Involvement of FOXO Transcription Factors, TRAIL-FasL/Fas, and Sirtuin Proteins Family in Canine Coronavirus Type II-Induced Apoptosis

n our previous study, we have shown that canine coronavirus type II (CCoV-II) activates both extrinsic and intrinsic apoptotic pathway in a canine fibrosarcoma cell line (A-72 cells). Herein we investigated the role of Sirtuin and Forkhead box O (FOXO) families in this experimental model using Nortern Blot and Western Blot analysis. Our results demonstrated that mitochondrial SIRT3 and SIRT4 protein expression increased from 12 and 24 h post infection (p.i.) onwards, respectively, whereas the nuclear SIRT1 expression increased during the first 12 h p.i. followed by a decrease after 36 h p.i., reaching the same level of control at 48 h p.i. Sirtuins interact with/and regulate the activity of FOXO family proteins, and we herein observed that FOXO3A and FOXO1 expression increased significantly and stably from 12 h p.i. onwards. In addition, CCoV-II induces a remarkable increase in the expression of TNF-related apoptosis-inducing ligand (TRAIL), while we observed a slight up-regulation of FasL/Fas at 36 p.i. with a decrease of both proteins at the end of infection. Furthermore, we found that virus infection increased both bax translocation into mitochondria and decreased bcl-2 expression in cytosol in a time-dependent manner

Expression of SIRT1, SIRT3 and SIRT4 cDNA of mock-infected and infected cells at the indicated times at different post infection.

<p>(<b>A</b>) The RT-PCR products were examined by 1.6% agarose gel electrophoresis. (<b>B</b>) Densitometric analysis of the corresponding band to SIRT1, SIRT3 and SIRT4. The bars represent the mean ± SEM of the results from three separate experiments. Significant differences between CCoV-II-infected cells and control cells are indicated by probability <i>P</i>. **<i>P</i> <0.01 and ***<i>P</i> <0.001.</p

CCoV-II infection induces bax translocation into the mitochondria and bcl-2 decrease.

<p>(<b>A</b>) A-72 cells were mock-infected (lane cc, control cells) and CCoV-II infected (4, 8, 12, 24, 36 and 48 h p.i.). The bax and bcl-2 expression was evaluated in the cytosol and in the mitochondrial fractions during the infection; equal amounts of proteins from each sample were subjected to Western blot analysis and probed for bax and bcl-2. β-actin and TIM50 were used as a loading control. The molecular weight (KDa) of protein size standards is shown on the left hand side. Blot is representative of three separate experiments. (<b>B</b>) Densitometric analysis of bax and (<b>C</b>) of bcl-2 are shown below the blots. The bars represent the mean ± SEM of the results from three separate experiments. Significant differences between CCoV-II-infected cells and control cells are indicated by probability <i>P</i>. **<i>P</i> <0.01 and ***<i>P</i> <0.001.</p

CCoV-II infection modulates the gene regulation of SIRT3 and SIRT4.

<p> (<b>A</b>) To perform Northern blot assay, RNA was extracted from mock-infected (lanes cc, control cells) and infected cells at the indicated times, electrophoresed and hybridized with a labelled probe as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027313#s2" target="_blank">Material and Methods</a>. β-actin was used as loading control. Blot is representative of three separate experiments. (<b>B</b>) Densitometric analysis of blots relative to SIRT3. The bars represent the mean ± SEM of the results from three separate experiments. Significant differences between CCoV-II-infected cells and control cells are indicated by probability <i>P</i>. **<i>P</i> <0.01, and ***<i>P</i> <0.001. (<b>C</b>) Densitometric analysis of blots relative to SIRT4. The bars represent the mean ± SEM of the results from three separate experiments. Significant differences between CCoV-II-infected cells and control cells are indicated by probability <i>P</i>. **<i>P</i> <0.01.</p

CCoV-II infection modulates SIRT3 protein expression in mitochondrial fraction.

<p>(<b>A</b>) Cell lysates were collected at the indicated times p.i., and equal amounts of proteins from each sample were subjected to Western blot analysis and probed for SIRT3. TIM50 was used as a loading control. The molecular weight (KDa) of protein size standards is shown on the left hand side. Blot is representative of three separate experiments. (<b>B</b>) Densitometric analysis of blots relative to SIRT3. The bars represent the mean ± SEM of the results from three separate experiments. Significant differences between CCoV-II-infected cells and control cells are indicated by probability <i>P</i>. *<i>P</i> <0.05, **<i>P</i> <0.01, and ***<i>P</i> <0.001.</p

CCoV-II infection modulates SIRT4 protein expression in mitochondrial fraction.

<p>(<b>A</b>) Cell lysates were collected at the indicated times p.i., and equal amounts of proteins from each sample were subjected to Western blot analysis and probed for SIRT4. TIM50 was used as a loading control. The molecular weight (KDa) of protein size standards is shown on the left hand side. Blot is representative of three separate experiments. (<b>B</b>) Densitometric analysis of blots relative to SIRT4. The bars represent the mean ± SEM of the results from three separate experiments. Significant differences between CCoV-II-infected cells and control cells are indicated by probability <i>P</i>. **<i>P</i> <0.01 and ***<i>P</i> <0.001.</p