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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 expression potentiates BMP9-induced early and late osteogenic markers in MSCs, which is diminished by silencing Creld2 expression.(A) Construction of the recombinant adenovirus expressing Creld2 or silencing Creld2 expression in MSCs. The recombinant adenovirus AdR-Creld2 and AdR-simCreld2 were shown to effectively transduce MSCs, such as C3H10T1/2 (a). For adenovirus-mediated exogenous Creld2 expression or silencing Creld2 expression, C3H10T1/2 cells were infected with Ad-RFP, AdR-Creld2, or AdR-simCreld2. Total RNA was isolated at 72 h post infection and subjected to sqPCR using primer pairs specific for mouse Creld2 (b). (B) Creld2 potentiates BMP9-induced ALP activity in MSCs. Subconfluent C3H10T1/2 cells were co-infected with AdBMP9, AdR-Creld2, AdR-simCreld2, and/or AdGFP. ALP activity was measured at day 7 by histochemical staining (a) and chemiluminescent assays (b). (C) Silencing Creld2 expression inhibits BMP9-induced ALP activity in MSCs. C3H10T1/2 cells were infected with AdBMP9 or AdGFP and escalating titers of AdR-simCrelds virus. At the indicated time points post infection, the ALP activity was assessed. “**”, p<0.001; “*”, p<0.05. Each assay condition was done in triplicate and/or carried out at least in three independent experiments. Representative results are shown.
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pone-0073086-g002: Exogenous Creld2 expression potentiates BMP9-induced early and late osteogenic markers in MSCs, which is diminished by silencing Creld2 expression.(A) Construction of the recombinant adenovirus expressing Creld2 or silencing Creld2 expression in MSCs. The recombinant adenovirus AdR-Creld2 and AdR-simCreld2 were shown to effectively transduce MSCs, such as C3H10T1/2 (a). For adenovirus-mediated exogenous Creld2 expression or silencing Creld2 expression, C3H10T1/2 cells were infected with Ad-RFP, AdR-Creld2, or AdR-simCreld2. Total RNA was isolated at 72 h post infection and subjected to sqPCR using primer pairs specific for mouse Creld2 (b). (B) Creld2 potentiates BMP9-induced ALP activity in MSCs. Subconfluent C3H10T1/2 cells were co-infected with AdBMP9, AdR-Creld2, AdR-simCreld2, and/or AdGFP. ALP activity was measured at day 7 by histochemical staining (a) and chemiluminescent assays (b). (C) Silencing Creld2 expression inhibits BMP9-induced ALP activity in MSCs. C3H10T1/2 cells were infected with AdBMP9 or AdGFP and escalating titers of AdR-simCrelds virus. At the indicated time points post infection, the ALP activity was assessed. “**”, p<0.001; “*”, p<0.05. Each assay condition was done in triplicate and/or carried out at least in three independent experiments. Representative results are shown.

Mentions: If Creld2 is an important target of BMP9-mediated osteogenic signaling, we hypothesized that exogenous expression of Creld2 could enhance BMP9-induced osteogenic differentiation of MSCs, whereas silencing Creld2 expression would inhibit BMP9-induced osteogenic signaling. To effectively introduce exogenous Creld2 or silence Creld2 expression in MSCs, we constructed recombinant adenovirus expressing mouse Creld2 (AdR-Creld2) or siRNAs targeting mouse Creld2 coding region (AdR-simCreld2) [50]. We demonstrated that both adenoviral vectors can transduce MSCs with high efficiency as both co-express RFP marker (Figure 2A, panel a). Adenovirus-mediated transgene expression and knockdown of endogenous Creld2 were verified by semi-quantitative RT-PCR (Figure 2A, panel b).


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 expression potentiates BMP9-induced early and late osteogenic markers in MSCs, which is diminished by silencing Creld2 expression.(A) Construction of the recombinant adenovirus expressing Creld2 or silencing Creld2 expression in MSCs. The recombinant adenovirus AdR-Creld2 and AdR-simCreld2 were shown to effectively transduce MSCs, such as C3H10T1/2 (a). For adenovirus-mediated exogenous Creld2 expression or silencing Creld2 expression, C3H10T1/2 cells were infected with Ad-RFP, AdR-Creld2, or AdR-simCreld2. Total RNA was isolated at 72 h post infection and subjected to sqPCR using primer pairs specific for mouse Creld2 (b). (B) Creld2 potentiates BMP9-induced ALP activity in MSCs. Subconfluent C3H10T1/2 cells were co-infected with AdBMP9, AdR-Creld2, AdR-simCreld2, and/or AdGFP. ALP activity was measured at day 7 by histochemical staining (a) and chemiluminescent assays (b). (C) Silencing Creld2 expression inhibits BMP9-induced ALP activity in MSCs. C3H10T1/2 cells were infected with AdBMP9 or AdGFP and escalating titers of AdR-simCrelds virus. At the indicated time points post infection, the ALP activity was assessed. “**”, p<0.001; “*”, p<0.05. Each assay condition was done in triplicate and/or carried out at least in three independent experiments. Representative results are shown.
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pone-0073086-g002: Exogenous Creld2 expression potentiates BMP9-induced early and late osteogenic markers in MSCs, which is diminished by silencing Creld2 expression.(A) Construction of the recombinant adenovirus expressing Creld2 or silencing Creld2 expression in MSCs. The recombinant adenovirus AdR-Creld2 and AdR-simCreld2 were shown to effectively transduce MSCs, such as C3H10T1/2 (a). For adenovirus-mediated exogenous Creld2 expression or silencing Creld2 expression, C3H10T1/2 cells were infected with Ad-RFP, AdR-Creld2, or AdR-simCreld2. Total RNA was isolated at 72 h post infection and subjected to sqPCR using primer pairs specific for mouse Creld2 (b). (B) Creld2 potentiates BMP9-induced ALP activity in MSCs. Subconfluent C3H10T1/2 cells were co-infected with AdBMP9, AdR-Creld2, AdR-simCreld2, and/or AdGFP. ALP activity was measured at day 7 by histochemical staining (a) and chemiluminescent assays (b). (C) Silencing Creld2 expression inhibits BMP9-induced ALP activity in MSCs. C3H10T1/2 cells were infected with AdBMP9 or AdGFP and escalating titers of AdR-simCrelds virus. At the indicated time points post infection, the ALP activity was assessed. “**”, p<0.001; “*”, p<0.05. Each assay condition was done in triplicate and/or carried out at least in three independent experiments. Representative results are shown.
Mentions: If Creld2 is an important target of BMP9-mediated osteogenic signaling, we hypothesized that exogenous expression of Creld2 could enhance BMP9-induced osteogenic differentiation of MSCs, whereas silencing Creld2 expression would inhibit BMP9-induced osteogenic signaling. To effectively introduce exogenous Creld2 or silence Creld2 expression in MSCs, we constructed recombinant adenovirus expressing mouse Creld2 (AdR-Creld2) or siRNAs targeting mouse Creld2 coding region (AdR-simCreld2) [50]. We demonstrated that both adenoviral vectors can transduce MSCs with high efficiency as both co-express RFP marker (Figure 2A, panel a). Adenovirus-mediated transgene expression and knockdown of endogenous Creld2 were verified by semi-quantitative RT-PCR (Figure 2A, panel b).

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