Suzuki Eiichi, Ochiai-Shino Hiromi, Aoki Hideto, Onodera Shoko, Saito Akiko, Saito Atsushi, Azuma Toshifumi
Department of Periodontology, Tokyo Dental College, Tokyo, Japan; Oral Health Science Center, Tokyo Dental College, Tokyo, Japan.
Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
PLoS One. 2014 Dec 3;9(12):e112566. doi: 10.1371/journal.pone.0112566. eCollection 2014.
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.
METHODOLOGY/PRINCIPAL FINDINGS: 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.
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.
我们之前报道过，用转化生长因子-β1（TGF-β1）反复处理人牙周膜细胞和小鼠前成骨细胞MC3T3-E1，会抑制它们的成骨细胞分化，原因是胰岛素样生长因子-1（IGF-1）分泌减少。我们还发现IGF-1/PI3K信号在TGF-β1处理诱导的成骨细胞分化中起重要作用；然而，控制这一过程的下游信号仍然未知。本研究的目的是调查Akt激活对于成骨细胞分化是否必要。
方法/主要发现：将MC3T3-E1细胞在含有或不含有0.1 ng/mL TGF-β1的成骨细胞分化培养基（OBM）中培养。每12小时更换一次含有TGF-β1的OBM，以实现TGF-β1的反复给药。MC3T3-E1细胞用表达组成型激活（CA）或显性负性（DN）-Akt的逆转录病毒载体感染。与单次TGF-β1处理相比，反复TGF-β1处理使碱性磷酸酶（ALP）活性和成骨细胞标志物mRNA水平显著降低。然而，CA-Akt的表达在TGF-β1处理后恢复了ALP活性。令人惊讶的是，随着TGF-β1给药次数增加，多次TGF-β1处理后ALP活性增加。Akt的激活显著增强了骨钙素的表达，但TGF-β1处理抑制了这一过程。在所有培养基条件下，CA-Akt的表达显著增强了MC3T3-E1细胞的矿化。外源性IGF-1恢复了反复TGF-β1给药导致的成骨细胞相关基因表达下调。然而，在表达DN-Akt的细胞中，无论是否进行IGF-1处理，这些水平仍然受到抑制。这些发现表明，Akt激活对于TGF-β1诱导的MC3T3-E1细胞成骨细胞分化的早期阶段是必要的。然而，Akt激活不足以逆转TGF-β1在成骨细胞分化后期的抑制作用。
根据Akt磷酸化状态，TGF-β1可能是MC3T3-E1细胞成骨细胞分化的诱导剂或抑制剂。我们的结果表明，Akt是TGF-β1诱导的MC3T3-E1细胞成骨细胞分化的分子开关。
