Ben Messaoud N and López JM (2024), p38α and p38β regulate osmostress-induced apopt...

XB-ART-61126

J Biol Chem 2024 Jan 07;3011:108061. doi: 10.1016/j.jbc.2024.108061.

Show Gene links Show Anatomy links

p38α and p38β regulate osmostress-induced apoptosis.

Ben Messaoud N , López JM .

???displayArticle.abstract???
Hyperosmotic shock induces cytochrome c release and caspase-3 activation in Xenopus oocytes. Different signaling pathways engaged by osmostress converge on the mitochondria to trigger cell death. The mitogen activated protein kinases (MAPKs) JNK1-1 and JNK1-2 are early activated by hyperosmotic shock and sustained activation of both isoforms accelerates the apoptotic program. Indeed, sustained activation of p38 accelerates osmostress-induced cell death, but the p38 isoforms involved are not well characterized. Here we study the expression and activation of Xenopus p38 isoforms in response to hyperosmotic stress. We find that p38α, p38β, and p38γ are early activated by hyperosmotic shock and sustained activation of p38α and p38β accelerates osmostress-induced apoptosis. Moreover, microinjection of cytochrome c in the oocytes induces caspase-3 activation and p38α and p38β phosphorylation suggesting that caspases and kinases are interlinked in a positive feedback loop to promote cell death. In summary, we present a more complete view of the mechanisms involved in osmostress-induced apoptosis.

???displayArticle.pubmedLink??? 39653241
???displayArticle.pmcLink??? PMC11757794
???displayArticle.link??? J Biol Chem

Genes referenced: mapk8

???attribute.lit??? ???displayArticles.show???

	Figure 1. Xp38α and Xp38γ are activated by hyperosmotic shock.A, C, and E, expression and phosphorylation of Xp38α, Xp38γ, and their mutants. Oocytes were injected with 50 nl of H2O or cRNAs (5 ng in 50 nl) Xp38α, Xp38α-AF, Xp38α-KR, Xp38γ, Xp38γ-DA, or MKK6-DD and 18 h later exposed to osmotic shock (300 mM sorbitol) for 1 h, 3 h, or non-treated. Expression of p38 isoforms was confirmed with Myc antibodies (A). MKK6-DD is a constitutively active mutant without Myc tag. pp38 and cytosolic cytochrome c (CC) levels were analyzed by Western blot and AMPK was used as a loading control. B, D, and F, caspase-3 activity was measured in all the conditions assayed, giving value 1 to non-treated water-injected oocytes. Expression of Xp38α, Xp38γ, or their inactive mutants did not modify hyperosmotic shock-induced apoptosis. Results in panels (B, D, and F) are the mean ± SD of three independent experiments. ∗p < 0.05 compared to water-injected oocytes (ANOVA and Dunnett’s test). Western blots are representative of three independent experiments (see Figs. S2 and S3 for additional experiments and blots quantification).
	Figure 2. Xp38β is activated by hyperosmotic shock.A, C, and E, expression and phosphorylation of p38 isoforms and mutants. Oocytes were injected with 50 nl of H2O or cRNAs (5 ng in 50 nl) Xp38β, Xp38β-AF, Xp38β-KR, Xp38δ, Xp38δ-AF, Xp38δ-KR, or MKK6-DD and 18 h later exposed to osmotic shock (300 mM sorbitol) for 1 h, 3 h, or non-treated. Expression of p38 isoforms was confirmed with Myc antibodies (A). pp38 and cytosolic cytochrome c (CC) levels were analyzed by Western blot and AMPK was used as a loading control. B, D, and F, caspase-3 activity was measured in all the conditions assayed, giving value 1 to non-treated water-injected oocytes. Expression Xp38β, Xp38δ, or their inactive mutants did not modify osmostress-induced apoptosis. Data in (B, D, and F) are represented as mean ± SD of three or four independent experiments. ∗p < 0.05 compared to water-injected oocytes (ANOVA and Dunnett’s test). Western blots are representative of three independent experiments (see Figs. S4 and S5 for additional experiments and blots quantification).
	Figure 3. Constitutively active Xp38α (CA) or Xp38β (DA/YL) expression accelerates hyperosmotic shock-induced apoptosis.A, C, and E, oocytes were injected with 50 nl of H2O or cRNAs (5 ng in 50 nl) Xp38α-CA, Xp38β-DA/YL, a combination of both, or MKK6-DD and 18 h later exposed to osmotic shock (300 mM sorbitol) for 1 h, 2 h, or non-treated. Expression of p38 mutants was confirmed with Myc antibodies (A). pp38 and cytosolic cytochrome c (CC) levels were analyzed by Western blot and AMPK was used as a loading control. B, D, and F, caspase-3 activity was measured in all the conditions assayed, giving value 1 to non-treated water-injected oocytes. Results represent the mean ± SD of seven independent experiments. ∗p < 0.05, ∗∗p < 0.01 compared to water-injected oocytes (ANOVA and Dunnett’s test). Western blots are representative of three independent experiments (see Figs. S7 and S8 for additional experiments and blots quantification).
	Figure 4. Cytochrome c injection in Xenopus oocytes induces caspase-3 activation and p38α and p38β phosphorylation.A, oocytes were injected with 50 nl of MBS or cRNAs (5 ng in 50 nl), Xp38α, Xp38β, Xp38γ, or Xp38δ and 18 h later injected with MBS (as a control) or horse cytochrome c (CC) (0.5 μM final concentration) dissolved in MBS and pools of 20 oocytes were lysed 1 h later to analyze Myc, pp38, and AMPK by Western blot. A representative blot of two independent experiments is shown (see Fig. S9 for additional experiment). B, blots were quantified with Image J and the ratio pp38/AMPK represented. Results are the mean ± SD of two independent experiments. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to oocytes of the same condition but non-injected with cytochrome c (ANOVA and Fisher’s LSD test). C, caspase-3 activity was determined in all the extracts and represented as arbitrary units (AU), giving value 1 to oocytes injected with MBS. Results are the mean ± SD of two independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to oocytes of the same condition but non-injected with cytochrome c (ANOVA and Fisher’s LSD test).
	Figure 5. Caspase-3 inhibition reduces phosphorylation of p38α and p38β induced by cytochrome c injection.A, oocytes were injected with MBS, horse cytochrome c (CC) (0.5 μM final intracellular concentration), or CC (0.5 μM) plus caspase-3 inhibitor Ac-DEVD-CHO (0.1 μM final intracellular concentration) and pools of 20 oocytes were lysed 1 h later to analyze Myc, pp38, and β-Actin by Western blot. Caspase-3 activity was determined as previously described (see values at the bottom of the blot). B, pp38 and Myc levels were quantified from the blot with Image J and the ratio pp38/Myc represented in arbitrary units (AU), giving value 100% to oocytes expressing Xp38α or Xp38β injected with cytochrome C. C and D, oocytes were treated and analyzed as described above, but Ac-DEVD-CHO was injected at 1 μM final concentration. Note that caspase-3 activity was markedly reduced in C compared with B.
	Figure 6. Model for osmostress-induced apoptosis. Like a heavy rain, hyperosmotic shock rapidly activates several signaling pathways that converge in the mitochondria to increase their permeability releasing cytochrome c to the cytosol. In Xenopus oocytes, hyperosmotic shock induces rapid calpain activation, Smac/DIABLO release from the mitochondria, cleavage of small amounts of Bid by an unknown protease, and activation of JNK1-1, JNK1-2, p38α, p38β, and p38γ. Sustained activation of JNK1-1, JNK1-2, p38α, and p38β in combination with Smac/DIABLO, t-Bid, and calpains converge on the mitochondria to induce the release of cytochrome c into the cytosol, which in turn activates caspase-3. The activation of caspase-3 induces JNK1-2 and Bid proteolysis, as well as p38α and p38β phosphorylation. These events, in turn, promote additional cytochrome c release and caspase-3 activation engaging at least three positive feedback loops (L1-L3) to complete the cell death program.