Rho kinase regulates phagocytosis, surface expression of GlcNAc, and Golgi fragmentation of apoptotic PC12 cells.

Apoptotic cells undergo a number of changes to prepare for phagocytosis; most occur during the execution phase of apoptosis, when dying cells undergo shrinkage and/or fragmentation into apoptotic bodies and express phagocytic markers on their surface. Although events during the execution phase are important to prepare corpses for phagocytosis, the mechanisms that control most execution phase events are unknown. To understand regulation of execution events we focused on Rho kinase (ROCK), because one isoform of ROCK, ROCK-I, is constitutively activated by caspases during execution. Using apoptotic PC12 cells as a model, we find that inhibition of ROCK activity during apoptosis decreases surface expression of GlcNAc, a carbohydrate known to function as a phagocytic marker. In addition, inhibition of ROCK blocks Golgi fragmentation in apoptotic cells, and constitutively active ROCK induces Golgi fragmentation in the absence of apoptosis. Importantly, PC12 cells dying in the presence of a ROCK inhibitor are less efficiently phagocytized than those dying without the inhibitor. These data highlight the role of ROCK in multiple processes in the execution phase of apoptosis, and suggest that ROCK plays an important role in controlling the outcome of apoptosis, that is, preparation of corpses for phagocytosis.

Rho kinase regulates fragmentation and phagocytosis of apoptotic cells.

During the execution phase of apoptosis, a cell undergoes cytoplasmic and nuclear changes that prepare it for death and phagocytosis. The end-point of the execution phase is condensation into a single apoptotic body or fragmentation into multiple apoptotic bodies. Fragmentation is thought to facilitate phagocytosis; however, mechanisms regulating fragmentation are unknown. An isoform of Rho kinase, ROCK-I, drives membrane blebbing through its activation of actin-myosin contraction; this raises the possibility that ROCK-I may regulate other execution phase events, such as cellular fragmentation. Here, we show that COS-7 cells fragment into a number of small apoptotic bodies during apoptosis; treating with ROCK inhibitors (Y-27632 or H-1152) prevents fragmentation. Latrunculin B and blebbistatin, drugs that interfere with actin-myosin contraction, also inhibit fragmentation. During apoptosis, ROCK-I is cleaved and activated by caspases, while ROCK-II is not activated, but rather translocates to a cytoskeletal fraction. siRNA knock-down of ROCK-I but not ROCK-II inhibits fragmentation of dying cells, consistent with ROCK-I being required for apoptotic fragmentation. Finally, cells dying in the presence of the ROCK inhibitor Y-27632 are not efficiently phagocytized. These data show that ROCK plays an essential role in fragmentation and phagocytosis of apoptotic cells.