Original articleAngiotensin(1–7) attenuated Angiotensin II-induced hepatocyte EMT by inhibiting NOX-derived H2O2-activated NLRP3 inflammasome/IL-1β/Smad circuit
Graphical abstract
Introduction
Epithelial-mesenchymal transition (EMT) is a dynamic cellular process in which polarized epithelial cells lose their epithelial properties, acquiring mesenchymal characteristics and becoming more migratory [1]. EMT has been well studied in organ fibrosis (type 2 EMT), such as of the kidneys [2], liver [3], and lungs [4]. In vitro, resident liver epithelial cells, including hepatocytes, have been shown to undergo EMT induced by TGF-β and to gain mesenchymal markers and migratory capacity [5]. Furthermore, using a lineage tracing technique, Zeisberg et al. [6] found that hepatocyte-derived fibroblasts were a lineage line of mesenchymal cells that might contribute to liver fibrogenesis. However, the occurrence of hepatocyte EMT in vivo has been seriously challenged by other research groups [7]. Myofibroblasts from fibrotic liver tissue were shown not to be originated from the geneticly labeled epithelial cells [8], [9]. More recently, two studies further identified hepatic stellate cells (HSCs) as a resident population of inherently plastic cells that could be reprogramed by mesenchymal- epithelial transition (MET)-EMT to differentiate into hepatocytes or ductular cells and replace adult liver epithelial cells [10], [11], suggesting EMT is still a subject worthy of study in liver fibrosis. These conflicting data could be associated with factors such as the pitfalls of lineage tracing techniques, unspecific cell-type markers, missing of the time window for identifying phenotypic transition, and limitations of current immunohistochemistry [12]. Hence, it is premature to conclude that EMT/MET does not occur in human liver fibrosis.
Many molecules have been documented to initiate EMT in organ fibrosis. TGF-β-activated Smad, Rho GTPases signaling pathways have been shown to stimulate type 2 EMT [13]. NAPDH oxidase (NOX)/mitochondria-derived reactive oxygen species (ROS) [14], Rac1 [15], and NOX isoforms [16] have been shown to mediate the triggering of EMT. However, the role of the downstream molecules involved in oxidative stress as a modulator of EMT remains unclear.
There are numerous downstream molecules of oxidative stress, among which NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome has been identified as a novel mechanism of liver injury and fibrosis [17]. NLRP3 inflammasome consists of NLRP3, apoptosis-associated speck like protein (ASC), and the effector molecule pro-caspase-1. NLRP3 inflammasome activation promotes the cleavage and activation of caspase-1 resulting in maturation of pro-inflammatory cytokines such as pro-interleukin-1β (pro-IL-1β) and pro-IL18 [18]. It was claimed that IL-1β promoted EMT [19]. Interestingly, emerging evidence has suggested that inflammasome-independent NLRP3 augmented TGF-β-induced EMT in the kidney epithelium, while IL-1β and caspase-1 were dispensable [20]. Whether NLRP3 inflammasome promotes hepatocyte EMT remains unknown. Our recent study [21] and Zhou [22] found that both NOX-derived ROS [21] and mitochondrial ROS [22] contribute to NLRP3 inflammasome activation. Hence we hypothesized that NOX-derived ROS triggers hepatocyte EMT by activating NLRP3 inflammasome.
The renin-angiotensin system (RAS) plays an important role in hepatic fibrosis [23]. Angiotensin II (AngII), the main effector molecule of the RAS, exerts physical effects through its type 1 receptor (AT1R). AngII induced mitochondrial ROS in cardiac hypertrophy [24]. AngII-induced EMT in tubular epithelial cells was mediated by NOX-derived ROS [25]. However, whether (and how) AngII induces hepatocyte EMT has remained unknown until now. Our recent study proved that the Ang II-induced liver fibrosis by activating NLRP3 inflammasome in HSCs [21], indicating the potential activation of AngII on inflammasome. Hence, we hypothesized that AngII initiates hepatocyte EMT by activating NOX-derived ROS-induced NLRP3 inflammasome.
The discovery of the angiotensin converting enzyme (ACE) homolog ACE2 has added new complexity to the RAS. ACE2 degrades AngII to Angiotensin-(1–7) [Ang-(1–7)], which exerts physical effects through its receptor, Mas [26]. The alternative ACE2/Ang-(1–7)/Mas axis of the RAS can be viewed as the principal counter-regulatory mechanism for the classic ACE/AngII/AT1R axis. Emerging evidence has suggested that the ACE2/Ang-(1–7)/Mas axis possesses antioxidant capability, which protects against liver injury induced by oxidative stress [27]. It was reported that Ang-(1–7) inhibited high glucose or AngII-induced EMT via Mas receptor in renal epithelial cells [28]. Also in our recent study, Ang-(1–7) could inhibit the Ang II-induced activation of NLRP3 inflammasome in HSCs [21]. Does Ang-(1–7) attenuate AngII-induced hepatocyte EMT by inhibiting NLRP3 inflammasome activation? There has been no answer until now, and further investigation is warranted.
Consequently, we hypothesized that AngII initiates hepatocyte EMT by activating NOX-derived ROS-induced NLRP3 inflammasome. Ang-(1–7) attenuates AngII-induced hepatocyte EMT by inhibiting NLRP3 inflammasome activation.
We firstly demonstrated that AngII initiated hepatocyte EMT by activating NOX4-dependent hydrogen peroxide ( H2O2)-induced NLRP3 inflammasome. Ang-(1–7) attenuated AngII-induced hepatocyte EMT by inhibiting NLRP3 inflammasome activation.
Section snippets
Reagents
AngII, Ang-(1–7), A779, DPI, catalase, Z-YVAD-FMK, and Ehop were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). IL-1 receptor antagonist (IL-1Ra), anakinra was purchased from Amgen (Thousand Oaks, CA). NOX4 siRNA, NLRP3 siRNA and Smad4 siRNA were provided by Gene Pharma (Shanghai, China).
Human liver
We retrospectively studied paraffin-embedded specimens of liver tissue from 12 patients. Normal liver samples were collected from 6 patients undergoing partial liver resection for hepatic haemangioma.
AngII induced an EMT state and promoted the motility in hepatocytes
Using immunofluorescent staining, we found that the protein expression of e-cadherin was markedly decreased, while that of Col1A1 was significantly increased in AngII-treated cells (Fig. 1A). Similarly, western blot analysis showed that the vimentin protein level of hepatocytes was elevated after incubation with AngII for 24 h, while the reduction of e-cadherin and albumin protein levels in AngII-treated hepatocytes occurred at 72 h, out of sync with the change in vimentin at 24 h (Fig. 1C). In
Discussion
In this study, for the first time, we demonstrated that Ang II initiated hepatocyte EMT by activating the NOX-derived H2O2-mediated NLRP3 inflammasome/IL-1ß/Smad circuit. Ang-(1–7) attenuated Ang II-induced hepatocyte EMT by inhibiting NLRP3 inflammasome activation. The principal findings obtained included the following: (1) NOX-derived H2O2 was involved in AngII-induced EMT in hepatocytes; (2) the NLRP3 inflammasome/IL-1ß/Smad circuit, activated by NOX-derived H2O2, mediated AngII-induced EMT
Conflict of interest
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Acknowledgment
We thank Prof. Pingsheng Wu, Prof. Zhenshu Zhang, and Prof. Xishan Yang for their kind help in this study. This study was supported by research grants from the National Natural Science Foundation of China (81070338) and Bao-en Wang Liver Fibrosis Research Foundation (20090018). We also thank Guangzhou Pilot Project of Clinical and Translational Research Center (early gastrointestinal cancers, No. 7415696196402) and Guangdong Provincial Bio-engineering Research Center for Gastroenterology
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These authors contributed equally to this work.