activity to sodium azide in RTHD cells. Western blot analysis of phosphorylated p44ERK after 30 min incubation in L-15ex with increasing concentration of NaN3 (N=1).

 

Figure 3
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  		Fig. 3. Time-dependent inhibition of p44ERK activity during chemical anoxia and activation during reoxygenation in RTHD fibroblasts. RTHDF cells were challenged with chemical anoxia and recovery for the indicated periods of time. Chemical anoxia was induced by incubation in L-15ex, containing 10 mmol l–1 sodium azide for 30 min. Recovery was achieved by incubating the cells in azide-free L-15ex. (A) Top: western blot analysis of phospho-p44ERK during anoxia (N=4) and total p44ERK (N=1). Bottom: quantification of band intensity showed significant inhibition of p44ERK activity after 30 min, relative to the normoxic control. (B) Top: western blot analysis of phospho-p44ERK during recovery (N=3) and total p44ERK (N=1). Bottom: quantification of band intensity showed that recovery resulted in significant activation of p44ERK after 10 min, relative to anoxic cells. *P<0.005.

 

Figure 4
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  		Fig. 4. Activation of p44ERK during recovery is MEK dependent. RTHDF cells were challenged with chemical anoxia (A) and recovery (R) in the absence and presence of 10 µmol l–1 of the MEK1/2 inhibitor PD98059 in the recovery medium. A representative western blot and quantification of band intensity is shown, demonstrating the inhibitory effect of 10 µmol l–1 PD98059 (N=3, *P<0.05).

 

Figure 5
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  		Fig. 5. Time-dependent inhibition of p44ERK activity during nitrogen-mediated anoxia and activation during recovery in RTHD fibroblasts. RTHD fibroblasts were exposed to nitrogen-induced anoxia and recovery for the indicated periods of time. Anoxia was obtained by flushing supplemented Leibovitz' L-15 with nitrogen until the oxygen was removed. Recovery occurred by incubating the cells in atmospheric air. (A) Top: western blot analysis of phospho-p44ERK during anoxia (N=3) and total p44ERK (N=1). Bottom: quantification of band intensity showed significant inhibition of p44ERK activity after 3 h, relative to the normoxic control (*P<0.05). (B) Top: western blot analysis of phospho-p44ERK during recovery (N=3) and total p44ERK (N=1). Bottom: quantification of band intensity showed recovery resulted in significant activation of p44ERK after 5 min, relative to anoxic cells (*P<0.05). The value at 120 min recovery was not significant (P=0.059) because of one very low experimental value.

 

Figure 6
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  		Fig. 6. p38MAPK mediates inhibition of p44ERK during chemical anoxia in RTHDF cells. Prior to 30 min chemical anoxia, RTHDF cells were pre-treated for 1 h with 10 µmol l–1 SB203580, inhibiting the MAPK p38{alpha}/ß. Cell extracts were analysed by western blotting (top) and the immunoreactive bands were quantified (bottom) (N=3, *P<0.05).

 

Figure 7
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  		Fig. 7. Reactivation of p44ERK under recovery requires PP1/PP2A activity. RTHDF cells were treated with 100 nmol l–1 calyculin A (Cal. A; an inhibitor of PP1/2) for 2 min prior to treatment. Cell extracts were analysed by western blotting (top) and the immunoreactive bands were quantified (bottom). (A) Effect of calyculin A on normoxic p44ERK activity. (B) Effect of calyculin A on p44ERK phosphorylation after chemical anoxia/recovery (A/R) (N=3, *P<0.05).

 

Figure 8
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  		Fig. 8. Activation of p44ERK by serum (FBS) is dependent on Raf-1, but activation of p44ERK during recovery is independent of Raf-1. (A) Western blot analysis (top) and quantification (bottom) of phospho-p44ERK after serum stimulation of serum-starved cells, in the absence and presence of 10 µmol l–1 Raf-1 inhibitor, RKI. (B) Western blot analysis (top) and quantification (bottom) of the effect of 10 µmol l–1 Raf-1 inhibitor on p44ERK phosphorylation during chemical anoxia and recovery (A/R) (N=3, *P<0.05).

 

Figure 9
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  		Fig. 9. Reactive oxygen species are required for reactivation of p44ERK activity during recovery. (A) Western blot analysis (top) and quantification (bottom) of basal p44ERK activity after 1 h treatment of RTHDF cells with 100 nmol l–1 DPI (N=3, *P<0.05). (B) Western blot analysis (top) and quantification (bottom) of phospho-ERK1-specific bands after 1 h treatment of RTHDF cells with 100 nmol l–1 DPI prior to chemical anoxia/recovery (A/R) (N=3, *P<0.05).

 

Figure 10
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  		Fig. 10. p44ERK is regulated at the level of MEK during chemical anoxia/recovery (A/R). Phosphorylation of MEK1 during A/R was analysed by western blotting (top) and phospho-specific bands were quantified (bottom). (A) Anoxia inhibits MEK in RTHD fibroblasts (N=4, *P<0.05). (B) Pre-treatment with 10 µmol l–1 SB203580 for 1 h prevented inhibition of MEK during anoxia (N=3, *P<0.05). (C) Pre-treatment with 100 nmol l–1 calyculin A (Cal. A) for 2 min blocked reactivation of MEK during recovery (N=4, *P<0.05).

 

Figure 11
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  		Fig. 11. Working model for the regulation of p44ERK activity in RTHD fibroblasts during chemical anoxia/recovery. Our data suggest that p38MAPK inhibits ERK activity at the level of MEK during chemical anoxia. During recovery, we demonstrated the requirement for ROS, produced via a NAD(P)H oxidase-like activity for reactivation of ERK in a Raf-independent manner. We speculate that ROS results in activation of Src, which in turn activates PKC upstream of MEKK1.

 




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