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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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BMP9 regulates Creld2 expression via Smad signaling pathway in MSCs.(A) Time course expression of Creld2 upon BMP9 stimulation using sqPCR. Subconfluent C3H10T1/2 cells were cultured in 1% FBS DMEM and infected with AdBMP9 or AdGFP. Total RNA was collected at the indicated time points and subjected to semi-quantitative RT-PCR analysis. All samples were normalized for GAPDH expression. (B) qPCR analysis of BMP9-induced Creld2 expression. RT-PCR samples prepared from (A) were used for SYBR Green-based qPCR analysis. The expression level of GAPDH was used as an internal control. (C) A schematic presentation of the 3.0 kb promoter region of mouse Creld2. (D) & (E) ChIP analysis of the mouse Creld2 promoter.C3H10T1/2 cells were infected with AdBMP9 or AdGFP for 36 h followed by formaldehyde cross-linking. The cross-linked cells were lysed and subjected to sonication and immunoprecipitation using anti-Smad1/5/8 (Santa Cruz biotechnology Inc., Cat# sc-6031-R) or control (rabbit) IgG. The recovered chromatin DNA fragments were used for PCR amplifications with two pairs of primers specific for the mouse Creld2 promoter. The expected PCR products are indicated by arrows.
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pone-0073086-g001: BMP9 regulates Creld2 expression via Smad signaling pathway in MSCs.(A) Time course expression of Creld2 upon BMP9 stimulation using sqPCR. Subconfluent C3H10T1/2 cells were cultured in 1% FBS DMEM and infected with AdBMP9 or AdGFP. Total RNA was collected at the indicated time points and subjected to semi-quantitative RT-PCR analysis. All samples were normalized for GAPDH expression. (B) qPCR analysis of BMP9-induced Creld2 expression. RT-PCR samples prepared from (A) were used for SYBR Green-based qPCR analysis. The expression level of GAPDH was used as an internal control. (C) A schematic presentation of the 3.0 kb promoter region of mouse Creld2. (D) & (E) ChIP analysis of the mouse Creld2 promoter.C3H10T1/2 cells were infected with AdBMP9 or AdGFP for 36 h followed by formaldehyde cross-linking. The cross-linked cells were lysed and subjected to sonication and immunoprecipitation using anti-Smad1/5/8 (Santa Cruz biotechnology Inc., Cat# sc-6031-R) or control (rabbit) IgG. The recovered chromatin DNA fragments were used for PCR amplifications with two pairs of primers specific for the mouse Creld2 promoter. The expected PCR products are indicated by arrows.

Mentions: We previously demonstrated that BMP9 is one of the most osteogenic factors for inducing osteoblastic differentiation in MSCs [3], [6], [10]–[12]. Through gene expression profiling analysis, we have identified several downstream targets that may play important roles in mediating BMP9-induced osteogenic signaling [12], [27]–[30]. Nonetheless, the exact molecular mechanisms underlying BMP9 functions in MSCs remain to be fully elucidated. Here, we investigated if Creld2 plays any role in BMP9 osteogenic signaling in MSCs, as Creld2 was identified as one of the top up-regulated genes in MSCs upon BMP9 stimulation (Table S1) [29]. Using sqPCR analysis, we found that Creld2 was up-regulated in MSC line C3H10T1/2 cells at 24 h, peaked at day 5 post BMP9 transduction (Figs. 1A and 1B). Similar results were obtained in other MSCs, including primary bone marrow stromal cells and iMEFs [58] (data not shown). These results confirm the microarray analysis data and suggest that Creld2 may function as a downstream target of BMP9 signaling.


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)

BMP9 regulates Creld2 expression via Smad signaling pathway in MSCs.(A) Time course expression of Creld2 upon BMP9 stimulation using sqPCR. Subconfluent C3H10T1/2 cells were cultured in 1% FBS DMEM and infected with AdBMP9 or AdGFP. Total RNA was collected at the indicated time points and subjected to semi-quantitative RT-PCR analysis. All samples were normalized for GAPDH expression. (B) qPCR analysis of BMP9-induced Creld2 expression. RT-PCR samples prepared from (A) were used for SYBR Green-based qPCR analysis. The expression level of GAPDH was used as an internal control. (C) A schematic presentation of the 3.0 kb promoter region of mouse Creld2. (D) & (E) ChIP analysis of the mouse Creld2 promoter.C3H10T1/2 cells were infected with AdBMP9 or AdGFP for 36 h followed by formaldehyde cross-linking. The cross-linked cells were lysed and subjected to sonication and immunoprecipitation using anti-Smad1/5/8 (Santa Cruz biotechnology Inc., Cat# sc-6031-R) or control (rabbit) IgG. The recovered chromatin DNA fragments were used for PCR amplifications with two pairs of primers specific for the mouse Creld2 promoter. The expected PCR products are indicated by arrows.
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pone-0073086-g001: BMP9 regulates Creld2 expression via Smad signaling pathway in MSCs.(A) Time course expression of Creld2 upon BMP9 stimulation using sqPCR. Subconfluent C3H10T1/2 cells were cultured in 1% FBS DMEM and infected with AdBMP9 or AdGFP. Total RNA was collected at the indicated time points and subjected to semi-quantitative RT-PCR analysis. All samples were normalized for GAPDH expression. (B) qPCR analysis of BMP9-induced Creld2 expression. RT-PCR samples prepared from (A) were used for SYBR Green-based qPCR analysis. The expression level of GAPDH was used as an internal control. (C) A schematic presentation of the 3.0 kb promoter region of mouse Creld2. (D) & (E) ChIP analysis of the mouse Creld2 promoter.C3H10T1/2 cells were infected with AdBMP9 or AdGFP for 36 h followed by formaldehyde cross-linking. The cross-linked cells were lysed and subjected to sonication and immunoprecipitation using anti-Smad1/5/8 (Santa Cruz biotechnology Inc., Cat# sc-6031-R) or control (rabbit) IgG. The recovered chromatin DNA fragments were used for PCR amplifications with two pairs of primers specific for the mouse Creld2 promoter. The expected PCR products are indicated by arrows.
Mentions: We previously demonstrated that BMP9 is one of the most osteogenic factors for inducing osteoblastic differentiation in MSCs [3], [6], [10]–[12]. Through gene expression profiling analysis, we have identified several downstream targets that may play important roles in mediating BMP9-induced osteogenic signaling [12], [27]–[30]. Nonetheless, the exact molecular mechanisms underlying BMP9 functions in MSCs remain to be fully elucidated. Here, we investigated if Creld2 plays any role in BMP9 osteogenic signaling in MSCs, as Creld2 was identified as one of the top up-regulated genes in MSCs upon BMP9 stimulation (Table S1) [29]. Using sqPCR analysis, we found that Creld2 was up-regulated in MSC line C3H10T1/2 cells at 24 h, peaked at day 5 post BMP9 transduction (Figs. 1A and 1B). Similar results were obtained in other MSCs, including primary bone marrow stromal cells and iMEFs [58] (data not shown). These results confirm the microarray analysis data and suggest that Creld2 may function as a downstream target of BMP9 signaling.

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