Limits...
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

RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM Bafilomycin A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A). Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM Bafilomycin A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A). Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).

Mentions: Dominant negative RhoA-T19N, inhibitors and siRNA techniques were used to elucidate the relationships between previously identified molecules involved in ARV S1133-mediated autophagy and apoptosis. The overexpression of dominant negative RhoA and the inhibition of ROCK by Y-27632 or ROCK1 siRNA led to a remarkable reduction in MDC positive and LC3-GFP punctuate cells: importantly, pan-caspase inhibitor, Z-VAD-FMK, did not affect the percentage of autophagic cells. Autophagy inhibitor Beclin-1 siRNA, 3-Methyladenine (3-MA), and Bafilomycin A1 also reduced the number of cells undergoing autophagy (Figure 4A).Figure 4


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

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

RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM Bafilomycin A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A). Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: RhoA and ROCK1 are essential for ARV S1133-induced autophagy, apoptosis, and the conversion of autophagy to apoptosis. (A) Vero cells transfected with RhoA-T19N and incubated for 24 hr, before being transfected with siRNA for 72 hr, pre-treated with 5 μM Y-27632, 10 μM Z-VAD-FMK, 50 μM 3-MA and 0.1 μM Bafilomycin A1 at non-toxic concentrations for 4 hr, and then infected with ARV S1133 at an MOI of 5 for an additional 18-hr incubation. Autophagic vacuoles were stained with MDC, and the percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (black bar). Vero cells transfected with LC3-GFP, together with RhoA-T19N, and incubated for 24 hr, or transfected with siRNA for 48 hr then transfected with LC3-GFP, or pretreated with inhibitor for 4 hr then transfected with LC3-GFP. 24 hr after LC3-GFP transfection, ARV S1133 was added for an additional 18-hr incubation period. GFP-positive cells containing more than 3 dots were counted as positive LC3-GFP cells. The percentage of positive cells was calculated in 20 independent fields at a magnification of 200× (white bar). (B) ARV S1133 infected cells at an MOI of 5 with an incubation period of 36 hr with identical treatment timings to those described in (A). Three apoptosis assays were performed. The left y-axis represents the percentage of Hoechst 33258 positive cells and the caspase-3 activity in relative light units (RLUs). The right y-axis represents the OD 405 nm values from an apoptotic ELISA assay. All experiments were performed three times, each in duplicate. The data are presented as the mean ± SD. (C) Punctate dots of GFP indicating autophagosomes are shown by the white arrows (400x magnification).
Mentions: Dominant negative RhoA-T19N, inhibitors and siRNA techniques were used to elucidate the relationships between previously identified molecules involved in ARV S1133-mediated autophagy and apoptosis. The overexpression of dominant negative RhoA and the inhibition of ROCK by Y-27632 or ROCK1 siRNA led to a remarkable reduction in MDC positive and LC3-GFP punctuate cells: importantly, pan-caspase inhibitor, Z-VAD-FMK, did not affect the percentage of autophagic cells. Autophagy inhibitor Beclin-1 siRNA, 3-Methyladenine (3-MA), and Bafilomycin A1 also reduced the number of cells undergoing autophagy (Figure 4A).Figure 4

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