Proteomic analysis of shear stress-mediated protection from TNF-alpha in endothelial cells

Abstract

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.

Fig. 1

A, Quantitative densitometry comparing cleaved caspase 3 and poly-ADP-Ribose-Polymerase (PARP) protein levels in non-treated control cells, cells treated with 10ng/ml of TNF-alpha for 12hrs, cells exposed to shear for 18hrs (10 dynes/cm²), and cells first exposed to shear (10 dynes/cm² for 18hrs) then treated with 10ng/ml of TNF-alpha for 12hrs (mean+/−SEM, n=5). B, Cells treated with 1mM LNMA for 12hrs, cells treated with 10ng/ml of TNF-alpha for 12hrs, cells treated with 1mM LNMA and 10ng/ml TNF-alpha for 12hrs simultaneously, cells first exposed to shear (10 dynes/cm² for 18hrs) then treated with 10ng/ml of TNF-alpha for 12hrs, and cells first incubated with 1mM LNMA for 1hr then exposed to shear (10 dynes/cm² for 18hrs) followed by TNF-alpha treatment (10ng/ml for 12hrs) (mean+/−SEM, n=5). C, Control cells treated with cycloheximide alone, (0.36μmol/L, 18hrs) cells first incubated with cycloheximide (0.36μmol/L) followed by TNF-alpha, (10ng/mL, 12hrs) cells incubated with cycloheximide (0.36μmol/L) followed by shear stress exposure, (10 dynes/cm² for 18hrs) and cells incubated with cycloheximide (0.36μmol/L) then exposed to shear stress (10 dynes/cm² for 18hrs) followed by TNF-alpha treatment (10ng/mL, 12hrs) (mean+/−SEM, n=6). D, Representative images of Western blots for each experiment A-C. (*) Significant difference vs. no treatment group, p<0.05.

Fig. 2

A, Relative ratios of the 15 quantifiable proteins from the ¹⁸O isotopic labeling experiment plotted as a ratio histogram under a normal distribution. One of the quantifiable proteins, CARD9, is shown as an outlier of the group. B, Normalized average ratio for CARD9 compared to the combined average ratio for all other identified proteins (top 15 - CARD9). C, Representative mass spectra showing both the heavy and light isotopic peaks from a CARD9 peptide, KVTGKEPAR, m/z of 985.57.

Fig. 3

Quantitative densitometry comparing the amount of CARD9 in cells treated with TNF-alpha (10ng/mL, 12hrs) and in cells first exposed to shear stress (10 dynes/cm² for 18hrs) followed by TNF-alpha treatment (10ng/mL, 12hrs). (n=6). (*) Significant difference, p<0.05. (mean +/− SEM)

Fig. 4

A, Quantitative densitometry comparing both Fadd and Tradd protein levels in non-treated control cells, cells treated with 10ng/ml of TNF-alpha for 12hrs, cells exposed to shear (10 dynes/cm² for 18hrs), and cells first exposed to shear (10 dynes/cm² for 18hrs) followed by TNF-alpha treatment (10ng/ml for 12hrs) (n=6). (mean+/−SEM) B, Phosphorylated IκB-α: total IκB in non-treated control cells, cells treated with 10ng/ml of TNF-alpha for 12hrs, cells exposed to shear (10 dynes/cm² for 18hrs), and cells first exposed to shear (10 dynes/cm² for 18hrs) followed by TNF-alpha treatment (10ng/ml for 12hrs). (n=6) (mean+/−SEM) (*) Significant difference vs. no treatment p<0.05.

Fig. 5

Diagram illustrating the signaling mechanism of how CARD9 may be inhibiting apoptosis from TNF-alpha. Upon binding of TNF-alpha to TNFR1, the receptor trimerizes and activates 1 of 2 signaling cascades. Activation of the FADD pathway results in further activation of a caspase cascade resulting in apoptosis. Activation of the TRADD pathway results in activation of NF-KB which signals to the nucleus to increase the number of survival proteins resulting in cell proliferation. Shear stress is known to result in phosphorylation of IκB, thus releasing NF-KB from its inhibitory state in order to signal to the nucleus. Our study suggests that CARD9 is one of the survival proteins that may be interacting with the caspase-induced pathway of TNF-alpha, thus protecting endothelial cells from apoptosis during chronic shear.