[Bax inhibitor 1 inhibits vascular calcification in mice by activating optic atrophy 1 expression].

OBJECTIVE: To investigate the effects of Bax inhibitor 1 (BI- 1) and optic atrophy protein 1 (OPA1) on vascular calcification (VC). METHODS: Mouse models of VC were established in ApoE-deficient (ApoE-/-) diabetic mice by high-fat diet feeding for 12 weeks followed by intraperitoneal injections with Nε-carboxymethyl-lysine for 16 weeks. ApoE-/- mice (control group), ApoE-/- diabetic mice (VC group), ApoE-/- diabetic mice with BI-1 overexpression (VC + BI-1TG group), and ApoE-/- diabetic mice with BI-1 overexpression and OPA1 knockout (VC+BI-1TG+OPA1-/- group) were obtained for examination of the degree of aortic calcification using von Kossa staining. The changes in calcium content in the aorta were analyzed using ELISA. The expressions of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP-2) were detected using immunohistochemistry, and the expression of cleaved caspase-3 was determined using Western blotting. Cultured mouse aortic smooth muscle cells were treated with 10 mmol/L ß-glycerophosphate for 14 days to induce calcification, and the changes in BI-1 and OPA1 protein expressions were examined using Western blotting and cell apoptosis was detected using TUNEL staining. RESULTS: ApoE-/- mice with VC showed significantly decreased expressions of BI-1 and OPA1 proteins in the aorta (P=0.0044) with obviously increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P= 0.0041). Overexpression of BI-1 significantly promoted OPA1 protein expression and reduced calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P=0.0006). OPA1 knockdown significantly increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 in the aorta (P=0.0007). CONCLUSION: BI-1 inhibits VC possibly by promoting the expression of OPA1, reducing calcium deposition and inhibiting osteogenic differentiation and apoptosis of the vascular smooth muscle cells.

Overexpression of MFN2 alleviates sorafenib-induced cardiomyocyte necroptosis via the MAM-CaMKIIδ pathway in vitro and in vivo.

Background: The continued success of oncological therapeutics is dependent on the mitigation of treatment-related adverse events, particularly cardiovascular toxicities. As such, there is an important need to understand the basic mechanisms of drug toxicities in the process of antitumor therapy. Our aim in this study was to elucidate the underlying mechanisms of sorafenib (sor)-induced cardiomyocyte damage. Methods: Primary mouse cardiomyocytes were prepared and treated with sor and various other treatments. Cardiomyocyte necroptosis was detected by flow cytometry, western blotting, and CCK8 assays. Mitochondrial Ca2+ uptake was detected by the Rhod-2 probe using confocal imaging. Morphological changes in mitochondria and mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) were imaged using transmission electron microscopy (TEM) and confocal microscopy. Cardiac perfusion was performed to detect cardiac specific role of MFN2 overexpression in vivo. Results: We reported that mitochondrial Ca2+ overload, the subsequent increase in calmodulin-dependent protein kinase II delta (CaMKIIδ) and RIP3/MLKL cascade activation, contributed to sor-induced cardiac necroptosis. Excess MAM formation and close ER-mitochondria contact were key pathogenesis of sor-induced Ca2+ overload. Sor mediated MFN2 downregulation in a concentration-dependent manner. Furthermore, we found that reduced mitofusin-2 (MFN2) level augmented sor-mediated elevated MAM biogenesis and increased mitochondria-MAM tethering in cardiomyocytes. Sor-induced Mammalian Target of Rapamycin (mTOR) inactivation, followed by the activation and nuclear translocation of Transcription Factor EB (TFEB), contributed to mitophagy and MFN2 degradation. In an in vivo model, mice subjected to sor administration developed cardiac dysfunction, autophagy activation and necroptosis; our investigation found that global and cardiac-specific overexpression of MFN2 repressed cardiac dysfunction, and sor-induced cardiomyocyte necroptosis via repressing the MAM-CaMKIIδ-RIP3/MLKL pathway. Conclusion: Sorafenib mediated cardiomyocyte necroptosis through the MFN2-MAM-Ca2+-CaMKIIδ pathway in vitro and in vivo. The overexpression of MFN2 could rescue sor-induced cardiomyocyte necroptosis without disturbing the anti-tumor effects.

Metastable biomolecular condensates of interferon-inducible antiviral Mx-family GTPases: A paradigm shift in the last three years.

Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. We summarize a paradigm shift over the last 3 years in the field of interferon (IFN)-inducible antiviral Mx-family GTPases. Expression of the 'myxovirus resistance proteins' MxA in human cells and its ortholog Mx1 in murine cells is increased 50- to 100-fold by Type I (IFN-α and -ß) and III IFNs (IFN-λ). Human MxA forms cytoplasmic structures, while murine Mx1 forms nuclear bodies. Since 2002, it has been widely thought that human (Hu) MxA is associated with the membraneous smooth endoplasmic reticulum (ER). In a paradigm shift, our recent data showed that HuMxA formed membraneless phase-separated biomolecular condensates in the cytoplasm. Some of the HuMxA condensates adhered to intermediate filaments generating a reticular pattern. Murine (Mu) Mx1, which was predominantly nuclear, was also confirmed to be in phase-separated nuclear biomolecular condensates. A subset of Huh7 cells showed association of GFP-MuMx1 with intermediate filaments in the cytoplasm. While cells with cytoplasmic GFP-HuMxA condensates and cytoplasmic GFP-MuMx1 filaments showed an antiviral phenotype towards vesicular stomatitis virus (VSV), those with only nuclear GFP-MuMx1 bodies did not. The new data bring forward the paradigm that both human MxA and murine Mx1 give rise to phase-separated biomolecular condensates, albeit in different subcellular compartments, and that differences in the subcellular localization of condensates of different Mx proteins determines the spectrum of their antiviral activity.

m6A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection.

Increasing evidence supports that N6-methyladenosine (m6A) mRNA modification may play an important role in regulating immune responses. Intestinal epithelial cells orchestrate gastrointestinal mucosal innate defense to microbial infection, but underlying mechanisms are still not fully understood. In this study, we present data demonstrating significant alterations in the topology of host m6A mRNA methylome in intestinal epithelial cells following infection by Cryptosporidium parvum, a coccidian parasite that infects the gastrointestinal epithelium and causes a self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients. Altered m6A methylation in mRNAs in intestinal epithelial cells following C. parvum infection is associated with downregulation of alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 and the fat mass and obesity-associated protein with the involvement of NF-кB signaling. Functionally, m6A methylation statuses influence intestinal epithelial innate defense against C. parvum infection. Specifically, expression levels of immune-related genes, such as the immunity-related GTPase family M member 2 and interferon gamma induced GTPase, are increased in infected cells with a decreased m6A mRNA methylation. Our data support that intestinal epithelial cells display significant alterations in the topology of their m6A mRNA methylome in response to C. parvum infection with the involvement of activation of the NF-кB signaling pathway, a process that modulates expression of specific immune-related genes and contributes to fine regulation of epithelial antimicrobial defense.

A Novel Role of Claudin-5 in Prevention of Mitochondrial Fission Against Ischemic/Hypoxic Stress in Cardiomyocytes.

BACKGROUND: Downregulation of claudin-5 in the heart is associated with the end-stage heart failure. However, the underlying mechanism ofclaudin-5 is unclear. Here we investigated the molecular actions of claudin-5 in perspective of mitochondria in cardiomyocytes to better understand the role of claudin-5 in cardioprotection during ischemia. METHODS: Myocardial ischemia/reperfusion (I/R; 30 min/24 h) and hypoxia/reoxygenation (H/R; 24 h/4 h) were used in this study. Confocal microscopy and transmission electron microscope (TEM) were used to observe mitochondrial morphology. RESULTS: Claudin-5 was detected in murine heart tissue and neonatal rat cardiomyocytes (NRCM). Its protein level was severely decreased after myocardial I/R or H/R. Confocal microscopy showedclaudin-5 presented in the mitochondria of NRCM. H/R-induced claudin-5 downregulation was accompanied by mitochondrial fragmentation. The mitofusin 2 (Mfn2) expressionwas dramatically decreased while the dynamin-related protein (Drp) 1 expression was significantly increased after H/R. The TEM indicatedH/R-induced mitochondrial swelling and fission. Adenoviral claudin-5 overexpression reversed these structural disintegration of mitochondria. The mitochondria-centered intrinsic pathway of apoptosis triggered by H/R and indicated by the cytochrome c and cleaved caspase 3 in the cytoplasm of NRCMs was also reduced by overexpressing claudin-5. Claudin-5 overexpression in mouse heart also significantly decreased cleaved caspase 3 and the infarct size in ischemic heart with improved systolic function. CONCLUSION: We demonstrated for the first time the presence of claudin-5 in the mitochondria in cardiomyocytes and provided the firm evidence for the cardioprotective role of claudin-5 in the preservation of mitochondrial dynamics and cell fate against hypoxia- or ischemia-induced stress.

Growing Up Under Constant Light: A Challenge to the Endocrine Function of the Leydig Cells.

The factors influencing Leydig cell maturity and the acquisition of functional capacity are incompletely defined. Here we analyzed the constant light (LL) influence on Leydig cells' endocrine function during reproductive maturation. Rats were exposed to LL from P21 to P90. Data were collected at juvenile (P35), peri/pubertal (P42, P49), and adult (P90) stages of life. The results proved the effect of LL on rats' physiology by changing of bimodal voluntary activity pattern into free-running. Additionally, the peripheral clock in Leydig cells changed in LL condition, indicating disturbed rhythm: the positive element (Bmal1) increased in pre-/pubertal but decreased in the adult period, while negative elements (Per2 and Reverba) were increased. The effects of LL were most prominent in puberty: pituitary genes encoding gonadotropic hormones (Cga, Lhb, Fshb) decreased; serum corticosterone increased, while serum androgens and mass of testicular and sex accessory organs reduced; markers of Leydig cells maturity/differentiation (Insl3, Lhcgr) and steroidogenesis-related genes (Scarb1, Star, Cyp11a1, Cyp17a1) decreased; the steroidogenic and energetic capacity of the Leydig cell mitochondria decreased; the mtDNA copy number reduced, and mitochondrial dynamics markers changed: fusion decreased (Opa1 and Mfn2), and mitophagy increased (Pink1). In adults, the negative effect of LL on mitochondrial function and steroidogenic capacity persists in adult Leydig cells while other parameters reached control values. Altogether, the results indicate that LL slows down Leydig cells' maturation by reducing the endocrine and energy capacity of cells leading to the delay of reproductive development.

Study of long non-coding RNA and mitochondrial dysfunction in diabetic rats.

Diabetes mellitus (DM) is a worldwide health problem. The Micro- and macro-vascular complications are the major causes of morbidity and mortality of DM. Molecular regulation of mitochondrial fission/fusion cycles is being studied, but the results were not conclusive. The aim of this study is to investigate the possible functional role of lncRNA H19 and its relation to mitofusin-2 (Mfn-2) gene expression in diabetic rats with cardiac and renal complications. Streptozotocin-induced diabetic male, albino rats and a matched control group were investigated. Cardiac weights, blood pressure and ECG were recorded. Biochemical evaluation of cardiac and renal functions was performed. Molecular determination of lncRNA H19 and Mfn-2 gene expression and histological examination by light and electron microscopy for cardiac and renal tissues were performed. Diabetic rats showed a significant increase of left ventricle weight/whole body weight ratio, R wave voltage, and a significant decrease of blood pressure, heart rate, and P wave voltage. At the molecular level, lncRNA H19 and Mfn-2 mRNA showed altered expression with a statistically significant downregulation of Mfn-2 mRNA expression in renal tissues. In conclusion, the changes in lncRNA H19 and Mfn-2 mRNA expression may help better understanding of the pathogenesis of cardiac and renal dysfunctions associated with type 1 DM.

Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5.

Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and several reports have suggested that mitochondrial abnormalities are involved in its etiology. We recruited 9 sIBM patients and found significant histological changes and an elevation of growth differential factor 15 (GDF15), a marker of mitochondrial disease, strongly suggesting the involvement of mitochondrial dysfunction. Bioenergetic analysis of sIBM patient myoblasts revealed impaired mitochondrial function. Decreased ATP production, reduced mitochondrial size and reduced mitochondrial dynamics were also observed in sIBM myoblasts. Cell vulnerability to oxidative stress also suggested the existence of mitochondrial dysfunction. Mitochonic acid-5 (MA-5) increased the cellular ATP level, reduced mitochondrial ROS, and provided protection against sIBM myoblast death. MA-5 also improved the survival of sIBM skin fibroblasts as well as mitochondrial morphology and dynamics in these cells. The reduction in the gene expression levels of Opa1 and Drp1 was also reversed by MA-5, suggesting the modification of the fusion/fission process. These data suggest that MA-5 may provide an alternative therapeutic strategy for treating not only mitochondrial diseases but also sIBM.

TLR4 knockout upregulates the expression of Mfn2 and PGC-1α in a high-fat diet and ischemia-reperfusion mice model of liver injury.

AIMS: Patients with nonalcoholic fatty liver disease (NAFLD) have less tolerance to ischemia-reperfusion injury (IRI) of the liver than those with the healthy liver; hence have a higher incidence of severe complications after surgery. This study aimed to investigate the dynamics of the liver and mitochondrial damage and the impact of TLR4 knockout (TLR4KO) on Mfn2 expression in the composite model of NAFLD and IRI. MAIN METHODS: We performed high-fat diet (HFD) feeding and ischemia reperfusion (IR) on wild type (WT) and TLR4 knockout TLR4KO mice. KEY FINDINGS: The degree of structural and functional injuries to the liver and mitochondria (NAFLD and IRI) is greater than that caused by a single factor (NAFLD or IRI) or a simple superposition of both. The IL-6 and TNF-α expressions were significantly suppressed (P < .05), while PGC-1α and Mfn2 expressions were up-regulated considerably (P < .05) after TLR4KO. Furthermore, mitochondrial fusion increased, while ATP consumption and ROS production decreased significantly after TLR4KO (P < .05). The degree of reduction of compound injury by TLR4KO is more significant than the reduction degree of single factor injury. Also, TNF-α and IL-6 levels can be used predictive markers for mitochondrial damage and liver tolerance to NAFLD and IRI. SIGNIFICANCE: TLR4KO upregulates the expression of Mfn2 and PGC-1α in the composite model of NAFLD and IRI. This pathway may be related to IL-6 and TNF-α. This evidence provides theoretical and experimental basis for the subsequent Toll-like receptor 4 (TLR4) receptor targeted therapy.

Neuroprotective actions of leptin facilitated through balancing mitochondrial morphology and improving mitochondrial function.

Mitochondrial dysfunction has a recognised role in the progression of Alzheimer's disease (AD) pathophysiology. Cerebral perfusion becomes increasingly inefficient throughout ageing, leading to unbalanced mitochondrial dynamics. This effect is exaggerated by amyloid ß (Aß) and phosphorylated tau, two hallmark proteins of AD pathology. A neuroprotective role for the adipose-derived hormone, leptin, has been demonstrated in neuronal cells. However, its effects with relation to mitochondrial function in AD remain largely unknown. To address this question, we have used both a glucose-serum-deprived (CGSD) model of ischaemic stroke in SH-SY5Y cells and a Aß1-42 -treatment model of AD in differentiated hippocampal cells. Using a combination of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and MitoRed staining techniques, we show that leptin prevents depolarisation of the mitochondrial membrane and excessive mitochondrial fragmentation induced by both CGSD and Aß1-42 . Thereafter, we used ELISAs and a number of activity assays to reveal the biochemical underpinnings of these processes. Specifically, leptin was seen to inhibit up-regulation of the mitochondrial fission protein Fis1 and down-regulation of the mitochondrial fusion protein, Mfn2. Furthermore, leptin was seen to up-regulate the expression and activity of the antioxidant enzyme, monoamine oxidase B. Herein we provide the first demonstration that leptin is sufficient to protect against aberrant mitochondrial dynamics and resulting loss of function induced by both CGSD and Aß1-42 . We conclude that the established neuroprotective actions of leptin may be facilitated through regulation of mitochondrial dynamics.

Inhibitor 1 of Protein Phosphatase 1 Regulates Ca2+/Calmodulin-Dependent Protein Kinase II to Alleviate Oxidative Stress in Hypoxia-Reoxygenation Injury of Cardiomyocytes.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), regulated by inhibitor 1 of protein phosphatase 1 (I1PP1), is vital for maintaining cardiovascular homeostasis. However, the role and mechanism of I1PP1 against hypoxia-reoxygenation (H/R) injury in cardiomyocytes remain a question. In our study, after I1PP1 overexpression by adenovirus infection in the neonatal cardiomyocytes followed by hypoxia for 4 h and reoxygenation for 12 h, the CaMKIIδ alternative splicing subtype, ATP content, and lactate dehydrogenase (LDH) release were determined. CaMKII activity was evaluated by phosphoprotein phosphorylation at Thr17 (p-PLB Thr17), CaMKII phosphorylation (p-CaMKII), and CaMKII oxidation (ox-CaMKII). Reactive oxygen species (ROS), mitochondrial membrane potential, dynamin-related protein 1 (DRP1), and optic atrophy 1 (OPA1) expressions were assessed. Our study verified that I1PP1 overexpression attenuated the CaMKIIδ alternative splicing disorder; suppressed PLB phosphorylation at Thr17, p-CaMKII, and ox-CaMKII; decreased cell LDH release; increased ATP content; attenuated ROS production; increased mitochondrial membrane potential; and decreased DRP1 expression but increased OPA1 expression in the cardiomyocytes after H/R. Contrarily, CaMKIIδ alternative splicing disorder, LDH release, ATP reduction, and ROS accumulation were aggravated after H/R injury with the I1PP1 knockdown. Collectively, I1PP1 overexpression corrected disorders of CaMKIIδ alternative splicing, inhibited CaMKII phosphorylation, repressed CaMKII oxidation, suppressed ROS production, and attenuated cardiomyocyte H/R injury.

OPA1 overexpression ameliorates mitochondrial cristae remodeling, mitochondrial dysfunction, and neuronal apoptosis in prion diseases.

Prion diseases caused by the cellular prion protein (PrPC) conversion into a misfolded isoform (PrPSc) are associated with multiple mitochondrial damages. We previously reported mitochondrial dynamic abnormalities and cell death in prion diseases via modulation of a variety of factors. Optic atrophy 1 (OPA1) is one of the factors that control mitochondrial fusion, mitochondrial DNA (mtDNA) maintenance, bioenergetics, and cristae integrity. In this study, we observed downregulation of OPA1 in prion disease models in vitro and in vivo, mitochondria structure damage and dysfunction, loss of mtDNA, and neuronal apoptosis. Similar mitochondria findings were seen in OPA1-silenced un-infected primary neurons. Overexpression of OPA1 not only alleviated prion-induced mitochondrial network fragmentation and mtDNA loss, decrease in intracellular ATP, increase in ADP/ATP ratio, and decrease in mitochondrial membrane potential but also protected neurons from apoptosis by suppressing the release of cytochrome c from mitochondria to cytosol and activation of the apoptotic factor, caspase 3. Our results demonstrated that overexpression of OPA1 alleviates prion-associated mitochondrial network fragmentation and cristae remodeling, mitochondrial dysfunction, mtDNA depletion, and neuronal apoptosis, suggesting that OPA1 may be a novel and effective therapeutic target for prion diseases.

MiR-195-5p Promotes Cardiomyocyte Hypertrophy by Targeting MFN2 and FBXW7.

Cardiac hypertrophy mainly predicts heart failure and is highly linked with sudden loss of lives. MicroRNAs (miRNAs) play essential roles in the development of cardiac hypertrophy through binding to corresponding mRNA targets. In this study, in order to investigate the roles of two mature forms of miRNA-195, miR-195-3p, and miR-195-5p, in vitro and in vivo models of cardiac hypertrophy were established by applying angiotensin II (Ang II) to H9c2 cardiomyocytes and infusing chronic Ang II to mice, respectively. We found that miR-195-5p was evidently equally upregulated in the in vitro and in vivo studies of cardiac hypertrophy induced by Ang II. High expressed miR-195-5p could adequately promote hypertrophy, whereas the suppression of miR-195-5p prevented hypertrophy of H9c2 cardiomyocytes under Ang II treatment. Furthermore, the luciferase reporter system demonstrated that MFN2 and FBWX7 were target genes of miR-195-5p, which negatively regulated the expression of these two genes in H9c2 cells. By contrast, in both models, expression of miR-195-3p was only slightly changed without statistical significance. In addition, we observed a trend towards decreased expression of hypertrophic markers by overexpressing miR-195-3p in AngII-treated H9c2 cardiomyocytes in vitro. Taken together, our study indicates that miR-195-5p promotes cardiac hypertrophy via targeting MFN2 and FBXW7 and may provide promising therapeutic strategies for interfering cardiac hypertrophy.

Targeting Mitochondrial Structure Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment.

The BCL2 family plays important roles in acute myeloid leukemia (AML). Venetoclax, a selective BCL2 inhibitor, has received FDA approval for the treatment of AML. However, drug resistance ensues after prolonged treatment, highlighting the need for a greater understanding of the underlying mechanisms. Using a genome-wide CRISPR/Cas9 screen in human AML, we identified genes whose inactivation sensitizes AML blasts to venetoclax. Genes involved in mitochondrial organization and function were significantly depleted throughout our screen, including the mitochondrial chaperonin CLPB. We demonstrated that CLPB is upregulated in human AML, it is further induced upon acquisition of venetoclax resistance, and its ablation sensitizes AML to venetoclax. Mechanistically, CLPB maintains the mitochondrial cristae structure via its interaction with the cristae-shaping protein OPA1, whereas its loss promotes apoptosis by inducing cristae remodeling and mitochondrial stress responses. Overall, our data suggest that targeting mitochondrial architecture may provide a promising approach to circumvent venetoclax resistance. SIGNIFICANCE: A genome-wide CRISPR/Cas9 screen reveals genes involved in mitochondrial biological processes participate in the acquisition of venetoclax resistance. Loss of the mitochondrial protein CLPB leads to structural and functional defects of mitochondria, hence sensitizing AML cells to apoptosis. Targeting CLPB synergizes with venetoclax and the venetoclax/azacitidine combination in AML in a p53-independent manner.See related commentary by Savona and Rathmell, p. 831.This article is highlighted in the In This Issue feature, p. 813.

MTG1 couples mitoribosome large subunit assembly with intersubunit bridge formation.

Mammalian mitochondrial ribosomes (mitoribosomes) synthesize 13 proteins, essential components of the oxidative phosphorylation system. They are linked to mitochondrial disorders, often involving cardiomyopathy. Mitoribosome biogenesis is assisted by multiple cofactors whose specific functions remain largely uncharacterized. Here, we examined the role of human MTG1, a conserved ribosome assembly guanosine triphosphatase. MTG1-silencing in human cardiomyocytes and developing zebrafish revealed early cardiovascular lesions. A combination of gene-editing and biochemical approaches using HEK293T cells demonstrated that MTG1 binds to the large subunit (mtLSU) 16S ribosomal RNA to facilitate incorporation of late-assembly proteins. Furthermore, MTG1 interacts with mtLSU uL19 protein and mtSSU mS27, a putative guanosine triphosphate-exchange factor (GEF), to enable MTG1 release and the formation of the mB6 intersubunit bridge. In this way, MTG1 establishes a quality control checkpoint in mitoribosome assembly. In conclusion, MTG1 controls mitochondrial translation by coupling mtLSU assembly with intersubunit bridge formation using the intrinsic GEF activity acquired by the mtSSU through mS27, a unique occurrence in translational systems.

Mitofusin-2 Expression Is Implicated in Cervical Cancer Pathogenesis.

BACKGROUND/AIM: It remains unclear whether mitofusin-2 (MFN2) functions as a tumour suppressor or oncogene in cancer progression. In this study we, therefore, aimed to investigate the effect of MFN2 on the pathogenesis of cervical cancer. MATERIALS AND METHODS: MFN2 expression was detected in seven healthy cervical, 64 cervical intraepithelial neoplasia (CIN), and 120 cervical squamous cell carcinoma (SCC) tissues by immunohistochemistry. Moreover, biological function of MFN2 in cervical cancer was investigated in vitro. RESULTS: MFN2 levels exhibited a tendency to gradually increase from healthy cervical tissue to CIN to SCC. Moreover, MFN2 expression was significantly associated with higher T-stage (p=0.008) and lymph node metastasis (p<0.001). The proliferative, migratory, and invasive abilities of MFN2-knockdown cells were significantly lower (p<0.001, p<0.001, and p<0.001, respectively) than control cells. CONCLUSION: MFN2 may be involved in cervical cancer pathogenesis as an oncogene and might serve as a biomarker of cervical SCC.

MicroRNA-29a Functions as a Tumor Suppressor and Increases Cisplatin Sensitivity by Targeting NRAS in Lung Cancer.

MicroRNAs have been reported to play an important role in diverse biological processes and progression of various cancers. MicroRNA-29a has been observed to be downregulated in human lung cancer tissues, but the function of microRNA-29a in lung cancer has not been well investigated. In this study, we demonstrated that the expression levels of microRNA-29a were significantly downregulated in 38 pairs of lung cancer tissues when compared to adjacent normal tissues. Overexpression of microRNA-29a inhibited the activity of cell proliferation and colony formation of lung cancer cells, H1299 and A549. Furthermore, microRNA-29a targeted NRAS proto-oncogene in lung cancer cells. In human clinical specimens, NRAS proto-oncogene was highly expressed in human lung cancer tissues compared to normal tissues. More interestingly, microRNA-29a also sensitizes lung cancer cells to cisplatin (CDDP[Please replace "CDDP" with its expansion in the abstract and also provide expansion for the same in its first occurrence in text, if appropriate.]) via its target, NRAS proto-oncogene. Thus, our results in this study demonstrated that microRNA-29a acted as a tumor suppressor microRNA, which indicated potential application of microRNAs for the treatment of human lung cancer in the future.

Quercetogetin protects against cigarette smoke extract-induced apoptosis in epithelial cells by inhibiting mitophagy.

Recent studies demonstrate that the autophagy-dependent turnover of mitochondria (mitophagy) mediates pulmonary epithelial cell death in response to cigarette smoke extract (CSE) exposure, and contributes to emphysema development in vivo during chronic cigarette smoke (CS)-exposure, although the underlying mechanisms remain unclear. Here, we investigated the role of mitophagy in regulating apoptosis in CSE-exposed human lung bronchial epithelial cells. Furthermore, we investigated the potential of the polymethoxylated flavone antioxidant quercetogetin (QUE) to inhibit CSE-induced mitophagy-dependent apoptosis. Our results demonstrate that CSE induces mitophagy in epithelial cells via mitochondrial dysfunction, and causes increased expression levels of the mitophagy-regulator protein PTEN-induced putative kinase-1 (PINK1) and the mitochondrial fission protein dynamin-1-like protein (DRP-1). CSE induced epithelial cell death and increased the expression of the apoptosis-related proteins cleaved caspase-3, -8 and -9. Caspase-3 activity was significantly increased in Beas-2B cells exposed to CSE, and decreased by siRNA-dependent knockdown of DRP-1. Treatment of epithelial cells with QUE inhibited CSE-induced mitochondrial dysfunction and mitophagy by inhibiting phospho (p)-DRP-1 and PINK1 expression. QUE suppressed mitophagy-dependent apoptosis by inhibiting the expression of cleaved caspase-3, -8 and -9 and downregulating caspase activity in human bronchial epithelial cells. These findings suggest that QUE may serve as a potential therapeutic in CS-induced pulmonary diseases.

A novel ADOA-associated OPA1 mutation alters the mitochondrial function, membrane potential, ROS production and apoptosis.

Autosomal dominant optic atrophy (ADOA) is a dominantly inherited optic neuropathy, affecting the specific loss of retinal ganglion cells (RGCs). The majority of affected cases of ADOA are associated with mutations in OPA1 gene. Our previous investigation identified the c.1198C > G (p.P400A) mutation in the OPA1 in a large Han Chinese family with ADOA. In this report, we performed a functional characterization using lymphoblostoid cell lines derived from affected members of this family and control subjects. Mutant cell lines exhibited the aberrant mitochondrial morphology. A ~24.6% decrease in the mitochondrial DNA (mtDNA) copy number was observed in mutant cell lines, as compared with controls. Western blotting analysis revealed the variable reductions (~45.7%) in four mtDNA-encoded polypeptides in mutant cell lines. The impaired mitochondrial translation caused defects in respiratory capacity. Furthermore, defects in mitochondrial ATP synthesis and mitochondrial membrane potential (ΔΨm) were observed in mutant cell lines. These abnormalities resulted in the accumulation of oxidative damage and increasing of apoptosis in the mutant cell lines, as compared with controls. All those alterations may cause the primary degeneration of RGCs and subsequent visual loss. These data provided the direct evidence for c.1198C > G mutation leading to ADOA. Our findings may provide new insights into the understanding of pathophysiology of ADOA.

TNFR2 Stimulation Promotes Mitochondrial Fusion via Stat3- and NF-kB-Dependent Activation of OPA1 Expression.

RATIONALE: Mitochondria are important cellular organelles and play essential roles in maintaining cell structure and function. Emerging evidence indicates that in addition to having proinflammatory and proapoptotic effects, TNFα (tumor necrosis factor α) can, under certain circumstances, promote improvements in mitochondrial integrity and function, phenomena that can be ascribed to the existence of TNFR2 (TNFα receptor 2). OBJECTIVE: The present study aimed to investigate whether and how TNFR2 activation mediates the effects of TNFα on mitochondria. METHODS AND RESULTS: Freshly isolated neonatal mouse cardiac myocytes treated with shRNA targeting TNFR1 were used to study the effects of TNFR2 activation on mitochondrial function. Neonatal mouse cardiac myocytes exhibited increases in mitochondrial fusion, a change that was associated with increases in mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption capacity. Importantly, TNFR2 activation-induced increases in OPA1 (optic atrophy 1) protein expression were responsible for the above enhancements, and these changes could be attenuated using siRNA targeting OPA1. Moreover, both Stat3 and RelA bound to the promoter region of OPA1 and their interactions synergistically upregulated OPA1 expression at the transcriptional level. Stat3 acetylation at lysine 370 or lysine 383 played a key role in the ability of Stat3 to form a supercomplex with RelA. Meanwhile, p300 modulated Stat3 acetylation in HEK293T (human embryonic kidney 293T) cells, and p300-mediated Stat3/RelA interactions played an indispensable role in OPA1 upregulation. Finally, TNFR2 activation exerted beneficial effects on OPA1 expression in an in vivo transverse aortic constriction model, whereby TNFR1-knockout mice exhibited better outcomes than in mice with both TNFR1 and TNFR2 knocked out. CONCLUSIONS: TNFR2 activation protects cardiac myocytes against stress by upregulating OPA1 expression. This process was facilitated by p300-mediated Stat3 acetylation and Stat3/RelA interactions, leading to improvements in mitochondrial morphology and function.