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, 43 (1), 58-65

Fat Mass and Obesity-Associated (FTO) Stimulates Osteogenic Differentiation of C3H10T1/2 Cells by Inducing Mild Endoplasmic Reticulum Stress via a Positive Feedback Loop With p-AMPK

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Fat Mass and Obesity-Associated (FTO) Stimulates Osteogenic Differentiation of C3H10T1/2 Cells by Inducing Mild Endoplasmic Reticulum Stress via a Positive Feedback Loop With p-AMPK

Hyo-Eun Son et al. Mol Cells.

Abstract

Fat mass and obesity-associated (FTO) gene helps to regulate energy homeostasis in mammals by controlling energy expenditure. In addition, FTO functions in the regulation of obesity and adipogenic differentiation; however, a role in osteogenic differentiation is unknown. This study investigated the effects of FTO on osteogenic differentiation of C3H10T1/2 cells and the underlying mechanism. Expression of osteogenic and endoplasmic reticulum (ER) stress markers were characterized by reverse-transcriptase polymerase chain reaction and western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity. BMP2 treatment increased mRNA expression of osteogenic genes and FTO. Overexpression of FTO increased expression of the osteogenic genes distal-less homeobox5 (Dlx5) and runt-related transcription factor 2 (Runx2). Activation of adenosine monophosphate-activated protein kinase (AMPK) increased FTO expression, and there was a positive feedback loop between FTO and p-AMPK. p-AMPK and FTO induced mild ER stress; however, tunicamycin-induced severe ER stress suppressed FTO expression and AMPK activation. In summary, FTO induces osteogenic differentiation of C3H10T1/2 cells upon BMP2 treatment by inducing mild ER stress via a positive feedback loop with p-AMPK. FTO expression and AMPK activation induce mild ER stress. By contrast, severe ER stress inhibits osteogenic differentiation by suppressing FTO expression and AMPK activation.

Keywords: C3H10T1/2 cells; adenosine monophosphate-activated protein kinase; endoplasmic reticulum stress; fat mass and obesity-associated; osteoblast.

Conflict of interest statement

Disclosure

The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. BMP2 treatment induces FTO expression in C3H10T1/2 cells
(A and B) RT-PCR and real-time PCR analyses were performed using total RNA isolated from C3H10T1/2 cells treated with BMP2 (+, 0.125 μg/ml; ++, 0.25 μg/ml) for 1 day. (C and D) RT-PCR and real-time PCR analyses were performed using total RNA isolated from C3H10T1/2 cells treated with 0.25 μg/ml BMP2 for 12 or 24 h. (E and F) C3H10T1/2 cells were treated with BMP2 for the indicated durations and harvested for western blot analysis using the indicated antibodies. *P < 0.05, **P < 0.001, and ***P < 0.005 compared with untreated control cells. Data represent the mean ± SEM of three individual experiments. All experiments were independently repeated at least three times.
Fig. 2
Fig. 2. Overexpression of FTO induces osteogenic differentiation of C3H10T1/2 cells
(A–C) C3H10T1/2 cells were transfected with pcDNA3.1 (2 μg) or pCMV-FTO (+, 1 μg; ++, 2 μg) for 6 h and treated with BMP2 (0.25 μg/ml) for 1 day. (A) RT-PCR analysis was performed using total RNA isolated from cells and primers targeting FTO, Dlx5, Runx2, and β-actin. (B) Real-time PCR was performed using total RNA isolated from cells. (C) Western blot analysis was performed using the indicated antibodies. (D) C3H10T1/2 cells were transfected with pCMV-FTO (+, 0.2 μg; ++, 0.4 μg) or treated with BMP2 (0.25 μg/ml) for 4 days. *P < 0.05, **P < 0.001, and ***P < 0.005 compared with untreated control cells. Data represent the mean ± SEM of three individual experiments. All experiments were independently repeated at least three times.
Fig. 3
Fig. 3. Knock-down of FTO attenuates BMP2-induced osteogenic differentiation of C3H10T1/2 cells
C3H10T1/2 cells were transfected with siFTO (+, 100 nM; ++, 200 nM) for 6 h and then treated with BMP2 (0.25 μg/ml) for 1 day. (A) RT-PCR analysis was performed using total RNA isolated from cells. (B) The real-time PCR analysis was performed using total RNA isolated from cells. (C) Western blot analysis was performed using the indicated antibodies. (D) C3H10T1/2 cells were transfected with siFTO for 6 h and then treated with BMP2 (0.25 μg/ml) for 4 days. ALP staining was performed. All experiments were independently repeated at least three times. Data represent the mean ± SEM of three individual experiments. *P < 0.05 and **P < 0.001 compared with untreated control cells. #P < 0.05, ##P < 0.01, and ###P < 0.005 compared with BMP2 treatment cells. All experiments were independently repeated at least three times.
Fig. 4
Fig. 4. Activation of AMPK enhances FTO expression in C3H10T1/2 cells
(A and B) C3H10T1/2 cells were transfected with pcDNA3.0-AMPK (2 μg) for 6 h and then incubated for a further 12 h. (A) RT-PCR analysis was performed using total RNA extracted from cells. (B) Western blot analysis was performed using the indicated antibodies. (C and D) C3H10T1/2 cells were treated with BMP2 (0.25 μg/ml) for 24 h. Com.C (+, 0.1 μM; ++, 1 μM) was added 3 h prior to harvest. (C) RT-PCR analysis was performed using total RNA extracted from cells. (D) Western blot analysis was performed using the indicated antibodies. (E) C3H10T1/2 cells were transiently transfected with pc-DNA3.0AMPK (0.4 μg/well) and/or treated with BMP2 (0.25 μg/ml) for 4 days, and then stained with BCIP/NBT liquid substrate. **P < 0.001 compared with untreated control cells. #P < 0.05 compared with CA-AMPK transfected cells. (F) C3H10T1/2 cells were treated with or without BMP2 (0.25 μg/ml) in the presence of Com.C (1 μM) for 4 days and then ALP staining was performed. *P < 0.05 compared with untreated control cells. #P < 0.05 compared with BMP2 treated cells. (G) C3H10T1/2 cells were transfected with FTO-Luc (2 μg), pc-DNA3.0-AMPK (2 μg) and b-galactosidase plasmids. After 24 h, cultures were treated with BMP2 (0.25 μg/ml) or Com.C (1 μM) for 48 h. Luciferase activity was measured and presented as the mean ± SEM of individual experiments. ***P < 0.005 compared with untreated control cells. &&&P < 0.005 compared with BMP2 treated cells. ###P < 0.005 compared with pc-DNA-3.0-AMPK-transfected cells. All experiments were independently repeated at least three times.
Fig. 5
Fig. 5. FTO induces phosphorylation of AMPK in C3H10T1/2 cells
(A) C3H10T1/2 cells were transfected with pCMV-FTO (2 μg) for 6 h and then incubated for a further 12 h. Western blot assay analysis was performed using the indicated antibodies. (B) C3H10T1/2 cells were transfected with siFTO for 6 h and then treated with BMP2 (0.25 μg/ml) for 12 h. Western blot analysis was performed using the indicated antibodies. All experiments were independently repeated at least three times.
Fig. 6
Fig. 6. FTO and p-AMPK induce mild ER stress
(A) C3H10T1/2 cells were transfected with siFTO for 6 h and then treated with BMP2 (0.25 μg/ml) and Com.C (1 μM) for 1 day. RT-PCR was performed using total RNA isolated from cells. (B and D) C3H10T1/2 cells were transfected with pCMV-FTO (2 μg) for 6 h and then treated with Com.C (1 μM) for 24 h. (C and E) C3H10T1/2 cells were transfected with pc-DNA3.0-AMPK (2 μg) and siFTO for 6 h and then incubated for a further 12 h. RT-PCR (B and C) and western blot (D and E) analyses of ER stress markers (ATF4 and CHOP) were performed. All experiments were independently repeated at least three times.
Fig. 7
Fig. 7. Severe ER stress induced by TM abrogates BMP2-induced upregulation of FTO and p-AMPK
(A) C3H10T1/2 cells were treated with BMP2 (0.25 μg/ml) and/or TM (100 ng/ml) for 24 h. RT-PCR was performed using total RNA isolated from cells. (B) C3H10T1/2 cells were treated with BMP2 (0.25 μg/ml) and/or TM (100 ng/ml) for 12 h. Western blotting was performed with the indicated antibodies. (C) C3H10T1/2 cells were treated with BMP2 (0.25 μg/ml) and/or TM (100 ng/ml) for 4 days. ALP staining was performed. **P < 0.001 compared with untreated control cells. ##P < 0.001 compared with BMP2 treated cells. All experiments were independently repeated at least three times. (D) FTO induces osteogenic differentiation of C3H10T1/2 cells via a positive feedback loop with p-AMPK. FTO and p-AMPK stimulate osteogenic differentiation by inducing mild ER stress; however, TM-induced severe ER stress suppresses FTO expression and AMPK activation.

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References

    1. Canalis E., Economides A.N., Gazzerro E. Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev. 2003;24:218–235. doi: 10.1210/er.2002-0023. - DOI - PubMed
    1. Fischer J., Koch L., Emmerling C., Vierkotten J., Peters T., Bruning J.C., Ruther U. Inactivation of the Fto gene protects from obesity. Nature. 2009;458:894–898. doi: 10.1038/nature07848. - DOI - PubMed
    1. Frayling T.M., Timpson N.J., Weedon M.N., Zeggini E., Freathy R.M., Lindgren C.M., Perry J.R., Elliott K.S., Lango H., Rayner N.W., et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science. 2007;316:889–894. doi: 10.1126/science.1141634. - DOI - PMC - PubMed
    1. Gething M.J., Sambrook J. Protein folding in the cell. Nature. 1992;355:33–45. doi: 10.1038/355033a0. - DOI - PubMed
    1. Ghemrawi R., Battaglia-Hsu S.F., Arnold C. Endoplasmic reticulum stress in metabolic disorders. Cells. 2018;7:E63. doi: 10.3390/cells7060063. - DOI - PMC - PubMed
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