bims-noxint 2021-11-14 papers

Issue of 2021‒11‒14
six papers selected by
Laia Caja Puigsubira
Uppsala University

J Mol Cell Cardiol. 2021 Oct 28. pii: S0022-2828(21)00198-X. [Epub ahead of print]163 147-155

Inositol 1,4,5-trisphosphate receptor - reactive oxygen signaling domain regulates excitation-contraction coupling in atrial myocytes.

Disha Varma, Jonathas F Q Almeida, Jaime DeSantiago, Lothar A Blatter, Kathrin Banach.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is up-regulated in patients with atrial fibrillation (AF) and InsP3-induced Ca2+ release (IICR) is linked to pro-arrhythmic spontaneous Ca2+ release events. Nevertheless, knowledge of the physiological relevance and regulation of InsP3Rs in atrial muscle is still limited. We hypothesize that InsP3R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP3R agonist affinity and thereby Ca2+ release. To quantitate the contribution of IICR to atrial excitation-contraction coupling (ECC) atrial myocytes (AMs) were isolated from wild type and NOX2 deficient (Nox2-/-) mice and changes in the cytoplasmic Ca2+ concentration ([Ca2+]i; fluo-4/AM, indo-1) or ROS (2',7'-dichlorofluorescein, DCF) were monitored by fluorescence microscopy. Superfusion of AMs with Angiotensin II (AngII: 1 μmol/L) significantly increased diastolic [Ca2+]i (F/F0, Ctrl: 1.00 ± 0.01, AngII: 1.20 ± 0.03; n = 7; p < 0.05), the field stimulation induced Ca2+ transient (CaT) amplitude (ΔF/F0, Ctrl: 2.00 ± 0.17, AngII: 2.39 ± 0.22, n = 7; p < 0.05), and let to the occurrence of spontaneous increases in [Ca2+]i. These changes in [Ca2+]i were suppressed by the InsP3R blocker 2-aminoethoxydiphenyl-borate (2-APB; 1 μmol/L). Concomitantly, AngII induced an increase in ROS production that was sensitive to the NOX2 specific inhibitor gp91ds-tat (1 μmol/L). In NOX2-/- AMs, AngII failed to increase diastolic [Ca2+]i, CaT amplitude, and the frequency of spontaneous Ca2+ increases. Furthermore, the enhancement of CaTs by exposure to membrane permeant InsP3 was abolished by NOX inhibition with apocynin (1 μM). AngII induced IICR in Nox2-/- AMs could be restored by addition of exogenous ROS (tert-butyl hydroperoxide, tBHP: 5 μmol/L). In saponin permeabilized AMs InsP3 (5 μmol/L) induced Ca2+ sparks that increased in frequency in the presence of ROS (InsP3: 9.65 ± 1.44 sparks*s-1*(100μm)-1; InsP3 + tBHP: 10.77 ± 1.5 sparks*s-1*(100μm)-1; n = 5; p < 0.05). The combined effect of InsP3 + tBHP was entirely suppressed by 2-APB and Xestospongine C (XeC). Changes in IICR due to InsP3R glutathionylation induced by diamide could be reversed by the reducing agent dithiothreitol (DTT: 1 mmol/L) and prevented by pretreatment with 2-APB, supporting that the ROS-dependent post-translational modification of the InsP3R plays a role in the regulation of ECC. Our data demonstrate that in AMs the InsP3R is under dual control of agonist induced InsP3 and ROS formation and suggest that InsP3 and NOX2-derived ROS co-regulate atrial IICR and ECC in a defined InsP3R/NOX2 signaling domain.

Keywords: Angiotensin II; Atrial excitation contraction coupling; Inositol 1,4,5-trisphosphate receptor induced Ca(2+) release; NADPH oxidase 2; Signaling domain

DOI: https://doi.org/10.1016/j.yjmcc.2021.10.006

Cancer Lett. 2021 Nov 03. pii: S0304-3835(21)00537-1. [Epub ahead of print]525 146-157

CYLD destabilizes NoxO1 protein by promoting ubiquitination and regulates prostate cancer progression.

Saba Haq, Neha Sarodaya, Janardhan Keshav Karapurkar, Bharathi Suresh, Jung Ki Jo, Vijai Singh, Yun Soo Bae, Kye-Seong Kim, Suresh Ramakrishna.

The NADPH oxidase (Nox) family of enzymes is solely dedicated in the generation of reactive oxygen species (ROS). ROS generated by Nox are involved in multiple signaling cascades and a myriad of pathophysiological conditions including cancer. As such, ROS seem to have both detrimental and beneficial roles in a number of cellular functions, including cell signaling, growth, apoptosis and proliferation. Regulatory mechanisms are required to control the activity of Nox enzymes in order to maintain ROS balance within the cell. Here, we performed genome-wide screening for deubiquitinating enzymes (DUBs) regulating Nox organizer 1 (NoxO1) protein expression using a CRISPR/Cas9-mediated DUB-knockout library. We identified cylindromatosis (CYLD) as a binding partner regulating NoxO1 protein expression. We demonstrated that the overexpression of CYLD promotes ubiquitination of NoxO1 protein and reduces the NoxO1 protein half-life. The destabilization of NoxO1 protein by CYLD suppressed excessive ROS generation. Additionally, CRISPR/Cas9-mediated knockout of CYLD in PC-3 cells promoted cell proliferation, migration, colony formation and invasion in vitro. In xenografted mice, injection of CYLD-depleted cells consistently led to tumor development with increased weight and volume. Taken together, these results indicate that CYLD acts as a destabilizer of NoxO1 protein and could be a potential tumor suppressor target for cancer therapeutics.

Keywords: CRISPR/Cas9; Colon cancer; Deubiquitination; Proteasomal degradation; Proteolysis; ROS

DOI: https://doi.org/10.1016/j.canlet.2021.10.032

Eur J Cell Biol. 2021 Nov 03. pii: S0171-9335(21)00032-7. [Epub ahead of print]100(7-8): 151181

ROS-Nrf2 pathway mediates the development of TGF-β1-induced epithelial-mesenchymal transition through the activation of Notch signaling.

Kai Yazaki, Yosuke Matsuno, Kazufumi Yoshida, Mingma Sherpa, Masayuki Nakajima, Masashi Matsuyama, Takumi Kiwamoto, Yuko Morishima, Yukio Ishii, Nobuyuki Hizawa.

Epithelial-mesenchymal transition (EMT) is a cellular process by which epithelial cells transform to acquire mesenchymal phenotypes. Accumulating evidence indicate the involvement of EMT in the progression of malignant diseases. Notch signaling mediates TGF-β1-induced EMT through direct transcriptional activation of Snai1. The molecular mechanism how TGF-β1 activates Notch signaling, however, remains unknown. In this study, we show a pivotal role for reactive oxygen species (ROS)-Nrf2 pathway in TGF-β1-induced Notch signaling activation and EMT development. TGF-β1 induces Nrf2 activation through ROS production. Inhibiting Nrf2 activation either by reducing ROS levels by N-acetylcysteine or by knocking down of Nrf2 by small interfering RNA attenuated both Notch signaling activation and EMT development. TGF-β1 induced the transcription of Notch4 via Nrf2-dependent promoter activation. In conclusion, our study indicates the ROS-Nrf2 pathway mediates the development of TGF-β1-induced EMT through the activation of Notch signaling.

Keywords: A549; EMT; Notch signaling; Nrf2; ROS; TGF-β1

DOI: https://doi.org/10.1016/j.ejcb.2021.151181

Cancers (Basel). 2021 Oct 21. pii: 5279. [Epub ahead of print]13(21):

Sulfatase-2 Regulates Liver Fibrosis through the TGF-β Signaling Pathway.

Ikuo Nakamura, Faizal Z Asumda, Catherine D Moser, Yoo Na N Kang, Jin-Ping Lai, Lewis R Roberts.

Transforming growth factor-β (TGF-β) activates hepatic stellate cells (HSCs), which drive liver fibrosis via the production and deposition of extracellular matrix (ECM). We aimed to elucidate the mechanistic role of sulfatase-2 (SULF2) in liver fibrosis. To this end, we induced liver fibrosis in wild-type (WT) and SULF2 knockout (Sulf2-KO) mice (6-8 weeks-old) via bile duct ligation (BDL), intraperitoneal injection of carbon tetrachloride (CCl4) or thioacetamide (TAA). The levels of fibrosis in the liver sections were assessed via Sirius red and Masson's trichrome staining, immunohistochemistry and immunoblotting for α-smooth muscle actin (α-SMA) and hydroxyproline. To evaluate the interaction between TGF-β and SULF2, we transfected human HSCs with scrambled control shRNA and shRNA constructs targeting SULF2 and measured α-SMA expression following treatment with TGF-β1 ligand. We show here that knockout of SULF2 significantly decreases collagen content, as well as bands of bridging fibrosis, as demonstrated by Sirius red, Masson's trichrome and α-SMA staining after BDL, CCl4 and TAA injection in Sulf2-KO versus WT mice. In all three models of liver fibrosis, we observed significantly lower levels of hydroxyproline in the Sulf2-KO mice compared to the WT mice. HSCs with reduced levels of SULF2 failed to significantly express α-SMA and collagen type I following treatment with TGF-β1. Furthermore, SULF2 co-localizes with TGFBR3 and the in vitro knockdown of SULF2 in HSCs decreases the release of TGF-β1 from TGFBR3. Together, these data suggest that SULF2 regulates liver fibrosis via the TGF-β signaling pathway. Pharmacologic inhibition of SULF2 may represent a novel therapeutic approach to improve liver fibrosis.

Keywords: SULF2; cirrhosis; hepatocellular carcinoma; liver fibrosis; transforming growth factor-β

DOI: https://doi.org/10.3390/cancers13215279

Int J Mol Sci. 2021 Oct 30. pii: 11816. [Epub ahead of print]22(21):

LncRNA MALAT1 Modulates TGF-β1-Induced EMT in Keratinocyte.

Liping Zhang, Junyi Hu, Bahar I Meshkat, Kenneth W Liechty, Junwang Xu.

One of the major complications in diabetes is impaired wound healing. Unfortunately, effective therapies are currently lacking. Epithelial to mesenchymal transition (EMT) is a critical process involved in cutaneous wound healing. In response to injury, EMT is required to activate and mobilize stationary keratinocytes in the skin toward the wound bed, which allows for re-epithelialization. This process is stalled in diabetic wounds. In this study, we investigate the role of long non-coding RNA (lncRNA), MALAT1, in transforming growth factor beta 1(TGF-β1)-induced EMT of human keratinocyte (HaCaT) cells. Initially, we detected MALAT1 and TGF-β1 expression in non-diabetic and diabetic wounds and found that these expression are significantly up-regulated in diabetic wounds. Then, HaCaT cells were cultured and exposed to TGF-β1. The EMT of HaCaT cells were confirmed by the increased expression of CDH2, KRT10, and ACTA2, in addition to the down-regulation of CDH1. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA). MALAT1 silencing attenuates TGFβ1-induced EMT. Mechanistically, MALAT1 is involved in TGF-β1 mediated EMT through significantly induced ZEB1 expression, a critical transcription factor for EMT. In summary, lncRNA MALAT1 is involved in TGFβ1-induced EMT of human HaCaT cells and provides new understanding for the pathogenesis of diabetic wounds.

Keywords: MALAT1; diabetic wounds; epithelial mesenchymal transition; long non-coding RNA

DOI: https://doi.org/10.3390/ijms222111816

Int J Mol Sci. 2021 Oct 27. pii: 11575. [Epub ahead of print]22(21):

Smad7 Deficiency in Myeloid Cells Does Not Affect Liver Injury, Inflammation or Fibrosis after Chronic CCl4 Exposure in Mice.

Ludmilla Unrau, Jessica Endig, Diane Goltz, Paulina Sprezyna, Hanna Ulrich, Julia Hagenstein, Bernd Geers, Karina Kaftan, Lukas Carl Heukamp, Gisa Tiegs, Linda Diehl.

Myeloid cells play an essential role in the maintenance of liver homeostasis, as well as the initiation and termination of innate and adaptive immune responses. In chronic hepatic inflammation, the production of transforming growth factor beta (TGF-β) is pivotal for scarring and fibrosis induction and progression. TGF-β signalling is tightly regulated via the Smad protein family. Smad7 acts as an inhibitor of the TGF-β-signalling pathway, rendering cells that express high levels of it resistant to TGF-β-dependent signal transduction. In hepatocytes, the absence of Smad7 promotes liver fibrosis. Here, we examine whether Smad7 expression in myeloid cells affects the extent of liver inflammation, injury and fibrosis induction during chronic liver inflammation. Using the well-established model of chronic carbon tetrachloride (CCl4)-mediated liver injury, we investigated the role of Smad7 in myeloid cells in LysM-Cre Smadfl/fl mice that harbour a myeloid-specific knock-down of Smad7. We found that the chronic application of CCl4 induces severe liver injury, with elevated serum alanine transaminase (ALT)/aspartate transaminase (AST) levels, centrilobular and periportal necrosis and immune-cell infiltration. However, the myeloid-specific knock-down of Smad7 did not influence these and other parameters in the CCl4-treated animals. In summary, our results suggest that, during long-term application of CCl4, Smad7 expression in myeloid cells and its potential effects on the TGF-β-signalling pathway are dispensable for regulating the extent of chronic liver injury and inflammation.

Keywords: CCl4; Smad7; TGF-β; chronic liver injury; fibrosis; inflammation; myeloid cell

DOI: https://doi.org/10.3390/ijms222111575