As a potential tumor suppressor protein, Phb1 was originally cloned based on its ability to induce a G1/S arrest. It had been proposed that Phb1 was involved in many cellular processes. But up to date, most studies focused on the role of Phb1 in some kinds of tumors and little is known about the role of Phb1 during LR. In recent years, some studies have explored the role of Phb1 in liver injury and liver cancer(8, 22–25). However, there is no study on the role of Phb1 in LR.
In this study, we found Phb1 mRNA and protein expression showed concordant changes during LR after 70% PHx. Comparing with sham-operation control groups, in 70% PHx test groups, Phb1 mRNA and protein expression were down-regulated at 24 h, up-regulated at 72 h and 168 h (Fig. 1). Previous study found that the gene encoding Phb1 might have additional anti-proliferative effects that do not require translation(9). And Manjeshwar et al. reported that the 3’UTR of the Phb1 gene encoded a functional RNA that arrested cell-cycle proliferation between the G1 and S phases(26). In combination with previous reports, we suggested that Phb1 might regulate cell proliferation during LR in a complex manner, which could involve mechanisms mediated by both Phb1 mRNA and protein.
The best-characterized function of Phb1 is as a chaperone involved in the stabilization of mitochondrial proteins. The mitochondrial-localized Phb1 is confirmed as a high-molecular-weight hetero-complex (ring-shaped structure) by single particle structures(5). Moreover, the interaction of nonassembled respiratory chain subunits with the Phb1 complex has led to the proposal of a chaperone activity of Phb1 during the biogenesis of the respiratory chain(27). Recently, Phb1 was reported to be essential for normal mitochondrial development, and Phb1 deficiency was showed to be associated with deficient mitochondrial biogenesis(28). PHBi showed enhanced sensitivity to anthralin-induced cell death due to enhanced loss of mitochondrial membrane potential in psoriatic lesions(29). Mitochondria are the center of energy metabolism and play a crucial role in the regulation of cell life. A number of stimuli induce dysfunction and structural injury in mitochondria which causes a series of cellular events and ultimately leads to apoptosis or necrosis. We found Phb1 was mainly located in mitochondria in rat liver, and its abundance was 0.47-fold reduction at 24 h, 1.47-fold induction at 72 h and nearly recovered to normal level at 168 h after 70% PHx (Fig. 2B). Interestingly, mitochondria showed marked changes in the ultrastructure at 24 h and nearly recovered to normal at 168 h after 70% PHx (Fig. 3B). The reduced mitochondrial mass also nearly recovered to normal at 168 h after 70% PHx. Mitochondrial membrane potential is an important parameter of mitochondrial function. In our previous study, we found that knockdown of Phb1 in BRL-3A cells resulted in disruption of mitochondrial membrane potential, implying a potential role of Phb1 in maintaining mitochondrial integrity(4). Ross et al. also reported that siRNA -mediated knockdown of Phb1 in Kit225 cells resulted in disruption of mitochondrial membrane potential and Phb1 proteins were novel phosphoproteins up-regulated during T cell activation that function to maintain mitochondrial integrity(30). In this study, using PHBi, we also observed that Phb1 knockdown cells showed a 1.56-fold increase in the number of apoptotic cells (Fig. 4C). Although this result provided evidence for a functional role of Phb1 in suppressing apoptosis in rat liver cells, the molecular mechanism(s) involved are currently unknown. It is likely that the mechanism by which knockdown of Phb1 resulted in apoptosis appears to be targeting of the mitochondria in agreement with previous findings(20). Altogether, these results suggested that Phb1 has a role in regulating stabilization of mitochondria during LR, which might affect mitochondrial function.
Although it has been reported that Phb1 is primarily in mitochondria(10, 28, 31, 32), others have also reported that Phb1 is located in the nucleus(33, 34). In this study, we found Phb1 was located in nucleus as well as mitochondria in rat liver and its abundance was increased during LR (Fig. 2B). Previous study reported that Phb1 was present in the nucleus and to interact with transcription factors important in cell cycle progression(33, 34). In this study, using PHBi, we observed that Phb1 knockdown cells showed an increased S-phase entry (Fig. 4D). The involvement of Phb1 in cell cycle was also observed in a prostate cancer cell line, where down-regulation of Phb1 led to a slight increment of cell cycle entry from G1 to S(28). Although in most situations data would suggest that Phb1 has an antiproliferative effect by interacting with both the p53 and pRb pathways in the nucleus(7, 35), it appears that Phb1 can also have anti-apoptotic effects. In osteosarcoma cells, Phb1 was identified as a gene with down-regulated expression in response to cytotoxic drugs, and the transient overexpression of the Phb1 coding sequence significantly reduced cytotoxic drug-induced apoptosis in these cells(36). In this study, we also observed that Phb1 knockdown cells showed an increase in the number of apoptotic cells (Fig. 4C). It was reported that the subcellular localization of Phb1 may depend on cell type examined and physiological status, and Phb1 might have distinct but overlapping functions in each of these cellular compartments(37). Although there is controversy concerning the function of nuclear-localized Phb1, in combination with previous reports, we suggested that the up-regulated Phb1 in nucleus in rat liver cells might have a function, at least in part, in regulating cell cycle progression of rat liver cells and might regulate the balance between proliferation and apoptosis during LR after 70% PHx, which need further investigation.
Taken together, our results show that Phb1 has two roles in the LR process: one is to regulate cell cycle and apoptosis, and the other is to regulate and maintain mitochondrial stability. Whether the two effects are directly linked or show two different effects is still unclear. In-depth studies will help us better understand the complexities and roles of Phb1 in the LR process.