Adenovirus E3-6.7K maintains calcium homeostasis and prevents apoptosis and arachidonic acid release

Abstract

E3-6.7K is a small and hydrophobic membrane glycoprotein encoded by the E3 region of subgroup C adenovirus. Recently, E3-6.7K has been shown to be required for the downregulation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors by the adenovirus E3/10.4K and E3/14.5K complex of proteins. We demonstrate here that E3-6.7K has additional protective roles, independent of other virus proteins. In transfected Jurkat T-cell lymphoma cells, E3-6.7K was found to maintain endoplasmic reticulum-Ca(2+) homeostasis and inhibit the induction of apoptosis by thapsigargin. The presence of E3-6.7K also lead to a reduction in the TNF-induced release of arachidonic acid from transfected U937 human histiocytic lymphoma cells. In addition, E3-6.7K protected cells against apoptosis induced through Fas, TNF receptor, and TRAIL receptors. Therefore, E3-6.7K confers a wide range of protective effects against both Ca(2+) flux-induced and death receptor-mediated apoptosis.

FIG. 1.

E3-6.7K protects against apoptosis induced by TNF in U937 cells. U937-neo (a to d) and U937-6.7K (e to h) cells were stimulated for 7 h with medium alone, 100 ng of TNF per ml, 10 μg of CHX per ml, or a combination of 100 ng of TNF and 10 μg of CHX per ml. For each cell population, a sample treatment was stained with annexin V-FITC and analyzed with a FACS. A second sample was analyzed with a FACS in the absence of annexin V-FITC in order to determine the background fluorescence, which corresponds to the fluorescence associated with the annexin V-negative cell population from each sample. The AI was calculated as (number of annexin V-positive viable cells/total number of viable cells) × 100. The results are representative of three repeat experiments.

FIG. 2.

E3-6.7K protects against apoptosis induced by Fas in Jurkat-neo and Jurkat-6.7K cells. To study Fas-induced apoptosis, cells were stimulated for 12 h with 1 μg of the anti-human Fas monoclonal antibody DX2 (Pharmingen) per ml and 2 μg of goat anti-mouse polyclonal antibody per ml. (A) Apoptotic cells were stained with annexin V-Alexa-488, indicating externalization of phosphatidyl serine; AI = (number of annexin V-positive, CytoxGreen-negative cells/total number of CytoxGreen negative cells) × 100. The V sector indicates the nonapoptotic, viable cell population, the A sector indicates the apoptotic, viable cell population, and the N sector indicates the necrotic cell population. (B) Alternatively, cells were stained with YO-PRO-1, indicating an increase in membrane permeability; AI = (number of YO-PRO-1-positive, PI-negative cells/total number of PI-negative cells) × 100. The lower left quadrant is the nonapoptotic, viable cell population, the lower right quadrant (A) represents the apoptotic, viable cell population, and the upper right quadrant (N) represents the necrotic cell population.

FIG. 3.

E3-6.7K protects against apoptosis induced by TRAIL in Jurkat-neo and Jurkat-6.7K cells. To study TRAIL-induced apoptosis, cells were stimulated for 12 h with 10 ng of a recombinant fusion protein containing the extracellular domain of TRAIL (Upstate Biotechnology) per ml and a potentiating reagent (5 μg/ml; Upstate Biotechnology), which consists of a monoclonal antibody against the tag present in purified TRAIL. (A) The apoptotic cells were stained with annexin V-Alexa-488, indicating externalization of phosphatidyl serine; AI = (number of annexin V-positive, CytoxGreen-negative cells/total number of CytoxGreen-negative cells) × 100. The V sector indicates the nonapoptotic, viable cell population, the A sector indicates the apoptotic, viable cell population, and the N sector indicates the necrotic cell population. (B) Alternatively, cells were stained with YO-PRO-1, indicating an increase in membrane permeability; AI = (number of YO-PRO-1-positive, PI-negative cells/total number of PI-negative cells) × 100. The lower left quadrant is the nonapoptotic, viable cell population, the lower right quadrant (A) represents the apoptotic, viable cell population, and the upper right quadrant (N) represents the necrotic cell population.

FIG. 4.

The presence of E3-6.7K results in the reduction of the thapsigargin-induced Ca²⁺ flux and apoptosis. (A) Effect of E3-6.7K on thapsigargin-induced apoptosis in transfected Jurkat cells. Jurkat-6.7K and Jurkat-neo cells were stimulated with medium containing 1% dimethyl sulfoxide (top),or 1 or 10 μM thapsigargin (TG) for 24 h, and then they were stained with YO-PRO-1 to examine the increase in the membrane permeability of apoptotic cells. The AI excluded the necrotic cell population and was calculated as (number of YO-PRO-1-positive, PI-negative apoptotic cells/total number of PI-negative, viable cells) × 100. The A region represents the apoptotic, viable population, and V represents the nonapoptotic, viable population; AI = A/(A+V). The experiment was repeated with similar results: 23.2% ± 1.4% and 47.8% ± 4.7% for Jurkat-neo cells stimulated with 1 and 10 μM TG, respectively, and 10.3% ± 1.3% and 21.9% ± 6.6% for Jurkat-6.7K cells stimulated with 1 and 10 μM TG, respectively (P < 0.05). (B) Kinetic analysis of the effect of E3-6.7K on the release of Ca²⁺ from the ER in response to TG. Jurkat-6.7K and Jurkat-neo cells were loaded for 90 min at 37°C with 0.5 μM Indo-1 AM ester and examined on a UV-equipped FACS-Vantage flow cytometer (Becton Dickinson) for 5 min to establish the baseline fluorescence, equivalent to the resting level of intracellular calcium. At 5 min, cells were treated with 5 nM TG and analyzed for 25 min for a total of 5 × 10⁵ events. The experiment was repeated three times with similar results.

FIG. 5.

Effect of E3-6.7K on the induction of procaspase-3 processing and PARP cleavage during TNF-induced apoptosis in vivo. Cell extracts were obtained from U937-neo and U937-6.7K cells that had been treated with 10 ng of TNF and 0.5 μg of CHX per ml for various lengths of time. Lysates containing equivalent amounts of protein based on the bicinchoninic acid (Pierce) protein concentration assay were loaded in each lane. After electrophoresis and transfer to PVDF membranes, blots were incubated with anti-caspase-3 rabbit antiserum that recognizes the 17- and 11-kDa subunits of the active, processed protein (A) and anti-PARP mouse monoclonal antibody that recognizes both the active 116-kDa and inactive 85-kDa forms of the protein (B). The blots were developed with a secondary antibody and visualized by chemiluminescence (Pierce Chemical). Similar results were obtained in two repeat experiments.

FIG. 6.

Analysis of the effect of E3-6.7K on the inducible release of radiolabeled arachidonic acid. U937-neo and U937-6.7K cells were stimulated with medium alone, 90 ng of TNF per ml, 2 μg of CHX per ml, or a combination of 90 ng of TNF and 2 μg of CHX per ml. After 20 h, the amount (in counts per minute) of [³H]arachidonic acid released in the medium was expressed as a percentage of the total incorporated [³H]arachidonic acid. The assay was set up in triplicate; however, due to their relatively small values, the standard deviations of the values for the samples from U937-6.7K cells treated with medium alone (6.72% ± 0.037%) and U937-6.7K treated with CHX (5.74% ± 0.027%) are difficult to observe on this graph. These results are representative of six repeat experiments.