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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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Exogenous Creld2 enhances, while silencing Creld2 inhibits BMP9-induced ectopic bone formation.C3H10T1/2 cells were co-transduced with BMP9, RFP, Creld2 and/or simCreld2 adenoviruses for 16 h (A) and collected for subcutaneous injections into the flanks of athymic nude mice. Bony masses were collected at 4 weeks and subjected to microCT analysis to determine the 3-dimensional iso-surface (B) and the mean mineralization density (C). No masses were detected in the subcutaneous injections, in which the implanted cells were transduced with RFP, Creld2, or simCreld2 without BMP9. The p-values were calculated by comparing the results from BMP9/Creld2 or BMP9/simCreld2 group with that the BMP9/RFP’s. Representative images are shown.
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pone-0073086-g004: Exogenous Creld2 enhances, while silencing Creld2 inhibits BMP9-induced ectopic bone formation.C3H10T1/2 cells were co-transduced with BMP9, RFP, Creld2 and/or simCreld2 adenoviruses for 16 h (A) and collected for subcutaneous injections into the flanks of athymic nude mice. Bony masses were collected at 4 weeks and subjected to microCT analysis to determine the 3-dimensional iso-surface (B) and the mean mineralization density (C). No masses were detected in the subcutaneous injections, in which the implanted cells were transduced with RFP, Creld2, or simCreld2 without BMP9. The p-values were calculated by comparing the results from BMP9/Creld2 or BMP9/simCreld2 group with that the BMP9/RFP’s. Representative images are shown.

Mentions: While the above in vitro studies established that Creld2 may play an important role in BMP9-mediated osteogenic signaling, it was essential to determine if Creld2 plays such a role in vivo. We chose to use our previously established stem implantation assay [25], [30], [53], [57], [58]. MSCs were first transduced with BMP9 and Creld2, simCreld2, or RFP for 16 h (Figure 4A). The cells were collected and injected subcutaneously into the flanks of athymic nude mice for 4 weeks. MSCs transduced with RFP, Creld2 or simCreld2 alone did not form any detectable masses (data not shown). We found that Creld2 over-expression significantly augmented BMP9-induced bony mass formation whereas simCreld2 inhibited BMP9-induced bone formation (Figure 4B). The gross size differences were quantitatively assessed by iso-surface 3-dimensional analysis of the µCT imaging data (Figure 4C). Conversely, silencing Creld2 expression significantly decreased the average bone volume (Figures 4B and 4C).


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)

Exogenous Creld2 enhances, while silencing Creld2 inhibits BMP9-induced ectopic bone formation.C3H10T1/2 cells were co-transduced with BMP9, RFP, Creld2 and/or simCreld2 adenoviruses for 16 h (A) and collected for subcutaneous injections into the flanks of athymic nude mice. Bony masses were collected at 4 weeks and subjected to microCT analysis to determine the 3-dimensional iso-surface (B) and the mean mineralization density (C). No masses were detected in the subcutaneous injections, in which the implanted cells were transduced with RFP, Creld2, or simCreld2 without BMP9. The p-values were calculated by comparing the results from BMP9/Creld2 or BMP9/simCreld2 group with that the BMP9/RFP’s. Representative images are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760886&req=5

pone-0073086-g004: Exogenous Creld2 enhances, while silencing Creld2 inhibits BMP9-induced ectopic bone formation.C3H10T1/2 cells were co-transduced with BMP9, RFP, Creld2 and/or simCreld2 adenoviruses for 16 h (A) and collected for subcutaneous injections into the flanks of athymic nude mice. Bony masses were collected at 4 weeks and subjected to microCT analysis to determine the 3-dimensional iso-surface (B) and the mean mineralization density (C). No masses were detected in the subcutaneous injections, in which the implanted cells were transduced with RFP, Creld2, or simCreld2 without BMP9. The p-values were calculated by comparing the results from BMP9/Creld2 or BMP9/simCreld2 group with that the BMP9/RFP’s. Representative images are shown.
Mentions: While the above in vitro studies established that Creld2 may play an important role in BMP9-mediated osteogenic signaling, it was essential to determine if Creld2 plays such a role in vivo. We chose to use our previously established stem implantation assay [25], [30], [53], [57], [58]. MSCs were first transduced with BMP9 and Creld2, simCreld2, or RFP for 16 h (Figure 4A). The cells were collected and injected subcutaneously into the flanks of athymic nude mice for 4 weeks. MSCs transduced with RFP, Creld2 or simCreld2 alone did not form any detectable masses (data not shown). We found that Creld2 over-expression significantly augmented BMP9-induced bony mass formation whereas simCreld2 inhibited BMP9-induced bone formation (Figure 4B). The gross size differences were quantitatively assessed by iso-surface 3-dimensional analysis of the µCT imaging data (Figure 4C). Conversely, silencing Creld2 expression significantly decreased the average bone volume (Figures 4B and 4C).

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