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

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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 induces IGFBP expression. (A) The altered expression of growth factors was examined using an antibody array. Hypoxia (2%, 24 h) upregulated the protein expression of IGFBP families in ASCs; (B) the mRNA level of IGFBP families under hypoxia was measured. Hypoxia significantly upregulated IGFBP3–6. Normoxia: black bars, hypoxia: white bars. ** p < 0.01.
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ijms-17-01389-f002: Hypoxia induces IGFBP expression. (A) The altered expression of growth factors was examined using an antibody array. Hypoxia (2%, 24 h) upregulated the protein expression of IGFBP families in ASCs; (B) the mRNA level of IGFBP families under hypoxia was measured. Hypoxia significantly upregulated IGFBP3–6. Normoxia: black bars, hypoxia: white bars. ** p < 0.01.

Mentions: We have previously reported that hypoxia up-regulates diverse growth factors expression [1,2,22,23]. We therefore examined the altered expression of growth factors by antibody array, and found that hypoxia up-regulated the protein expression of IGFBP families in ASCs (Figure 2A). We further examined the mRNA expression of IGF, IGF receptor and IGFBP families in ASCs. As summarized in Table 1, IGF-1, IGF-2, and their receptors are poorly expressed in ASCs. IGFBP1 and IGFBP2 are not expressed; however, IGFBP3–7 is highly expressed in ASCs. Therefore, we measured the mRNA expression of IGFBP3–7 under hypoxia, which led to significant up-regulation of IGFBP3–6 (Figure 2B).


Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation
Hypoxia induces IGFBP expression. (A) The altered expression of growth factors was examined using an antibody array. Hypoxia (2%, 24 h) upregulated the protein expression of IGFBP families in ASCs; (B) the mRNA level of IGFBP families under hypoxia was measured. Hypoxia significantly upregulated IGFBP3–6. 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-f002: Hypoxia induces IGFBP expression. (A) The altered expression of growth factors was examined using an antibody array. Hypoxia (2%, 24 h) upregulated the protein expression of IGFBP families in ASCs; (B) the mRNA level of IGFBP families under hypoxia was measured. Hypoxia significantly upregulated IGFBP3–6. Normoxia: black bars, hypoxia: white bars. ** p < 0.01.
Mentions: We have previously reported that hypoxia up-regulates diverse growth factors expression [1,2,22,23]. We therefore examined the altered expression of growth factors by antibody array, and found that hypoxia up-regulated the protein expression of IGFBP families in ASCs (Figure 2A). We further examined the mRNA expression of IGF, IGF receptor and IGFBP families in ASCs. As summarized in Table 1, IGF-1, IGF-2, and their receptors are poorly expressed in ASCs. IGFBP1 and IGFBP2 are not expressed; however, IGFBP3–7 is highly expressed in ASCs. Therefore, we measured the mRNA expression of IGFBP3–7 under hypoxia, which led to significant up-regulation of IGFBP3–6 (Figure 2B).

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