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Different Roles of GRP78 on Cell Proliferation and Apoptosis in Cartilage Development.

Xiong Z, Jiang R, Li X, Liu Y, Guo F - Int J Mol Sci (2015)

Bottom Line: Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot.In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo.Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing 400016, China. zyxiong38@sina.com.

ABSTRACT
Eukaryotic cells possess several mechanisms to adapt to endoplasmic reticulum (ER) stress and thereby survive. ER stress activates a set of signaling pathways collectively termed as the unfolded protein response (UPR). We previously reported that Bone morphogenetic protein 2 (BMP2) mediates mild ER stress and activates UPR signal molecules in chondrogenesis. The mammalian UPR protects the cell against the stress of misfolded proteins in the endoplasmic reticulum. Failure to adapt to ER stress causes the UPR to trigger apoptosis. Glucose regulated protein 78 (GRP78), as an important molecular chaperone in UPR signaling pathways, is responsible for binding to misfolded or unfolded protein during ER stress. However the influence on GRP78 in BMP2-induced chondrocyte differentiation has not yet been elucidated and the molecular mechanism underlyng these processes remain unexplored. Herein we demonstrate that overexpression of GRP78 enhanced cell proliferation in chondrocyte development with G1 phase advance, S phase increasing and G2-M phase transition. Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot. In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that GRP78 knockdown via siRNA activated the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.

No MeSH data available.


Related in: MedlinePlus

Cellular proliferation analysis by FCM. (A) Flow cytometry images with propidium iodide staining and analysis on cell cycle distribution. Micromass culture of ATDC5 cells and C3H10T1/2 were treated with BMP2 (300 ng/mL)/BMP2 + Ad-GFP/BMP2 + Ad-siRFP / BMP2 + Ad-GRP78 / BMP2 + Ad-siGRP78. Flow cytometry analysis showed that the percentage of the BMP2 + Ad-GRP78 ATDC5 cells in S phase were increased significantly compared to those in BMP2 controls, whereas the percentage of the BMP2 + Ad-siGRP78 ATDC5 cells in S phase were dramatically decreased compared with BMP2 control. The result of C3H10T1/2 is the same. Experiments were repeated three times, and samples were analyzed by Student’s t-test and statistical significance with p < 0.05. Representative images were shown; (B) Flow cytometry assay on the percentages of the ATDC5 and C3H10T1/2 cells in G2/M phase after treatment with BMP2 (300 ng/mL)/BMP2 + Ad-GFP / BMP2 + Ad-siRFP / BMP2 + Ad-GRP78 / BMP2 + Ad-siGRP78. * p < 0.05 compared with control; (C) Flow cytometry analysis showed that the percentages of the ATDC5 and C3H10T1/2 Ad-GRP78 cells in S phase were increased significantly, whereas the percentages of the ATDC5 and C3H10T1/2 Ad-siGRP78 cells in S phase were decreased compared with those in their controls. * p < 0.05 compared with control.
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ijms-16-21153-f004: Cellular proliferation analysis by FCM. (A) Flow cytometry images with propidium iodide staining and analysis on cell cycle distribution. Micromass culture of ATDC5 cells and C3H10T1/2 were treated with BMP2 (300 ng/mL)/BMP2 + Ad-GFP/BMP2 + Ad-siRFP / BMP2 + Ad-GRP78 / BMP2 + Ad-siGRP78. Flow cytometry analysis showed that the percentage of the BMP2 + Ad-GRP78 ATDC5 cells in S phase were increased significantly compared to those in BMP2 controls, whereas the percentage of the BMP2 + Ad-siGRP78 ATDC5 cells in S phase were dramatically decreased compared with BMP2 control. The result of C3H10T1/2 is the same. Experiments were repeated three times, and samples were analyzed by Student’s t-test and statistical significance with p < 0.05. Representative images were shown; (B) Flow cytometry assay on the percentages of the ATDC5 and C3H10T1/2 cells in G2/M phase after treatment with BMP2 (300 ng/mL)/BMP2 + Ad-GFP / BMP2 + Ad-siRFP / BMP2 + Ad-GRP78 / BMP2 + Ad-siGRP78. * p < 0.05 compared with control; (C) Flow cytometry analysis showed that the percentages of the ATDC5 and C3H10T1/2 Ad-GRP78 cells in S phase were increased significantly, whereas the percentages of the ATDC5 and C3H10T1/2 Ad-siGRP78 cells in S phase were decreased compared with those in their controls. * p < 0.05 compared with control.

Mentions: To investigate whether GRP78 can influence the cell cycle profile in chondrogenesis, flow cytometry analysis was undertaken to determine the effect of Ad-GRP78 and Ad-siGRP78 on cell cycle in BMP2-induced ATDC5 cells and C3H10T1/2 cells. As shown in Figure 4A,C, the data showed that the cell number in S phase was reduced in BMP2 + Ad-siGRP78 micromass culture of ATDC5 cells comparing with BMP2 and BMP2 + siRFP control. The cell number in S phase was 27.82% in BMP2 + Ad-siGRP78 ATDC5 cells and 23.67% in BMP2 + Ad-siGRP78 C3H10T1/2 cells. However, the cell number in S phase was increased in BMP2 + Ad-GRP78 micromass culture of ATDC5 cells comparing with BMP2 and BMP2 + AdGFP control. The cell number in S phase was 54.72% in BMP2 + Ad-GRP78 ATDC5 cells and 51.09% in BMP2 + Ad-GRP78 C3H10T1/2 cells. The differences between treatment groups and control groups have statistical significance (p < 0.05). These data indicate that GRP78 can influence cell cycle distribution. Overexpression of GRP78 enhances the cell number in S phase, while GRP78 knockdown decreases the cell number in S phase in chondrocyte differentiation. In addition, the data also showed that in ATDC5 cells, the percentage of G2 phase was reduced in BMP2 + Ad-siGRP78 group (14.78% ± 0.78%) compared with BMP2 + siRFP control and BMP2 group, while increased in BMP2 + Ad-GRP78 group (26.21% ± 0.81%) compared with BMP2 + AdGFP control and BMP2 group. In addition, in micromass culture of C3H10T1/2 cells, the percentage of G2 phase was reduced in BMP2+Ad-siGRP78 group (12.27% ± 0.62%) compared with BMP2 + siRFP control and BMP2 group, while increased in BMP2 + Ad-GRP78 group (27.08% ± 0.92%) compared with BMP2 + AdGFP control and BMP2 group (Figure 4B). The differences between treatment groups and control groups have statistical significance (p < 0.05). These data indicate that GRP78 can influence cell cycle distribution in chondrocyte differentiation. Overexpression of GRP78 enhanced, while GRP78 knockdown inhibited, the S phase cells in chondrogenesis.


Different Roles of GRP78 on Cell Proliferation and Apoptosis in Cartilage Development.

Xiong Z, Jiang R, Li X, Liu Y, Guo F - Int J Mol Sci (2015)

Cellular proliferation analysis by FCM. (A) Flow cytometry images with propidium iodide staining and analysis on cell cycle distribution. Micromass culture of ATDC5 cells and C3H10T1/2 were treated with BMP2 (300 ng/mL)/BMP2 + Ad-GFP/BMP2 + Ad-siRFP / BMP2 + Ad-GRP78 / BMP2 + Ad-siGRP78. Flow cytometry analysis showed that the percentage of the BMP2 + Ad-GRP78 ATDC5 cells in S phase were increased significantly compared to those in BMP2 controls, whereas the percentage of the BMP2 + Ad-siGRP78 ATDC5 cells in S phase were dramatically decreased compared with BMP2 control. The result of C3H10T1/2 is the same. Experiments were repeated three times, and samples were analyzed by Student’s t-test and statistical significance with p < 0.05. Representative images were shown; (B) Flow cytometry assay on the percentages of the ATDC5 and C3H10T1/2 cells in G2/M phase after treatment with BMP2 (300 ng/mL)/BMP2 + Ad-GFP / BMP2 + Ad-siRFP / BMP2 + Ad-GRP78 / BMP2 + Ad-siGRP78. * p < 0.05 compared with control; (C) Flow cytometry analysis showed that the percentages of the ATDC5 and C3H10T1/2 Ad-GRP78 cells in S phase were increased significantly, whereas the percentages of the ATDC5 and C3H10T1/2 Ad-siGRP78 cells in S phase were decreased compared with those in their controls. * p < 0.05 compared with control.
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Related In: Results  -  Collection

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ijms-16-21153-f004: Cellular proliferation analysis by FCM. (A) Flow cytometry images with propidium iodide staining and analysis on cell cycle distribution. Micromass culture of ATDC5 cells and C3H10T1/2 were treated with BMP2 (300 ng/mL)/BMP2 + Ad-GFP/BMP2 + Ad-siRFP / BMP2 + Ad-GRP78 / BMP2 + Ad-siGRP78. Flow cytometry analysis showed that the percentage of the BMP2 + Ad-GRP78 ATDC5 cells in S phase were increased significantly compared to those in BMP2 controls, whereas the percentage of the BMP2 + Ad-siGRP78 ATDC5 cells in S phase were dramatically decreased compared with BMP2 control. The result of C3H10T1/2 is the same. Experiments were repeated three times, and samples were analyzed by Student’s t-test and statistical significance with p < 0.05. Representative images were shown; (B) Flow cytometry assay on the percentages of the ATDC5 and C3H10T1/2 cells in G2/M phase after treatment with BMP2 (300 ng/mL)/BMP2 + Ad-GFP / BMP2 + Ad-siRFP / BMP2 + Ad-GRP78 / BMP2 + Ad-siGRP78. * p < 0.05 compared with control; (C) Flow cytometry analysis showed that the percentages of the ATDC5 and C3H10T1/2 Ad-GRP78 cells in S phase were increased significantly, whereas the percentages of the ATDC5 and C3H10T1/2 Ad-siGRP78 cells in S phase were decreased compared with those in their controls. * p < 0.05 compared with control.
Mentions: To investigate whether GRP78 can influence the cell cycle profile in chondrogenesis, flow cytometry analysis was undertaken to determine the effect of Ad-GRP78 and Ad-siGRP78 on cell cycle in BMP2-induced ATDC5 cells and C3H10T1/2 cells. As shown in Figure 4A,C, the data showed that the cell number in S phase was reduced in BMP2 + Ad-siGRP78 micromass culture of ATDC5 cells comparing with BMP2 and BMP2 + siRFP control. The cell number in S phase was 27.82% in BMP2 + Ad-siGRP78 ATDC5 cells and 23.67% in BMP2 + Ad-siGRP78 C3H10T1/2 cells. However, the cell number in S phase was increased in BMP2 + Ad-GRP78 micromass culture of ATDC5 cells comparing with BMP2 and BMP2 + AdGFP control. The cell number in S phase was 54.72% in BMP2 + Ad-GRP78 ATDC5 cells and 51.09% in BMP2 + Ad-GRP78 C3H10T1/2 cells. The differences between treatment groups and control groups have statistical significance (p < 0.05). These data indicate that GRP78 can influence cell cycle distribution. Overexpression of GRP78 enhances the cell number in S phase, while GRP78 knockdown decreases the cell number in S phase in chondrocyte differentiation. In addition, the data also showed that in ATDC5 cells, the percentage of G2 phase was reduced in BMP2 + Ad-siGRP78 group (14.78% ± 0.78%) compared with BMP2 + siRFP control and BMP2 group, while increased in BMP2 + Ad-GRP78 group (26.21% ± 0.81%) compared with BMP2 + AdGFP control and BMP2 group. In addition, in micromass culture of C3H10T1/2 cells, the percentage of G2 phase was reduced in BMP2+Ad-siGRP78 group (12.27% ± 0.62%) compared with BMP2 + siRFP control and BMP2 group, while increased in BMP2 + Ad-GRP78 group (27.08% ± 0.92%) compared with BMP2 + AdGFP control and BMP2 group (Figure 4B). The differences between treatment groups and control groups have statistical significance (p < 0.05). These data indicate that GRP78 can influence cell cycle distribution in chondrocyte differentiation. Overexpression of GRP78 enhanced, while GRP78 knockdown inhibited, the S phase cells in chondrogenesis.

Bottom Line: Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot.In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo.Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing 400016, China. zyxiong38@sina.com.

ABSTRACT
Eukaryotic cells possess several mechanisms to adapt to endoplasmic reticulum (ER) stress and thereby survive. ER stress activates a set of signaling pathways collectively termed as the unfolded protein response (UPR). We previously reported that Bone morphogenetic protein 2 (BMP2) mediates mild ER stress and activates UPR signal molecules in chondrogenesis. The mammalian UPR protects the cell against the stress of misfolded proteins in the endoplasmic reticulum. Failure to adapt to ER stress causes the UPR to trigger apoptosis. Glucose regulated protein 78 (GRP78), as an important molecular chaperone in UPR signaling pathways, is responsible for binding to misfolded or unfolded protein during ER stress. However the influence on GRP78 in BMP2-induced chondrocyte differentiation has not yet been elucidated and the molecular mechanism underlyng these processes remain unexplored. Herein we demonstrate that overexpression of GRP78 enhanced cell proliferation in chondrocyte development with G1 phase advance, S phase increasing and G2-M phase transition. Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot. In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that GRP78 knockdown via siRNA activated the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.

No MeSH data available.


Related in: MedlinePlus