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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.

No MeSH data available.


IGFBP3 overexpression reduces the osteogenic differentiation of ASCs. (A) Overexpression of IGFBP3 using lentiviral vectors; (B) IGFBP3 overexpression inhibited the osteogenic differentiation of ASCs at 7 and 14 days (40×); (C) quantification of ARS staining was measured at 7 days; (D,E) in addition, IGFBP3 overexpression significantly inhibited the expression of osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 days (D) and 14 days (E). Control: black bars, IGFBP3 overexpression: white bars. ** p < 0.01.
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ijms-17-01389-f004: IGFBP3 overexpression reduces the osteogenic differentiation of ASCs. (A) Overexpression of IGFBP3 using lentiviral vectors; (B) IGFBP3 overexpression inhibited the osteogenic differentiation of ASCs at 7 and 14 days (40×); (C) quantification of ARS staining was measured at 7 days; (D,E) in addition, IGFBP3 overexpression significantly inhibited the expression of osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 days (D) and 14 days (E). Control: black bars, IGFBP3 overexpression: white bars. ** p < 0.01.

Mentions: In addition, we examined the gain-of-function by IGFBP3 overexpression. Transfection of IGFBP3 with lentiviral vectors significantly up-regulated IGFBP3 expression (Figure 4A), and IGFBP3 overexpression inhibited the osteogenic differentiation of ASCs at 7 and 14 days (Figure 4B,C). In addition, IGFBP3 overexpression significantly inhibited the expression of osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 days (Figure 4D) and 14 days (Figure 4E).


Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation
IGFBP3 overexpression reduces the osteogenic differentiation of ASCs. (A) Overexpression of IGFBP3 using lentiviral vectors; (B) IGFBP3 overexpression inhibited the osteogenic differentiation of ASCs at 7 and 14 days (40×); (C) quantification of ARS staining was measured at 7 days; (D,E) in addition, IGFBP3 overexpression significantly inhibited the expression of osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 days (D) and 14 days (E). Control: black bars, IGFBP3 overexpression: 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-f004: IGFBP3 overexpression reduces the osteogenic differentiation of ASCs. (A) Overexpression of IGFBP3 using lentiviral vectors; (B) IGFBP3 overexpression inhibited the osteogenic differentiation of ASCs at 7 and 14 days (40×); (C) quantification of ARS staining was measured at 7 days; (D,E) in addition, IGFBP3 overexpression significantly inhibited the expression of osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 days (D) and 14 days (E). Control: black bars, IGFBP3 overexpression: white bars. ** p < 0.01.
Mentions: In addition, we examined the gain-of-function by IGFBP3 overexpression. Transfection of IGFBP3 with lentiviral vectors significantly up-regulated IGFBP3 expression (Figure 4A), and IGFBP3 overexpression inhibited the osteogenic differentiation of ASCs at 7 and 14 days (Figure 4B,C). In addition, IGFBP3 overexpression significantly inhibited the expression of osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 days (Figure 4D) and 14 days (Figure 4E).

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.