Proteomic analysis of shear stress-mediated protection from TNF-alpha in endothelial cells.
Previous studies have shown that physiological levels of shear stress can protect endothelial cells (ECs) from apoptotic stimuli. Here, we differentiate between acute and chronic protection and demonstrate the use of proteomic technologies to uncover mechanisms associated with chronic protection of ECs. We hypothesized that changes in abundance of proteins associated with the TNF-alpha signaling cascade orchestrate shear stress-mediated protection from TNF-alpha when cells are preconditioned with shear prior to the exposure of apoptotic stimuli. Detection of cleaved caspase 3 through Western blot analysis confirmed chronic shear stress-mediated protection from TNF-alpha. In the presence of the nitric oxide synthase inhibitor, LNMA (N(omega)-monomethyl-l-arginine), chronic protection remained. Treatment with a de novo protein synthesis inhibitor, cycloheximide, eliminated this protective effect. Isotopic-labeling experiments, coupled with LC-MS/MS (liquid chromatography-tandem mass spectrometry) of isolated components of the TNF-alpha pathway revealed that CARD9, a known activator of the NF-kappaB pathway, was increased (60%) in sheared cells versus nonsheared cells. This result was confirmed through Western blot analysis. Our data suggest that de novo formation of proteins is required for protection from TNF-alpha in ECs chronically exposed to shear stress, and that CARD9 is a candidate protein in this response.
Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Julie K
JK
Freed
Andrew S
AS
Greene
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053468:CARD Signaling Adaptor Proteins / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D040901:Proteomics; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013314:Stress, Mechanical; D053199:TNF Receptor-Associated Death Domain Protein / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D053468:CARD Signaling Adaptor Proteins; D004791:Enzyme Inhibitors; C505535:Fadd protein, rat; D053200:Fas-Associated Death Domain Protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D053199:TNF Receptor-Associated Death Domain Protein; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase