Effects of inhibiting PDK‑1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation <em>in vitro</em>

Bai,Yiguang; Zhang,Qiong; Zhou,Quan; Zhang,Yanan; Nong,Haibin; Liu,Mingfu; Shi,Zhuohua; Zeng,Gaofeng; Zong,Shaohui

doi:10.3892/mmr.2020.11757

Effects of inhibiting PDK‑1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation in vitro

Authors:
- Yiguang Bai
- Qiong Zhang
- Quan Zhou
- Yanan Zhang
- Haibin Nong
- Mingfu Liu
- Zhuohua Shi
- Gaofeng Zeng
- Shaohui Zong
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Affiliations: Department of Spine Osteopathia, The First Affiliated Hospital of Guangxi Medical University, Guangxi Medical University, Nanning, Guangxi 530021, P.R. China, Department of Nutrition and Food Hygiene, College of Public Hygiene of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China, Collaborative Innovation Center of Guangxi Biological Medicine, Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
Published online on: December 4, 2020 https://doi.org/10.3892/mmr.2020.11757
Article Number: 118
Copyright: © Bai et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Osteoblasts are the main functional cells in bone formation, which are responsible for the synthesis, secretion and mineralization of bone matrix. The PI3K/AKT signaling pathway is strongly associated with the differentiation and survival of osteoblasts. The 3‑phosphoinositide‑dependent protein kinase‑1 (PDK‑1) protein is considered the master upstream lipid kinase of the PI3K/AKT cascade. The present study aimed to investigate the role of PDK‑1 in the process of mouse osteoblast differentiation in vitro. In the BX‑912 group, BX‑912, a specific inhibitor of PDK‑1, was added to osteoblast induction medium (OBM) to treat bone marrow mesenchymal stem cells (BMSCs), whereas the control group was treated with OBM alone. Homozygote PDK1^flox/flox mice were designed and generated, and were used to obtain BMSCs^{PDK1flox/flox}. Subsequently, an adenovirus containing Cre recombinase enzyme (pHBAd‑cre‑EGFP) was used to disrupt the PDK‑1 gene in BMSCs^{PDK1flox/flox}; this served as the pHBAd‑cre‑EGFP group and the efficiency of the disruption was verified. Western blot analysis demonstrated that the protein expression levels of phosphorylated (p)‑PDK1 and p‑AKT were gradually increased during the osteoblast differentiation process. Notably, BX‑912 treatment and disruption of the PDK‑1 gene with pHBAd‑cre‑EGFP effectively reduced the number of alkaline phosphatase (ALP)‑positive cells and the optical density value of ALP activity, as well as the formation of cell mineralization. The mRNA expression levels of PDK‑1 in the pHBAd‑cre‑EGFP group were significantly downregulated compared with those in the empty vector virus group on days 3‑7. The mRNA expression levels of the osteoblast‑related genes RUNX2, osteocalcin and collagen I were significantly decreased in the BX‑912 and pHBAd‑cre‑EGFP groups on days 7 and 21 compared with those in the control and empty vector virus groups. Overall, the results indicated that BX‑912 and disruption of the PDK‑1 gene in vitro significantly inhibited the differentiation and maturation of osteoblasts. These experimental results provided an experimental and theoretical basis for the role of PDK‑1 in osteoblasts.

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