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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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Hypoxia suppresses the spontaneous mineralization and osteogenic differentiation of ASCs. ASCs were cultured in α-MEM and osteogenic differentiation medium (ODM) for 7 and 14 days, and then stained with Alizarin Red S (ARS). (A,B) Hypoxia (2% O2) significantly reduced the ARSstaining of ASCs (red) in α-MEM and ODM (40×), and ARS staining was measured; (C) osteogenic differentiation markers such as RUNX2, osteocalcin, and osterix were significantly reduced under hypoxia. Normoxia: black bars, Hypoxia: white bars. **p< 0.01.
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ijms-17-01389-f001: Hypoxia suppresses the spontaneous mineralization and osteogenic differentiation of ASCs. ASCs were cultured in α-MEM and osteogenic differentiation medium (ODM) for 7 and 14 days, and then stained with Alizarin Red S (ARS). (A,B) Hypoxia (2% O2) significantly reduced the ARSstaining of ASCs (red) in α-MEM and ODM (40×), and ARS staining was measured; (C) osteogenic differentiation markers such as RUNX2, osteocalcin, and osterix were significantly reduced under hypoxia. Normoxia: black bars, Hypoxia: white bars. **p< 0.01.

Mentions: Hypoxia significantly reduced the spontaneous mineralization and osteogenic differentiation of ASCs (Figure 1A). ARS staining of ASCs was quantified in α-MEM and ODM (Figure 1B). In addition, osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced under hypoxia (Figure 1C). Collectively, these results indicate that hypoxia suppress the spontaneous mineralization and osteogenic differentiation of ASCs.


Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation
Hypoxia suppresses the spontaneous mineralization and osteogenic differentiation of ASCs. ASCs were cultured in α-MEM and osteogenic differentiation medium (ODM) for 7 and 14 days, and then stained with Alizarin Red S (ARS). (A,B) Hypoxia (2% O2) significantly reduced the ARSstaining of ASCs (red) in α-MEM and ODM (40×), and ARS staining was measured; (C) osteogenic differentiation markers such as RUNX2, osteocalcin, and osterix were significantly reduced under hypoxia. Normoxia: black bars, Hypoxia: white bars. **p< 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01389-f001: Hypoxia suppresses the spontaneous mineralization and osteogenic differentiation of ASCs. ASCs were cultured in α-MEM and osteogenic differentiation medium (ODM) for 7 and 14 days, and then stained with Alizarin Red S (ARS). (A,B) Hypoxia (2% O2) significantly reduced the ARSstaining of ASCs (red) in α-MEM and ODM (40×), and ARS staining was measured; (C) osteogenic differentiation markers such as RUNX2, osteocalcin, and osterix were significantly reduced under hypoxia. Normoxia: black bars, Hypoxia: white bars. **p< 0.01.
Mentions: Hypoxia significantly reduced the spontaneous mineralization and osteogenic differentiation of ASCs (Figure 1A). ARS staining of ASCs was quantified in α-MEM and ODM (Figure 1B). In addition, osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced under hypoxia (Figure 1C). Collectively, these results indicate that hypoxia suppress the spontaneous mineralization and osteogenic differentiation of ASCs.

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