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.
Orlando
Kelly A
KA
Department of Pharmacology, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, PA 19104, USA.
Stone
Nicole L
NL
Pittman
Randall N
RN
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D019556:COS Cells; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D053938:DNA Fragmentation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008387:Marine Toxins / Q000494:pharmacology; D009119:Muscle Contraction; D009218:Myosins / Q000378:metabolism; D016716:PC12 Cells; D010587:Phagocytosis; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D054460:rho-Associated Kinases
C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D000199:Actins; D000577:Amides; D019086:Bridged Bicyclo Compounds, Heterocyclic; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D006576:Heterocyclic Compounds, 4 or More Rings; D047908:Intracellular Signaling Peptides and Proteins; D008387:Marine Toxins; D011725:Pyridines; D034741:RNA, Small Interfering; D013844:Thiazoles; D053778:Thiazolidines; C108830:Y 27632; C472645:blebbistatin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020169:Caspases; D009218:Myosins; C037068:latrunculin B