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• Research Paper Volume 12, Issue 20 pp 20152-20162

  Polymerase I and transcript release factor transgenic mice show impaired function of hematopoietic stem cells

  Relevance score: 11.982862

  Lin Bai, Ying Lyu, Guiying Shi, Keya Li, Yiying Huang, Yuanwu Ma, Yu-Sheng Cong, Lianfeng Zhang, Chuan Qin

  Keywords: PTRF, HSCs, caveolin-1

  Published in Aging on October 21, 2020

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  The age-dependent decline in stem cell function plays a critical role in aging, although the molecular mechanisms remain unclear. PTRF/Cavin-1 is an essential component in the biogenesis and function of caveolae, which regulates cell proliferation, endocytosis, signal transduction and senescence. This study aimed to analyze the role of PTRF in hematopoietic stem cells (HSCs) senescence using PTRF transgenic mice. Flow cytometry was used to detect the frequency of immune cells and hematopoietic stem/progenitor cells (HSCs and HPCs). The results showed than the HSC compartment was significantly expanded in the bone marrow of PTRF transgenic mice compared to age-matched wild-type (WT) mice, and exhibited the senescent phenotype characterized by G1 cell cycle arrest, increased SA-β-Gal activity and high levels of reactive oxygen species (ROS). The PTRF-overexpressing HSCs also showed significantly lower self-renewal and ability to reconstitute hematopoiesis in vitro and in vivo. Real-time PCR was performed to analyze the expression levels of senescence-related genes. PTRF induced HSCs senescence via the ROS-p38-p16 and caveolin-1-p53-p21 pathways. Furthermore, the PTRF⁺cav-1^-/- mice showed similar HSCs function as WT mice, indicating that PTRF induces senescence in HSCs partly through caveolin-1. Thus PTRF impaired HSCs aging partly via caveolin-1.

• Research Paper Volume 11, Issue 13 pp 4323-4337

  HMGB1 and Caveolin-1 related to RPE cell senescence in age-related macular degeneration

  Relevance score: 9.705855

  Shuo Sun, Bincui Cai, Yao Li, Wenqi Su, Xuzheng Zhao, Boteng Gong, Zhiqing Li, Xiaomin Zhang, Yalin Wu, Chao Chen, Stephen H. Tsang, Jin Yang, Xiaorong Li

  Keywords: A2E, HMGB1, Caveolin-1, RPE cell senescence, AMD

  Published in Aging on July 7, 2019

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  Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is considered a major cause of RPE dysfunction and senescence in age-related macular degeneration (AMD), and N-retinylidene-N-retinylethanolamine (A2E) is the main fluorophore identified in lipofuscin from aged human eyes. Here, human-induced pluripotent stem cell (iPSC)-RPE was generated from healthy individuals to reveal proteomic changes associated with A2E-related RPE cell senescence. A novel RPE cell senescence-related protein, high-mobility group box 1 (HMGB1), was identified based on proteomic mass spectrometry measurements on iPSC-RPE with A2E treatment. Furthermore, HMGB1 upregulated Caveolin-1, which also was related RPE cell senescence. To investigate whether changes in HMGB1 and Caveolin-1 expression under A2E exposure contribute to RPE cell senescence, human ARPE-19 cells were stimulated with A2E; expression of HMGB1, Caveolin-1, tight junction proteins and senescent phenotypes were verified. HMGB1 inhibition alleviated A2E induced cell senescence. Migration of RPE cells was evaluated. Notably, A2E less than or equal to 10μM induced both HMGB1 and Caveolin-1 protein upregulation and HMGB1 translocation, while Caveolin-1 expression was downregulated when there was more than 10μM A2E. Our data indicate that A2E-induced upregulation of HMGB1、Caveolin-1 and HMGB1 release may relate to RPE cell senescence and play a role in the pathogenesis of AMD.

  

  Proteomic mass spectrometry-based measurement of differential expression of HMGB1. (A) The flow chart of shotgun mass spectrometry. (B) Volcano plot illustrating significant differential abundant proteins based on quantitative analysis. The -log₁₀ (P value) was plotted against log₂(fold change A2E treatment/Control). Proteins were significantly upregulated (red dots) or downregulated (green dots) between the A2E treatment and control. The red arrowhead indicates HMGB1.

  
  
  

  

  Experimental validation that blue light exposure of A2E-treated ARPE-19 cells induces HMGB1 upregulation and translocation. (A) An MTT assay was performed on RPE cells treated with different concentrations of A2E with or without blue light photosensitization. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, *** indicates a p value < 0.001, compared to the control, n=3. (B) FDA/PI staining of RPE cells after in vitro culture for 48 h with 10 μM A2E + blue light (10 min). Most living RPE cells were stained green by fluorescein diacetate (FDA); a few dead cells were stained red bypropidium iodide (PI). (C) Western blot analyses showed that HMGB1 protein expression was higher in 10μM A2E + blue light-treated cells compared to the control and also higher in the blue light treatment, as quantified by densitometry; the results are expressed as a ratio with β-actin. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (D) HMGB1 localization in RPE cells was assessed by confocal microscopy after 10μM A2E + blue light treatment. HMGB1 moved from the nucleus (arrow) to the cytoplasm (star) after 10μM A2E + blue light treatment. Nuclei are labelled with DAPI (blue); HMGB1 is stained green.

  
  
  

  

  HMGB1 upregulation and release increase the expression of Caveolin-1. (A) (i) Western blot analyses showed that overexpression of HMGB1 upregulated Caveolin-1; β-actin was used as the loading control; Western blot results were quantified by densitometry, and the results are expressed as a ratio with β-actin. (ii) qPCR analyses showed that overexpression of HMGB1 upregulated Caveolin-1. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (iii) Expression of EGFP and Caveolin-1 was assessed by immunofluorescence in HMGB1-overexpressing RPE cells and negative-control RPE cells. (B) Protein interaction between HMGB1 and Caveolin-1 was revealed by the STRING version 9.1 program. (C) Relative Caveolin-1expression in RPE cell incubated with normal medium, 1μg/ml rHMGB1, 100μM GA, or 1μg/ml rHMGB1+100μM GA, Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (D) Western blot analyses showed that knock-down of HMGB1 downregulated Caveolin-1; Tublin was used as the loading control, western blot results were quantified by densitometry, and the results are expressed as a ratio with Tublin. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3.

  
  
  

  

  Overexpression of Caveolin-1 induced ARPE-19 cell senescence and inhibited migration and invasion. (A) Western blot analyses showed that overexpression of Caveolin-1 upregulated Zo-1 and β-catenin; β-actin was used as the loading control. (B) Western blot results were quantified by densitometry, and the results are expressed as a ratio with β-actin. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, *** indicates a p value < 0.001, n=3. (C) qPCR analyses showed that overexpression of Caveolin-1 upregulated Zo-1 and β-catenin. Data are presented as means ± SD; * indicates a p value < 0.05, n=3. (D) Expression of EGFP, Zo-1 and β-catenin was assessed by immunofluorescence in Caveolin-1-overexpressing RPE cells and negative-control RPE cells. (E) Representative microscopic images of β-galactosidase staining in RPE cells showed overexpression of Caveolin-1 in RPE cells compared with that in negative-control RPE cells. Quantification of percentage of cells with positive SA-β-gal staining.Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (F) (i) Wound-healing assays in Caveolin-1-overexpressing RPE cells. (ii). Transwell invasion assays in Caveolin-1-overexpressing RPE cells. (G) (i) The rate of cell migration in different groups was measured at different time points. Note that cell migration was decreased in Caveolin-1-overexpressing RPE cells. (ii) The mean number of invaded cells was assessed in 5 fields. Note that cell invasion was decreased in Caveolin-1-overexpressing RPE cells. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, *** indicates a p value < 0.001, n=3.

  
  
  

  

  Blue light exposure of A2E-treated ARPE-19 cells increased HMGB1 and Caveolin-1 expression. (A) Western blot assay for HMGB1 and Caveolin-1 in RPE cells treated with a concentration gradient of A2E with or without blue light, quantified by densitometry, and the results are expressed as a ratio with β-actin. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (B) Representative microscopic images of β-galactosidase staining in RPE cells with various concentrations of A2E. Quantification of percentage of cells with positive SA-β-gal staining.Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (C) The release of HMGB1 induced by A2E treatment were detected by ELISA assays.

  
  
  

  

  Glycyrrhizic acid alleviated A2E induced cell senescence. (A) An MTT assay was performed on RPE cells treated with different concentrations of GA. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (B)The release of HMGB1 induced by different concentrations of A2E+BL with or without 100μM GA were detected by ELISA assays. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (C) Representative microscopic images of β-galactosidase staining in RPE cells induced by different concentrations of A2E+BL with or without 100μM GA. (D) Quantification of percentage of cells with positive SA-β-gal staining. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (E) Proposed schematic model for strategies for HMGB1 inhibition in response to A2E treatment.

  
  
  

• Research Paper Volume 11, Issue 7 pp 2138-2150

  MiR-204 reduces cisplatin resistance in non-small cell lung cancer through suppression of the caveolin-1/AKT/Bad pathway

  Relevance score: 11.151142

  Gang Huang, Tianzheng Lou, Jiongwei Pan, Zaiting Ye, Zhangyong Yin, Lu Li, Wei Cheng, Zhuo Cao

  Keywords: miR-204, caveolin-1, cisplatin, resistance, NSCLC

  Published in Aging on April 12, 2019

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  Non-small cell lung cancer (NSCLC) is the most common and lethal human malignant tumor worldwide. Platinum-based chemotherapy is still the mainstay of treatment for NSCLC. However, long-term chemotherapy usually induces serious drug resistance in NSCLC cells. Accordingly, treatment strategies that reverse the resistance of NSCLC cells against platinum-based drugs may have considerable clinical value. In the present study, we observed significant upregulation of CAV-1 expression and a significant decrease of miR-204 expression in cisplatin-resistant A549 (CR-A549) and cisplatin-resistant PC9 (CR-PC9) cells compared to their parental A549 and PC9 cells. Furthermore, we demonstrated that the downregulation of miR-204 expression was responsible for CAV-1 overexpression in these cisplatin-resistant NSCLC cells. We then found that enforced expression of miR-204 can resensitize CR-A549 and CR-PC9 cells to cisplatin-induced mitochondrial apoptosis through suppression of the caveolin-1/AKT/Bad pathway. We demonstrated that dysregulation of miR-204/caveolin-1 axis is an important mechanism for NSCLC cells to develop the chemoresistance.

  

  Cisplatin resistance of CR-A549 and CR-PC9 cells. (A) After treatment with different concentrations of cisplatin (0–60 μM), viability of A549 and CR-A549 cells was detected by using MTT assays. *P<0.05 vs. A549 cells. (B) After treatment with different concentrations of cisplatin (0–60 μM), viability of PC9 and CR-PC9 cells was detected by using MTT assays. *P<0.05 vs. PC9 cells.

  
  
  

  

  Role of CAV-1 in regulating cisplatin sensitivity in NSCLC. (A) Expression of CAV-1 in A549, CR-A549, PC9, and CR-PC9 cells was detected by western blot analysis. (B) Effect of the CAV-1 plasmid (2 μg/ml) and CAV-1 siRNA (50 pmol/ml) on the expression level of CAV-1 in A549, CR-A549, PC9, and CR-PC9 cells. (C) Effect of CAV-1 siRNA (50 pmol/ml) on the sensitivity of CR-A549 and CR-PC9 cells to cisplatin (8 μM) treatment. *P<0.05 vs. Cisplatin+NCO group. (D) Effect of the CAV-1 plasmid (2 μg/ml) on the sensitivity of A549 and PC9 cells to cisplatin (8 μM) treatment. *P<0.05 vs. Cisplatin+NCO group.

  
  
  

  

  miR-204 targets CAV-1 in NSCLC. (A) Seed region of the CAV-1 3′ UTR paired with miR-204. (B) Expression of miR-204 in A549, CR-A549, PC9, and CR-PC9 cell lines. *P<0.05. (C) Transfection efficiency of miR-204 in CR-A549 and CR-PC9 cells. *P<0.05 vs. NCO group. (D) Effect of miR-204 (50 pmol/ml) on the expression level of CAV-1 in CR-A549 and CR-PC9 cells. (E) Luciferase activities in CR-A549 and CR-PC9 cells were measured using the Dual-Luciferase Reporter Assay System. *P<0.05 vs. NCO group.

  
  
  

  

  Role of the miR-204/CAV-1 axis in regulating cisplatin sensitivity in NSCLC. (A) Effect of miR-204 (50 pmol/ml) on the sensitivity of CR-A549 cells to cisplatin treatment (0–60 μM). *P<0.05 vs. NCO group. (B) Effect of miR-204 (50 pmol/ml) on the sensitivity of CR-PC9 cells to cisplatin treatment (0–60 μM). *P<0.05 vs. NCO group. (C) Effect of the CAV-1 plasmid (2 μg/ml) on protecting the CR-A549 and CR-PC9 cells that were co-treated with cisplatin (8 μM) and miR-204 (50 pmol/ml). *P<0.05 vs. Cisplatin+NCO group. ^#P<0.05 vs. Cisplatin+miR-204 group. (D) Effect of the anti-miR-204 (50 pmol/ml) on protecting the A549 and PC9 cells that were co-treated with cisplatin (8 μM). *P<0.05 vs. Cisplatin+NCO group.

  
  
  

  

  miR-204/CAV-1 axis regulates the AKT/Bad pathway in cisplatin-resistant NSCLC cells. (A) Effect of miR-204 (50 pmol/ml), cisplatin (8 μM), and the CVA-1 plasmid (2 μg/ml) on the phosphorylation of AKT and Bad in CR-A549 and CR-PC9 cells. (B) A co-immunoprecipitation assay was performed to evaluate interactions with Bad and Bcl-xl/Bcl-2 after treatment with miR-204 (50 pmol/ml), cisplatin (8 μM), and the CVA-1 plasmid (2 μg/ml). (C) Effect of miR-204 (50 pmol/ml), cisplatin (8 μM), and the CVA-1 plasmid (2 μg/ml) on the mitochondrial membrane potential (ΔΨ_m) of CR-A549 and CR-PC9 cells.

  
  
  

  

  miR-204/CAV-1 axis regulates the mitochondrial apoptosis pathway in cisplatin-resistant NSCLC cells. (A) After mitochondria removal, the protein level of cytochrome c in cytosol was measured using western blot analysis. (B) Cleavage of caspase-9 and caspase-3 in CR-A549 and CR-PC9 cells was detected using western blot analysis. (C) Effect of miR-204 (50 pmol/ml), cisplatin (8 μM), and the CVA-1 plasmid (2 μg/ml) on the apoptotic rate of CR-A549 and CR-PC9 cells. *P<0.05 vs. Cisplatin+NCO group. ^#P<0.05 vs. Cisplatin+miR-204 group.

  
  
  

  

  miR-204 enhances the anti-tumor effect of cisplatin on cisplatin-resistant NSCLC in vivo. (A) Nude mice were inoculated with CR-A549/control or CR-A549/miR-204 cells before treatment with cisplatin (5 mg/kg) twice a week. Tumor volumes were detected every three days until the experimental end-point (28 days post-injection). (B) Expression of miR-204 in tumor tissues was measured by qRT-PCR analysis. *P<0.05 vs. CR-A549/control group. ^#P<0.05 vs. CR-A549/control+cisplatin group. (C) Expression of CAV-1 and phosphorylation of AKT and Bad were evaluated by western blot analysis.

  
  
  

• Research Paper Volume 8, Issue 10 pp 2355-2369

  Caveolin-1 controls mitochondrial function through regulation of m-AAA mitochondrial protease

  Relevance score: 10.616009

  Daniela Volonte, Zhongmin Liu, Sruti Shiva, Ferruccio Galbiati

  Keywords: caveolin-1, caveolae, oxidative stress, mitochondria, glycolysis

  Published in Aging on October 4, 2016

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  Mitochondrial proteases ensure mitochondrial integrity and function after oxidative stress by providing mitochondrial protein quality control. However, the molecular mechanisms that regulate this basic biological function in eukaryotic cells remain largely unknown. Caveolin-1 is a scaffolding protein involved in signal transduction. We find that AFG3L2, a m-AAA type of mitochondrial protease, is a novel caveolin-1-interacting protein in vitro. We show that oxidative stress promotes the translocation of both caveolin-1 and AFG3L2 to mitochondria, enhances the interaction of caveolin-1 with AFG3L2 in mitochondria and stimulates mitochondrial protease activity in wild-type fibroblasts. Localization of AFG3L2 to mitochondria after oxidative stress is inhibited in fibroblasts lacking caveolin-1, which results in impaired mitochondrial protein quality control, an oxidative phosphorylation to aerobic glycolysis switch and reduced ATP production. Mechanistically, we demonstrate that a lack of caveolin-1 does not alter either mitochondrial number or morphology but leads to the cytoplasmic and proteasome-dependent degradation of complexes I, III, IV and V upon oxidant stimulation. Restoration of mitochondrial respiratory chain complexes in caveolin-1 null fibroblasts reverts the enhanced glycolysis observed in these cells. Expression of a mutant form of AFG3L2, which has reduced affinity for caveolin-1, fails to localize to mitochondria and promotes degradation of complex IV after oxidative stress. Thus, caveolin-1 maintains mitochondrial integrity and function when cells are challenged with free radicals by promoting the mitochondrial localization of m-AAA protease and its quality control functions.

  

  AFG3L2 directly interacts with caveolin-1 in vitro. (A) Schematic diagram showing the consensus caveolin-binding-domain (CBD) and the CBD of human AFG3L2 (amino acids 138-146 and 147-154). Φ represents an aromatic amino acid and X represents any amino acid. (B) Caveolin-1-GST fusion proteins [GST-Cav-1(82-101), GST-Cav-1(1-101) and GST-Cav-1-FL] were used in pull down assays with cell lysates from NIH 3T3 cells transiently transfected with AFG3L2-myc. Pull-down assays with GST alone was used as internal control.

  
  
  

  

  AFG3L2 interacts with caveolin-1 after oxidative stress in vivo. Wild type and caveolin-1 null mouse embryonic fibroblasts (MEFs) were treated with sublethal doses of hydrogen peroxide (150 μM) for 2 hours. Cells were then recovered in complete medium for 7 days. We have chosen these conditions because we have previously shown that they upregulate caveolin-1 expression [20,22,23] and activate caveolin-1-mediated signaling [19,21,24,28,29]. Untreated cells (-H₂O₂) were used as control. (A) Mitochondrial fractions were isolated and the expression levels of caveolin-1, prohibitin-1 and AFG3L2 were measured by immunoblotting analysis. (B) Mitochondrial fractions were isolated from untreated and hydrogen peroxide-treated wild type MEFs and immunoprecipitated using an antibody probe specific for caveolin-1 (Cav-1); immunoprecipitates were then subjected to immunoblotting analysis with anti-AFG3L2 and prohibitin-1 IgGs. (C) Mitochondria were isolated and mitochondrial protease activity was quantified using the Protease Fluorescent Detection Kit from Sigma-Aldrich (St. Louis, MO) (PF0100). Values in (C) represent mean ± SEM; *^,#P<0.001.

  
  
  

  

  A lack of caveolin-1 promotes bioenergetic defects in ROS-treated fibroblasts. Wild type and caveolin-1 null mouse embryonic fibroblasts (MEFs) were treated with sublethal doses of hydrogen peroxide (150 μM) for 2 hours. Cells were then recovered in complete medium for 7 days. Untreated cells (-H₂O₂) were used as control. Bioenergetic profile was determined using the Seahorse Metabolic Analyzer, which simultaneously measures oxygen consumption rate (OCR) (A) and extracellular acidification rate (ECAR) (B). Viability was assessed by crystal violet staining. OCR and ECAR were normalized to cell number. Values in (A) and (B) represent mean ± SEM; *P<0.001.

  
  
  

  

  Lactate production is increased and ATP synthesis is inhibited in caveolin-1-lacking fibroblasts following oxidative stress. (A-B) Wild type and caveolin-1 null mouse embryonic fibroblasts (MEFs) were treated with sublethal doses of hydrogen peroxide (150 μM) for 2 hours. Cells were then recovered in complete medium for different periods of time (3 days and 7 days). Untreated cells (-H₂O₂) were used as control. (A) Lactate production was quantified using the Lactate Assay Kit from Sigma-Aldrich (MAK064). (B) ATP production was quantified using the Adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit from Sigma-Aldrich (FL-AA). Values were normalized to cell number. (C) Wild type and caveolin-1 null mouse embryonic fibroblasts (MEFs) were treated with sublethal doses of hydrogen peroxide (150 μM) for 2 hours in the presence or absence of 2-deoxy-D-glucose (2-DG; 5mM). Cells were recovered in complete medium for 3 days in the presence or absence of 2-DG. Untreated cells (-H₂O₂) were used as control. Cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values in (A-C) represent mean ± SEM; *P<0.001.

  
  
  

  

  Oxidative stress promotes degradation of mitochondrial respiratory chain complexes in caveolin-1 null MEFs. Wild type and caveolin-1 null mouse embryonic fibroblasts (MEFs) were treated with sublethal doses of hydrogen peroxide (150 μM) for 2 hours. Cells were then recovered in complete medium for different periods of time. Untreated cells (-H₂O₂) were used as control. (A) The ratio of mitochondrial to nuclear DNA was quantified by performing RT-PCR analysis for the mitochondrial gene ND1 and the nuclear encoded gene Histone 19 using gene-specific primers. (B) Cells were incubated with Mitotracker Green FM (Thermo Fisher Scientific; Waltham, MA) at a concentration of 100 nM in DMEM. Cells were incubated at 37°C for 30 min, washed with PBS and imaged using a Zeiss Confocal Microscope (LSM 5 Pascal; Carl Zeiss, Jena, Germany). (C) The expression level of complex I, complex II, complex III, complex IV, complex V and caveolin-1 was determined by immunoblotting analysis using specific antibody probes. Immunoblotting with anti-β-actin IgGs was performed as internal control. (D) RT-PCR analysis for complex I, complex II, complex III, complex IV and complex V was performed using gene-specific primers. RT-PCR analysis using primers for LR32 was performed as internal control.

  
  
  

  

  Proteasome inhibition rescues the expression of respiratory chain complexes in ROS-treated caveolin-1 null MEFs. Wild type and caveolin-1 null mouse embryonic fibroblasts (MEFs) were treated with sublethal doses of hydrogen peroxide (150 μM) for 2 hours in the presence or absence of 0.1 μM MG-132. Cells were then recovered in complete medium for 7 days in the presence or absence of 0.1 μM MG-132. Untreated cells (-H₂O₂) were used as control. (A) Total expression of complex I, complex II, complex III and complex IV was determined by immunoblotting analysis using specific antibody probes. Immunoblotting with anti-β-actin IgGs was performed as internal control. (B) Mitochondria were isolated and the expression of complex IV was determined using anti-complex IV IgGs. (C) Lactate production was quantified using the Lactate Assay Kit from Sigma-Aldrich (MAK064). Values were normalized to cell number. Values in (C) represent mean ± SEM; *P<0.001.

  
  
  

  

  Φ→A-AFG3L2 poorly interacts with caveolin-1, does not accumulate in mitochondria and promotes degradation of complex IV after oxidative stress. (A) GST-Cav-1(82-101) was used in pull down assays with cell lysates from NIH 3T3 cells transiently transfected with either wild type AFG3L2-myc or Φ→A-AFG3L2-myc. Pull-down assays with GST alone was used as internal control. (B) Wild type mouse embryonic fibroblasts (MEFs) were infected with a lentiviral vector (pLVX) expressing either WT-AFG3L2-myc or Φ→A-AFG3L2-myc. After 48 hours, cells were treated with sublethal doses of hydrogen peroxide (150 μM) for 2 hours. Cells were then recovered in complete medium for 7 days. Mitochondrial fractions (MITO) were isolated and the expression levels of AFG3L2-myc and complex IV were measured by immunoblotting analysis. Total expression (TOT) of WT-AFG3L2-myc and Φ→A-AFG3L2-myc is shown in the upper panel.

  
  
  

  

  Schematic diagram summarizing the control of mitochondrial functions by caveolin-1 through the regulation of AFG3L2. Under resting conditions (-ROS), both wild type and caveolin-1 null cells possess functional respiratory chain complexes and generate energy mostly through oxidative phosphorylation. Upon oxidative stress, the caveolin-1-dependent localization of AFG3L2 to mitochondria in wild type cells prevents ROS-mediated mitochondrial damage by providing mitochondrial protein quality control. As a consequence, functional respiratory chain complexes are maintained. After oxidative stress but in the absence of caveolin-1, AFG3L2 fails to localize to mitochondria and the AFG3L2-mediated protective mechanism is lost, leading to the degradation of respiratory chain proteins. Under these conditions, oxidative phosphorylation is impaired and caveolin-1 null cells rely on enhanced glycolysis for their bioenergetic requirements.

  
  
  

• Research Paper Volume 2, Issue 4 pp 185-199

  Transcriptional evidence for the "Reverse Warburg Effect" in human breast cancer tumor stroma and metastasis: Similarities with oxidative stress, inflammation, Alzheimer's disease, and "Neuron-Glia Metabolic Coupling"

  Relevance score: 7.033004

  Stephanos Pavlides, Aristotelis Tsirigos, Iset Vera, Neal Flomenberg, Philippe G. Frank, Mathew C. Casimiro, Chenguang Wang, Richard G. Pestell, Ubaldo E. Martinez-Outschoorn, Anthony Howell, Federica Sotgia, Michael P. Lisanti

  Keywords: caveolin-1, tumor stroma, oxidative stress, hypoxia, inflammation, mitochondrial dysfunction, Alzheimer's disease, neuron-glia metabolic coupling

  Published in Aging on March 31, 2010

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  Caveolin-1 (-/-) null stromal cells are a novel genetic model for cancer-associated fibroblasts and myofibroblasts. Here, we used an unbiased informatics analysis of transcriptional gene profiling to show that Cav-1 (-/-) bone-marrow derived stromal cells bear a striking resemblance to the activated tumor stroma of human breast cancers. More specifically, the transcriptional profiles of Cav-1 (-/-) stromal cells were most closely related to the primary tumor stroma of breast cancer patients that had undergone lymph-node (LN) metastasis. This is consistent with previous morphological data demonstrating that a loss of stromal Cav-1 protein (by immuno-histochemical staining in the fibroblast compartment) is significantly associated with increased LN-metastasis. We also provide evidence that the tumor stroma of human breast cancers shows a transcriptional shift towards oxidative stress, DNA damage/repair, inflammation, hypoxia, and aerobic glycolysis, consistent with the "Reverse Warburg Effect". Finally, the tumor stroma of "metastasis-prone" breast cancer patients was most closely related to the transcriptional profiles derived from the brains of patients with Alzheimer's disease. This suggests that certain fundamental biological processes are common to both an activated tumor stroma and neuro-degenerative stress. These processes may include oxidative stress, NO over-production (peroxynitrite formation), inflammation, hypoxia, and mitochondrial dysfunction, which are thought to occur in Alzheimer's disease pathology. Thus, a loss of Cav-1 expression in cancer-associated myofibroblasts may be a protein biomarker for oxidative stress, aerobic glycolysis, and inflammation, driving the "Reverse Warburg Effect" in the tumor micro-environment and cancer cell metastasis.

  

  A HeatMap containing 205 intersecting genes is shown (FC >1.5; p <0.05). See also Supplementary Tables. FC, fold-change.

  
  
  

  

  In "Neuron-Glia Metabolic Coupling", astrocytes take up more glucose, shift towards aerobic glycolyis, secrete pyruvate and lactate, which is then taken up by adjacent neurons and then "feeds" into the neuronal TCA cycle, resulting in increased neuronal oxidative mitochondrial metabolism, and higher ATP production in neurons. In essence, the astrocytes function as support cells to "feed" the adjacent neuronal cells. This schematic diagram shows that "Neuron-Glia Metabolic Coupling" and the "Reverse Warburg Effect" are analogous biological processes, where the astrocytes are the cancer-associated fibroblasts and the neurons are the epithelial tumor cells. Thus, the "Reverse Warburg Effect" could also be more generally termed "Epithelial-Stromal Metabolic Coupling" or "Epithelial-Fibroblast Metabolic Coupling". This figure was partially re-drawn from Bonucelli et al. 2010, with permission [24]. MCT, mono-carboxylate transporter.