Clusterin protects against Cr(VI)-induced oxidative stress-associated hepatotoxicity by mediating the Akt-Keap1-Nrf2 signaling pathway.

Hexavalent chromium [Cr(VI)] is a serious environmental pollutant that threatens human life. Cr(VI) is widely used in industrial processes such as metallurgy, leather processing, and electroplating, which can enter the human body through the respiratory or digestive tracts, thus causing a number of human disease, including inflammation and cancer. Although it has been confirmed that oxidative stress is one of the primary mechanism of liver injury caused by Cr(VI) exposure, the related toxic target and effective intervention measures have not been found. Clusterin (CLU) is an acute phase response protein with cytoprotective and apoptosis-delaying effects, and its expression has been confirmed to increase significantly after exposure to Cr(VI). In this study, our data clearly indicates that Cr(VI) is capable of causing hepatocytes damage through the production of large amounts of reactive oxygen species (ROS), causing an increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In contrast, over expression of CLU was able to inhibit ROS production and alleviate Cr(VI)-induced liver injury. The specific mechanisms are that CLU acts on the protein kinase B (PKB/Akt)-Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor E2-related factor 2 (Nrf2) signaling pathway to release Nrf2 into the nucleus. This is to initiate the expression of a downstream protein, heme oxygenase 1 (HO-1), thereby attenuating the ubiquitination ability of Keap1 with Nrf2. We also demonstrated that CLU could affect oxidative stress through the Akt/Nrf2 pathway, which reduced the production of ROS induced by Cr(VI) and protected against Cr(VI)-induced oxidative stress-associated hepatotoxicity. This study demonstrates a mechanism of Cr(VI)-induced hepatotoxicity and indicates that CLU as an intervention target of oxidative stress can provide valuable experimental basis for the prevention and treatment of occupational diseases in Cr(VI)-exposed population. Under the state of Cr(VI)-induced oxidative stress, CLU though phosphorylation Akt, leading to Nrf2 dissociation from Keap1. Activated Nrf2 entered the nucleus and formed the next step, thus binding to the structure of the antioxidant response element ARE, which activated HO-1, resulting in the decrease in intracellular ROS.

Polystyrene microplastics trigger hepatocyte apoptosis and abnormal glycolytic flux via ROS-driven calcium overload.

Human health could be affected by the spread of microplastics in the food chain. Our previous research has indicated that microplastics accumulated in the liver and subsequently induce oxidative damage. However, the molecular events linking oxidative stress to calcium ion (Ca2+) signaling during microplastics stress remains elusive. The present research demonstrated that up-regulation of Orai 1 and stromal interaction molecule 1 (Stim1) expression participated in the microplastics-triggered Ca2+ overload, accompanied with the down-regulation of arcoplasmic reticulum Ca2+ ATPase (SERCA). However, when the protein expression of Stim1/SERCA is restored, microplastics-induced Ca2+ overload is ameliorated. Further analysis revealed that inhibiting the microplastics-induced Ca2+ overload was integral to prevent hepatocyte apoptosis and S phase arrest in the L02 hepatocyte. Simultaneously, we observed that inhibiting microplastics-evoked reactive oxygen species (ROS) could alleviate Ca2+ overload via reversing expression of store-operated Ca2+ channels (SOCs). These changes were accompanied by restoration of glycolytic flux, likely due to the regulation of AMP-activated protein kinase (AMPK)-PGC-1α signaling. Our findings highlight the role of SOCs at microplastics-evoked ROS in Ca2+ overload, and its a crucial step in triggering hepatocyte death. Collectively, this study reveals a regulatory paradigm that links ROS with AMPK and Ca2+ signaling in microplastics-triggered hepatotoxicity.

Clusterin inhibits Cr(VI)-induced apoptosis via enhancing mitochondrial biogenesis through AKT-associated STAT3 activation in L02 hepatocytes.

Improper treatment of a large amount of industrial waste makes hexavalent chromium [Cr(VI)] seriously pollute the atmosphere, soil and water, and enter the food chain, seriously affecting the health of workers and local residents. We previously proved that Clusterin (CLU) can inhibit the apoptosis of L02 hepatocytes induced by Cr(VI) through mitochondrial pathway, but the associated molecular mechanism has not been further studied. Mitochondrial biogenesis is an important step in mitochondrial damage repair, but the mechanism of mitochondrial biogenesis in Cr(VI)-induced liver toxicity is still unclear. We demonstrated in the present study that Cr(VI) triggered mitochondrial biogenesis dysfunction-associated apoptosis, and CLU delayed Cr(VI)-induced apoptosis by enhancing mitochondrial biogenesis. Signal transducer and activator of transcription 3 (STAT3) was down-regulated in Cr(VI)-induced apoptosis, and CLU may regulate STAT3 via protein kinase B (PKB/AKT) in Cr(VI)-exposed hepatocytes. We used the STAT3 inhibitor C188-9 and the AKT inhibitor Uprosertib to eliminate the anti-apoptotic effect of CLU, and found that CLU inhibited Cr(VI)-induced apoptosis by up-regulating AKT/STAT3 signal. Based on the fact that both AKT and STAT3 are closely related to mitochondrial biogenesis and mitochondrial pathway-associated apoptosis, this study is the first time to link CLU, STAT3, AKT and mitochondrial biogenesis function after Cr(VI) exposure, to further enrich the experimental basis of Cr(VI)-induced hepatotoxicity, clarify the molecular mechanism of CLU helping cells to escape apoptosis, and also suggest that new ways can be sought to prevent and treat Cr(VI)-induced hepatotoxicity by regulating mitochondrial biosynthesis.

Role of Clusterin/NF-κB in the secretion of senescence-associated secretory phenotype in Cr(VI)-induced premature senescent L-02 hepatocytes.

Hexavalent chromium [Cr(VI)] and its compounds have caused serious environmental pollution and health damage. Senescent cells can actively change the surrounding environment by secreting some factors, which are called senescence associated secretory phenotype (SASP). Our previous work has confirmed that premature senescent hepatocytes induced by Cr(VI) expressed high level of Clusterin (CLU) and secrete interleukin-6 (IL-6) and IL-8. CLU is involved in the regulation of tumor development and drug resistance, but whether CLU regulates SASP components and participates in Cr(VI)-induced malignant transformation is unclear. In this study we demonstrated that Cr(VI) induced the secretion of tumor promoting components of SASP such as IL-6, IL-8, and granulocyte-macrophage colony stimulating factor (GM-CSF) in senescent L-02 hepatocytes, while the levels of the anti-tumor components of SASP such as chemokine (c-x-c motif) ligand-1 (CXCL-1) and monocyte chemoattractant protein-1 (MCP-1) were not altered. CLU shRNA interference significantly reduced the levels of IL-6, IL-8, and GM-CSF in the culture medium of senescent cells, suggesting CLU may regulate SASP. The NF-κB inhibitor PDTC significantly alleviated Cr(VI)-induced increase of IL-6, IL-8, and GM-CSF, confirming that NF-κB can regulate the tumor promoting components of SASP. CLU shRNA interference aggravated the inhibitory effect of PDTC on SASP secretion, indicating that CLU regulated the secretion of SASP in Cr(VI)-induced senescent hepatocytes through the NF-κB signaling. We speculated that SASP secreted by Cr(VI)-induced premature senescent hepatocytes was tightly related to the carcinogenic effect of Cr(VI). Therefore, elucidation of upstream regulatory mechanism of SASP is of great significance. In addition to further clarifying the carcinogenic mechanisms associated with Cr(VI), we could also seek out new targets for treatment of Cr(VI)-related cancer.

The role of PKA/PP2B-mediated Drp1 phosphorylation and the subsequent EGFR inhibition in Cr(VI)-induced premature senescence.

In recent years, frequent hexavalent chromium [Cr(VI)] pollution incidents have severely damaged the ecology and endangered the public health. It is well known that cell senescence could promote the carcinogenesis, thus the related research on the occurrence of premature senescence is of great significance to the elucidation of the carcinogenic mechanism of Cr(VI). We previously confirmed that long-term low-dose Cr(VI) exposure induced premature senescence, but the key molecular events that determine the occurrence of premature senescence are still unclear. In the present study, we found that Cr(VI) induced phosphorylation of dynamin-relatedprotein 1 (Drp1)-S637 site in premature senescent cells, which was accompanied with the decrease of mitochondrial fission. We also demonstrated that the phosphorylation status of Drp1-S637 after Cr(VI) exposure was related to the antagonism of PKA/PP2B, and continuous dephosphorylation of Drp1-S637 attenuated premature senescence caused by Cr(VI). The epidermal growth factor receptor (EGFR) overexpression significantly alleviated the occurrence of premature senescence, and the expressions of EFGR and its downstream molecules were related to the phosphorylation status of Drp1-S637. In brief, we revealed the role of PKA/PP2B-mediated Drp1 phosphorylation and the subsequent EGFR inhibition in Cr(VI)-induced premature senescence. This study is the first time to link the phosphorylation of Drp1 with Cr(VI)-induced premature senescence, in order to find the key molecular events that determine the occurrence of premature senescence and demonstrate the molecular mechanism of abnormal elongated mitochondria formation in the senescence process. The significance of this study is to explore the carcinogenesis of Cr(VI) and provide new ideas and strategies for the targeted treatment of Cr(VI)-related cancers.

Identification and functional analysis of senescence-associated secretory phenotype of premature senescent hepatocytes induced by hexavalent chromium.

Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant that can cause a number of human disease, including inflammation and cancer. Senescent cells can secrete a variety of molecules known as senescence-associated secretory phenotype (SASP). Our previous studies have confirmed that Cr(VI) can induce premature senescence in L02 hepatocytes, but the composition and the function of the related SASP are still unknown. In order to understand the components of SASP secreted by senescent L02 hepatocytes under the action of Cr(VI), we applied LC-MS/MS-based label-free protein quantification. We found that three SASP components including Coactosin-like protein 1 (COTL1), Alpha-enolase (ENO1), and Peroxiredoxin 2 (PRDX2) were up-regulated, which were confirmed by western blotting and qRT-PCR. Evidence suggested that SASP may promote the development of tumor through chronic inflammatory response, therefore we identified and analyzed the potential biological functions and signaling pathways of these three SASP components using GO and KEGG methods. The interaction between SASP components was analyzed by STRING, and verified by Co-IP. We also found that ENO1 and PRDX2, which have direct interaction, can inhibit the growth and proliferation of wildtype hepatocytes and premature senescent hepatocytes, but can promote the proliferation and behavioral changes of liver tumor cells. The present study provides valuable clues for elucidation of the carcinogenic mechanism of Cr(VI), especially for further prevention and targeted treatment of Cr(VI)-related cancer.

Cellular and molecular mechanisms of xenobiotics-induced premature senescence.

Premature senescence, which share common features with replicative senescence such as morphology, senescence-associated galactosidase (SA-ß-gal) activity, cell cycle regulation, and gene expression, can be triggered by the exposure of various xenobiotics including environmental pollutant, peroxides, and anticancer drugs. The exact mechanisms underlying the senescence onset and stabilization are still obscure. In this review, we summarized the possible cellular and molecular mechanisms of xenobiotics-induced premature senescence, including induction of reactive oxygen species (ROS), tumor suppressors, and DNA damage; disequilibrium of calcium homeostasis; activation of transforming growth factor-ß (TGF-ß); and blockage of aryl hydrocarbon receptor (AHR) pathway. The deeper understanding of the molecular mechanisms underlying xenobiotics-induced senescence may shed light on new therapeutic strategies for age-related pathologies and extend healthy lifespan.

Clusterin alleviates Cr(VI)-induced mitochondrial apoptosis in L02 hepatocytes via inhibition of Ca2+-ROS-Drp1-mitochondrial fission axis.

Hexavalent chromium [Cr(VI)] is ubiquitous in the environment and is commonly used in various industrial processes. Clusterin (CLU) is an extracellular chaperone protein which exerts the anti-apoptotic function. In this study, we aimed to explore the effect of CLU on Cr(VI)-induced mitochondrial fission and apoptosis. We revealed that the apoptosis rate of L02 hepatocytes treated with Cr (VI) was increased. CLU over-expression could protect the hepatocytes from Cr(VI)-induced mitochondrial apoptosis. Furthermore, Cr(VI) triggered the intracellular calcium overload, resulting in the activation of xanthine oxidase (XO). Cr(VI) induced reactive oxygen species (ROS) overproduction, led to dynamin-related protein 1 (Drp1) translocation to mitochondria and the subsequent mitochondrial fission, contributing to the caspase-3-dependent mitochondrial apoptosis as evidenced by higher mitochondrial permeability transition pore (mPTP) opening rate, lower mitochondrial membrane potential (MMP), and more alanine transaminase (ALT)/aspartate transaminase (AST) leakage into the culture medium. However, CLU over-expression could trigger the AMP-activated protein kinase (AMPK) pathway, which was followed by the increase of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) expression. CLU-induced AMPK/SERCA2a activation attenuated calcium overload, caspase-3 activation, and ultimate mitochondrial apoptosis. All in all, the present study demonstrated that Cr(VI) induced hepatocytes apoptosis via Ca2+-ROS-Drp1-mitochondrial fission axis and CLU alleviated the mitochondrial apoptosis through activation of the AMPK/SERCA2a pathway.

ROS-mediated miR-21-5p regulates the proliferation and apoptosis of Cr(VI)-exposed L02 hepatocytes via targeting PDCD4.

Although much has been determined about the molecular mechanisms of hexavalent chromium [Cr(VI)]-induced hepatotoxicity, more remains to be explored. In particular, explicit epigenetic alterations of microRNAs (miRNAs) which can negatively regulate mRNAs at post transcriptional level remain understudied. In the present study, cell apoptosis was determined using Annexin V/propidium iodide (PI) staining, while proliferative growth was analyzed by colony formation assay and proliferating cell nuclear antigen (PCNA) detection. miRNA microarray was performed to compare the global miRNAs expression patterns. miR-21-5p mimics (mi)/inhibitor (in), and PDCD4-siRNAs were transfected into L02 hepatocytes. Our results revealed that Cr(VI) induced apoptosis and inhibited proliferation in L02 hepatocytes via reactive oxygen species (ROS), the formation of which is closely related to mitochondrial damage, especially the inhibition of mitochondrial respiratory chain complex (MRCC). We also confirmed that ROS-mediated miR-21-5p inhibition participated in cell apoptosis and proliferative inhibition induced by Cr(VI). Furthermore, programmed cell death protein 4 (PDCD4), the up-regulation of which was related to ROS over-production, was predicted and verified as a target of miR-21-5p. Transcription factor PDCD4 silencing suppressed apoptosis and stimulated cell proliferation. In conclusion, from the perspective of epigenetics, the present study revealed that ROS-mediated miR-21-5p regulated the proliferation and apoptosis of Cr(VI)-exposed L02 hepatocytes via targeting PDCD4, which provided the new targets for molecular intervention and treatment of liver damage in Cr(VI)-exposed population.

Blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr(VI)-induced mitochondrial dysfunction in L02 hepatocytes.

Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant widely used in various industrial fields. It is well known that mitochondria are the most vulnerable targets of heavy metals, but the key molecule/event that directly mediated mitochondrial dysfunction after Cr(VI) exposure is still unclear. The present study was aimed to explore whether Cr(VI) exposure could affect the mitochondrial fission/fusion process, and whether the related abnormal mitochondrial dynamics have been implicated in Cr(VI)-induced mitochondrial dysfunction. We found that the mitochondrial dysfunction caused by Cr(VI) exposure was characterized by decreased mitochondrial respiratory chain complex (MRCC) I/II activities and levels, collapsed mitochondrial membrane potential (MMP), depleted ATP, and increased reactive oxygen species (ROS) level. Cr(VI) induced abnormal mitochondrial fission/fusion events, the antioxidant Nacetyl-L-cysteine (NAC) restored the abnormal mitochondrial function as well as the fission/fusion dynamics. ROS was the up-stream regulator of extracellular regulated protein kinases (ERK) signaling, and the application of a specific ERK1/2 inhibitor PD98059 confirmed that activation of ERK1/2 signaling was associated with the abnormal mitochondrial fission/fusion and mitochondrial dysfunction. We also demonstrated that treatment with dynamic-like protein 1 (DLP1)-siRNA rescued mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes. We reached the conclusion that blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr(VI)-induced mitochondrial dysfunction in L02 hepatocytes, which may provide the new avenue for developing effective strategies to protect against Cr(VI)-induced hepatotoxicity.