Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 May 20;20(10):2481.
doi: 10.3390/ijms20102481.

Phloretin Suppresses Bone Morphogenetic Protein-2-Induced Osteoblastogenesis and Mineralization via Inhibition of Phosphatidylinositol 3-kinases/Akt Pathway

Affiliations
Free PMC article

Phloretin Suppresses Bone Morphogenetic Protein-2-Induced Osteoblastogenesis and Mineralization via Inhibition of Phosphatidylinositol 3-kinases/Akt Pathway

Ayumu Takeno et al. Int J Mol Sci. .
Free PMC article

Abstract

Phloretin has pleiotropic effects, including glucose transporter (GLUT) inhibition. We previously showed that phloretin promoted adipogenesis of bone marrow stromal cell (BMSC) line ST2 independently of GLUT1 inhibition. This study investigated the effect of phloretin on osteoblastogenesis of ST2 cells and osteoblastic MC3T3-E1 cells. Treatment with 10 to 100 µM phloretin suppressed mineralization and expression of osteoblast differentiation markers, such as alkaline phosphatase (ALP), osteocalcin (OCN), type 1 collagen, runt-related transcription factor 2 (Runx2), and osterix (Osx), while increased adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid-binding protein 4, and adiponectin. Phloretin also inhibited mineralization and decreased osteoblast differentiation markers of MC3T3-E1 cells. Phloretin suppressed phosphorylation of Akt in ST2 cells. In addition, treatment with a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor, LY294002, suppressed the mineralization and the expression of osteoblast differentiation markers other than ALP. GLUT1 silencing by siRNA did not affect mineralization, although it decreased the expression of OCN and increased the expression of ALP, Runx2, and Osx. The effects of GLUT1 silencing on osteoblast differentiation markers and mineralization were inconsistent with those of phloretin. Taken together, these findings suggest that phloretin suppressed osteoblastogenesis of ST2 and MC3T3-E1 cells by inhibiting the PI3K/Akt pathway, suggesting that the effects of phloretin may not be associated with glucose uptake inhibition.

Keywords: MC3T3-E1 cell; PI3K/Akt pathway; ST2 cell; adipogenesis; bone marrow stromal cell; glucose uptake; osteoblast; osteoblastogenesis; phloretin.

Conflict of interest statement

The authors declare no conflict of interest. Eli Lilly Japan had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
The effects of phloretin on osteoblast differentiation and mineralization in marrow stromal ST2 cells. (A) After reaching confluence, ST2 cells were incubated in osteoblast differentiation medium with 0, 10, 50, and 100 µM phloretin, and von Kossa staining and Alizarin red staining were performed at day 14. The results are representative of three different experiments. (BK) After reaching confluence, the cells were incubated in osteoblast differentiation medium with 0–100 µM phloretin. The mRNA expression of osteoblast differentiation markers (Alp, Col-1, Ocn, Runx2, and Osx) was examined on day 3 and day 5 by real-time PCR. The results are expressed as mean ± SE (n ≥ 7). * p < 0.05, ** p < 0.01, *** p < 0.001. Phl; phloretin.
Figure 2
Figure 2
The effects of phloretin on the expression of adipocyte differentiation markers during BMP-2-induced osteoblastogenesis in ST2 cells. (AJ) ST2 cells were incubated in osteoblast differentiation medium with 0–100 µM phloretin, and the mRNA expression of adipogenic markers, Pparγ, C/ebpα, Fas, Fabp4, and Apn, was examined on day 3 and day 5 by real-time PCR. The results are expressed as mean ± SE (n ≥ 5). * p < 0.05, ** p < 0.01, *** p < 0.001. Phl; phloretin.
Figure 3
Figure 3
The effects of phloretin on mineralization and expression of osteoblastogenic and adipogenic markers in MC3T3-E1 cells. (A,B) After reaching confluence, MC3T3-E1 cells were incubated in osteoblast differentiation medium with 0, 10, and 50 µM phloretin, and von Kossa staining, Alizarin red staining, and its quantification were performed on day 14. Quantification results are expressed as mean ± SE (n = 6). *** p < 0.001. (CL) After reaching confluence, MC3T3-E1 cells were incubated in osteoblast differentiation medium with 0 and 50 µM phloretin. The mRNA expression of osteoblast differentiation markers (Alp, Col-1, Ocn, Runx2, and Osx) and adipocyte differentiation markers (Pparγ, C/ebpα, Fas, Fabp4, and Apn) was examined on day 3 by real-time PCR. The results are expressed as mean ± SE (n ≥ 5). *** p < 0.001. Phl; phloretin.
Figure 4
Figure 4
The involvement of suppression of PI3K/Akt pathway in the phloretin-induced downregulation of osteoblast differentiation markers and upregulation of adipocyte differentiation markers during BMP-2-induced osteoblastogenesis in ST2 cells. (AC) After reaching confluence, ST2 cells were incubated in osteoblast differentiation medium for 2 days. Thereafter, the cells were treated with 100 µM phloretin for up to 12 h, total protein was extracted, and western blot analysis was performed to examine the time-dependent effect of phloretin on Akt (A). To test dose-dependency, the cells were treated with phloretin (0 to 100 µM) for 12 h (B). Quantification of the bands was performed (C). The results are representative of three experiments. Quantification results are expressed as mean ± SE (n = 3). * p < 0.05, ** p < 0.01. (D) After reaching confluence, ST2 cells were incubated in osteoblast differentiation medium with 0 or 5 µM LY294002, and von Kossa staining and Alizarin red staining were performed on day 14. (EN) After reaching confluence, ST2 cells were incubated in osteoblast differentiation medium with 0 to 10 µM LY294002. Total mRNA was extracted on day 3, and the mRNA expression of osteoblastogenic markers (Alp, Col-1, Ocn, Runx2, and Osx) and adipogenic markers (Pparg, C/ebpa, Fas, Fabp4, and Apn) was examined by real-time PCR. The results are expressed as mean ± SE (n ≥ 5). * p < 0.05, ** p < 0.01, *** p < 0.001. Phl; phloretin, LY; LY294002.
Figure 5
Figure 5
The effects of Glut1 silencing on mineralization and the expression of osteoblast and adipocyte differentiation markers in ST2 cells. (A,CL) After transfection of siRNA for Glut1 and non-specific control, the cells were incubated in osteoblast differentiation medium for 3 days. Thereafter, the mRNA expression of osteoblastogenic markers (Alp, Col-1, Ocn, Runx2, and Osx) and adipogenic markers (Pparg, C/ebpa, Fas, Fabp4, and Apn) was examined by real-time PCR. 36b4 was used to normalize the differences in the efficiencies of reverse transcription. The results are expressed as mean ± SE (n ≥ 10). * p < 0.05, ** p < 0.01, *** p < 0.001. NC; negative control. (B) After transfection of siRNA, the cells were incubated in osteoblast differentiation medium, and the mineralization staining was performed on day 14.

Similar articles

See all similar articles

Cited by 1 article

References

    1. Chamberlain G., Fox J., Ashton B., Middleton J. Concise review: Mesenchymal stem cells: Their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 2007;25:2739–2749. doi: 10.1634/stemcells.2007-0197. - DOI - PubMed
    1. Meunier P., Aaron J., Edouard C., Vignon G. Osteoporosis and the replacement of cell populations of the marrow by adipose tissue. A quantitative study of 84 iliac bone biopsies. Clin. Orthop. Relat. Res. 1971;80:147–154. doi: 10.1097/00003086-197110000-00021. - DOI - PubMed
    1. Moerman E.J., Teng K., Lipschitz D.A., Lecka-Czernik B. Aging activates adipogenic and suppresses osteogenic programs in mesenchymal marrow stroma/stem cells: The role of PPAR-gamma2 transcription factor and TGF-beta/BMP signaling pathways. Aging Cell. 2004;3:379–389. doi: 10.1111/j.1474-9728.2004.00127.x. - DOI - PMC - PubMed
    1. Schwartz A.V., Sigurdsson S., Hue T.F., Lang T.F., Harris T.B., Rosen C.J., Vittinghoff E., Siggeirsdottir K., Sigurdsson G., Oskarsdottir D., et al. Vertebral bone marrow fat associated with lower trabecular BMD and prevalent vertebral fracture in older adults. J. Clin. Endocrinol. Metab. 2013;98:2294–2300. doi: 10.1210/jc.2012-3949. - DOI - PMC - PubMed
    1. Sui B., Hu C., Liao L., Chen Y., Zhang X., Fu X., Zheng C., Li M., Wu L., Zhao X., et al. Mesenchymal progenitors in osteopenias of diverse pathologies: Differential characteristics in the common shift from osteoblastogenesis to adipogenesis. Sci. Rep. 2016;6:30186. doi: 10.1038/srep30186. - DOI - PMC - PubMed
Feedback