TNF induces caspase-dependent inflammation in renal endothelial cells through a Rho- and myosin light chain kinase-dependent mechanism.
The pathogenesis of LPS-induced acute kidney injury (AKI) requires signaling through tumor necrosis factor-alpha (TNF) receptor 1 (TNFR1), which within the kidney is primarily located in the endothelium. We showed previously that caspase inhibition protected mice against LPS-induced AKI and in parallel significantly inhibited LPS-induced renal inflammation. Therefore we hypothesized that caspase activation amplifies TNF-induced inflammation in renal endothelial cells (ECs). In cultured renal ECs, TNF induced apoptosis through a caspase-8-dependent pathway. TNF caused translocation of the p65 subunit of NF-kappaB to the nucleus, resulting in upregulation of inflammatory markers such as adhesion molecules ICAM-1 and VCAM-1. However, the broad-spectrum caspase inhibitor Boc-d-fmk reduced NF-kB activation as assessed by gel shift assay, reduced phosphorylation of subunit IkappaBalpha, and significantly inhibited TNF-induced expression of ICAM-1 and VCAM-1 as assessed by both real-time PCR and flow cytometry. Broad-spectrum caspase inhibition markedly inhibited neutrophil adherence to the TNF-activated endothelial monolayer, supporting the functional significance of this effect. Specific inhibitors of caspases-8 and -3, but not of caspase-1, reduced TNF-induced NF-kappaB activation. Caspase inhibition also reduced TNF-induced myosin light chain (MLC)-2 phosphorylation, and activation of upstream regulator RhoA. Consistent with this, MLC kinase (MLCK) inhibitor ML-7 reduced TNF-induced NF-kappaB activation. Thus caspase activation influences NF-kappaB signaling via its affect on cytoskeletal changes occurring through RhoA and MLCK pathways. These cell culture experiments support a role for caspase activation in TNF-induced inflammation in the renal endothelium, a key event in LPS-induced AKI.
Section of Nephrology, University of Chicago, Chicago, Illinois 60637, USA.
Xiaoyan
X
Wu
Rongqing
R
Guo
Peili
P
Chen
Quan
Q
Wang
Patrick N
PN
Cunningham
D021581:Active Transport, Cell Nucleus; D000818:Animals; D017209:Apoptosis; D053181:Caspase 8 / Q000378:metabolism*; D061945:Caspase Inhibitors; D002448:Cell Adhesion; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D004789:Enzyme Activation; D020672:I-kappa B Proteins / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007668:Kidney / Q000098:blood supply*; D051379:Mice; D008810:Mice, Inbred C57BL; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D009393:Nephritis / Q000201:enzymology* / Q000276:immunology / Q000473:pathology / Q000517:prevention & control; D020556:Neutrophil Infiltration; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D051996:Transcription Factor RelA / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D020672:I-kappa B Proteins; C521958:Mylpf protein, mouse; D018994:Myosin Light Chains; C000606205:Nfkbia protein, mouse; D047428:Protein Kinase Inhibitors; C498144:Rela protein, mouse; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; D009219:Myosin-Light-Chain Kinase; C505526:Casp8 protein, mouse; D053181:Caspase 8; C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein