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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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Creld2 potentiates BMP9-induced terminal osteogenic differentiation and matrix mineralization.The retrieved samples were fixed, decalcified, paraffin-embedded, and subjected to histologic analysis. (A) H & E staining. Sections from the retrieved samples of BMP9/RFP (a), BMP9/Creld2 (b), and BMP9/simCreld2 (c) were subjected to H & E staining. The trabecular structures, including % of trabecular area of the total area (d), were quantitatively analyzed using the ImageJ software. The p-values were calculated by comparing the results from BMP9/Creld2 or BMP9/simCreld2 group with that the BMP9/RFP’s. (B) Alcian blue staining. CM, cartilage matrix. (C) Masson’s Trichrome staining. MM, mineralized matrix; OM, osteoid matrix. Magnification, 200×. Representative results are shown.
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pone-0073086-g005: Creld2 potentiates BMP9-induced terminal osteogenic differentiation and matrix mineralization.The retrieved samples were fixed, decalcified, paraffin-embedded, and subjected to histologic analysis. (A) H & E staining. Sections from the retrieved samples of BMP9/RFP (a), BMP9/Creld2 (b), and BMP9/simCreld2 (c) were subjected to H & E staining. The trabecular structures, including % of trabecular area of the total area (d), were quantitatively analyzed using the ImageJ software. The p-values were calculated by comparing the results from BMP9/Creld2 or BMP9/simCreld2 group with that the BMP9/RFP’s. (B) Alcian blue staining. CM, cartilage matrix. (C) Masson’s Trichrome staining. MM, mineralized matrix; OM, osteoid matrix. Magnification, 200×. Representative results are shown.

Mentions: The retrieved samples were further subjected to histologic analysis and other special staining. H & E staining revealed that Creld2 significantly enhanced BMP9-induced bone formation and mineralization and that silencing Creld2 inhibited BMP9-induced osteogenesis (Figure 5A panels a vs. b). Quantitative analysis indicates that co-expression of BMP9 and Creld2 significantly increased the average thickness of trabeculae and the percentage of trabecular area over total area (Fig, 5A, panel d), whereas knocking down Creld2 expression exhibited an inhibitory effect (Figure 5A, panels a vs. c). The alcian blue staining revealed that silencing Creld2 expression led to the accumulation of cartilaginous matrix in BMP9-transduced MSCs, compared with that of the BMP9 alone group, whereas the BMP9+Creld2 group exhibited a slight decrease in alcian blue staining (Figure 5B). These results suggest that Creld2 may facilitate BMP9-induced terminal osteogenic differentiation. This notion was further confirmed by Masson’s trichrome staining assays. We found that, in the presence of exogenous Creld2, BMP9 induced robust and highly mature bone matrix mineralization, while BMP9 failed to induce the formation of mature bone matrix from MSCs when Creld2 expression was silenced (Figure 5C). These in vivo findings are supported by the in vitro studies. Collectively, our results strongly indicate that Creld2 is an important mediator of BMP9-induced terminal osteogenic differentiation, and that exogenous Creld2 expression augments BMP9-induced osteogenic differentiation and hence produces more mature bone.


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)

Creld2 potentiates BMP9-induced terminal osteogenic differentiation and matrix mineralization.The retrieved samples were fixed, decalcified, paraffin-embedded, and subjected to histologic analysis. (A) H & E staining. Sections from the retrieved samples of BMP9/RFP (a), BMP9/Creld2 (b), and BMP9/simCreld2 (c) were subjected to H & E staining. The trabecular structures, including % of trabecular area of the total area (d), were quantitatively analyzed using the ImageJ software. The p-values were calculated by comparing the results from BMP9/Creld2 or BMP9/simCreld2 group with that the BMP9/RFP’s. (B) Alcian blue staining. CM, cartilage matrix. (C) Masson’s Trichrome staining. MM, mineralized matrix; OM, osteoid matrix. Magnification, 200×. Representative results are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760886&req=5

pone-0073086-g005: Creld2 potentiates BMP9-induced terminal osteogenic differentiation and matrix mineralization.The retrieved samples were fixed, decalcified, paraffin-embedded, and subjected to histologic analysis. (A) H & E staining. Sections from the retrieved samples of BMP9/RFP (a), BMP9/Creld2 (b), and BMP9/simCreld2 (c) were subjected to H & E staining. The trabecular structures, including % of trabecular area of the total area (d), were quantitatively analyzed using the ImageJ software. The p-values were calculated by comparing the results from BMP9/Creld2 or BMP9/simCreld2 group with that the BMP9/RFP’s. (B) Alcian blue staining. CM, cartilage matrix. (C) Masson’s Trichrome staining. MM, mineralized matrix; OM, osteoid matrix. Magnification, 200×. Representative results are shown.
Mentions: The retrieved samples were further subjected to histologic analysis and other special staining. H & E staining revealed that Creld2 significantly enhanced BMP9-induced bone formation and mineralization and that silencing Creld2 inhibited BMP9-induced osteogenesis (Figure 5A panels a vs. b). Quantitative analysis indicates that co-expression of BMP9 and Creld2 significantly increased the average thickness of trabeculae and the percentage of trabecular area over total area (Fig, 5A, panel d), whereas knocking down Creld2 expression exhibited an inhibitory effect (Figure 5A, panels a vs. c). The alcian blue staining revealed that silencing Creld2 expression led to the accumulation of cartilaginous matrix in BMP9-transduced MSCs, compared with that of the BMP9 alone group, whereas the BMP9+Creld2 group exhibited a slight decrease in alcian blue staining (Figure 5B). These results suggest that Creld2 may facilitate BMP9-induced terminal osteogenic differentiation. This notion was further confirmed by Masson’s trichrome staining assays. We found that, in the presence of exogenous Creld2, BMP9 induced robust and highly mature bone matrix mineralization, while BMP9 failed to induce the formation of mature bone matrix from MSCs when Creld2 expression was silenced (Figure 5C). These in vivo findings are supported by the in vitro studies. Collectively, our results strongly indicate that Creld2 is an important mediator of BMP9-induced terminal osteogenic differentiation, and that exogenous Creld2 expression augments BMP9-induced osteogenic differentiation and hence produces more mature bone.

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