Increased FoxO3a expression prevents osteoblast differentiation and matrix calcification

Abstract

Forkhead Box O transcription factors play important roles in bone metabolism by defending against oxidative stress and apoptosis. FoxO3a is of special interest as it is the predominant isoform expressed in bone. In osteoblasts, the administration of 1,25 dihydroxyvitamin D₃ (1,25D₃) increases FoxO3a expression, and alters calcium handling. We therefore queried whether FoxO3a participates in vitamin D-mediated regulation of calcium transport pathways or matrix calcification, independent of reactive oxygen species (ROS) formation. To examine this possibility, we differentiated MC3T3-E1 cells into mature osteoblast-like cells over 7 days. This coincided with an increased ability to mineralize extracellular matrix. FoxO3a expression increased throughout differentiation. 1,25D₃ enhanced both FoxO3a mRNA and protein expression. Immunofluorescence microscopy found increased FoxO3a nuclear localization with differentiation and after treatment with 1,25D₃. Live cell ratiometric imaging with Fura-2AM identified significant L-type calcium channel mediated calcium uptake that was enhanced by 1,25D₃. We observed expression of both Ca_v1.2 and Ca_v1.3, although expression decreased throughout differentiation and was not altered by 1,25D₃ treatment. FoxO3a overexpression reduced calcium uptake and calcium deposition. FoxO3a overexpression also prevented alterations in calcium channel expression and the cell differentiation associated decrease in expression of Runx2 and increased expression of osteocalcin, findings consistent with a failure for the cells to differentiate. Based on both our expression and functional data, we suggest that high levels of FoxO3a prevent osteoblast differentiation and matrix calcification.

Keywords: Calcium deposition; Forkhead Box O3; MC3T3-E1 cells; Matrix calcification; Osteoblast differentiation; Osteoblast mineralization.

Fig. 1

MC3T3-E1 cells can be differentiated into an osteoblast like cell line. A) Relative Runx2 and OCN mRNA expression, normalized to 18S. B). FoxO3a, RXRα, Txnip and VDR mRNA expression, normalized to 18S. C) Protein expression throughout differentiation and D) Pearson's correlation coefficient for FoxO3a and DAPI colocalization. E) Representative immunoblots from C. F) Representative immunofluorescence images of FoxO3a (orange), DAPI (blue) and phalloidin (green) throughout differentiation. Scale bar = 8 μM. ** represents p < 0.01, n is at least 6 for mRNA and protein expression studies. N is at least 15 independent images from at least 3 slides per condition for immunofluorescence studies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

An L-type calcium channel mediates calcium uptake into 7-day differentiated MC3T3-E1 cells. A) mRNA expression of mediators of calcium fluxes throughout differentiation of MC3T3-E1 cells, normalized to 18S. B–I) Fura-2AM ratiometric live cell fluorescence imaging of single differentiated cells assessed as the magnitude of uptake, Δpeak (B, D, F and H) or rate (C, E, G and I) in the presence of either LaCl₃ (B and C), ruthenium red (D and E), NNC (F and G) or Felodipine (H and I), normalized to the untreated condition. J) Calcium deposition by pre-osteoblasts (pOB) or 7 day differentiated MC3T3-E1 cells. K) Calcium deposition in 7-day differentiated cells in the presence of the amount of additional CaCl₂ added to culture media, listed below the x-axis, data is per well and normalized to pOB in J and 0 CaCl₂ added in K. * represents p < 0.05 and ** represents p < 0.01. For calcium flux studies, n is at least 6 separate coverslips, with at least 20 cells per coverslip analyzed per condition, scale bar = 8 μM. For calcium deposition studies n is >6 per condition.

Fig. 3

1,25D₃ increases FoxO3a expression and nuclear localization in 7-day differentiated MC3T3-E1 cells. FoxO3a, RXRα and VDR A) mRNA expression normalized to 18S and B) protein expression, normalized to beta actin before and after 24 h treatment with 100 nM 1,25D₃ after differentiation. Representative blots are presented in Supplemental materials. C) Representative immunofluorescence images of FoxO3a (orange), DAPI (blue) and phalloidin (green) before and after 1,25D₃ treatment and D) Pearson's correlation coefficient for FoxO3a and DAPI colocalization before and after 1,25D₃ treatment, scale bar = 20 μM. * represents p < 0.05 and ** represents p < 0.01, n is >6 per condition in A&B and at least 15 independent images from at least 3 slides per condition for the immunofluorescence studies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 1,25D₃ increases FoxO3a expression and nuclear localization in 7-day differentiated MC3T3-E1 cells. FoxO3a, RXRα and VDR A) mRNA expression normalized to 18S and B) protein expression, normalized to beta actin before and after 24 h treatment with 100 nM 1,25D₃ after differentiation. Representative blots are presented in Supplemental materials. C) Representative immunofluorescence images of FoxO3a (orange), DAPI (blue) and phalloidin (green) before and after 1,25D₃ treatment and D) Pearson's correlation coefficient for FoxO3a and DAPI colocalization before and after 1,25D₃ treatment, scale bar = 20 μM. * represents p < 0.05 and ** represents p < 0.01, n is >6 per condition in A&B and at least 15 independent images from at least 3 slides per condition for the immunofluorescence studies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

1,25D₃ increases calcium influx in 7-day differentiated MC3T3-E1 cells via an L-type calcium channel. A) mRNA expression of mediators of calcium fluxes before and after 1,25D₃ treatment, normalized to 18S B–G) Fura-2AM ratiometric live cell fluorescence imaging of differentiated cells assessed as the magnitude of uptake, Δpeak (B, D and F) or rate (C, E and G) in the presence of either 1,25D₃ (B and C) or 1,25D₃ and LaCl₃ (D and E) or 1,25D₃ and Felodipine (F and G), normalized to either untreated (B & C), or just 1,25D₃ (D-G) condition. * represents p < 0.05 and ** represents p < 0.01, n is >6 per condition throughout, with 20 cells per coverslip analyzed per condition.

Fig. 5

Characterization of MC3T3-E1 cells over-expressing FoxO3a. A) Representative immunofluorescence images of MC3T3-E1 cells over-expressing FoxO3a immunostained for FoxO3a (orange) DAPI (blue) or phalloidin (green), scale bar = 8 μM. B) Immunoblot of lysate form wild-type (WT) or cells over-expressing FoxO3a (OE), blotted for FoxO3a. FoxO3a, RXRα and VDR C) mRNA expression (normalized to 18S) and D and E) protein expression from MC3T3-E1 cells overexpressing FoxO3a throughout differentiation. * represents p < 0.05 and ** represents p < 0.01, n = 9 per condition for protein and mRNA quantification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

FoxO3a over-expression attenuates L-type calcium channel mediated calcium influx into MC3T3-E1 cells. A) mRNA expression of mediators of calcium fluxes throughout differentiation in MC3T3-E1 cells over-expressing FoxO3a. Data normalized to 18S as an internal control. B–E) Fura-2AM ratiometric live cell fluorescence imaging of MC3T3-E1 wild-type (WT) or cells over-expressing (OE) FoxO3a assessed as the magnitude of uptake, Δpeak (B and D) or rate (C and E), normalized to the wild-type cell response. L-type calcium channel activity was assessed by the addition of Felodipine (FE), (D and E), n is >6 per condition throughout.

Fig. 7

FoxO3a over-expression in MC3T3-E1 cells attenuates matrix calcification. A) Representative images of MC3T3-E1 cells stained with Alizarin red pre (pOB) and 7 days post (7d) differentiation. Data from wildtype cells were included for comparison. B) Quantification of Alizarin red staining from A) and after addition of the amount of calcium chloride written below the x-axis C), data is per well and normalized to pOB in B and 0 CaCl₂ added in C. D and E) Fura2 ratiometric live cell fluorescence imaging of MC3T3-E1 cells over-expressing (OE) FoxO3a assessed as the magnitude of uptake, Δpeak (D) or rate (E) in the absence and presence of 100 nM 1,25D₃ for 24 h, normalized to the untreated condition. * represents p < 0.05 and ** represents p < 0.01, n is = 9 per condition throughout. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

FoxO3a over-expression inhibits osteoblast differentiation. A) Runx2 mRNA expression and B) OCN mRNA expression in MC3T3-E1 cells throughout differentiation. Control is wild-type cells. All data normalized to 18S. * represents p < 0.05 and ** represents p < 0.01, n = 9 per cell type.