DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling

Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has an poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors, however its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.

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Titration of mitochondrial fusion rescues Mff-deficient cardiomyopathy

The Journal of Cell Biology ◽

10.1083/jcb.201507035 ◽

2015 ◽

Vol 211 (4) ◽

pp. 795-805 ◽

Cited By ~ 69

Author(s):

Hsiuchen Chen ◽

Shuxun Ren ◽

Clary Clish ◽

Mohit Jain ◽

Vamsi Mootha ◽

...

Keyword(s):

Respiratory Chain ◽

Fusion Gene ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Therapeutic Benefit ◽

Mitochondrial Fusion ◽

Tissue Integrity ◽

Cell Type Specific ◽

Respiratory Chain Activity ◽

First Time

Defects in mitochondrial fusion or fission are associated with many pathologies, raising the hope that pharmacological manipulation of mitochondrial dynamics may have therapeutic benefit. This approach assumes that organ physiology can be restored by rebalancing mitochondrial dynamics, but this concept remains to be validated. We addressed this issue by analyzing mice deficient in Mff, a protein important for mitochondrial fission. Mff mutant mice die at 13 wk as a result of severe dilated cardiomyopathy leading to heart failure. Mutant tissue showed reduced mitochondrial density and respiratory chain activity along with increased mitophagy. Remarkably, concomitant deletion of the mitochondrial fusion gene Mfn1 completely rescued heart dysfunction, life span, and respiratory chain function. Our results show for the first time that retuning the balance of mitochondrial fusion and fission can restore tissue integrity and mitochondrial physiology at the whole-organ level. Examination of liver, testis, and cerebellum suggest, however, that the precise balance point of fusion and fission is cell type specific.

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Alterations in Mitochondrial and Inflammasome Homeostasis by 2-Chloroethyl Ethyl Sulfide and their Mitigation by Curcumin: An In vitro Study

Iranian Journal of Allergy Asthma and Immunology ◽

10.18502/ijaai.v20i5.7411 ◽

2021 ◽

Author(s):

Azam Kia ◽

Mona Nadi ◽

Vahideh Hajhasan ◽

Jafar Salimian

Keyword(s):

Sulfur Mustard ◽

Fusion Gene ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

In Vitro Study ◽

Inhibitory Effect ◽

Epithelial Cell Line ◽

Complete Suppression ◽

Mitofusin 2

The mitochondrion has a substantial role in innate immunity and inflammasome signaling pathways. Sulfur mustard (SM) induces toxicity in cytoplasmic organelles. We aimed to evaluate the potential therapeutic effect of curcumin on the toxicity of SM analog through measuring gene expression levels of mitochondrial dynamics followed by induction of the inflammasome signaling pathway. After the treatment of pulmonary epithelial cell line (A549) by 2-chloroethyl ethyl sulfide (CEES) (2500 mM) for 48h, the transcriptional activity of mitochondrial fission and fusion genes such as dynamin-related protein 1 (Drp1), mitochondrial fission 1 protein (Fis1), mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and Dominant optic atrophy (Opa1) and inflammasome pathway genes including absent in melanoma 2 (AIM2), NLR family containing protein 3 (NLRP3), and Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) was measured. Furthermore, the inhibitory effect of curcumin (160 mM) concurrent with SM analog on the expression level of mitochondria and inflammasome genes was investigated. CEES was able to over-express the fission, fusion (Drp1 ~ 8, Fis1 4.5, Mfn2 15, and Opa1 16-fold) and inflammasome genes (AIM2, NLRP3, 8 and 6-fold, respectively), whereas Mfn1 was significantly decreased (0.5-fold) and a not statistically significant decrease was observed in the ASC gene. Curcumin could modulate the effect of CEES, mitigate the expression of fission, fusion, and inflammasome genes exceedingly. However, a major increase in the repairer fusion gene (Mfn1, 6-fold) and complete suppression of the ASC gene were the outcomes of using the curcumin. In conclusion, we suggest curcumin alleviates the disturbance of mitochondrial dynamics and downregulates the inflammasome genes exposed to the CEES.

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Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

Cell Death and Disease ◽

10.1038/s41419-021-03752-2 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Anthony R. Anzell ◽

Garrett M. Fogo ◽

Zoya Gurm ◽

Sarita Raghunayakula ◽

Joseph M. Wider ◽

...

Keyword(s):

Cortical Neurons ◽

Ischemia Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Ischemia Reperfusion ◽

Mitochondrial Fission ◽

Conditional Knockout ◽

Mitochondrial Morphology ◽

Primary Neurons ◽

Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

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Syntaxin-17-Dependent Mitochondrial Dynamics is Essential for Protection Against Oxidative-Stress-Induced Apoptosis

Antioxidants ◽

10.3390/antiox8110522 ◽

2019 ◽

Vol 8 (11) ◽

pp. 522 ◽

Cited By ~ 4

Author(s):

Wang ◽

Xiao ◽

Huang ◽

Liu

Keyword(s):

Oxidative Stress ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Autophagic Flux ◽

Wild Type ◽

Dual Roles ◽

Defective Mutant ◽

U2os Cells ◽

Induced Apoptosis

In this study, cell death induced by the oxidant tert-butylhydroperoxide (tBH) was observed in U2OS cells; this phenotype was rescued by Syntaxin 17 (STX17) knockout (KO) but the mechanism is unknown. STX17 plays dual roles in autophagosome–lysosome fusion and mitochondrial fission. However, the contribution of the two functions of STX17 to apoptosis has not been extensively studied. Here, we sought to dissect the dual roles of STX17 in oxidative-stress-induced apoptosis by taking advantage of STX17 knockout cells and an autophagosome–lysosome fusion defective mutant of STX17. We generated STX17 knockout U2OS cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and the STX17 knockout cells were reconstituted with wild-type STX17 and its autophagosome–lysosome fusion defective mutant. Autophagy was assessed by autophagic flux assay, Monomer red fluorescent protein (mRFP)–GFP–LC3 assay and protease protection assay. Golgi, endoplasmic reticulum (ER)/ER–Golgi intermediate compartment (ERGIC) and mitochondrial dynamics were examined by staining the different indicator proteins. Apoptosis was evaluated by caspase cleavage assay. The general reactive oxygen species (ROS) were detected by flow cytometry. In STX17 complete knockout cells, sealed autophagosomes were efficiently formed but their fusion with lysosomes was less defective. The fusion defect was rescued by wild-type STX17 but not the autophagosome–lysosome fusion defective mutant. No obvious defects in Golgi, ERGIC or ER dynamics were observed. Mitochondria were significantly elongated, supporting a role of STX17 in mitochondria fission and the elongation caused by STX17 KO was reversed by the autophagosome–lysosome fusion defective mutant. The clearance of protein aggregation was compromised, correlating with the autophagy defect but not with mitochondrial dynamics. This study revealed a mixed role of STX17 in autophagy, mitochondrial dynamics and oxidative stress response. STX17 knockout cells were highly resistant to oxidative stress, largely due to the function of STX17 in mitochondrial fission rather than autophagy.

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Non-alcoholic fatty liver disease in mice with hepatocyte-specific deletion of mitochondrial fission factor

Diabetologia ◽

10.1007/s00125-021-05488-2 ◽

2021 ◽

Author(s):

Yukina Takeichi ◽

Takashi Miyazawa ◽

Shohei Sakamoto ◽

Yuki Hanada ◽

Lixiang Wang ◽

...

Keyword(s):

Er Stress ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Primary Hepatocytes ◽

Alcoholic Fatty Liver ◽

Expression Of Genes ◽

Specific Deletion

Abstract Aims/hypothesis Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. Methods We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. Results MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. Conclusions/interpretation We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH. Graphical abstract

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A Comprehensive Review on Solitary Fibrous Tumor: New Insights for New Horizons

Cancers ◽

10.3390/cancers13122913 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2913

Author(s):

Javier Martin-Broto ◽

Jose L. Mondaza-Hernandez ◽

David S. Moura ◽

Nadia Hindi

Keyword(s):

Solitary Fibrous Tumor ◽

Fusion Gene ◽

Current Knowledge ◽

Fusion Transcript ◽

Chemotherapeutic Agents ◽

Comprehensive Review ◽

Molecular Events ◽

In Series ◽

Prospective Phase ◽

Fibrous Tumor

Solitary fibrous tumor (SFT) is a rare mesenchymal, ubiquitous tumor, with an incidence of 1 new case/million people/year. In the 2020 WHO classification, risk stratification models were recommended as a better tool to determine prognosis in SFT, to the detriment of “typical” or “malignant” classic terms. The risk for metastasis is up to 35–45%, or even greater, in series with a longer follow-up. Over the last few decades, advances in immunohistochemistry and molecular diagnostics identified STAT6 nuclear protein expression and the NAB2–STAT6 fusion gene as more precise tools for SFT diagnosis. Recent evidence taken from retrospective series and from two prospective phase II clinical trials showed that antiangiogenics are active and their sequential use from first line should be considered, except for dedifferentiated SFT for which chemotherapy is the best option. Since the fusion transcript driver’s first description in 2013, new insights have been brought on key molecular events in SFT. This comprehensive review mainly focuses on the superior efficacy of antiangiogenics over chemotherapeutic agents in SFT, provides the current knowledge of key molecules that could co-drive the SFT behavior, and suggests new target candidates that deserve to be explored in preclinical and clinical research in SFT.

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Selective Anti-Cancer Effects of Plasma-Activated Medium and Its High Efficacy with Cisplatin on Hepatocellular Carcinoma with Cancer Stem Cell Characteristics

International Journal of Molecular Sciences ◽

10.3390/ijms22083956 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3956

Author(s):

Yan Li ◽

Tianyu Tang ◽

Hae June Lee ◽

Kiwon Song

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Death ◽

Cell Lines ◽

Primary Liver Cancer ◽

Atmospheric Pressure Plasma ◽

Normal Liver ◽

Normal Liver Cell ◽

Ample Evidence ◽

Huh7 Cells ◽

Anti Cancer

Hepatocellular carcinoma (HCC) is a major histological subtype of primary liver cancer. Ample evidence suggests that the pathological properties of HCC originate from hepatic cancer stem cells (CSCs), which are responsible for carcinogenesis, recurrence, and drug resistance. Cold atmospheric-pressure plasma (CAP) and plasma-activated medium (PAM) induce apoptosis in cancer cells and represent novel and powerful anti-cancer agents. This study aimed to determine the anti-cancer effect of CAP and PAM in HCC cell lines with CSC characteristics. We showed that the air-based CAP and PAM selectively induced cell death in Hep3B and Huh7 cells with CSC characteristics, but not in the normal liver cell line, MIHA. We observed both caspase-dependent and -independent cell death in the PAM-treated HCC cell lines. Moreover, we determined whether combinatorial PAM therapy with various anti-cancer agents have an additive effect on cell death in Huh7. We found that PAM highly increased the efficacy of the chemotherapeutic agent, cisplatin, while enhanced the anti-cancer effect of doxorubicin and the targeted-therapy drugs, trametinib and sorafenib to a lesser extent. These findings support the application of CAP and PAM as anti-cancer agents to induce selective cell death in cancers containing CSCs, suggesting that the combinatorial use of PAM and some specific anti-cancer agents is complemented mechanistically.

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Fibrolamellar Carcinoma: 2012 Update

Scientifica ◽

10.6064/2012/743790 ◽

2012 ◽

Vol 2012 ◽

pp. 1-15 ◽

Cited By ~ 25

Author(s):

Michael Torbenson

Keyword(s):

Tumor Cells ◽

Primary Liver Cancer ◽

Neuroendocrine Differentiation ◽

Science Literature ◽

Unique Type ◽

Younger Age ◽

Genetic Abnormalities ◽

Fibrolamellar Carcinoma ◽

Prognostic Feature ◽

Children And Young Adults

Fibrolamellar carcinomas are a unique type of primary liver cancer. They occur most commonly in children and young adults. Their etiology remains a mystery, as they are not associated with chronic liver disease. Fibrolamellar carcinomas are not indolent tumors, but have an overall better prognosis than typical hepatocellular carcinomas, in part because of the younger age at presentation and the lack of cirrhosis. The most important prognostic feature is whether the tumor is resectable. Histologically, the tumor is made up of large cells that contain abundant mitochondria. The nuclei of the tumor cells have prominent nucleoli. The tumor cells induce the formation of extensive intratumoral fibrosis, which often grows in parallel, or lamellar bands. The tumor cells clearly show hepatocellular features but are also unique in showing both biliary and neuroendocrine differentiation. The uniqueness of fibrolamellar carcinoma extends to their molecular findings. While the genetic abnormalities that lead to fibrolamellar carcinomas are not yet known, studies have shown that they lack mutations in the genes most commonly mutated in typical hepatocellular carcinoma (TP53andCTNNB1). In this paper, the clinical, pathological, and basic science literature on fibrolamellar carcinoma is comprehensively reviewed. Key areas of needed research are also discussed.

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High glucose induces Drp1-mediated mitochondrial fission via the Orai1 calcium channel to participate in diabetic cardiomyocyte hypertrophy

Cell Death and Disease ◽

10.1038/s41419-021-03502-4 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Qing-Rui Wu ◽

Dan-Lin Zheng ◽

Pei-Ming Liu ◽

Hui Yang ◽

Lu-An Li ◽

...

Keyword(s):

Calcium Channel ◽

Mitochondrial Dysfunction ◽

High Glucose ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Calcium Handling ◽

Cardiomyocyte Hypertrophy ◽

Mitochondrial Morphology ◽

Calmodulin Binding ◽

Downstream Target

AbstractMitochondrial dysfunction and impaired Ca2+ handling are involved in the development of diabetic cardiomyopathy (DCM). Dynamic relative protein 1 (Drp1) regulates mitochondrial fission by changing its level of phosphorylation, and the Orai1 (Ca2+ release-activated calcium channel protein 1) calcium channel is important for the increase in Ca2+ entry into cardiomyocytes. We aimed to explore the mechanism of Drp1 and Orai1 in cardiomyocyte hypertrophy caused by high glucose (HG). We found that Zucker diabetic fat rats induced by administration of a high-fat diet develop cardiac hypertrophy and impaired cardiac function, accompanied by the activation of mitochondrial dynamics and calcium handling pathway-related proteins. Moreover, HG induces cardiomyocyte hypertrophy, accompanied by abnormal mitochondrial morphology and function, and increased Orai1-mediated Ca2+ influx. Mechanistically, the Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1) prevents cardiomyocyte hypertrophy induced by HG by reducing phosphorylation of Drp1 at serine 616 (S616) and increasing phosphorylation at S637. Inhibition of Orai1 with single guide RNA (sgOrai1) or an inhibitor (BTP2) not only suppressed Drp1 activity and calmodulin-binding catalytic subunit A (CnA) and phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) expression but also alleviated mitochondrial dysfunction and cardiomyocyte hypertrophy caused by HG. In addition, the CnA inhibitor cyclosporin A and p-ERK1/2 inhibitor U0126 improved HG-induced cardiomyocyte hypertrophy by promoting and inhibiting phosphorylation of Drp1 at S637 and S616, respectively. In summary, we identified Drp1 as a downstream target of Orai1-mediated Ca2+ entry, via activation by p-ERK1/2-mediated phosphorylation at S616 or CnA-mediated dephosphorylation at S637 in DCM. Thus, the Orai1–Drp1 axis is a novel target for treating DCM.

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Dynamin-related protein 1 deficiency accelerates lipopolysaccharide-induced acute liver injury and inflammation in mice

Communications Biology ◽

10.1038/s42003-021-02413-6 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Lixiang Wang ◽

Xin Li ◽

Yuki Hanada ◽

Nao Hasuzawa ◽

Yoshinori Moriyama ◽

...

Keyword(s):

Liver Disease ◽

Liver Injury ◽

Er Stress ◽

Disease Progression ◽

Acute Liver Injury ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Mitochondrial Fusion ◽

Related Protein ◽

Liver Disease Progression

AbstractMitochondrial fusion and fission, which are strongly related to normal mitochondrial function, are referred to as mitochondrial dynamics. Mitochondrial fusion defects in the liver cause a non-alcoholic steatohepatitis-like phenotype and liver cancer. However, whether mitochondrial fission defect directly impair liver function and stimulate liver disease progression, too, is unclear. Dynamin-related protein 1 (DRP1) is a key factor controlling mitochondrial fission. We hypothesized that DRP1 defects are a causal factor directly involved in liver disease development and stimulate liver disease progression. Drp1 defects directly promoted endoplasmic reticulum (ER) stress, hepatocyte death, and subsequently induced infiltration of inflammatory macrophages. Drp1 deletion increased the expression of numerous genes involved in the immune response and DNA damage in Drp1LiKO mouse primary hepatocytes. We administered lipopolysaccharide (LPS) to liver-specific Drp1-knockout (Drp1LiKO) mice and observed an increased inflammatory cytokine expression in the liver and serum caused by exaggerated ER stress and enhanced inflammasome activation. This study indicates that Drp1 defect-induced mitochondrial dynamics dysfunction directly regulates the fate and function of hepatocytes and enhances LPS-induced acute liver injury in vivo.

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