Akt Activation is Required for TGF-β1-Induced Osteoblast Differentiation of MC3T3-E1 Pre-Osteoblasts

<div><p>Background</p><p>We have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation.</p><p>Methodology/Principal Findings</p><p>MC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation.</p><p>Conclusions</p><p>TGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells.</p></div

Abrogation of PI3K signaling inhibits TGF-β1-mediated osteoblast differentiation in MC3T3-E1 cells.

<p>(A) Confluent MC3T3-E1 cells were cultured in OBM with and without 0.1 ng/mL TGF-β1 in the absence or presence of the PI3K inhibitor LY294002 (5, 10, or 25 µM) for 72 h. ALP activity was visualized by ALP staining of cells. (B) Cells were cultured in OBM with and without 0.1 ng/mL TGF-β1 in the absence or presence of 10 µM LY294002 for 72 h, and ALP activity was measured. Each experiment was performed in triplicate, and the data represent the means ± S.E. (<i>n</i> = 3). The Bonferroni correction for multiple comparisons was applied. *, <i>p</i><0.001.</p

Overexpression of CA-Akt reverses the inhibition of ALP activity induced by repeated administration of TGF-β1.

<p>MC3T3-E1 cells were infected with CA-Akt vector or Mock vector. Phosphorylation of Akt is evident in CA-Akt cells cultured in α-MEM (A). Then cells were treated with or without repeated administration of 0.1 ng/mL TGF-β1 for 72 h in OBM. The levels of phosphorylated Akt in these cells were detected by western blot analysis (B). ALP staining (C) and ALP activity (D) were assessed following repeated TGF-β1 administration. Figures shown represent at least three independent experiments. Values represent mean ± S.E. (<i>n</i> = 4). Bonferroni correction for multiple comparisons was applied. *, <i>p</i><0.001.</p

Primers used for qRT-PCR.

<p>Primers used for qRT-PCR.</p

Forced expression of dominant-negative Akt abrogates the pro-differentiation effect of IGF-1.

<p>MC3T3-E1 cells were infected with DN-Akt vector or Mock vector (A), and then cells were cultured in OBM with and without a triple administration of 0.1 ng/mL TGF-β1 for 72 h. Exogenous IGF-1 (200 ng/mL) was applied in conjunction with administration of TGF-β1 and the expression of <i>Alp</i> and <i>Oc</i> mRNA levels measured in Mock (B, C) and DN-Akt (D, E) cells. Gene expression was analyzed by qRT-PCR, and mRNA levels were normalized to that of 18S rRNA and measured in triplicate. Values represent mean ± S.E. (<i>n</i> = 3). Bonferroni correction for multiple comparisons was applied. *, <i>p</i><0.001; ***, <i>p</i><0.05.</p

Repeated TGF-β1 treatment inhibits expression of osteoblast differentiation markers in MC3T3-E1 cells.

<p>Confluent MC3T3-E1 cells were treated for 72 h with OBM, OBM with a single administration of 0.1 ng/mL TGF-β1, and OBM with a triple administration of 0.1 ng/mL TGF-β1. Repeated TGF-β1 treatment significantly decreased the expression of <i>Alp</i> (A), <i>Oc</i> (C), and <i>Bsp</i> (D). The decrease in the expression of <i>Igf-1</i> (B) was not statistically significant. Expression of these genes was analyzed by qRT-PCR, and their mRNA levels were normalized to that of 18S rRNA and measured in triplicate. Values represent mean ± S.E. (<i>n</i> = 3). Bonferroni correction for multiple comparisons was applied. *, <i>p</i><0.001; **, <i>p</i><0.01; ***, <i>p</i><0.05.</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

Characterization of ALP-positive cells derived from human iPSCs.

<p>(a) Single cells from hEBs were cultured with various cytokines for 2 weeks and stained for ALP activity. The cells were cultured in α-MEM containing 10% FBS (α-MEM); α-MEM containing 10% FBS, ascorbic acid, and β-glycerophosphate (β-GP) (OBM); OBM with FGF-2 and TGF-β1 (FGF2 + TGF); OBM with FGF-2, TGF-β1, and IGF-1 (FGF2 + TGF + IGF); OBM with FGF-2 and BMP-2/-7 (FGF2 + BMP); or OBM with FGF-2, BMP-2/-7, and IGF-1 (FGF2 + BMP + IGF). The percentages shown indicate the frequency of ALP-positive cells determined by FACS analysis. (b) FACS analysis for the isolation of ALP-positive cells (right) and isotype control (left). (c) Expression of ALP isoenzymes: germ cell-specific ALP (G), placenta-specific ALP (P), intestine-specific ALP (I), tissue-nonspecific ALP (T), and β-actin (B) in parental hiPSCs, isolated ALP-positive cells, and isolated ALP-negative cells. (d) FACS analysis of CD90 and E-cadherin in the TNAP-positive population. (e) Morphology of TNAP-positive (TNAP+) and TNAP-negative (TNAP−) cells.</p

SEM and TEM images of TNAP-positive cells induced to differentiate into osteocyte-like cells.

<p>After isolation by FACS, TNAP-positive and -negative cells were cultured in OBM for 120 days. (a) SEM images of TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells. (b) Images of toluidine blue-stained semi-thin sections of TNAP-positive cells are shown at low (left) and high (right) magnification with light microscopy. (c) TEM image of TNAP-positive cells. Arrowheads indicate cytoplasmic processes.</p

TNAP-positive cells express various osteocyte markers.

<p>(a) Osteogenic differentiation was confirmed by Alizarin Red staining after 40 days in OBM. The upper panels are whole-well images and lower panels are magnified images. (b) Phase-contrast images of TNAP-negative and -positive cells derived from hiPSCs at day 40 of culture in OBM (upper panel). The black box in the upper images represents the region shown in the middle and lower images. (c) Comparison of the expression of osteocyte marker genes between TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells by qRT-PCR. Osteogenic-differentiated HPDLCs (ODH) were used as a control. (d) TNAP-positive cells were treated with vehicle (white bars), 10 nM vitamin D3 (gray bars), or 50 nM vitamin D3 (black bars) for 6 days. (e) Comparison of expression of osteocyte marker genes between TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells by RT-PCR. Abbreviations: SOST, sclerostin; RELN, reelin; NPY, neuropeptide Y. Expression of these genes was analyzed by qRT-PCR, and mRNA levels of the genes were normalized to that of <i>GAPDH</i>. The experiments were performed in triplicate. Values represent mean ± S.D. (<i>n</i>  =  4). *<i>p</i><0.05, **<i>p</i><0.01.</p