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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 osteogenic differentiation of clonal BM-MSCs. (A) Hypoxia (2%) induced the mRNA expression of IGFBP3 in clonal BM-MSCs; (B) hypoxia suppressed the osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (40×); (C) quantification of ARS staining was measured at 7 days; (D) osteogenic induction markers such as RUNX2, osteocalcin, and osterixwere significantly reduced by hypoxia in DMEM and ODM; (E) siRNA-mediated inhibition of IGFBP3 significantly attenuated the hypoxia-suppressed osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (40×); (F) quantification of ARS staining was measured at 7 days; (G,H) osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced by hypoxia at 7 days (G) and 14 days (H). Normoxia: black bars, hypoxia: white bars. * p < 0.5, ** p < 0.01.
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ijms-17-01389-f006: Hypoxia suppresses the osteogenic differentiation of clonal BM-MSCs. (A) Hypoxia (2%) induced the mRNA expression of IGFBP3 in clonal BM-MSCs; (B) hypoxia suppressed the osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (40×); (C) quantification of ARS staining was measured at 7 days; (D) osteogenic induction markers such as RUNX2, osteocalcin, and osterixwere significantly reduced by hypoxia in DMEM and ODM; (E) siRNA-mediated inhibition of IGFBP3 significantly attenuated the hypoxia-suppressed osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (40×); (F) quantification of ARS staining was measured at 7 days; (G,H) osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced by hypoxia at 7 days (G) and 14 days (H). Normoxia: black bars, hypoxia: white bars. * p < 0.5, ** p < 0.01.

Mentions: Since osteogenic differentiation is also important for BM-MSCs, we examined the effect of hypoxia on their osteogenic differentiation, as well as the involvement of IGFBP3 during osteogenesis. We used clonal BM-MSCs derived using our previously reported novel method of isolation [24,25] to examine the effect of hypoxia on osteogenic differentiation. As expected, hypoxia (2%) induced the mRNA expression of IGFBP3 in clonal BM-MSCs (Figure 6A). Hypoxia suppressed the osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (Figure 6B). Osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced by hypoxia in DMEM and ODM (Figure 6D). In addition, siRNA-mediated inhibition of IGFBP3 significantly attenuated the hypoxia-suppressed osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (Figure 6E,F). Osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced by hypoxia at 7 days (Figure 6G) and 14 days (Figure 6H).


Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation
Hypoxia suppresses the osteogenic differentiation of clonal BM-MSCs. (A) Hypoxia (2%) induced the mRNA expression of IGFBP3 in clonal BM-MSCs; (B) hypoxia suppressed the osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (40×); (C) quantification of ARS staining was measured at 7 days; (D) osteogenic induction markers such as RUNX2, osteocalcin, and osterixwere significantly reduced by hypoxia in DMEM and ODM; (E) siRNA-mediated inhibition of IGFBP3 significantly attenuated the hypoxia-suppressed osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (40×); (F) quantification of ARS staining was measured at 7 days; (G,H) osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced by hypoxia at 7 days (G) and 14 days (H). Normoxia: black bars, hypoxia: white bars. * p < 0.5, ** p < 0.01.
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getmorefigures.php?uid=PMC5037669&req=5

ijms-17-01389-f006: Hypoxia suppresses the osteogenic differentiation of clonal BM-MSCs. (A) Hypoxia (2%) induced the mRNA expression of IGFBP3 in clonal BM-MSCs; (B) hypoxia suppressed the osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (40×); (C) quantification of ARS staining was measured at 7 days; (D) osteogenic induction markers such as RUNX2, osteocalcin, and osterixwere significantly reduced by hypoxia in DMEM and ODM; (E) siRNA-mediated inhibition of IGFBP3 significantly attenuated the hypoxia-suppressed osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (40×); (F) quantification of ARS staining was measured at 7 days; (G,H) osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced by hypoxia at 7 days (G) and 14 days (H). Normoxia: black bars, hypoxia: white bars. * p < 0.5, ** p < 0.01.
Mentions: Since osteogenic differentiation is also important for BM-MSCs, we examined the effect of hypoxia on their osteogenic differentiation, as well as the involvement of IGFBP3 during osteogenesis. We used clonal BM-MSCs derived using our previously reported novel method of isolation [24,25] to examine the effect of hypoxia on osteogenic differentiation. As expected, hypoxia (2%) induced the mRNA expression of IGFBP3 in clonal BM-MSCs (Figure 6A). Hypoxia suppressed the osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (Figure 6B). Osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced by hypoxia in DMEM and ODM (Figure 6D). In addition, siRNA-mediated inhibition of IGFBP3 significantly attenuated the hypoxia-suppressed osteogenic differentiation of clonal BM-MSCs at 7 and 14 days (Figure 6E,F). Osteogenic induction markers such as RUNX2, osteocalcin, and osterix were significantly reduced by hypoxia at 7 days (Figure 6G) and 14 days (Figure 6H).

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