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Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation

View Article: PubMed Central - PubMed

ABSTRACT

Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O2) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.

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Related in: MedlinePlus

ROS up-regulates IGFBP3 expression. (A) mRNA expression of IGFBP3 was measured after NAC or DPI treatment. These treatments attenuated hypoxia-induced IGFBP3 upregulation; (B) chemical inhibition of Akt and ERK by LY294002 and U0126, respectively, significantly reduced the hypoxia-induced IGFBP3 mRNA level; (C) siRNA transfection of NF-κB and Elk1 significantly attenuated the IGFBP3 levels under hypoxia. Normoxia: black bars, hypoxia: white bars. ** p < 0.01.
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ijms-17-01389-f005: ROS up-regulates IGFBP3 expression. (A) mRNA expression of IGFBP3 was measured after NAC or DPI treatment. These treatments attenuated hypoxia-induced IGFBP3 upregulation; (B) chemical inhibition of Akt and ERK by LY294002 and U0126, respectively, significantly reduced the hypoxia-induced IGFBP3 mRNA level; (C) siRNA transfection of NF-κB and Elk1 significantly attenuated the IGFBP3 levels under hypoxia. Normoxia: black bars, hypoxia: white bars. ** p < 0.01.

Mentions: We previously demonstrated that certain growth factor expression in ASCs was mediated by ROS generation [8]. Therefore, we investigated whether IGFBP3 expression is regulated by ROS signaling. First, we measured the mRNA expression of IGFBP3 after N-acetyl-cysteine (NAC, ROS scavenger) or diphenyleneiodonium chloride (DPI, Noxinhibitor) treatment, which attenuated hypoxia-induced IGFBP3 up-regulation (Figure 5A). These results indicate that ROS generation under hypoxia mediates IGFBP3 expressionin ASCs. Since ROS generation primarily induced Akt and ERK phosphorylation, followed by NF-κB and Elk1 phosphorylation, we detected the alteration of IGFBP3 expression using inhibitors of these signaling pathways [2,9]. Chemical inhibition of Akt and ERK by LY294002 and U0126, respectively, significantly reduced hypoxia-induced IGFBP3 mRNA levels (Figure 5B). In addition, siRNA transfection of NF-κB and Elk1 significantly attenuated IGFBP3 levels under hypoxia (Figure 5C). These results collectively indicate that ROS generation, as well as the PI3K/Akt and MAPK pathways are involved in IGFBP3 expression under hypoxia.


Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation
ROS up-regulates IGFBP3 expression. (A) mRNA expression of IGFBP3 was measured after NAC or DPI treatment. These treatments attenuated hypoxia-induced IGFBP3 upregulation; (B) chemical inhibition of Akt and ERK by LY294002 and U0126, respectively, significantly reduced the hypoxia-induced IGFBP3 mRNA level; (C) siRNA transfection of NF-κB and Elk1 significantly attenuated the IGFBP3 levels under hypoxia. Normoxia: black bars, hypoxia: white bars. ** p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037669&req=5

ijms-17-01389-f005: ROS up-regulates IGFBP3 expression. (A) mRNA expression of IGFBP3 was measured after NAC or DPI treatment. These treatments attenuated hypoxia-induced IGFBP3 upregulation; (B) chemical inhibition of Akt and ERK by LY294002 and U0126, respectively, significantly reduced the hypoxia-induced IGFBP3 mRNA level; (C) siRNA transfection of NF-κB and Elk1 significantly attenuated the IGFBP3 levels under hypoxia. Normoxia: black bars, hypoxia: white bars. ** p < 0.01.
Mentions: We previously demonstrated that certain growth factor expression in ASCs was mediated by ROS generation [8]. Therefore, we investigated whether IGFBP3 expression is regulated by ROS signaling. First, we measured the mRNA expression of IGFBP3 after N-acetyl-cysteine (NAC, ROS scavenger) or diphenyleneiodonium chloride (DPI, Noxinhibitor) treatment, which attenuated hypoxia-induced IGFBP3 up-regulation (Figure 5A). These results indicate that ROS generation under hypoxia mediates IGFBP3 expressionin ASCs. Since ROS generation primarily induced Akt and ERK phosphorylation, followed by NF-κB and Elk1 phosphorylation, we detected the alteration of IGFBP3 expression using inhibitors of these signaling pathways [2,9]. Chemical inhibition of Akt and ERK by LY294002 and U0126, respectively, significantly reduced hypoxia-induced IGFBP3 mRNA levels (Figure 5B). In addition, siRNA transfection of NF-κB and Elk1 significantly attenuated IGFBP3 levels under hypoxia (Figure 5C). These results collectively indicate that ROS generation, as well as the PI3K/Akt and MAPK pathways are involved in IGFBP3 expression under hypoxia.

View Article: PubMed Central - PubMed

ABSTRACT

Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O2) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-&kappa;B through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.

No MeSH data available.


Related in: MedlinePlus