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Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex
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Hyperosmotic activation of the CD95 system.

Roland Reinehr1, Dieter Häussinger

  • 1Clinic for Gastroenterology, Hepatology, and Infectiology, Heinrich-Heine-University Düsseldorf, Germany.

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Hyperosmotic cell shrinkage in hepatocytes activates the CD95 death receptor system, leading to apoptosis. This process involves reactive oxygen species, EGFR, and JNK signaling, ultimately sensitizing cells to CD95 ligand-induced cell death.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Programmed cell death, or apoptosis, is characterized by cell shrinkage, nuclear condensation, DNA fragmentation, and apoptotic body formation.
  • Apoptotic volume decrease (AVD) is an early and universal event in apoptosis.
  • In hepatocytes, hyperosmotic cell shrinkage activates the CD95 death receptor system, sensitizing them to CD95 ligand-induced apoptosis.

Purpose of the Study:

  • To elucidate the early signaling events involved in CD95 receptor activation by hyperosmolarity in hepatocytes.
  • To investigate the role of reactive oxygen species (ROS), epidermal growth factor receptor (EGFR), and c-Jun-N-terminal kinase (JNK) in hyperosmolarity-induced CD95 activation.
  • To determine the threshold of hyperosmotic stress required for apoptotic cell death induction in hepatocytes.

Main Methods:

  • Hepatocytes were exposed to varying degrees of hyperosmotic stress.
  • Measurements included endosomal acidification, ceramide generation, ROS production, protein phosphorylation (tyrosine, serine/threonine), receptor-ligand interactions, and caspase activation.
  • Inhibitors of anion channels, vacuolar-type H(+)-ATPase, and specific kinases were used to dissect signaling pathways.

Main Results:

  • Hyperosmotic exposure induced rapid endosomal acidification, increasing intracellular ceramide and ROS production via NADPH oxidase.
  • ROS formation activated Src-family kinase Yes, leading to EGFR activation, JNK activation, and subsequent CD95 tyrosine phosphorylation.
  • CD95 tyrosine phosphorylation mediated CD95 oligomerization, translocation to the plasma membrane, and DISC formation, sensitizing cells to CD95L-induced apoptosis. Severe hyperosmolarity (>505 mosmol/liter) induced apoptosis.
  • Mild hyperosmolarity (405 mosmol/liter) induced DISC formation and caspase activation but did not reduce cell viability.

Conclusions:

  • Hyperosmotic stress triggers a signaling cascade involving ASM, ceramide, ROS, EGFR, and JNK, culminating in CD95 activation and sensitization to apoptosis in hepatocytes.
  • The degree of hyperosmotic stress determines whether hepatocytes are sensitized to apoptosis or undergo direct apoptotic cell death.
  • Covalent modifications like tyrosine nitration or serine/threonine phosphorylation of CD95 can inhibit its activation.