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RhoA/ROCK1 regulates Avian Reovirus S1133-induced switch from autophagy to apoptosis.

Lin PY, Chang CD, Chen YC, Shih WL - BMC Vet. Res. (2015)

Bottom Line: Conversely, inhibition of caspase-3 did not affect the level of autophagy.A co-immunoprecipitation assay demonstrated that the formation of a RhoA, ROCK1 and Beclin-1 complex coincided with the induction of autophagy.Our results demonstrate that RhoA/ROCK1 signaling play critical roles in the transition of cell activity from autophagy to apoptosis in ARV S1133-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Pingtung, 91201, Taiwan. likelego1980@yahoo.com.tw.

ABSTRACT

Background: Autophagy is an essential process in the control of cellular homeostasis. It enables cells under certain stress conditions to survive by removing toxic cellular components, and may protect cells from apoptosis. In the present study, the signaling pathways involved in ARV S1133 regulated switch from autophagy to apoptosis were investigated.

Results: ARV S1133 infection caused autophagy in the early to middle infectious stages in Vero and DF1 cells, and apoptosis in the middle to late stages. Conversion of the autophagy marker LC3-I to LC3-II occurred earlier than cleavage of the apoptotic marker caspase-3. ARV S1133 also activated the Beclin-1 promoter in the early to middle stages of infection. Levels of RhoA-GTP and ROCK1 activity were elevated upon ARV S1133 infection, while inhibition of RhoA and ROCK1 reduced autophagy and subsequent apoptosis. Conversely, inhibition of caspase-3 did not affect the level of autophagy. Beclin-1 knockdown and treatment with autophagy inhibitors, 3-MA and Bafilomycin A1, suppressed ARV S1133-induced autophagy and apoptosis simultaneously, suggesting the shift from autophagy to apoptosis. A co-immunoprecipitation assay demonstrated that the formation of a RhoA, ROCK1 and Beclin-1 complex coincided with the induction of autophagy.

Conclusion: Our results demonstrate that RhoA/ROCK1 signaling play critical roles in the transition of cell activity from autophagy to apoptosis in ARV S1133-infected cells.

No MeSH data available.


Related in: MedlinePlus

Endogenous gene knockdown efficiency confirmed by Western blotting analysis. The soluble cell lysates of Vero cells transfected with (A) RhoA-T19N for 48 hrs or (B) and (C) siRNA for 72 hrs were subjected to SDS-PAGE followed by antibody detection.
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Fig6: Endogenous gene knockdown efficiency confirmed by Western blotting analysis. The soluble cell lysates of Vero cells transfected with (A) RhoA-T19N for 48 hrs or (B) and (C) siRNA for 72 hrs were subjected to SDS-PAGE followed by antibody detection.

Mentions: Next, the apoptotic characteristics of the cells were analyzed by Hoechst 33258 staining, the caspase-3 activity was assessed by chemiluminescence ELISA, and apoptotic DNA was measured by ELISA following inhibition of RhoA/ROCK1 signaling, inhibition of autophagy by Beclin-1 siRNA, and blocking of autophagosome formation by 3-MA and Bafilomycin A1, which is a known inhibitor of the late phase of autophagy. The results of these three experiments showed that the level of apoptosis was also inhibited when the autophagic process was blocked (Figure 4B). Figure 4C shows the LC3-GFP punctate pattern of ARV S1133-infected Vero cells in the presence of various inhibitors or siRNA. The level of LC3-GFP dots significantly increased in ARV S1133-infected cells (Figure 4C, (B)), as well as in cells in the presence of caspase-3 inhibitor Z-VAD-FMK (Figure 4C, (C)). Upon inhibition of the RhoA/ROCK1 pathway, the LC3-GFP positive signals dramatically reduced (Figure 4C, (D)(E)(F)). Inhibition of autophagy mediators by Beclin-1 siRNA, 3-MA, and Bafilomycin A1, and inhibition of the RhoA/ROCK1 pathway also significantly reduced the number of apoptotic cells, as evidenced by Hoechst 33258 staining and the immunofluorescence assay using an antibody against cleaved caspase-3 (Figure 5). The efficiency of siRNA inhibition and overexpression of dominant negative RhoA were demonstrated by Western blotting analysis (Figure 6). Taken together, these experiments clearly demonstrate that RhoA/ROCK1 signaling plays an important role in the processes of autophagy and apoptosis that occur in the early and late stages of ARV S1133 infection of cultured Vero cells.Figure 5


RhoA/ROCK1 regulates Avian Reovirus S1133-induced switch from autophagy to apoptosis.

Lin PY, Chang CD, Chen YC, Shih WL - BMC Vet. Res. (2015)

Endogenous gene knockdown efficiency confirmed by Western blotting analysis. The soluble cell lysates of Vero cells transfected with (A) RhoA-T19N for 48 hrs or (B) and (C) siRNA for 72 hrs were subjected to SDS-PAGE followed by antibody detection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4430033&req=5

Fig6: Endogenous gene knockdown efficiency confirmed by Western blotting analysis. The soluble cell lysates of Vero cells transfected with (A) RhoA-T19N for 48 hrs or (B) and (C) siRNA for 72 hrs were subjected to SDS-PAGE followed by antibody detection.
Mentions: Next, the apoptotic characteristics of the cells were analyzed by Hoechst 33258 staining, the caspase-3 activity was assessed by chemiluminescence ELISA, and apoptotic DNA was measured by ELISA following inhibition of RhoA/ROCK1 signaling, inhibition of autophagy by Beclin-1 siRNA, and blocking of autophagosome formation by 3-MA and Bafilomycin A1, which is a known inhibitor of the late phase of autophagy. The results of these three experiments showed that the level of apoptosis was also inhibited when the autophagic process was blocked (Figure 4B). Figure 4C shows the LC3-GFP punctate pattern of ARV S1133-infected Vero cells in the presence of various inhibitors or siRNA. The level of LC3-GFP dots significantly increased in ARV S1133-infected cells (Figure 4C, (B)), as well as in cells in the presence of caspase-3 inhibitor Z-VAD-FMK (Figure 4C, (C)). Upon inhibition of the RhoA/ROCK1 pathway, the LC3-GFP positive signals dramatically reduced (Figure 4C, (D)(E)(F)). Inhibition of autophagy mediators by Beclin-1 siRNA, 3-MA, and Bafilomycin A1, and inhibition of the RhoA/ROCK1 pathway also significantly reduced the number of apoptotic cells, as evidenced by Hoechst 33258 staining and the immunofluorescence assay using an antibody against cleaved caspase-3 (Figure 5). The efficiency of siRNA inhibition and overexpression of dominant negative RhoA were demonstrated by Western blotting analysis (Figure 6). Taken together, these experiments clearly demonstrate that RhoA/ROCK1 signaling plays an important role in the processes of autophagy and apoptosis that occur in the early and late stages of ARV S1133 infection of cultured Vero cells.Figure 5

Bottom Line: Conversely, inhibition of caspase-3 did not affect the level of autophagy.A co-immunoprecipitation assay demonstrated that the formation of a RhoA, ROCK1 and Beclin-1 complex coincided with the induction of autophagy.Our results demonstrate that RhoA/ROCK1 signaling play critical roles in the transition of cell activity from autophagy to apoptosis in ARV S1133-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Pingtung, 91201, Taiwan. likelego1980@yahoo.com.tw.

ABSTRACT

Background: Autophagy is an essential process in the control of cellular homeostasis. It enables cells under certain stress conditions to survive by removing toxic cellular components, and may protect cells from apoptosis. In the present study, the signaling pathways involved in ARV S1133 regulated switch from autophagy to apoptosis were investigated.

Results: ARV S1133 infection caused autophagy in the early to middle infectious stages in Vero and DF1 cells, and apoptosis in the middle to late stages. Conversion of the autophagy marker LC3-I to LC3-II occurred earlier than cleavage of the apoptotic marker caspase-3. ARV S1133 also activated the Beclin-1 promoter in the early to middle stages of infection. Levels of RhoA-GTP and ROCK1 activity were elevated upon ARV S1133 infection, while inhibition of RhoA and ROCK1 reduced autophagy and subsequent apoptosis. Conversely, inhibition of caspase-3 did not affect the level of autophagy. Beclin-1 knockdown and treatment with autophagy inhibitors, 3-MA and Bafilomycin A1, suppressed ARV S1133-induced autophagy and apoptosis simultaneously, suggesting the shift from autophagy to apoptosis. A co-immunoprecipitation assay demonstrated that the formation of a RhoA, ROCK1 and Beclin-1 complex coincided with the induction of autophagy.

Conclusion: Our results demonstrate that RhoA/ROCK1 signaling play critical roles in the transition of cell activity from autophagy to apoptosis in ARV S1133-infected cells.

No MeSH data available.


Related in: MedlinePlus