HIF-1α Regulates Glucocorticoid-Induced Osteoporosis Through PDK1/AKT/mTOR Signaling Pathway

Abstract

Long-term and high dose glucocorticoid treatment can cause decreased viability and function of osteoblasts, which leads to osteoporosis and osteonecrosis. In this study, we investigated the role and mechanism of action of HIF-1α in glucocorticoid-induced osteogenic inhibition in MC3T3-E1 cells. Our results showed that HIF-1α protein expression was reduced when MC3T3-E1 cells were exposed to dexamethasone (Dex) at varying concentrations ranging from 10^-9 to 10^-6 M. PDK1 expression was also decreased in MC3T3-E1 cells after dexamethasone treatment. MC3T3-E1 cells when treated with the glucocorticoid receptor antagonist RU486 along with dexamethasone showed enhanced HIF-1α expression. In addition, upregulated expression of HIF-1α was capable of promoting the osteogenic ability of MC3T3-E1 cells and PDK1 expression. However, the HIF-1α antagonist 2-methoxyestradiol (2-ME) had a reverse effect in MC3T3-E1 cells exposed to dexamethasone. Furthermore, the PDK1 antagonist dichloroacetate could repress the osteogenic ability of MC3T3-E1 cells, although HIF-1α was upregulated when transduced with adenovirus-HIF-1α construct. The PDK1 agonist PS48 was able to promote the osteogenic ability of MC3T3-E1 cells treated with dexamethasone. Importantly, the protein levels of p-AKT and p-mTOR were increased in MC3T3-E1 cells treated with dexamethasone after PS48 treatment. in vivo, the PDK1 agonist PS48 could maintain the bone mass of mice treated with dexamethasone. This study provides a new understanding of the mechanism of glucocorticoid-induced osteoporosis.

Keywords: HIF-1α; PDK1; glucocorticoid; osteoblast; osteoporosis.

Figure 1

Dexamethasone inhibited the osteogenic function of MC3T3-E1 cells via regulating HIF-1α expression. (A) The protein expression levels of HIF-1α, PDK1, OCN, Runx2, and GAPDH were determined by western blotting in MC3T3-E1 cells treated with a varying concentration of dexamethasone for 14 days. (B) ALP and Alizarin red staining were performed to detect the osteogenic function of MC3T3-E1 cells treated with vehicle and dexamethasone. (C) Western blotting was performed to detect the protein expression levels of HIF-1α, PDK1, Runx2, and GAPDH in MC3T3-E1 cells treated with vehicle, dexamethasone, and dexamethasone + RU486, respectively. Differences were considered as statistically significant when p < 0.05. ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 2

HIF-1α mediated dexamethasone-induced osteogenic inhibition of osteoblasts via regulating PDK1 expression. (A) Osteogenic ability was confirmed by ALP and Alizarin red staining in MC3T3-E1 cells treated with Ad-NC, Ad- HIF-1α + dexamethasone, Ad-NC + dexamethasone and Ad-HIF-1α + dexamethasone, respectively. Scale Bar = 20 μm. (B) The protein expression levels of HIF-1α, PDK1, Runx2, and GAPDH were detected by western blotting in MC3T3-E1 cells treated with Ad-NC, Ad-HIF-1α + dexamethasone, Ad-NC + dexamethasone and Ad-HIF-1α + dexamethasone, respectively. (C) Osteogenic ability was confirmed by ALP staining in MC3T3-E1 cells treated with empty vector, dexamethasone, 2-ME and dexamethasone + 2-ME, respectively. Scale Bar = 20 μm. (D) The protein expression levels of HIF-1α, PDK1, Runx2, OCN, and GAPDH were detected by western blotting in MC3T3-E1 cells treated with empty vector, dexamethasone, 2-ME and dexamethasone + 2-ME, respectively. Differences were considered as statistically significant when p < 0.05. ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 3

Repression of PDK1 reversed the protective effect of Adenovirus-HIF-1α on glucocorticoid-induced osteogenic inhibition in MC3T3-E1 cells. (A) The protein expression levels of osteogenic markers Runx2, OCN, ALP, and GAPDH were determined by western Blotting in MC3T3-E1 cells exposed to empty vector, dexamethasone, dexamethasone + Ad-HIF-1α, and dexamethasone + Ad-HIF-1α+ DCA, respectively. (B) ALP and Alizarin red Staining were performed to determine the osteogenic ability of MC3T3-E1 cells exposed to empty vector, dexamethasone, dexamethasone + Ad-HIF-1α, dexamethasone + Ad-HIF-1α+ DCA, respectively. Scale Bar = 20 μm. Differences were considered as statistically significant when p < 0.05. ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 4

Upregulation of PDK1 promoted the osteogenic ability of MC3T3-E1 cells. (A) Western blotting was performed to detect the protein expression levels of PDK1 and osteogenic markers Runx2, OCN, ALP, PDK1, and GAPDH in MC3T3-E1 cells exposed to empty vector, dexamethasone, and dexamethasone + PS48, respectively. (B) ALP and Alizarin red staining were performed to determine the osteogenic ability of MC3T3-E1 cells exposed to empty vector, dexamethasone, and dexamethasone + PS48, respectively. Scale Bar = 20 μm. Differences were considered as statistically significant when p < 0.05. ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 5

Upregulation of PDK1 by Adenovirus-PDK1 promoted the osteogenic ability of MC3T3-E1 cells by activating the AKT/mTOR pathway. (A) The expression of Adenovirus-PDK1 was confirmed by qRT-PCR. (B) The effect of Adenovirus-PDK1 was confirmed by qRT-PCR. (C) Western blotting was performed to detect the protein expression levels of p-AKT, total-AKT, p-mTOR, total-mTOR, and GAPDH in MC3T3-E1 cells exposed to empty vector, dexamethasone, and dexamethasone + Adenovirus-PDK1, respectively. (D) ALP and Alizarin red staining were performed to determine the osteogenic ability of MC3T3-E1 cells treated with empty vector, dexamethasone, and dexamethasone + Adenovirus-PDK1, respectively. Scale Bar = 20 μm. Differences were considered as statistically significant when p < 0.05. ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 6

Upregulation of PDK1 improved the bone mass of mice treated with dexamethasone. (A) Representative μCT images and quantification analysis of bone parameters in femurs of control, dexamethasone and dexamethasone + Adenovirus-PDK1 mice. (B) Representative images of HE staining to examine the structure of femurs. (C) Immunohistochemical staining for detecting the protein expression of PDK1 in femurs of mice. Isotype control showed that the binding of the primary antibody was specific. Scale Bar = 5 μm. BMD, Bone Mineral Density; BV/TV, bone volume over total volume; Tb. Th, trabecular thickness; Tb. N, trabecular number; Tb. Sp, trabecular separation, Tt. Ar, Total area; Ma. Ar, Marrow area; Ct. Ar, Cortical area, Cortical bone thickness (Ct. Th) Differences were considered as statistically significant when p < 0.05. ***p < 0.001, **p < 0.01, *p < 0.05.