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Endoplasmic reticulum (ER) stress inducible factor cysteine-rich with EGF-like domains 2 (Creld2) is an important mediator of BMP9-regulated osteogenic differentiation of mesenchymal stem cells.

Zhang J, Weng Y, Liu X, Wang J, Zhang W, Kim SH, Zhang H, Li R, Kong Y, Chen X, Shui W, Wang N, Zhao C, Wu N, He Y, Nan G, Chen X, Wen S, Zhang H, Deng F, Wan L, Luu HH, Haydon RC, Shi LL, He TC, Shi Q - PLoS ONE (2013)

Bottom Line: We confirm that Creld2 is up-regulated by BMP9 in MSCs.We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation.Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Ministry of Education Key Laboratory of Diagnostic Medicine and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.

ABSTRACT
Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

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Knockdown of Creld2 expression diminishes BMP9-induced late osteogenic markers and matrix mineralization of MSCs in vitro, which are augmented by Creld2 overexpression.(A) Silencing Creld2 blunts BMP9-induced expression of OPN and OCN. Subconfluent C3H10T1/2 cells were co-infected with the indicated adenoviral vectors. At 7 days post post infection, total RNA was isolated for sqPCR analysis using primers specific for mouse OPN, OCN, and GAPDH (as a control). (B) Silencing Creld2 inhibits BMP9-induced matrix mineralization. C3H10T1/2 cells were co-infected with the indicated adenoviral vectors. Alizarin Red S staining was conducted at 10 days after infection (a). The staining was further quantitatively analyzed (b). Each assay condition was done in triplicate and/or carried out at least in three independent experiments. “**”, p<0.001; Representative results are shown.
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pone-0073086-g003: Knockdown of Creld2 expression diminishes BMP9-induced late osteogenic markers and matrix mineralization of MSCs in vitro, which are augmented by Creld2 overexpression.(A) Silencing Creld2 blunts BMP9-induced expression of OPN and OCN. Subconfluent C3H10T1/2 cells were co-infected with the indicated adenoviral vectors. At 7 days post post infection, total RNA was isolated for sqPCR analysis using primers specific for mouse OPN, OCN, and GAPDH (as a control). (B) Silencing Creld2 inhibits BMP9-induced matrix mineralization. C3H10T1/2 cells were co-infected with the indicated adenoviral vectors. Alizarin Red S staining was conducted at 10 days after infection (a). The staining was further quantitatively analyzed (b). Each assay condition was done in triplicate and/or carried out at least in three independent experiments. “**”, p<0.001; Representative results are shown.

Mentions: We further analyzed the effect of Creld2 on the expression of late osteogenic markers osteopotin (OPN) and osteocalcin (OCN) in BMP9-stimulated MSCs. While exogenous Creld2 expression slightly augmented the expression of both OPN and OCN, silencing Creld2 expression effectively reduced the expression levels of OPN and OCN (Figure 3A). Furthermore, we found Creld2 expression significantly increased the BMP9-induced matrix mineralization as assessed by alizarin red staining, where silencing Creld2 led to a decrease in alizarin red staining (Figure 3B, panel a). Quantitative analysis of the alizarin red staining indicates that the BMP9-induced matrix mineralization was significantly enhanced by Creld2 overexpression while inhibited by silencing Creld2 (p<0.01) (Figure 3B, panel b). Taken these in vitro results together, Creld2 has been shown to potentiate BMP9-induced osteoblastic commitment and terminal differentiation of MSCs in vitro.


Endoplasmic reticulum (ER) stress inducible factor cysteine-rich with EGF-like domains 2 (Creld2) is an important mediator of BMP9-regulated osteogenic differentiation of mesenchymal stem cells.

Zhang J, Weng Y, Liu X, Wang J, Zhang W, Kim SH, Zhang H, Li R, Kong Y, Chen X, Shui W, Wang N, Zhao C, Wu N, He Y, Nan G, Chen X, Wen S, Zhang H, Deng F, Wan L, Luu HH, Haydon RC, Shi LL, He TC, Shi Q - PLoS ONE (2013)

Knockdown of Creld2 expression diminishes BMP9-induced late osteogenic markers and matrix mineralization of MSCs in vitro, which are augmented by Creld2 overexpression.(A) Silencing Creld2 blunts BMP9-induced expression of OPN and OCN. Subconfluent C3H10T1/2 cells were co-infected with the indicated adenoviral vectors. At 7 days post post infection, total RNA was isolated for sqPCR analysis using primers specific for mouse OPN, OCN, and GAPDH (as a control). (B) Silencing Creld2 inhibits BMP9-induced matrix mineralization. C3H10T1/2 cells were co-infected with the indicated adenoviral vectors. Alizarin Red S staining was conducted at 10 days after infection (a). The staining was further quantitatively analyzed (b). Each assay condition was done in triplicate and/or carried out at least in three independent experiments. “**”, p<0.001; Representative results are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760886&req=5

pone-0073086-g003: Knockdown of Creld2 expression diminishes BMP9-induced late osteogenic markers and matrix mineralization of MSCs in vitro, which are augmented by Creld2 overexpression.(A) Silencing Creld2 blunts BMP9-induced expression of OPN and OCN. Subconfluent C3H10T1/2 cells were co-infected with the indicated adenoviral vectors. At 7 days post post infection, total RNA was isolated for sqPCR analysis using primers specific for mouse OPN, OCN, and GAPDH (as a control). (B) Silencing Creld2 inhibits BMP9-induced matrix mineralization. C3H10T1/2 cells were co-infected with the indicated adenoviral vectors. Alizarin Red S staining was conducted at 10 days after infection (a). The staining was further quantitatively analyzed (b). Each assay condition was done in triplicate and/or carried out at least in three independent experiments. “**”, p<0.001; Representative results are shown.
Mentions: We further analyzed the effect of Creld2 on the expression of late osteogenic markers osteopotin (OPN) and osteocalcin (OCN) in BMP9-stimulated MSCs. While exogenous Creld2 expression slightly augmented the expression of both OPN and OCN, silencing Creld2 expression effectively reduced the expression levels of OPN and OCN (Figure 3A). Furthermore, we found Creld2 expression significantly increased the BMP9-induced matrix mineralization as assessed by alizarin red staining, where silencing Creld2 led to a decrease in alizarin red staining (Figure 3B, panel a). Quantitative analysis of the alizarin red staining indicates that the BMP9-induced matrix mineralization was significantly enhanced by Creld2 overexpression while inhibited by silencing Creld2 (p<0.01) (Figure 3B, panel b). Taken these in vitro results together, Creld2 has been shown to potentiate BMP9-induced osteoblastic commitment and terminal differentiation of MSCs in vitro.

Bottom Line: We confirm that Creld2 is up-regulated by BMP9 in MSCs.We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation.Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Ministry of Education Key Laboratory of Diagnostic Medicine and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.

ABSTRACT
Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

Show MeSH
Related in: MedlinePlus