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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

GRP78 decreases the expression of the XBP1S in BMP2-induced chondrogenesis. (A) Western blotting assay of the expression of ER stress associated molecules in C3H10T1/2 cells induced by BMP2 (300 ng/mL) for different times (1, 3, 5 days). Cell lysates prepared from micromass culture of C3H10T1/2 cells induced by BMP2, as indicated, were subjected to SDS-PAGE and detected with anti-BiP, anti-XBP1S, anti-IRE1α and anti-tubulin (serving as an internal control), respectively. Protein levels of IRE1α, XBP1S, ATF3 and tubulin (internal control) are shown; (B) Ad-GRP78 reduces, while siGRP78 increases, the level of XBP1S mRNA. C3H10T1/2 cells infected with Ad-GRP78 and control Ad-GFP, siGRP78 and control siRFP, were cultured for 48 h and endogenous XBP1S gene expression was determined by real-time PCR. The normalized values were then calibrated against the control value. The units are arbitrary, and the left bar indicates a relative level of XBP1S mRNA of 1; * p < 0.05; (C) Ad-GRP78 reduces, while siGRP78 increases, the level of XBP1S protein level in C3H10T1/2 cells induced by BMP2. C3H10T1/2 cells infected with Ad-GRP78 and control Ad-GFP, siGRP78 and control siRFP, were cultured for 48 h, respectively, and the endogenous XBP1S protein level was determined by Western blotting. siGRP78, an siRNA adenovirus targeting GRP78. Tubulin protein served as an internal control; (D) Semi-quantification of relative levels of XBP1S in micromass culture of C3H10T1/2 cells induced by BMP2. Levels were normalized against those of tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad), data were expressed as means ± S.D. (n = 3). Every treatment group was compared with control groups respectively, * p < 0.05; (E) Ad-GRP78 decreases the level of XBP1S protein spliced by IRE1α in chondrocytes induced by BMP2. Micromass cultures of C3H10T1/2 cells were treated with 300 ng/mL BMP2, then C3H10T1/2 cells were infected with either Ad-GFP (serving as a control), siRFP (serving as a control), or Ad-IRE1α, siIRE1α, or Ad-IRE1α + Ad-GRP78, as indicated. Five or six days later, the cell lysates were used to detect the protein level of XBP1S by Western blotting. siIRE1α, an siRNA adenoviruse targeting IRE1α. Tubulin protein served as an internal control; (F) Semi-quantification of relative levels of XBP1S in micromass culture of C3H10T1/2 cells induced by BMP2. Levels were normalized against those of Tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad), data were expressed as means ± S.D. (n = 3). Every treatment group was compared with control groups respectively, * p < 0.05. Error bars, S.D.
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ijms-16-21153-f003: GRP78 decreases the expression of the XBP1S in BMP2-induced chondrogenesis. (A) Western blotting assay of the expression of ER stress associated molecules in C3H10T1/2 cells induced by BMP2 (300 ng/mL) for different times (1, 3, 5 days). Cell lysates prepared from micromass culture of C3H10T1/2 cells induced by BMP2, as indicated, were subjected to SDS-PAGE and detected with anti-BiP, anti-XBP1S, anti-IRE1α and anti-tubulin (serving as an internal control), respectively. Protein levels of IRE1α, XBP1S, ATF3 and tubulin (internal control) are shown; (B) Ad-GRP78 reduces, while siGRP78 increases, the level of XBP1S mRNA. C3H10T1/2 cells infected with Ad-GRP78 and control Ad-GFP, siGRP78 and control siRFP, were cultured for 48 h and endogenous XBP1S gene expression was determined by real-time PCR. The normalized values were then calibrated against the control value. The units are arbitrary, and the left bar indicates a relative level of XBP1S mRNA of 1; * p < 0.05; (C) Ad-GRP78 reduces, while siGRP78 increases, the level of XBP1S protein level in C3H10T1/2 cells induced by BMP2. C3H10T1/2 cells infected with Ad-GRP78 and control Ad-GFP, siGRP78 and control siRFP, were cultured for 48 h, respectively, and the endogenous XBP1S protein level was determined by Western blotting. siGRP78, an siRNA adenovirus targeting GRP78. Tubulin protein served as an internal control; (D) Semi-quantification of relative levels of XBP1S in micromass culture of C3H10T1/2 cells induced by BMP2. Levels were normalized against those of tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad), data were expressed as means ± S.D. (n = 3). Every treatment group was compared with control groups respectively, * p < 0.05; (E) Ad-GRP78 decreases the level of XBP1S protein spliced by IRE1α in chondrocytes induced by BMP2. Micromass cultures of C3H10T1/2 cells were treated with 300 ng/mL BMP2, then C3H10T1/2 cells were infected with either Ad-GFP (serving as a control), siRFP (serving as a control), or Ad-IRE1α, siIRE1α, or Ad-IRE1α + Ad-GRP78, as indicated. Five or six days later, the cell lysates were used to detect the protein level of XBP1S by Western blotting. siIRE1α, an siRNA adenoviruse targeting IRE1α. Tubulin protein served as an internal control; (F) Semi-quantification of relative levels of XBP1S in micromass culture of C3H10T1/2 cells induced by BMP2. Levels were normalized against those of Tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad), data were expressed as means ± S.D. (n = 3). Every treatment group was compared with control groups respectively, * p < 0.05. Error bars, S.D.

Mentions: BMP2 is known to activate the unfolded protein response of ER stress [5,6,7]. We next did Western blotting to examine the expression profiling of UPR signaling molecules in BMP2-induced chondrocyte differentiation. For this purpose, Western blot analysis was performed in micromass culture of C3H10T1/2 cells with 300 ng/mL BMP2. The result showed that ER stress-associated molecules, such as IRE1α, XBP1S and ATF3, were activated and expressed in BMP2-treated cells (Figure 3A). Interestingly, with the BMP2 stimulation, the expression of IRE1α, XBP1S and ATF3 was also increased. The result showed that UPR signal pathway of ER stress has been activated after treatment with BMP2.


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)

GRP78 decreases the expression of the XBP1S in BMP2-induced chondrogenesis. (A) Western blotting assay of the expression of ER stress associated molecules in C3H10T1/2 cells induced by BMP2 (300 ng/mL) for different times (1, 3, 5 days). Cell lysates prepared from micromass culture of C3H10T1/2 cells induced by BMP2, as indicated, were subjected to SDS-PAGE and detected with anti-BiP, anti-XBP1S, anti-IRE1α and anti-tubulin (serving as an internal control), respectively. Protein levels of IRE1α, XBP1S, ATF3 and tubulin (internal control) are shown; (B) Ad-GRP78 reduces, while siGRP78 increases, the level of XBP1S mRNA. C3H10T1/2 cells infected with Ad-GRP78 and control Ad-GFP, siGRP78 and control siRFP, were cultured for 48 h and endogenous XBP1S gene expression was determined by real-time PCR. The normalized values were then calibrated against the control value. The units are arbitrary, and the left bar indicates a relative level of XBP1S mRNA of 1; * p < 0.05; (C) Ad-GRP78 reduces, while siGRP78 increases, the level of XBP1S protein level in C3H10T1/2 cells induced by BMP2. C3H10T1/2 cells infected with Ad-GRP78 and control Ad-GFP, siGRP78 and control siRFP, were cultured for 48 h, respectively, and the endogenous XBP1S protein level was determined by Western blotting. siGRP78, an siRNA adenovirus targeting GRP78. Tubulin protein served as an internal control; (D) Semi-quantification of relative levels of XBP1S in micromass culture of C3H10T1/2 cells induced by BMP2. Levels were normalized against those of tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad), data were expressed as means ± S.D. (n = 3). Every treatment group was compared with control groups respectively, * p < 0.05; (E) Ad-GRP78 decreases the level of XBP1S protein spliced by IRE1α in chondrocytes induced by BMP2. Micromass cultures of C3H10T1/2 cells were treated with 300 ng/mL BMP2, then C3H10T1/2 cells were infected with either Ad-GFP (serving as a control), siRFP (serving as a control), or Ad-IRE1α, siIRE1α, or Ad-IRE1α + Ad-GRP78, as indicated. Five or six days later, the cell lysates were used to detect the protein level of XBP1S by Western blotting. siIRE1α, an siRNA adenoviruse targeting IRE1α. Tubulin protein served as an internal control; (F) Semi-quantification of relative levels of XBP1S in micromass culture of C3H10T1/2 cells induced by BMP2. Levels were normalized against those of Tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad), data were expressed as means ± S.D. (n = 3). Every treatment group was compared with control groups respectively, * p < 0.05. Error bars, S.D.
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ijms-16-21153-f003: GRP78 decreases the expression of the XBP1S in BMP2-induced chondrogenesis. (A) Western blotting assay of the expression of ER stress associated molecules in C3H10T1/2 cells induced by BMP2 (300 ng/mL) for different times (1, 3, 5 days). Cell lysates prepared from micromass culture of C3H10T1/2 cells induced by BMP2, as indicated, were subjected to SDS-PAGE and detected with anti-BiP, anti-XBP1S, anti-IRE1α and anti-tubulin (serving as an internal control), respectively. Protein levels of IRE1α, XBP1S, ATF3 and tubulin (internal control) are shown; (B) Ad-GRP78 reduces, while siGRP78 increases, the level of XBP1S mRNA. C3H10T1/2 cells infected with Ad-GRP78 and control Ad-GFP, siGRP78 and control siRFP, were cultured for 48 h and endogenous XBP1S gene expression was determined by real-time PCR. The normalized values were then calibrated against the control value. The units are arbitrary, and the left bar indicates a relative level of XBP1S mRNA of 1; * p < 0.05; (C) Ad-GRP78 reduces, while siGRP78 increases, the level of XBP1S protein level in C3H10T1/2 cells induced by BMP2. C3H10T1/2 cells infected with Ad-GRP78 and control Ad-GFP, siGRP78 and control siRFP, were cultured for 48 h, respectively, and the endogenous XBP1S protein level was determined by Western blotting. siGRP78, an siRNA adenovirus targeting GRP78. Tubulin protein served as an internal control; (D) Semi-quantification of relative levels of XBP1S in micromass culture of C3H10T1/2 cells induced by BMP2. Levels were normalized against those of tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad), data were expressed as means ± S.D. (n = 3). Every treatment group was compared with control groups respectively, * p < 0.05; (E) Ad-GRP78 decreases the level of XBP1S protein spliced by IRE1α in chondrocytes induced by BMP2. Micromass cultures of C3H10T1/2 cells were treated with 300 ng/mL BMP2, then C3H10T1/2 cells were infected with either Ad-GFP (serving as a control), siRFP (serving as a control), or Ad-IRE1α, siIRE1α, or Ad-IRE1α + Ad-GRP78, as indicated. Five or six days later, the cell lysates were used to detect the protein level of XBP1S by Western blotting. siIRE1α, an siRNA adenoviruse targeting IRE1α. Tubulin protein served as an internal control; (F) Semi-quantification of relative levels of XBP1S in micromass culture of C3H10T1/2 cells induced by BMP2. Levels were normalized against those of Tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad), data were expressed as means ± S.D. (n = 3). Every treatment group was compared with control groups respectively, * p < 0.05. Error bars, S.D.
Mentions: BMP2 is known to activate the unfolded protein response of ER stress [5,6,7]. We next did Western blotting to examine the expression profiling of UPR signaling molecules in BMP2-induced chondrocyte differentiation. For this purpose, Western blot analysis was performed in micromass culture of C3H10T1/2 cells with 300 ng/mL BMP2. The result showed that ER stress-associated molecules, such as IRE1α, XBP1S and ATF3, were activated and expressed in BMP2-treated cells (Figure 3A). Interestingly, with the BMP2 stimulation, the expression of IRE1α, XBP1S and ATF3 was also increased. The result showed that UPR signal pathway of ER stress has been activated after treatment with BMP2.

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