MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

Abstract

Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3' UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo.

Fig. 1.

Osteoblast differentiation of hMSC. hMSCs were induced to osteoblast differentiation. (A) Osteoblast differentiation was confirmed by qRT-PCR analysis of osteoblast marker genes (RUNX2, ALP, and OC normalized to β-actin). (B) ALP activity was measured during the course of differentiation. miR-138 expression was measured during osteoblast differentiation. White bars represent noninduced samples, and black bars represent induced samples. ***P < 0.001 between noninduced and induced samples. (C) ALP and Alizarin Red staining were performed at day 15. (D) miR-138 expression (n = 3 for all experiments).

Fig. 2.

miR-138 inhibits osteoblast differentiation. To study the effect of miR-138 on osteoblast differentiation, hMSCs transfected with 25 nM miR-C, premiR-138, or antimiR-138 were induced to osteoblast differentiation for 15 d. (A) Osteoblast differentiation was evaluated with qRT-PCR analysis of osteoblast marker genes (RUNX2, OSX, ALP, and OC normalized to β-actin) at day 15. (B) Alkaline phosphatase activity was measured at day 10 of osteoblast differentiation. (C) ALP and Alizarin Red staining were performed at day 15. *P < 0.05; ***P < 0.001 (n = 3 for all experiments).

Fig. 3.

miR-138 inhibits ectopic bone formation in vivo. hMSCs were transfected with 25 nM miR-C, premiR-138, or antimiR-138 and implanted into NOD/SCID mice. (A) qRT-PCR analysis of osteoblast marker genes (RUNX2, OSX, ALP, and OC normalized to β-actin) was performed after 1 wk of implantation. (B) H&E staining was performed after 8 wk of implantation. Bone formation was quantified as total bone volume per total volume from H&E staining, and it was expressed as fold change of miR-C. Four implants per treatment were engrafted into mice, and three sections of each implant were quantified to minimize variations within the implants. *P < 0.05; **P < 0.01.

Fig. 4.

FAK is a potential target of miR-138. (A) PTK2 gene expression profile during osteoblast differentiation of hMSCs was quantified with qRT-PCR. (B) Computational analysis was performed for the complementarities of miR-138 seed sequence to the 3′ UTR of PTK2 and conservation of the putative binding site in vertebrates. (C) Huh7 cells were transfected with 20 ng empty PsiCHECK-2 or PsiCHECK-2-PTK2 vector. Cells were cotransfected with 0, 10, or 20 nM of the premiR-138 or 2 0nM of a negative control. Firefly and Renilla luciferases were measured in cell lysates, and values are normalized to the PsiCHECK vector and presented as fold change of miR-C. **P < 0.01; ***P < 0.001.

Fig. 5.

miR-138 targets FAK and inhibits ERK pathway. hMSCs were transfected with 25 nM miR-C, premiR-138, or antimiR-138 and induced to osteoblast differentiation. (A) PTK2 gene expression at day 2. (B) Western blot analysis for FAK protein and phosphorylation of FAK and ERK1/2 were performed on day 2. Graphs represent quantifications of Western blot results relative to tubulin. (C) Phosphorylation of Runx2 was analyzed by Western blot at day 10. qRT-PCR analysis of OSX gene expression at day 10.

Fig. 6.

A proposed model for miR-138–mediated suppression of osteoblast differentiation. miR-138 is expressed in undifferentiated MSC and suppresses FAK translation, thereby decreases phosphorylation of FAK and its downstream target ERK1/2. Inhibition of the cascade results in decreased phosphorylation of Runx2 and expression of OSX; subsequently, this results in suppression of osteoblast differentiation of MSC.