Defective apoptotic cell contractility provokes sterile inflammation, leading to liver damage and tumour suppression
- Cancer Research United Kingdom Beatson Institute, Garscube Estate, United Kingdom
- Institute of Cancer Sciences, University of Glasgow, United Kingdom
- Department of Chemistry and Biology, Ryerson University, Canada
- Electron Microscopy Facility, School of Life Sciences, University of Glasgow, United Kingdom
- Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, United Kingdom
Figures
Figure 1 with 3 supplements
ROCK1 cleavage is essential for increased myosin light chain (MLC) phosphorylation during apoptosis.
(A) Representative western blot showing Myc-tagged ROCK1 immunoprecipitated from transfected HEK293T cells assayed in vitro for MLC phosphorylation (pMLC) without (-) or with (+) cleavage by …
Figure 1—figure supplement 1
Uncropped western blots and in-cell westerns.
Figure 1—figure supplement 2
Homologous recombination targeting strategy to generate ROCK1 non-cleavable allele.
(A) Line diagram of mouse genomic Rock1 wild-type (ROCK1wt) locus with numbered Rock1 exons indicated by blue boxes (top). Targeting vector homology arms to Rock1 wild-type locus is indicated by …
Figure 1—figure supplement 3
Example fluorescence-activated cell sorting (FACS) determination of ROCK1wt and ROCK1nc mouse embryo fibroblast (MEF) apoptosis.
Representative FACS dot plots of ROCK1wt and ROCK1nc MEFs that were untreated or treated with tumour necrosis factor α (TNFα) and cycloheximide (CHX) for 4 hr. Cells were stained with …
Figure 2 with 1 supplement
Apoptotic ROCK1nc mouse embryo fibroblasts (MEFs) do not undergo forceful contraction.
(A) Homozygous ROCK1wt (upper panels) or ROCK1nc (lower panels) MEFs were serum-starved overnight, then treated with tumour necrosis factor α (TNFα) (50 ng/mL) plus cycloheximide (CHX) (10 µg/mL) to …
Figure 2—figure supplement 1
Characterization of ROCK1wt and ROCK1nc mouse embryo fibroblasts (MEFs).
(A) Cell numbers after plating 4 × 103 ROCK1wt and ROCK1nc MEFs at daily intervals up to 4 days. Means ± standard deviation, n = 5 independent replicates. (B) Example images used for high content …
Figure 3 with 2 supplements
Elevated chemically induced liver damage in ROCK1nc mice.
(A) Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining of apoptotic cells in representative liver sections from ROCK1wt or ROCK1nc mice 72 hr after diethylnitrosamine (DEN…
Figure 3—figure supplement 1
Absence of indications of autoimmunity in aged ROCK1wt or ROCK1nc kidneys or spleens.
(A) Representative periodic acid-Schiff (PAS)-stained kidney sections from ROCK1wt and ROCK1nc mice showing no observable differences in basement membrane thickening (white arrows). Scale bars …
Figure 3—figure supplement 2
Ageing does not affect blood cells, liver or spleen masses differently in ROCK1wt or ROCK1nc mice.
Numbers of (A) red blood cells, (B) white blood cells, (C) neutrophils, (D) lymphocytes, (E) monocytes, (F) eosinophils, or (G) platelets in ROCK1wt or ROCK1nc mice 10 weeks (ROCK1wt n = 11 mice; …
Figure 4
Higher neutrophil infiltration in chemically damaged ROCK1nc livers.
(A) Immunohistochemical (IHC) staining with F4/80 antibody of formalin-fixed paraffin-embedded (FFPE) liver sections from homozygous ROCK1wt (WT) or ROCK1nc (NC) mice 72 hr after diethylnitrosamine (…
Figure 5
Inhibition of high-mobility group B1 (HMGB1) signalling reduces diethylnitrosamine (DEN)-induced liver damage.
(A) Immunohistochemical (IHC) staining with anti-HMGB1 antibody of formalin-fixed paraffin-embedded (FFPE) liver sections from homozygous ROCK1wt (WT) or ROCK1nc (NC) mice 72 hr after DEN injection. …
Figure 6
Diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) tumour numbers are lower in ROCK1nc mice.
(A) Timeline of DEN-induced HCC protocol. (B) Macroscopic view of representative livers from ROCK1wt and ROCK1nc mice 9 months after HCC initiation with DEN. Scale bars = 1 cm. (C) Tumour numbers …
Figure 7 with 1 supplement
Similar liver mass and tumour grades in diethylnitrosamine (DEN)-treated ROCK1wt and ROCK1nc mice.
(A) Liver mass, (B) body mass and (C) liver to body ratios for ROCK1wt and ROCK1nc mice (n = 12 mice for each genotype) 9 months after hepatocellular carcinoma (HCC) initiation with DEN. Boxes …
Figure 7—figure supplement 1
Histopathological features of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC).
Representative H&E-stained liver sections from mice with DEN-induced HCC showing (A) tumour border (dotted line), (B) trabecular pattern (arrow), (C) macrotrabecular structures (arrow), (D) compact …
Figure 8 with 1 supplement
Glycyrrhizin (GLZ) increases diethylnitrosamine (DEN)-induced liver tumour number in ROCK1nc mice.
(A) Timeline of the DEN-induced hepatocellular carcinoma (HCC) and GLZ treatment protocol. (B) The number of liver tumours in ROCK1nc mice 9 months after treatment with GLZ alone (n = 10 mice) or …
Figure 8—figure supplement 1
Similar liver mass in ROCK1nc mice treated with diethylnitrosamine (DEN) and glycyrrhizin (GLZ).
(A) Liver mass, (B) body mass and (C) liver to body mass ratios for ROCK1nc mice 9 months after treatment with GLZ alone (n = 10 mice) or following hepatocellular carcinoma (HCC) initiation with DEN …
Videos
Video 1
Time lapse of apoptotic ROCKwt mouse embryo fibroblasts (MEFs).
ROCK1wt MEFs were plated on 6-well glass-bottom dishes at a density of 2 × 106 cells per well. Cells were serum-starved overnight prior to induction of apoptosis with tumour necrosis factor α (TNFα) …
Video 2
Time lapse of apoptotic ROCKnc mouse embryo fibroblasts (MEFs).
ROCK1nc MEFs were plated on 6-well glass-bottom dishes at a density of 2 × 106 cells per well. Cells were serum-starved overnight prior to induction of apoptosis with tumour necrosis factor α (TNFα) …
Tables
Table 1
Conservation of ROCK1 caspase cleavage site.
| Mammals | |
| Homo sapiens | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Chimp | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Gorilla | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Orangutan | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Gibbon | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Rhesus | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Crab-eating macaque | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Baboon | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Green monkey | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Marmoset | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Squirrel monkey | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Bushbaby | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Chinese tree shrew | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Squirrel | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Lesser Egyptian jerboa | QLRAKLLDLSDSTSVASFPSADETDGSLP |
| Prairie vole | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Chinese hamster | QLRAKLLDLSDSTSIASFPSADETDGNLP |
| Golden hamster | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Mouse | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Rat | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Naked mole-rat | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Guinea pig | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Chinchilla | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Brush-tailed rat | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Rabbit | QLRAKLSDLSDSTSVASFPSADETDGNLP |
| Pika | QLRAKLLDLSDSTSVASFPSTDEaDGNLP |
| Pig | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Alpaca | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Bactrian camel | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Dolphin | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Killer whale | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Tibetan antelope | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Cow | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Sheep | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Domestic goat | QLRAKLLDLSDSTSVASFPSADETDGNLS |
| Horse | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| White rhinoceros | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Cat | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Dog | QLRAKLLDLSDSTSIASFPSADETDGNLP |
| Ferret | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Panda | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Pacific walrus | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Weddell seal | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Black flying-fox | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Megabat | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| David’s myotis (bat) | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Microbat | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Big brown bat | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Hedgehog | QLRAKLLDLADSTSVASFPSADETDGNLP |
| Shrew | QLRSKLLDLSDSTSVASFPSADETDGTLP |
| Star-nosed mole | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Elephant | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Cape elephant shrew | QLRAKLLDLSDSTSVASFPSADETDGSLP |
| Manatee | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Cape golden mole | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Tenrec | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Aardvark | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Armadillo | QLRAKLLDLSDSTSVASFPSADETDGNLP |
| Opossum | QLRAKLLDLSDSTSVTSFPSADETDGNLP |
| Tasmanian devil | QLRAKLLDLSDSTSVTSFPSADETDGNLP |
| Wallaby | QLRSKLSDLLDSGSVASL-S-DETDGNLV |
| platypus | QLRRKILDLLDSTSVASLQ--DETDGNLS |
| Birds | |
| Saker falcon | QLRRKILDLLDSTSVASLQ--DETDGNLS |
| Peregrine falcon | QLRRKIMDLLDSTSVASLP--DEmDGNLP |
| Collared flycatcher | QLRRKIMDLLDSTSVASLP--DETDGNLS |
| White-throated sparrow | QLRRKIMDLLDSTSVASLP--DETDGNLS |
| Medium ground finch | QLRRKIMDLLDSTSVASLP--DETDGNLS |
| Zebra finch | QLRRKIMDLLDSTSVASLP--DETDGNLS |
| Tibetan ground jay | QLRRKILDLLDSTSVASLQ--DETDGNLS |
| Budgerigar | QLRRKILDLLDSTSVASLQ--DETDGNLS |
| Parrot | QLRRKILDLLDSTSVASLQ--DETDGNLS |
| Scarlet macaw | QLRRKILDLLDSTSVASLQ--DETDGNLS |
| Rock pigeon | QLRRKILDLLDSTSVASLP--DETDGNLS |
| Mallard duck | QLRRKILDLLDSTSVASLQ--DETDGNLS |
| Chicken | QLRRKILDLLDSTSVSSLQ--DEiDGNLS |
| Turkey | QLRRKILDLLDSTSVSSLQ--DEiDGNLS |
| Sarcopterygii | |
| American alligator | QLRSKILDLLDSTSVASLQ--DETDGNVS |
| Green seaturtle | QLRSKILDLLDSTSVASLQ--DETDGNLT |
| Painted turtle | QLRSKLLDLLDSTSVASLQ--DETDGNLT |
| Chinese softshell turtle | QLRSKILDLLDSASVASLQ--DETDGNLT |
| Spiny softshell turtle | QLRSKILDLLDSASVASLQ--DETDGNLT |
| Lizard | QLRSKILDLLDSTSVASLP--DETDGNIV |
| X. tropicalis | QLRARLADLLDSTSVASLQ--DdTDGIIG |
| Coelacanth | QLRAKLMDLLDNTSVASLQ--DEvDGNVI |
| Fish | |
| Tetraodon | QLREKLNDLLDNSSITSLQ--DETDSNIA |
| Fugu | QLREKLNDLLDNSSITSLQ--DETDSNIA |
| Yellowbelly pufferfish | QLREKLNDLLDNSSITSLQ--DETDSNIA |
| Nile tilapia | QLREKLNDLLDNSSVTSLQ--DETDSNIA |
| Princess of Burundi | QLREKLNDLLDNSSVTSLQ--DETDSNIA |
| Burton's mouthbreeder | QLREKLNDLLDNSSVTSLQ--DETDSNIA |
| Zebra mbuna | QLREKLNDLLDNSSVTSLQ--DETDSNIA |
| Pundamilia nyererei | QLREKLNDLLDNSSVTSLQ--DETDSNIA |
| Medaka | QLREKLNDMLENSSITSLQ--DETDSNIA |
| Southern platyfish | QLREKLSDLLENSSVTSLQ--DETDSNTA |
| Stickleback | QLREKLNDLLDTSSVTSLQ--DETDGNIA |
| Atlantic cod | QLREKLNDLLDSSSVTSLL--DETDGNLA |
| Zebrafish | QLREKLNDLLDNSSVTSLQ--DElDSNIA |
| Spotted gar | QLREKLNDLLDNSSVASLQ--DEaDGNIA |
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Protein sequences orthologous to human ROCK1 were aligned in 93 vertebrate species using Multiz Alignment (Blanchette et al., 2004) in the UCSC Genome Browser. These include representative species from the major classes: 62 mammals, 14 birds, 8 Sarcopterygii and 14 fish. The conserved caspase cleavage site indicated with underlined bold typeface, with non-identical amino acids in lowercase.
