Ben Messaoud N et al. (2015), Basic Properties of the p38 Signaling Pathway i...

XB-ART-51228

PLoS One 2015 Sep 01;109:e0135249. doi: 10.1371/journal.pone.0135249.

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Basic Properties of the p38 Signaling Pathway in Response to Hyperosmotic Shock.

Ben Messaoud N , Katzarova I , López JM .

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Some properties of signaling systems, like ultrasensitivity, hysteresis (a form of biochemical memory), and all-or-none responses at a single cell level, are important to understand the regulation of irreversible processes. Xenopus oocytes are a suitable cell model to study these properties. The p38 MAPK (mitogen-activated protein kinase) pathway is activated by different stress stimuli, including osmostress, and regulates multiple biological processes, from immune response to cell cycle. Recently, we have reported that activation of p38 and JNK regulate osmostress-induced apoptosis in Xenopus oocytes and that sustained activation of p38 accelerates cytochrome c release and caspase-3 activation. However, the signaling properties of p38 in response to hyperosmotic shock have not been studied. Here we show, using Xenopus oocytes as a cell model, that hyperosmotic shock activates the p38 signaling pathway with an ultrasensitive and bimodal response in a time-dependent manner, and with low hysteresis. At a single cell level, p38 activation is not well correlated with cytochrome c release 2 h after hyperosmotic shock, but a good correlation is observed at 4 h after treatment. Interestingly, cytochrome c microinjection induces p38 phosphorylation through caspase-3 activation, and caspase inhibition reduces p38 activation induced by osmostress, indicating that a positive feedback loop is engaged by hyperosmotic shock. To know the properties of the stress protein kinases activated by hyperosmotic shock will facilitate the design of computational models to predict cellular responses in human diseases caused by perturbations in fluid osmolarity.

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Species referenced: Xenopus laevis
Genes referenced: casp3.2 grap2 mapk1 mapk14 mapk8 prkaa1 tspan31

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	Fig 2. p38 is ultrasensitive to hyperosmolar sorbitol.Oocytes were treated with increasing concentrations of sorbitol for 4 h and pools of 20 oocytes were collected and lysed to analyze pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, giving 100% value to the highest activity. Results are represented as mean ± SEM of four independent experiments. Hill coefficient (nH) was calculated with SAS 9.2 informatics program and represented with GraphPad Prism 4 program. A representative Western blot is shown. Caspase-3 activity was determined at different concentrations of sorbitol as reported before. Data are represented as mean ± SEM (n = 3), *p<0.05 (ANOVA and Dunnett’s test) comparing with non treated oocytes.
	Fig 3. Monostability in the p38 signaling system.Time course of p38 activation in oocytes treated with 300 mM sorbitol. After 4 h of treatment (arrow), oocytes were washed several times with MBS and maintained in MBS without sorbitol. Pools of 20 oocytes were collected at different times to determine pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, given 100% value to the highest activity. Results are represented as mean ± SEM of three independent experiments. A representative Western blot is shown. Caspase-3 activity was determined as reported before. Data are represented as mean ± SEM (n = 3), *p<0.05 (ANOVA and Dunnett’s test) comparing with non treated oocytes.
	Fig 4. Bimodal and time-dependent response of the p38 system to hyperosmolar sorbitol.(A) Oocytes were incubated with 200 mM sorbitol for 2 h and pp38, p38, and cytochrome c (CC) were measured by Western blot in individual oocytes. p38 activity is represented as pp38/p38 ratio, taking as maximum activity (100% value) the oocytes treated with 400 mM sorbitol (M). Each box represents one individual oocyte. Results are the pool of three independent experiments. The Western blot shows a representative experiment. The arrow indicates no correlation between p38 activation and cytochrome c release at 2 h. (B) p38 activity in individual oocytes treated with 200 mM sorbitol for 4 h. Activity was measured by Western blot, as described previously, and the results expressed as pp38/p38, taking as maximum activity the value obtained with 400 mM sorbitol. (C) Correlation between pp38 and cytochrome c in oocytes incubated with 200 mM sorbitol for 2 h. pp38 and cytochrome c (CC) levels were determined by Western blot in individual oocytes (n = 50) from 5 independent experiments, giving 100% value to individual oocytes treated with 400 mM sorbitol. The data obtained for each oocyte was represented graphically and the correlation coefficient (Pearson r, Spearman r) calculated with GraphPad Prism4. (D) Correlation between pp38 and cytochrome c in oocytes incubated with 200 mM sorbitol for 4 h. pp38 and cytochrome c (CC) were determined by Western blot in individual oocytes (n = 50) as reported before, the data obtained represented graphically, and the correlation coefficients calculated with GraphPad Prism4.
	Fig 5. Microinjection of cytochrome c (CC) induces caspase-3 activation and p38 phosphorylation.(A) Caspase inhibitors reduce p38 phosphorylation induced by cytochrome c injection. Oocytes were injected with MBS, horse cytochrome c (CC) (0.5 μM final concentration), CC (0.5 μM) plus Z-VAD.fmk (5 μM), CC (0.5 μM) plus Ac-DEVD-CHO (100 nM), or non injected (control). Pools of 20 oocytes were collected at different times and caspase-3 activity was determined as the concentration of fluorescent AMC formation from Z-DEVD-AMC substrate, and represented as arbitrary units of caspase-3 activity, giving value 1 to MBS injected oocytes. pp38, p38 and AMPK (loading control) were analyzed by Western blot. The result presented is representative of three independent experiments. (B) Injection of BSA or cytochrome c from Saccharomyces cerevisiae (CC Yeast) does not induce caspase-3 activation and p38 phosphorylation. Oocytes were injected with MBS, horse cytochrome c (CC) (0.5 μM), yeast cytochrome c (CC Yeast) (0.5 μM), BSA (0.5 μM), or non injected (control). Pools of 20 oocytes were collected at different times and caspase-3 activity was determined as described before. pp38, p38 and AMPK were analyzed by Western blot. (C) DMSO injection does not interfere with p38 phosphorylation and caspase-3 activation induced by cytochrome c. Oocytes were injected with MBS plus DMSO (diluted 1:50 in MBS), horse cytochrome c (CC) (0.5 μM) plus DMSO (diluted 1:50 in CC), CC (0.5 μM) plus Z-VAD.fmk (50 μM), or CC (0.5 μM) plus Ac-DEVD-CHO (1 μM). Pools of 20 oocytes were collected at different times and caspase-3 activity was determined as described before. pp38, p38 and AMPK were analyzed by Western blot.
	Fig 6. Caspase-3 inhibitor reduces p38 phosphorylation induced by hyperosmotic shock.Oocytes were incubated with 200 mM sorbitol for 4 h in the presence or absence of caspase-3 inhibitor Z-DEVD.fmk (50 μM), or with 400 mM sorbitol for 1 h. Pools of 20 oocytes were collected and pp38, p38, cytochrome c (CC) and AMPK (loading control) were analyzed by Western blot. Caspase-3 activity was determined as the concentration of fluorescent AMC formation from Z-DEVD-AMC substrate, and represented as arbitrary units of caspase-3 activity, giving value 1 to non treated oocytes.
	Fig 1. Hyperosmotic shock activates p38 in Xenopus laevis oocytes.Oocytes (stage VI) were treated with sorbitol (400 mM) and pools of 20 oocytes were lysed at different times to analyze pp38, p38, cytochrome c (CC), and cleaved caspase-3 by Western blot. p38 activity is represented as pp38/p38 ratio, giving 100% value to the highest activity. Results are represented as mean ± SEM of four independent experiments. A representative Western blot is shown. Caspase-3 activity was determined at different times, using the synthetic peptide Z-DEVD-AMC as a substrate and giving value 1 to non treated oocytes. Data are represented as mean ± SEM (n = 3), *p<0.05 (ANOVA and Dunnett’s test) comparing with non treated oocytes.

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Bagowski, Bistability in the JNK cascade. 2001, Pubmed, Xenbase