Mitochondrial Transcription Factor A added to Osteocytes in a Stressed Environment has a Cytoprotective Effect

Abstract

The main precipitant of glucocorticoid-associated femoral head osteonecrosis is widely accepted to be an ischemic-hypoxic event, with oxidative stress also as an underlying factor. Mitochondrial DNA is more vulnerable to oxidative injury than the nucleus, and mitochondrial transcription factor A (TFAM), which plays roles in its function, preservation, and regulation is being increasingly investigated. In the present study we focused on the impact of TFAM on the relation between the oxidative injury induced by the addition of glucocorticoid to a hypoxic environment and osteocytic cell necrosis. Using cultured osteocytes MLO-Y4 in a 1% hypoxic environment (hypoxia) to which 1µM dexamethasone (Dex) was added (Dex(+)/hypoxia(+)), an immunocytochemical study was conducted using 8-hydroxy-2'-deoxyguanosine (8-OHdG), an index of oxidative stress, and hypoxia inducible factor-1α (HIF-1α), a marker of hypoxia. Next, after adding TFAM siRNA, TFAM knockdown, cultured for 24h, and mitochondrial membrane potential were measured, they were stained with ATP5A which labels adenosine triphosphate (ATP) production. Dex was added to MLO-Y4 to which TFAM had been added, and cultured for 24h in hypoxia. The ratio of dead cells to viable cells was determined and compared. Enhanced expression of 8-OHdG, HIF-1α was found in osteocytes following the addition of glucocorticoid in a hypoxic environment. With TFAM knockdown, as compared to normoxia, mitochondrial function significantly decreased. On the other hand, by adding TFAM, the incidence of osteocytic cell necrosis was significantly decreased as compared with Dex(+)/hypoxia(+). TFAM was confirmed to be important in mitochondrial function and preservation, inhibition of oxidative injury and maintenance of ATP production. Moreover, prevention of mitochondrial injury can best be achieved by decreasing the development of osteocytic cell necrosis.

Keywords: mitochondrial function; mitochondrial transcription factor A (TFAM); osteocytic cell necrosis; oxidative injury.

Figure 1

Expression of oxidative stress and hypoxia markers. A. Immunofluorescent staining of 8-OHdG and HIF-1α. In 20% O₂, the group without addition of Dex was named Dex(-)/normoxia, that subjected to addition of Dex alone Dex(+)/normoxia, that with hypoxia alone Dex(-)/hypoxia(+), and that subjected to the combination of Dex and hypoxia Dex(+)/hypoxia(+). Regarding 8-OHdG, in Dex(-)/normoxia almost no expression was found, while in Dex(-)/hypoxia(+) and Dex(+)/normoxia expression was found in the cytoplasm. In Dex(+)/hypoxia(+) intense expression was found. Regarding HIF-1α, there was almost no expression in Dex(-)/normoxia, in Dex(+)/normoxia expression was limited to the cytoplasm, while in Dex(-)/hypoxia(+) expression was found in the nucleus, with transition within the nucleus seen. In Dex(+)/hypoxia(+) as compared with Dex(-)/hypoxia(+) expression was enhanced. Each five independent experiments were carried out. (Scale bar: 20 µm) B. Western blotting of 8-OHdG (36kDa) and HIF-1α (95-120 kDa). By Western blotting no obvious expression was found in either Dex(-)/normoxia (N). Both 8-OHdG and HIF-1α in Dex(-)/hypoxia(+) (H) and Dex(+)/normoxia (D) showed slight expression, and in Dex(+)/hypoxia(+) (DH) expression was enhanced. Each three independent experiments were carried out.

Figure 2

Effects of TFAM knockdown using siRNA. A. Mitochondrial membrane potential (Mito Tracker Red) and expression of ATP5A. ATP5A which documents the existence of ATP production, a major mitochondrial function, was detected. In Dex(-)/normoxia (normoxia) the mitochondrial membrane potential was preserved, and ATP5A expression was also found. By TFAM knockdown the mitochondrial membrane potential was no longer preserved, and ATP5A expression was attenuated. Each five independent experiments were carried out. (Scale bar: 20 µm) B. Western blotting of TFAM siRNA-transfected cells. In WB TFAM (29kDa) expression was significantly inhibited by TFAM siRNA. Each three independent experiments were carried out. C. JC-1 staining. On JC-1 staining, the mitochondrial membrane potential decreased by the osteocyte apoptosis induced by TFAM knockdown. Each three independent experiments were carried out. Viable cells: red, apoptotic cells: green. (Scale bar: 200 µm).

Figure 3

Oxidative stress and hypoxia marker expression and number of osteocytic cell deaths after TFAM addition to osteocytes subjected to glucocorticoid administration in hypoxic environment. TFAM was added to Dex(+)/hypoxia(+) and cultured for 24h (TFAM(+)). A. Immunofluorescent staining of 8-OHdG and HIF-1α with the addition of TFAM. With the addition of TFAM, the expression of both 8-OHdG and HIF-1α was inhibited. Each five independent experiments were carried out. (Scale bar: 20 µm) B. Graph indicates the percentages of apoptotic and necrotic cells in the indicated conditions. The numbers of apoptotic or necrotic cells were counted and related to the total number of cells. Columns and bars indicate means and S.D. respectively (n=5). C. Immunofluorescent staining of apoptotic and necrotic cells using an Apoptotic/Necrotic Cells Detection Kit as described in Materials and Methods. With addition of TFAM a significant decrease in the number of osteocytic cell deaths was noted as compared with Dex(+)/hypoxia(+) (*p<0.01). (Scale bar: 100 µm).