bims-noxint Biomed News
on NADPH oxidases in tumorigenesis
Issue of 2021‒01‒24
eight papers selected by
Laia Caja Puigsubira
Uppsala University

  1. J Biol Chem. 2020 Nov 23. pii: S0021-9258(20)00023-X. [Epub ahead of print]296 100037
    Chen X, Zhao C, Xu Y, Huang K, Wang Y, Wang X, Zhou X, Pang W, Yang G, Yu T.
      With the improvement of people's living standards, the number of obese patients has also grown rapidly. It is reported that the level of oxidative stress in obese patients has significantly increased, mainly caused by the increase in reactive oxygen species (ROS) levels in adipose tissue. Studies have shown that the use of siRNA to interfere with bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) expression could promote adipocyte differentiation, and under hypoxic conditions, BAMBI could act as a regulator of HIF1α to regulate the polarity damage of epithelial cells. In view of these results, we speculated that BAMBI may regulate adipogenesis by regulating the level of ROS. In this study, we generated adipose-specific BAMBI knockout mice (BAMBI AKO) and found that compared with control mice, BAMBI AKO mice showed obesity when fed with high-fat diet, accompanied by insulin resistance, glucose intolerance, hypercholesterolemia, and increased inflammation in adipose tissue. Interestingly, adipose-specific deficiency of BAMBI could cause an increase in the expression level of Nox4, thereby promoting ROS production in cytoplasm and mitochondria and the DNA-binding activity of C/EBPβ and ultimately promoting adipogenesis. Consistently, our findings indicated that BAMBI may be a reactive oxygen regulator to affect adipogenesis, thereby controlling obesity and metabolic syndrome.
    Keywords:  BAMBI; NADPH oxidase 4; ROS production; adipogenesis; insulin resistance
  2. J Biol Chem. 2020 Aug 21. pii: S0021-9258(17)50073-3. [Epub ahead of print]295(34): 12130-12142
    Arrington ME, Temple B, Schaefer A, Campbell SL.
      The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K. Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K. As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.
    Keywords:  GTPase-activating protein (GAP); NADPH oxidase; NADPH oxidase (NOX2); RAS; Ras protein; Ras-related C3 botulinum toxin substrate 2 (RAC2); T-cell lymphoma and metastasis 1 (TIAM1); dedicator of cytokinesis (DOCK); guanine nucleotide exchange factor (GEF); immunodeficiency; molecular dynamics; molecular dynamics simulations; nucleotide exchange factor (GEF); p21-activated kinase (PAK); serine/threonine-protein kinase PAK1
  3. Cardiovasc Res. 2021 Jan 23. pii: cvab019. [Epub ahead of print]
    Mighiu AS, Recalde A, Ziberna K, Carnicer R, Tomek J, Bub G, Brewer AC, Verheule S, Shah AM, Simon JN, Casadei B.
      AIMS: Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility.METHODS AND RESULTS: NOX2-Tg mice showed a 2-2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (2-fold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1β, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype.
    CONCLUSIONS: Together, these data indicate that whilst atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.
    TRANSLATIONAL PERSPECTIVE: Atrial NOX2-derived superoxide is an independent predictor of postoperative AF in humans and contributes to atrial oxidative stress early after AF induction. Whilst experimental models have reported an association of NOX2 with AF-induced remodelling, a causal link between NOX2 activity and AF remains to be established. Here we show that overexpression of the human NOX2 gene in mice resulted in a 2-fold higher atrial superoxide production and a modest increase in AF susceptibility, independent of atrial electrical or structural remodelling. Short-term treatment with atorvastatin normalized atrial superoxide in NOX2-Tg mice without affecting AF susceptibility. Together these findings indicate that atrial NOX2-derived superoxide is more likely a biomarker of AF risk than a primary driver of AF development, and that NOX2 inhibition is unlikely to prevent the new-onset of AF.
    Keywords:  Arrhythmia (mechanisms); Atorvastatin; Atrial Fibrillation; NADPH oxidases; oxidant stress
  4. Int J Ophthalmol. 2021 ;14(1): 50-56
    Dong K, Yang N, Ding J, Yan YY, Lu L, Wang YS.
      AIM: To observe the effects of the inhibition of NADPH oxidase 4 (NOX4) expression on the retinal vascular barriers and visual function after retinal detachment (RD).METHODS: RD model was established 3wk after adeno-associaned virus vector injection. The retinal tissue was harvested 3d after RD, and the death of retinal vascular endothelial cells and photoreceptors was observed using electron microscopy. The NOX4 expression was detected by Western blot. Confocal microscopy was used to observe a retinal patch that had been perfused with Evans blue. A modified water maze test was used to detect the time required to find the platform on the water surface. The visual function of the rats was evaluated and reactive oxygen species (ROS) expression was detected by a fluorescence microplate reader.
    RESULTS: The retinal patch showed that NOX4 interference significantly reduced the destruction of the tight junctions between the retinal endothelium of RD rats and reduced leakage. Western blotting showed decreased expression of the NOX4 protein and decreased expression of ROS in retinal tissue; the Morris water maze test results showed that NOX4 interference significantly decreased the escape latency of the rats.
    CONCLUSION: NOX4 interference reduces the production of ROS in retinal vascular endothelial cells after experimental RD, thereby protecting the blood-retinal barrier and protecting visual function.
    Keywords:  NOX4; adeno-associated virus; blood-retinal barrier; reactive oxygen species; retinal detachment
  5. Antioxidants (Basel). 2021 Jan 19. pii: E134. [Epub ahead of print]10(1):
    Ammendola R, Parisi M, Esposito G, Cattaneo F.
      BACKGROUND: Formyl peptide receptor 2 (FPR2) is involved in the pathogenesis of chronic inflammatory diseases, being activated either by pro-resolving or proinflammatory ligands. FPR2-associated signal transduction pathways result in phosphorylation of several proteins and in NADPH oxidase activation. We, herein, investigated molecular mechanisms underlying phosphorylation of heat shock protein 27 (HSP27), oxidative stress responsive kinase 1 (OSR1), and myristolated alanine-rich C-kinase substrate (MARCKS) elicited by the pro-resolving FPR2 agonists WKYMVm and annexin A1 (ANXA1).METHODS: CaLu-6 cells or p22phoxCrispr/Cas9 double nickase CaLu-6 cells were incubated for 5 min with WKYMVm or ANXA1, in the presence or absence of NADPH oxidase inhibitors. Phosphorylation at specific serine residues of HSP27, OSR1, and MARCKS, as well as the respective upstream kinases activated by FPR2 stimulation was analysed.
    RESULTS: Blockade of NADPH oxidase functions prevents WKYMVm- and ANXA1-induced HSP-27(Ser82), OSR1(Ser339) and MARCKS(Ser170) phosphorylation. Moreover, NADPH oxidase inhibitors prevent WKYMVm- and ANXA1-dependent activation of p38MAPK, PI3K and PKCδ, the kinases upstream to HSP-27, OSR1 and MARCKS, respectively. The same results were obtained in p22phoxCrispr/Cas9 cells.
    CONCLUSIONS: FPR2 shows an immunomodulatory role by regulating proinflammatory and anti-inflammatory activities and NADPH oxidase is a key regulator of inflammatory pathways. The activation of NADPH oxidase-dependent pro-resolving downstream signals suggests that FPR2 signalling and NADPH oxidase could represent novel targets for inflammation therapeutic intervention.
    Keywords:  HSP-27; MARCKS (Myristolated Alanine-Rich C-Kinase Substrate); NADPH oxidase (Nicotinamide Adenine Dinucleotide Phosphate oxidase); OSR1 (Oxidative-Stress-Responsive kinase 1); annexin A1; formyl peptide receptors; inflammation; reactive oxygen species
  6. Autophagy. 2021 Jan 19. 1-20
    Meyer N, Henkel L, Linder B, Zielke S, Tascher G, Trautmann S, Geisslinger G, Münch C, Fulda S, Tegeder I, Kögel D.
      Increasing evidence suggests that induction of lethal macroautophagy/autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7 (autophagy related 5 and 7)-dependent type of cell death that is significantly reduced with cathepsin inhibitors and the lipid reactive oxygen species (ROS) scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of SMPD1/ASM (sphingomyelin phosphodiesterase 1) and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of CTSB (cathepsin B) into the cytosol in MZ-54 wild-type (WT) cells. Whereas LMP and cell death were significantly attenuated in ATG5 and ATG7 knockout (KO) cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM. Abbreviations: ACD: autophagic cell death; AKT1: AKT serine/threonine kinase 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; CERS1: ceramide synthase 1; CTSB: cathepsin B; CYBB/NOX2: cytochrome b-245 beta chain; ER: endoplasmatic reticulum; FBS: fetal bovine serum; GBM: glioblastoma; GO: gene ontology; HTR7/5-HT7: 5-hydroxytryptamine receptor 7; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; LMP: lysosomal membrane permeabilization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; RB1CC1: RB1 inducible coiled-coil 1; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SMPD1/ASM: sphingomyelin phosphodiesterase 1; VCP/p97: valosin containing protein; WT: wild-type.
    Keywords:  Acid sphingomyelinase; autophagy-dependent cell death; brain tumors; cholesterol metabolism; drug repurposing; er stress; lysophagy
  7. J Cell Mol Med. 2021 Jan 20.
    Wang F, Wang H, Liu X, Yu H, Huang X, Huang W, Wang G.
      Neuregulin-1 (NRG-1) is reported to be cardioprotective through the extracellular-regulated protein kinase (ERK) 1/2 pathway in myocardial ischaemia-reperfusion injury (MIRI). NOX4-induced ROS activated NLRP3 inflammasome and exacerbates MIRI. This study aims to investigate whether NRG-1 can suppress NOX4 by ERK1/2 and consequently inhibit the NLRP3/caspase-1 signal in MIRI. The myocardial infarct size (IS) was measured by TTC-Evans blue staining. Immunohistochemical staining, real-time quantitative PCR (RT-qPCR) and Western blotting were used for detection of the factors, such as NOX4, ERK1/2, NLRP3, caspase-1 and IL-1β .The IS in the NRG-1 (3 μg/kg, intravenous) group was lower than that in the IR group. Immunohistochemical analysis revealed NRG-1 decreased 4HNE and NOX4. The RT-qPCR and Western blot analyses revealed that NRG-1 mitigated the IR-induced up-regulation of NOX4 and ROS production. Compared with the IR group, the NRG-1 group exhibited a higher level of P-ERK1/2 and a lower level of NLRP3. In the Langendorff model, PD98059 inhibited ERK1/2 and up-regulated the expression of NOX4, NLRP3, caspase-1 and IL-1β, which exacerbated oxidative stress and inflammation. In conclusion, NRG-1 can reduce ROS production by inhibiting NOX4 through ERK1/2 and inhibit the NLRP3/caspase-1 pathway to attenuate myocardial oxidative damage and inflammation in MIRI.
    Keywords:  ERK1/2; NLRP3; NOX4; Neuregulin-1
  8. Exp Ther Med. 2021 Feb;21(2): 157
    Xiao X, Huang M, Fan C, Zuo F.
      Skin and in particular photoaging or premature aging, are caused by a variety of factors, including hormone imbalance and exposure to ultraviolet radiation. The aim of the present study was to explore the roles of Dual oxidase 2 (DUOX2) and related NF-κB signals in skin photoaging. Cell models of photoaging were constructed by irradiating human skin fibroblast lines (HSF2) with ultraviolet B (UVB) of different doses (0, 15, 30 and 60 mj/cm2). The cell counting kit-8 (CCK8) was used to determine cell proliferation. Flow cytometry was used to determine the production of reactive oxygen species (ROS). A biochemical method was to determine the content of hydrogen peroxide, and the quantitative PCR (qPCR) was used to determine the expression of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), Col-Ⅰ and α-SMA in the cells. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Western blot analysis was performed to determine the expression of DUOX2, p65 and p-p65. The results showed that,UVB irradiation dose- and time-dependently inhibited the proliferation of HSF2 cells. Cellular inflammatory response, ROS production and hydrogen peroxide increase was promoted. Col-Ⅰ and α-SMA were downregulated, MMP2 and MMP9 were upregulated, and the phosphorylation of NF-κB p65 was promoted. The above indicators were all reversed by interference with DUOX2. Overexpression of DUOX2 has an effect that is similar to UVB irradiation, but the effects can be significantly weakened by NF-κB inhibitor, NAC. Upregulation of DUOX2 expression plays a crucial role in UVB-induced aging of HSF2 cells. The specific mechanism is related to the promotion of ROS production and cellular inflammatory response and activation of NF-κB signals.
    Keywords:  DUOX2; NF-κB; ROS; inflammatory response; photoaging