Hypoxia-induced Mitochondrial Fission Research Articles

Hypoxia-induced mitochondrial fission regulates the fate of bone marrow mesenchymal stem cells by maintaining HIF1α stabilization

For mesenchymal stem cells derived from bone marrow, a controlled reduction in ambient oxygen concentration has been recognized as a facilitator of osteogenic differentiation and the formation of calcium nodules. However, the specific molecular mechanisms underlying this phenotype remain unclear. The aim of this study was to elucidate the impact of hypoxia on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and to explore the involvement of mitophagy and the regulation of mitochondrial dynamics mediated by the mitochondrial dynamic regulatory factor FUN14 domain-containing 1 (FUNDC1). Our findings suggest that FUNDC1 is required for promoting osteogenic differentiation in BMSCs under hypoxic conditions. However, this effect was not dependent on FUNDC1-mediated mitophagy but rather on FUNDC1-mediated regulation of mitochondrial fission. At the mechanistic level, FUNDC1 binds more DNM1L and less OPA1 under hypoxic conditions, leading to an upsurge in mitochondrial division. This heightened mitochondrial division culminates in the increased translocation of Parkin to mitochondria, diminishing its interactions with HIF1α in the cytoplasm and consequently facilitating HIF1α deubiquitination and stabilization. In summary, FUNDC1-regulated mitochondrial division in hypoxic culture emerges as a critical determinant for the translocation of Parkin to mitochondria, ultimately maintaining HIF1α stabilization and promoting osteogenic differentiation.

Hypoxia-induced SENP3 promotes chemosensitivity and mitochondrial fission via deSUMOylation of Drp1.

The study aimed to investigate the effect of the SUMOylation status of Drp1 on mitochondrial fission in CDDP-treated HNSCC cells cultured under hypoxic conditions. The effect of hypoxia on the chemosensitivity of HNCC cells was evaluated by flow cytometry and CCK-8 assays. The biological function of SUMO-specific peptidase 3 (SENP3) was evaluated by loss-of-function assays both in vitro and in vivo. SENP3-regulated deSUMOylation of Drp1 were performed with co-IP assays. SENP3 expression correlated with chemosensitivity in clinical HNSCC samples subjected to hypoxic conditions. Hypoxia-induced ROS increased HIF-1α/SENP3 expression and mitochondrial fission in CDDP-treated HNSCC cells, and these effects were reversed by NAC treatment. SENP3 knockdown reversed hypoxia-induced mitochondrial fission and inhibited HNSCC cell apoptosis, which decreased CDDP sensitivity. Furthermore, hypoxia-induced SENP3 deconjugated SUMO2 from Drp1. Our findings revealed that hypoxia-induced SENP3 facilitates CDDP sensitivity and mitochondrial fission via deSUMOylation of Drp1.

Down-regulation of the mitochondrial fusion protein Opa1/Mfn2 promotes cardiomyocyte hypertrophy in Su5416/hypoxia-induced pulmonary hypertension rats

BackgroundMaladaptive right ventricular (RV) remodeling is the most important pathological feature of pulmonary hypertension (PH), involving processes such as myocardial hypertrophy and fibrosis. A growing number of studies have shown that mitochondria-associated endoplasmic reticulum membranes (MAMs) are involved in various physiological and pathological processes, such as calcium homeostasis, lipid metabolism, inflammatory response, mitochondrial dynamics, and autophagy/mitophagy. The abnormal expression of MAMs-related factors is closely related to the occurrence and development of heart-related diseases. However, the role of MAM-related factors in the maladaptive RV remodeling of PH rats remains unclear. Methods and resultsWe first obtained the transcriptome data of RV tissues from PH rats induced by Su5416 combined with hypoxia treatment (SuHx) from the Gene Expression Omnibus (GEO) database. The results showed that two MAMs-related genes (Opa1 and Mfn2) were significantly down-regulated in RV tissues of SuHx rats, accompanied by significant up-regulation of cardiac hypertrophy-related genes (such as Nppb and Myh7). Subsequently, using the SuHx-induced PH rat model, we found that the downregulation of mitochondrial fusion proteins Opa1 and Mfn2 may be involved in maladaptive RV remodeling by accelerating mitochondrial dysfunction. Finally, at the cellular level, we found that overexpression of Opa1 and Mfn2 could inhibit hypoxia-induced mitochondrial fission and reduce ROS production in H9c2 cardiomyocytes, thereby retarded the progression of cardiomyocyte hypertrophy. ConclusionsThe down-regulation of mitochondrial fusion protein Opa1/Mfn2 can accelerate cardiomyocyte hypertrophy and then participate in maladaptive RV remodeling in SuHx-induced PH rats, which may be potential targets for preventing maladaptive RV remodeling.

Salidroside, a phenyl ethanol glycoside from Rhodiola crenulata, orchestrates hypoxic mitochondrial dynamics homeostasis by stimulating Sirt1/p53/Drp1 signaling

Ethnopharmacological relevanceRhodiola crenulata is clinically used to combat hypobaric hypoxia brain injury at high altitude with the function of invigorating Qi and promoting blood circulation in Tibetan medicine. Salidroside (Sal), an active compound identified from Rhodiola species, has been shown to exert neuroprotective effects against hypoxic brain injury. However, its mitochondrial protective mechanisms remain largely unknown. Aim of the studyThe present study aimed to explore the mitochondrial protection of Sal and the involved mechanisms related to mitochondrial dynamics homeostasis on hypoxia-induced injury of HT22 cells. Materials and methodsHypoxic condition was performed as cells cultured in a tri-gas incubator with 1% O2, 5% CO2 and 94% N2. We firstly investigated the effects of different concentrations of Sal on the viability of normal or hypoxic HT22 cells. Whereafter, the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), adenosine triphosphate (ATP) and Na+-K+-ATPase were tested by commercial kits. Meanwhile, mitochondrial superoxide, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were determined by specific labeled probes. Mitochondrial morphology was detected by mito-tracker green with confocal microscopy. Additionally, the potential interactions of Sal with Sirt1/p53/Drp1 signaling pathway-related proteins were predicted and tested by molecular docking and localized surface plasmon resonance (LSPR) techniques, respectively. Furthermore, the protein levels of Sirt1, p53, ac-p53, Drp1, p-Drp1(s616), Fis1 and Mfn2 were estimated by western blot analysis. ResultsSal alleviated hypoxia-induced oxidative stress in HT22 cells as evidenced by increased cell viability and SOD activity, while decreased LDH release and MDA content. The protected mitochondrial function by Sal treatment was indicated by the increases of ATP level, Na+-K+-ATPase activity and MMP. Miraculously, Sal reduced hypoxia-induced mitochondrial fission, while increased mitochondrial tubular or linear morphology. The results of molecular docking and LSPR confirmed the potential binding of Sal to proteins Sirt1, p53, Fis1 and Mfn2 with affinity values 1.38 × 10−2, 5.26 × 10−3, 6.46 × 10−3 and 7.26 × 10−3 KD, respectively. And western blot analysis further demonstrated that Sal memorably raised the levels of Sirt1 and Mfn2, while decreased the levels of ac-p53, Drp1, p-Drp1 (s616) and Fis1. ConclusionCollectively, our data confirm that Sal can maintain mitochondrial dynamics homeostasis by activating the Sirt1/p53/Drp1 signaling pathway.

Dichotomy in hypoxia-induced mitochondrial fission in placental mesenchymal cells during development and preeclampsia: consequences for trophoblast mitochondrial homeostasis

Dynamic changes in physiologic oxygen are required for proper placenta development; yet, when low-oxygen levels persist, placental development is halted, culminating in preeclampsia (PE), a serious complication of pregnancy. Considering mitochondria’s function is intimately linked to oxygen changes, we investigated the impact of oxygen on mitochondrial dynamics in placental mesenchymal stromal cells (pMSCs) that are vital for proper placental development. Transmission electron microscopy, proximity ligation assays for mitochondrial VDAC1 and endoplasmic reticulum IP3R, and immunoanalyses of p-DRP1 and OPA1, demonstrate that low-oxygen conditions in early 1st trimester and PE promote mitochondrial fission in pMSCs. Increased mitochondrial fission of mesenchymal cells was confirmed in whole PE placental tissue sections. Inhibition of DRP1 oligomerization with MDiVi-1 shows that low oxygen-induced mitochondrial fission is a direct consequence of DRP1 activation, likely via HIF1. Mitophagy, a downstream event prompted by mitochondrial fission, is a prominent outcome in PE, but not 1st trimester pMSCs. We also investigated whether mesenchymal–epithelial interactions affect mitochondrial dynamics of trophoblasts in PE placentae. Exposure of trophoblastic JEG3 cells to exosomes of preeclamptic pMSCs caused heightened mitochondrial fission in the cells via a sphingomyelin-dependent mechanism that was restored by MDiVi-1. Our data uncovered dichotomous regulation of mitochondrial fission and health in human placental mesenchymal cells under physiologic and pathologic hypoxic conditions and its impact on neighboring trophoblast cells.

Hypoxia-induced ROS promotes mitochondrial fission and cisplatin chemosensitivity via HIF-1α/Mff regulation in head and neck squamous cell carcinoma.

Chemotherapy based on cisplatin (CDDP) has been established as the treatment of choice for head and neck squamous cell carcinoma (HNSCC). Malignant tumors respond to microenvironmental alterations through a dynamic balance between mitochondrial fission and fusion. HNSCCs are known to exhibit hypoxic conditions, yet the respective effects and underlying mechanisms of hypoxia on chemosensitivity and mitochondrial dynamics remain to be resolved. The effect of hypoxia on the chemosensitivity of HNCC cells was determined by flow cytometry. Mitochondrial fission factor (Mff) expression was assessed by RT-PCR and Western blotting in hypoxic HNSCC cells, and further verified in primary CDDP-sensitive and CDDP-resistant HSNCC samples. The biological function of Mff was evaluated by loss of function and gain of function analyses, both in vitro and in vivo. We found that hypoxia promoted mitochondrial fission and CDDP sensitivity in HNSCC cells. Importantly, Mff was found to be correlated with chemosensitivity in primary clinical samples under hypoxic conditions. Hypoxia-inducible factor 1α (HIF-1α) was found to markedly increase Mff transcription and to directly bind to Mff. Hypoxia enhanced the release of reactive oxygen species (ROS) and upregulated the expression of Mff via HIF-1α in HNSCC cells. ROS depletion in HNSCC cells attenuated HIF-1α expression, Mff expression and mitochondrial fission. Moreover, Mff knockdown led to suppression of hypoxia-induced mitochondrial fission and to decreased CDDP chemosensitivity in vivo and in vitro. Our findings indicate that hypoxia-induced release of ROS can promote mitochondrial fission and CDDP chemosensitivity via HIF1α/Mff regulation in HNSCC cells, indicating that Mff may serve as a biomarker to predict neoadjuvant chemosensitivity in HNSCC patients and as a target for overcoming chemoresistance.

Activation of ERK–Drp1 signaling promotes hypoxia‐induced Aβ accumulation by upregulating mitochondrial fission and BACE1 activity

Hypoxia is a risk factor for Alzheimer's disease (AD). Besides, mitochondrial fission is increased in response to hypoxia. In this study, we sought to investigate whether hypoxia‐induced mitochondrial fission plays a critical role in regulating amyloid‐β (Aβ) production. Hypoxia significantly activated extracellular signal‐regulated kinase (ERK), increased phosphorylation of dynamin‐related protein 1 (Drp1) at serine 616, and decreased phosphorylation of Drp1 at serine 637. Importantly, hypoxia triggered mitochondrial dysfunction, elevated β‐secretase 1 (BACE1) and γ‐secretase activities, and promoted Aβ accumulation in HEK293 cells transfected with β‐amyloid precursor protein (APP) plasmid harboring the Swedish and Indiana familial Alzheimer's disease mutations (APPSwe/Ind HEK293 cells). Then, we investigated whether the ERK inhibitor PD325901 and Drp1 inhibitor mitochondrial division inhibitor‐1 (Mdivi‐1) would attenuate hypoxia‐induced mitochondrial fission and Aβ generation in APPSwe/Ind HEK293 cells. PD325901 and Mdivi‐1 inhibited phosphorylation of Drp1 at serine 616, resulting in reduced mitochondrial fission under hypoxia. Furthermore, hypoxia‐induced mitochondrial dysfunction, BACE1 activation, and Aβ accumulation were downregulated by PD325901 and Mdivi‐1. Our data demonstrate that hypoxia induces mitochondrial fission, impairs mitochondrial function, and facilitates Aβ generation. The ERK–Drp1 signaling pathway is partly involved in the hypoxia‐induced Aβ generation by regulating mitochondrial fission and BACE1 activity. Therefore, inhibition of hypoxia‐induced mitochondrial fission may prevent or slow the progression of AD.

USP19 promotes hypoxia-induced mitochondrial division via FUNDC1 at ER-mitochondria contact sites.

The ER tethers tightly to mitochondria and the mitochondrial protein FUNDC1 recruits Drp1 to ER-mitochondria contact sites, subsequently facilitating mitochondrial fission and preventing mitochondria from undergoing hypoxic stress. However, the mechanisms by which the ER modulates hypoxia-induced mitochondrial fission are poorly understood. Here, we show that USP19, an ER-resident deubiquitinase, accumulates at ER-mitochondria contact sites under hypoxia and promotes hypoxia-induced mitochondrial division. In response to hypoxia, USP19 binds to and deubiquitinates FUNDC1 at ER-mitochondria contact sites, which facilitates Drp1 oligomerization and Drp1 GTP-binding and hydrolysis activities, thereby promoting mitochondrial division. Our findings reveal a unique hypoxia response pathway mediated by an ER protein that regulates mitochondrial dynamics.

UCP2-dependent improvement of mitochondrial dynamics protects against acute kidney injury.

Acute kidney injury (AKI) is a public health concern, with high morbidity and mortality rates in hospitalized patients and because survivors have an increased risk of progression to chronic kidney disease. Mitochondrial damage is the critical driver of AKI-associated dysfunction and loss of tubular epithelial cells; however, the pathways that mediate these events are poorly defined. Here, in murine ischemia/reperfusion (I/R)-induced AKI, we determined that mitochondrial damage is associated with the level of renal uncoupling protein 2 (UCP2). In hypoxia-damaged proximal tubular cells, a disruption of mitochondrial dynamics demonstrated by mitochondrial fragmentation and disturbance between fusion and fission was clearly indicated. Ucp2-deficient mice (knockout mice) with I/R injury experienced more severe AKI and mitochondrial fragmentation than wild-type mice. Moreover, genetic or pharmacological treatment increased UCP2 expression, improved renal function, reduced tubular injury and limited mitochondrial fission. In cultured proximal tubular epithelial cells, hypoxia-induced mitochondrial fission was exacerbated in cells with UCP2 deletion, whereas an increase in UCP2 ameliorated the hypoxia-induced disturbance of the balance between mitochondrial fusion and fission. Furthermore, results following modulation of UCP2 suggested it has a role in preserving mitochondrial integrity by preventing loss of membrane potential and reducing subsequent mitophagy. Taken together, our results indicate that UCP2 is protective against AKI and suggest that enhancing UCP2 to improve mitochondrial dynamics has potential as a strategy for improving outcomes of renal injury. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Increased mitochondrial fission is critical for hypoxia-induced pancreatic beta cell death.

Hypoxia-mediated pancreatic beta cell death is one of the main causes of pancreatic beta celldeath, which leads to the loss of functional pancreatic beta cell mass and type 1 diabetes andtype 2 diabetes.However, the molecular mechanisms that control life and death of pancreatic beta cells remain poorly understood. Here we showed that mitochondrial fission was strongly induced in pancreatic beta cellsmainly due to an elevation of DRP1S616 phosphorylation through HIF-1αactivation and subsequent DRP1 mitochondrial translocation. Hypoxia-induced pancreatic beta cell death can be reversed by the inhibition of mitochondrial fission viaDRP1 knockdown. We further demonstrated that hypoxia-induced mitochondrial fission untightened the cristae formation, which subsequently triggers mitochondrial cytochrome c release and consequent caspase activation. Moreover, treatment with mitochondrial division inhibitor-1 (Mdivi-1), a specific inhibitor of DRP1-mediated mitochondrial fission, significantly suppressedbeta cell death in vitro, indicating a promising therapeutic strategy for treatment of diabetes.Taken together, our results reveal a crucial role for the DRP1-mediated mitochondrial fission in hypoxia-induced beta cell death, which provides a strong evidence for thisprocess as drug target indiabetestreatment.

Hypoxia-dependent mitochondrial fission regulates endothelial progenitor cell migration, invasion, and tube formation.

Tumor undergo uncontrolled, excessive proliferation leads to hypoxic microenvironment. To fulfill their demand for nutrient, and oxygen, tumor angiogenesis is required. Endothelial progenitor cells (EPCs) have been known to the main source of angiogenesis because of their potential to differentiation into endothelial cells. Therefore, understanding the mechanism of EPC-mediated angiogenesis in hypoxia is critical for development of cancer therapy. Recently, mitochondrial dynamics has emerged as a critical mechanism for cellular function and differentiation under hypoxic conditions. However, the role of mitochondrial dynamics in hypoxia-induced angiogenesis remains to be elucidated. In this study, we demonstrated that hypoxia-induced mitochondrial fission accelerates EPCs bioactivities. We first investigated the effect of hypoxia on EPC-mediated angiogenesis. Cell migration, invasion, and tube formation was significantly increased under hypoxic conditions; expression of EPC surface markers was unchanged. And mitochondrial fission was induced by hypoxia time-dependent manner. We found that hypoxia-induced mitochondrial fission was triggered by dynamin-related protein Drp1, specifically, phosphorylated DRP1 at Ser637, a suppression marker for mitochondrial fission, was impaired in hypoxia time-dependent manner. To confirm the role of DRP1 in EPC-mediated angiogenesis, we analyzed cell bioactivities using Mdivi-1, a selective DRP1 inhibitor, and DRP1 siRNA. DRP1 silencing or Mdivi-1 treatment dramatically reduced cell migration, invasion, and tube formation in EPCs, but the expression of EPC surface markers was unchanged. In conclusion, we uncovered a novel role of mitochondrial fission in hypoxia-induced angiogenesis. Therefore, we suggest that specific modulation of DRP1-mediated mitochondrial dynamics may be a potential therapeutic strategy in EPC-mediated tumor angiogenesis.

Abstract 4522: Hypoxia-induced alteration of mitochondrial dynamics is linked to chemoresistance in ovarian cancer

Abstract Hypoxia, commonly observed in the tumor microenvironment, has been shown to be related to the cancer hallmarks. Hypoxia generates reactive oxygen species (ROS), inducing changes in mitochondrial function and structure in various diseases. Mitochondria, dynamic intracellular organelles, go through incessant processes of fission and fusion for efficient generation of energy. We investigated alterations in mitochondrial networks under hypoxic conditions (< 1 % O2) and subsequent effects of the mitochondrial alteration on drug response in ovarian cancer cells. Mitochondrial fission was prevalent in cancer cells under hypoxic conditions. Translocation of phospho-Drp1 (Ser616), a biochemically active mitochondrial fission protein, to mitochondria was enhanced under hypoxic conditions. An increase in ROS production was accompanied under hypoxic conditions. ROS scavenging reversed the hypoxia-elicited mitochondrial fission, while exogenous H2O2 treatment promoted mitochondrial fission. These results strongly implicate that ROS are important modulators of mitochondrial dynamics under hypoxia. Interestingly, hypoxia-induced mitochondrial fission altered the response of cancer cells to cisplatin (CDDP). Ovarian cancer cells in hypoxic and normoxic conditions were differentially affected by CDDP when mitochondrial fission was inhibited. Taken together, the alteration of mitochondrial dynamics, a cellular adaptation to hypoxia, is associated with chemoresistance. Mitochondria adapted to the hypoxic tumor microenvironment could be a potential target for anti-cancer therapy. Citation Format: Youngjin Han, Boyun Kim, Yong Sang Song. Hypoxia-induced alteration of mitochondrial dynamics is linked to chemoresistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4522. doi:10.1158/1538-7445.AM2017-4522

Mitochondrial dynamics regulates hypoxia-induced migration and antineoplastic activity of cisplatin in breast cancer cells.

Mitochondria are high dynamic organelles with frequent fission and fusion. Here, we found hypoxia stimulated Drp1 expression, mitochondrial fission and migration in metastatic MDA-MB‑231 cells, but not in non-metastatic MCF-7 cells. Inhibition of Drp1-dependent mitochondrial fission by Mdivi-1 or silencing Drp1 attenuated hypoxia-induced mitochondrial fission and migration in MDA-MB‑231 cells. On the other hand, cisplatin induced significant apoptosis and mitochondrial fission in MDA-MB‑231 cells, but not in MCF-7 cells. Mdivi-1 and silencing Drp1 also efficiently prevented cisplatin-induced MMP decrease, ROS production and apoptosis in MDA-MB‑231 cells. Our data suggest that Drp1-dependent mitochondrial fission not only regulates hypoxia-induced migration of breast cancer cells, but also facilitates its sensitivity to chemotherapeutic agents. Thus, targeting Drp1-dependent mitochondrial dynamics may provide a novel strategy to suppress breast cancer metastasis and improve the chemotherapeutic effect in the future.

Involvement of Drp1 in hypoxia-induced migration of human glioblastoma U251 cells.

Glioblastoma is one of the most aggressive brain tumors with high morbidity and mortality. Hypoxia is often the common characteristic of tumor microenvironment, and hypoxia-inducible factor-1α (HIF-1α) is an essential factor regulating the migratory activity of cancer cells including glioblastoma. Recently, mitochondrial dynamics was found to be involved in the aggression of cancer cells. However, whether dynamin-related protein 1 (Drp1) contributes to the migration of human glioblastoma cells under hypoxia remains unknown. In the present study, hypoxia was found to upregulate the transcription and expression of Drp1, and stimulated mitochondrial fission in glioblastoma U251 cells. Inhibition of HIF-1α with echinomycin blocked hypoxia‑induced expression of Drp1. Notably, Drp1 inhibitor Mdivi-1 efficiently attenuated hypoxia-induced mitochondrial fission and migration of U251 cells. In addition, three U251 stable cell lines expressing GFP, GFP-Drp1 and dominant negative GFP-Drp1‑K38A were established to examine the direct role of Drp1 in hypoxia-induced migration. MTT assay showed that there was no significant difference in proliferation of three cell lines. Compared with the GFP cell line, exogenously expressed GFP-Drp1-K38A inhibited hypoxia-induced migration of U251 cells, while stable expression of GFP-Drp1 enhanced the migration of U251 cells under hypoxia. Therefore, this study indicates the involvement of Drp1 in hypoxia-induced migration of human glioblastoma U251 cells, and suggests Drp1 to be a potential therapeutic target to suppress the aggression of glioblastoma in the future.

Fine-Tuning of Drp1/Fis1 Availability by AKAP121/Siah2 Regulates Mitochondrial Adaptation to Hypoxia

Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C.elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.