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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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Intracellular IGFBP3 inhibits osteogenic differentiation. (A) siRNA knockdown of IGFBP3 induced ARS staining in ASCs at 7 and 14 days (40×); (B) quantification of ARS staining was measured at 7 days; (C,D) siRNA knockdown of IGFBP3 significantly attenuated hypoxia-reduced osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 and 14 days; (E) hypoxia increased the transfer of IGFBP3 into the nuclear region of ASCs, and IGFBP3 signal (green) is increased in the nuclear region (DAPI, blue) of ASCs. ** p < 0.01.
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ijms-17-01389-f003: Intracellular IGFBP3 inhibits osteogenic differentiation. (A) siRNA knockdown of IGFBP3 induced ARS staining in ASCs at 7 and 14 days (40×); (B) quantification of ARS staining was measured at 7 days; (C,D) siRNA knockdown of IGFBP3 significantly attenuated hypoxia-reduced osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 and 14 days; (E) hypoxia increased the transfer of IGFBP3 into the nuclear region of ASCs, and IGFBP3 signal (green) is increased in the nuclear region (DAPI, blue) of ASCs. ** p < 0.01.

Mentions: Since exogenous recombinant IGFBPs did not affect the osteogenic differentiation of ASCs, we further examined whether endogenous intracellular IGFBPs affected osteogenic differentiation. We thus examined whether siRNA-mediated inhibition of IGFBP proteins could attenuate hypoxia-induced suppression of osteogenic differentiation. We prepared siRNAs of IGFBP3–5, and transfected them into ASCs. siRNA transfection reduced the mRNA expression of IGFBP3–5 in ASCs (Figure S2A,B). Compared with other IGFBPs, IGFBP3 plays a pivotal role in osteogenic differentiation of ASCs (Figure S2C). For example, knockdown of IGFBP3 led to positive ARS staining in ASCs at 7 and 14 days (Figure 3A,B). In addition, inhibition of IGFBP3 significantly up-regulated the hypoxia-reduced osteogenic markers such as RUNX2, osteocalcin, and osterix at 7 and 14 days (Figure 3C,D). We also examined whether hypoxia increases the translocalization of IGFBP3 into the nuclear region of ASCs. Compared to controls, IGFBP3 expression (green) was increased in the nuclear region (DAPI, blue) of ASCs under hypoxia (Figure 3E).


Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation
Intracellular IGFBP3 inhibits osteogenic differentiation. (A) siRNA knockdown of IGFBP3 induced ARS staining in ASCs at 7 and 14 days (40×); (B) quantification of ARS staining was measured at 7 days; (C,D) siRNA knockdown of IGFBP3 significantly attenuated hypoxia-reduced osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 and 14 days; (E) hypoxia increased the transfer of IGFBP3 into the nuclear region of ASCs, and IGFBP3 signal (green) is increased in the nuclear region (DAPI, blue) of ASCs. ** p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01389-f003: Intracellular IGFBP3 inhibits osteogenic differentiation. (A) siRNA knockdown of IGFBP3 induced ARS staining in ASCs at 7 and 14 days (40×); (B) quantification of ARS staining was measured at 7 days; (C,D) siRNA knockdown of IGFBP3 significantly attenuated hypoxia-reduced osteogenic induction markers such as RUNX2, osteocalcin, and osterix at 7 and 14 days; (E) hypoxia increased the transfer of IGFBP3 into the nuclear region of ASCs, and IGFBP3 signal (green) is increased in the nuclear region (DAPI, blue) of ASCs. ** p < 0.01.
Mentions: Since exogenous recombinant IGFBPs did not affect the osteogenic differentiation of ASCs, we further examined whether endogenous intracellular IGFBPs affected osteogenic differentiation. We thus examined whether siRNA-mediated inhibition of IGFBP proteins could attenuate hypoxia-induced suppression of osteogenic differentiation. We prepared siRNAs of IGFBP3–5, and transfected them into ASCs. siRNA transfection reduced the mRNA expression of IGFBP3–5 in ASCs (Figure S2A,B). Compared with other IGFBPs, IGFBP3 plays a pivotal role in osteogenic differentiation of ASCs (Figure S2C). For example, knockdown of IGFBP3 led to positive ARS staining in ASCs at 7 and 14 days (Figure 3A,B). In addition, inhibition of IGFBP3 significantly up-regulated the hypoxia-reduced osteogenic markers such as RUNX2, osteocalcin, and osterix at 7 and 14 days (Figure 3C,D). We also examined whether hypoxia increases the translocalization of IGFBP3 into the nuclear region of ASCs. Compared to controls, IGFBP3 expression (green) was increased in the nuclear region (DAPI, blue) of ASCs under hypoxia (Figure 3E).

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