In the present study, we have investigated the protective ability of prohibitin overexpression in CSE exposure associated mitochondrial dysfunction, ROS generation, oxidative DNA damage and apoptosis in HPMECs.
CSE may induce apoptosis in bronchial epithelial cells, endothelial cells and human airway smooth muscle cells (ASMCs)[11–14] CSE may cause injury in mitochondrial morphology and function and induce apoptosis via an intrinsic apoptotic pathway, which involves mitochondrial fragmentation and the disruption of mitochondrial membrane integrity, inhibit of mitochondrial respiration and release of cytochrome c, as well as changed expression levels of pro-apoptotic and anti-apoptotic molecules[13, 15–18]. A previous study has demonstrated that prohibitin decrease the apoptosis of endothelial cells in response to glyLDL. Another study has shown that high prohibitin tumor expression is associated with poorer overall survival in patients with non-small cell lung cancer (NSCLC) and systemic delivery of PHB1 siRNA drastic inhibit tumor growth. These results suggest a pro-survival activity prohibitin.
Previous studies have shown a negative effect of oxidative stress like hydrogen peroxide (H2O2) on the expression of prohibitin in human renal proximal tubule epithelial cell line and adult retinal pigment epithelial cell line-19[21, 22]In agreement with these studies, we discovered that prohibitin expression is significantly reduced in endothelial cells in a CSE dose-dependent manner. The mRNA levels of prohibitin significantly decreased at 2.5% CSE for 12 hr, and the protein levels of prohibitin also decreased to some extent at 2.5% CSE. Therefore, the cells exposed for 2.5% CSE and 12 h were used for subsequent experiments. Furthermore, our results showed that the mRNA levels of prohibitin were reduced in endothelial cells exposed to CSE, indicating a defect in the transcriptional regulation of prohibitin. However, as microRNA may directly targeted prohibitin and phosphorylation of prohibitin plays a pivital role in cell survival[23, 24], a post-translational deimination of prohibitin in CSE-induced apoptosis in HPMECs also can’t be excluded.
We then investigated the contribution of prohibitin to mitochondrial function under CSE treatment. The term mitochondrial dysfunction is referred to as a complex display of cellular events. Mitochondrial injury causes change in mitochondrial morphology, impaired oxidative phosphorylation, reduced membrane potential, altered metabolic activity, increased mitochondrial superoxide levels including alterations in intracellular Ca2 + flux[25, 26]. We observed a protective effect of prohibitin overexpression on the decline in ATP levels and MMP in HPMECs. Prohibitin is a key regulator of mitochondrial quality control. As its uniquely complex structure in mitochondrial localization, prohibitin exert protection function by maintain mitochondrial structure and morphology. Importantly, prohibitin complex is essential for mitochondrial biogenesis and degradation, and responding to mitochondrial stress. As demonstrated by other studies, prohibitin overexpression prevents mitochondrial fragmentation, preserved mitochondrial respiratory function, attenuated mitochondrial complex I oxidative degradation of cells exposed to oxidative stress. Our data provide the evidence that CSE caused mitochondrial depolarization, decreased energy production and prohibitin modulation affects mitochondrial function in this type of the cell under CSE treatments.
In the current study, we also verified that CSE increased ROS production and oxidative DNA damage in HPMECs. The general idea of the interplay between depolarization of the mitochondrial membrane and ROS production is that, upon mitochondrial damage or dysfunction, mitochondrial pores open and allowing the influx of potassium and calcium cations, thereby depolarizing the mitochondrial membrane, which in turn induces ROS production and release. Meanwhile, excessive ROS directly causing the collapse of MMP and depletion of ATP, which later activates a series of signaling pathways that induce apoptosis. Several studies showed prohibitin acts as a coactivator for ARE-dependent gene expression and promotes endogenous antioxidant defense components under oxidative stress[30–32]. Prohibitin overexpression significantly reduced the level of ROS within HPMECs under CSE, suggesting that reduction of oxidative stress is one major mechanism of prohibitin-mediated endothelial cell protection. The oxidative damage in DNA by cigarette smoke is either caused directly, or through the generation of ROS. Prohibitin interact with the Nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase protein complex, which is essential for oxidoreductase activity and DNA repair within cells[34, 35]. Hence, prohibitin may act as an anti-oxidative agent by inhibiting CSE-induced oxidative damage and oxidative DNA damage.
mtTFA, a nucleus-encoded protein, regulates the transcription and replication of mtDNA and maintains mtDNA copy number. Massive apoptosis was found in mtTFA knockout embryos and in the heart of the tissue-specific mtTFA knockouts, and the respiratory chain deficiency caused by mtTFA knockout predisposes cells to apoptosis. TFAM overexpression inhibits mitochondrial ROS production and reduces oxidative stress to mtDNA[38, 39]. As demonstrated by our previous studies, in COPD patients with squamous cell lung cancer, and the level of mtTFA protein in lung tissue negatively correlates with pulmonary vascular endothelial apoptotic index and smoking index. Expression of mtTFA mRNA and protein was downregulated in CSE-treated HUVECs as a consequence of hypermethylation of the mtTFA promoter. Prohibitin subunits have been involved in the organization and stability of mitochondrial nucleoids together with mtDNA-binding proteins, mtTFA, and mitochondrial single-stranded DNA binding protein (mtSSB). In HeLa cells, prohibitin regulates copy number of mtDNA by stabilizing mtTFA protein, probably in a chaperone-like fashion. Our study also demonstrated that mtTFA is a downstream effector of prohibitin that modulates mitochondrial function in CSE-exposed HPMECs.
Since a previous study showed that NF-κB activation is considered a central event in endothelial cells’ response to inflammatory stimuli. We also found CSE exposure markedly increased IKKα/β phosphorylation and IκB-α degradation in HPMECs. Interstingly, prohibitin did not only attenuate CSE-induced phosphorylation of IKKα/β but also restore the level of IκB-α. However, the mechanism through which prohibitin affects NF-κB remains to be established. It has been shown that ROS can both activate and repress NF-κB signaling in a phase and context dependent manner. While overexpression of mtTFA significantly inhibited rotenone-induced mitochondrial ROS generation, mitochondrial DNA Damage and the subsequent NF-κB nuclear translocation. Interestingly, prohibitin overexpression decrease NF-κB transcription activity, even after the proinflammatory cytokine LPS or TNF-α stimulation [7, 44].
There are several open questions worth future study. First, we didn’t explore the phosphorylation or other post transcriptional modification state of prohibitin, since these epigenetic changes is far more important than relative expression for cell function. Second, it is interesting to explore if there is protein–protein interaction between mtTFA and prohibitin. Third, if changed NF-κB and mtTFA signal could insult the protective effect of prohibitin overexpression on HPMECs under CSE is unclear.