NLRP3 inflammasome inhibition attenuates silica-induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells

Silicosis is an incurable and progressive lung disease characterized by chronic inflammation and fibroblasts accumulation. Studies have indicated a vital role for epithelial-mesenchymal transition (EMT) in fibroblasts accumulation. NLRP3 inflammasome is a critical mediator of inflammation in response to a wide range of stimuli (including silica particles), and plays an important role in many respiratory diseases. However, whether NLRP3 inflammasome regulates silica-induced EMT remains unknown. Our results showed that silica induced EMT in human bronchial epithelial cells (16HBE cells) in a dose- and time-dependent manner. Meanwhile, silica persistently activated NLRP3 inflammasome as indicated by continuously elevated extracellular levels of interleukin-1β (IL-1β) and IL-18. NLRP3 inflammasome inhibition by short hairpin RNA (shRNA)-mediated knockdown of NLRP3, selective inhibitor MCC950, and caspase-1 inhibitor Z-YVAD-FMK attenuated silica-induced EMT. Western blot analysis indicated that TAK1-MAPK-Snail/NF-κB pathway involved NLRP3 inflammasome-mediated EMT. Moreover, pirfenidone, a commercially and clinically available drug approved for treating idiopathic pulmonary fibrosis (IPF), effectively suppressed silica-induced EMT of 16HBE cells in line with NLRP3 inflammasome inhibition. Collectively, our results indicate that NLRP3 inflammasome is a promising target for blocking or retarding EMT-mediated fibrosis in pulmonary silicosis. On basis of this mechanism, pirfenidone might be a potential drug for the treatment of silicosis.     

The central inflammasome adaptor protein ASC activates the inflammasome after transition from a soluble to an insoluble state

Apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC) is a 22 kDa protein that functions as the central adaptor for inflammasome assembly. ASC forms insoluble specks in monocytes undergoing pyroptosis, and the polymerization of ASC provides a template of CARDs that leads to proximity-mediated autoactivation of caspase-1 in canonical inflammasomes. However, specks are insoluble protein complexes, and solubility is typically important for protein function. Therefore, we sought to define whether ASC specks comprise active inflammasome complexes or are simply the end stage of exhausted ASC polymers. Using a THP-1 cell–lysing model of caspase-1 activation that is ASC dependent, we compared caspase-1 activation induced by preassembled insoluble ASC specks and soluble monomeric forms of ASC. Unexpectedly, after controlling for the concentration dependence of ASC oligomerization, we found that only insoluble forms of ASC promoted caspase-1 autocatalysis. This link to insolubility was recapitulated with recombinant ASC. We show that purified recombinant ASC spontaneously precipitated and was functional, whereas the maltose-binding protein–ASC fusion to ASC (promoting enhanced solubility) was inactive until induced to insolubility by binding to amylose beads. This functional link to insolubility also held true for the Y146A mutation of the CARD of ASC, which avoids insolubility and caspase-1 activation. Thus, we conclude that the role of ASC insolubility in inflammasome function is inextricably linked to its pyrin domain–mediated and CARD-mediated polymerizations. These findings will support future studies into the molecular mechanisms controlling ASC solubility.     

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.     

Protons sensitize epithelial cells to mesenchymal transition

Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1.     

Uric acid metabolism of kidney and intestine in a rat model of chronic kidney disease

ABSTRACT

Uric acid (UA) is a potential risk factor of the progression of chronic kidney disease (CKD). Recently, we reported that intestinal UA excretion might be enhanced via upregulation of the ATP-binding cassette transporter G2 (Abcg2) in a 5/6 nephrectomy (Nx) rat model. In the present study, we examined the mRNA and protein expressions of UA transporters, URAT1, GLUT9/URATv1, ABCG2 and NPT4 in the kidney and ileum in the same rat model. Additionally, we investigated the Abcg2 mRNA expression of ileum in hyperuricemic rat model by orally administering oxonic acid. Male Wistar rats were randomly assigned to three groups consisting of Nx group, oxonic acid-treated (Ox) group and sham-operated control group, and sacrificed at 8 weeks. Creatinine and UA were measured and the mRNA expressions of UA transporters in the kidney and intestine were evaluated by a real time PCR. UA transporters in the kidney sections were also examined by immunohistochemistry. Serum creatinine elevated in the Nx group whereas serum UA increased in the Ox group. Both the mRNA expression and the immunohistochemistry of the UA transporters were decreased in the Nx group, suggesting a marginal role in UA elevation in decreased kidney function. In contrast, the mRNA expression of Abcg2 in the ileum significantly increased in the Ox group. These results suggest that the upregulation of Abcg2 mRNA in the ileum triggered by an elevation of serum UA may play a compensatory role in increasing intestinal UA excretion.     

Benzophenone-3 increases metastasis potential in lung cancer cells via epithelial to mesenchymal transition

Exposure to compounds with cancer-potentiating effects can contribute to the progression of cancer. Herein we have discovered for the first time that benzophenone-3 (BP-3), a chemical used as sunscreen in various cosmetic products, enhances the ability of lung cancer cells to undergo metastasis. The exposure of the lung cancer cells to BP-3 at non-toxic concentrations significantly increased the number of anoikis resistant cells in a dose-dependent manner. Also, BP-3 increased the growth rate as well as the number of colonies accessed by anchorage-independent growth assay. We found that the underlying mechanisms of such behaviors were the epithelial to mesenchymal transition (EMT) process of cancer cells, and the increase in caveolin-1 (Cav-1) expression. As both mechanistic events mediated anoikis resistance via augmentation of cellular survival signals, our results further revealed that the BP-3 treatment significantly up-regulated extracellular-signal-regulated kinase (ERK). Also, such compounds increased the cellular levels of anti-apoptotic Bcl-2 and Mcl-1 proteins. As the presence of a substantial level of BP-3 in plasma of the consumers has been reported, this finding may facilitate further investigations that lead to better understanding and evidence concerning the safety of use in cancer patients.     

Immunoediting of cancers may lead to epithelial to mesenchymal transition

Tumors evade both natural and pharmacologically induced (e.g., vaccines) immunity by a variety of mechanisms, including induction of tolerance and immunoediting. Immunoediting results in reshaping the immunogenicity of the tumor, which can be accompanied by loss of Ag expression and MHC molecules. In this study, we evaluated immunoediting in the neu-transgenic mouse model of breast cancer. A tumor cell line that retained expression of rat neu was generated from a spontaneous tumor of the neu-transgenic mouse and, when injected into the non-transgenic parental FVB/N mouse, resulted in the development of a strong immune response, initial rejection, and ultimately the emergence of neu Ag-loss variants. Morphologic and microarray data revealed that the immunoedited tumor cells underwent epithelial to mesenchymal transition accompanied by an up-regulation of invasion factors and increased invasiveness characteristic of mesenchymal tumor cells. These results suggest that immunoediting of tumor results in cellular reprogramming may be accompanied by alterations in tumor characteristics including increased invasive potential. Understanding the mechanisms by which tumors are immunoedited will likely lead to a better understanding of how tumors evade immune detection.     

TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro

The aim of this study was to investigate whether transforming growth factor-beta1 (TGF-beta1) could induce alveolar epithelial to mesenchymal transition (EMT) in vitro. Alveolar epithelial cells (AECs) from SD rats were isolated by elastase cell dispersion and IgG panning. Expression of alpha-smooth muscle actin (alpha-SMA) was assayed using Western blotting and immunostaining analysis. Morphological changes, the markers of epithelial cell (E-cadherin), and stress fiber by actin reorganization were detected by an indirect immunostaining. The contents of collagen I were determined by spectrophotometry. The levels of endogenous TGF-beta1 were measured with ELISA. Incubation of AECs with TGF-beta1 (0.1 approximately 10 ng/mL) induced abundant expression of alpha-SMA protein, and alpha-SMA expression in AECs reached a plateau when TGF-beta1 was > 3 ng/mL. Furthermore, we found that TGF-beta1 (3 ng/mL) exposure of AECs induced an authentic EMT characterized by abundant expression of alpha-smooth muscle actin, transformation of myofibroblastic morphology, increased formation of stress fiber by actin reorganization, and loss of epithelial marker E-cadherin. Meanwhile, significant increase in the levels of collagen I from 32.0 +/- 6.6 mg/g in control to 98 +/- 10.8 mg/g in TGF-beta1-treated group was found over a 72 h incubation period. Moreover, following stimulated by TGF-beta1 (3 ng/mL), a marked and time-dependent increase in endogenous TGF-beta1 released from AECs was observed. At time points 72 h, TGF-beta1 release mounted to 3451 pg/ml, which was much enough to induce EMT in vitro. These results demonstrated that AECs, under stimulation of TGF-beta1, underwent a conversion process into myofibroblasts in vitro.     

An in vitro model that recapitulates the epithelial to mesenchymal transition (EMT) in human breast cancer

The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells down-regulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In human breast cancer, invasion into surrounding tissue is the first step in metastatic progression. Here, we devised an in vitro model using selected cell lines, which recapitulates many features of EMT as observed in human breast cancer. By comparing the gene expression profiles of claudin-low breast cancers with the experimental model, we identified a 9-gene signature characteristic of EMT. This signature was found to distinguish a series of breast cancer cell lines that have demonstrable, classical EMT hallmarks, including loss of E-cadherin protein and acquisition of N-cadherin and vimentin expression. We subsequently developed a three-dimensional model to recapitulate the process of EMT with these cell lines. The cells maintain epithelial morphology when encapsulated in a reconstituted basement membrane, but undergo spontaneous EMT and invade into surrounding collagen in the absence of exogenous cues. Collectively, this model of EMT in vitro reveals the behaviour of breast cancer cells beyond the basement membrane breach and recapitulates the in vivo context for further investigation into EMT and drugs that may interfere with it.     

上皮细胞向间充质细胞转变在肝癌进程中的作用

上皮细胞向间充质细胞转变(epithelial to mesenchymal transition,EMT)是细胞通过瞬时去分化为间充质表型,导致上皮细胞的可塑性发生变化的多步骤的生物学过程。EMT及其逆转MET多数发生在胚胎发生形态发生过程中。近期更多的证据显示EMT参与肝纤维化和肝癌进程。在肝癌转移的早期阶段,细胞由于E-钙粘蛋白的消融而丧失细胞-细胞接触抑制,迁移能力增强,得以扩散到周围或远处组织,故EMT在肝癌转移的早期阶段中起关键作用。此外,由于EMT的增强诱导剂如转移生长因子(transforming growth factor-β,TGF-β)具有协调肝纤维化及肝癌进程的作用,故肝癌进程中上皮细胞的可塑性研究显得尤为重要。在本综述中,作者将阐述EMT-MET在肝癌进程中的重要性,及EMT在肝癌进程中的作用机制。同时,概述最近在识别影响重要EMT转录因子的临床诊治方面取得的进展。     

Regulation of epithelial to mesenchymal transition by bone morphogenetic proteins

Epithelial to mesenchymal transition (EMT) is a process in which fully differentiated epithelial cells lose many of their epithelial characteristics and adopt features typical of mesenchymal cells, thus allowing cells to become migratory and invasive. EMT is a critical process in development and its role in cancer and fibrosis is becoming increasingly recognised. It is also becoming apparent that EMT is not just restricted to embryonic development and disease in adults, but in fact may be an important process for the maintenance and regeneration of adult tissue architecture. While transforming growth factor-β (TGF-β) is considered a prototypic inducer of EMT, relatively little is known about other signalling molecules that regulate EMT. Bone morphogenic proteins (BMPs) are members of the TGF-β superfamily and 20 different human BMPs have been identified. Originally named for their effects on bone, these proteins are now considered to be key morphogenetic signals that orchestrate tissue architecture throughout the body. BMP2, -4 and -7 are the best studied to date. There are disparate reports of the roles of BMPs in EMT during development, cancer and fibrosis. Here, we present an overview of this literature as well as the emerging role of EMT in tissue regeneration and the involvement of BMPs in regulating this process.     

Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils

Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.     

Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney

In the kidney, a unique plasticity exists between epithelial and mesenchymal cells. During kidney development, the metanephric mesenchyme contributes to emerging epithelium of the nephron via mesenchymal to epithelial transition (MET). In the injured adult kidney, renal epithelia contribute to the generation of fibroblasts via epithelial-mesenchymal transition, facilitating renal fibrosis. Recombinant human bone morphogenic protein (BMP)-7, a morphogen that is essential for the conversion of epithelia from condensing mesenchyme during kidney development, enhances the repair of tubular structures in the kidney. In this setting, BMP-7 inhibits epithelial-mesenchymal transition involving adult renal epithelial tubular cells and decreases secretion of type I collagen by adult renal fibroblasts. In search of a mechanism behind the ability of BMP-7 to repair damaged renal tubules, we hypothesized that systemic treatment with BMP-7 might induce MET involving adult renal fibroblasts in the injured kidney, generating functional epithelial cells. Here we report that BMP-7 induces formation of epithelial cell aggregates in adult renal fibroblasts associated with reacquisition of E-cadherin expression and decreased motility, mimicking the effect of BMP-7 on embryonic metanephric mesenchyme to generate epithelium. In addition, we provide evidence that BMP-7-mediated repair of renal injury is associated with MET involving adult renal interstitial fibroblasts in mouse models for renal fibrosis. Collectively, these findings suggest that adult renal fibroblasts might retain parts of their original embryonic imprint and plasticity, which can be re-engaged by systemic administration of BMP-7 to mediate repair of tubular injury in a fibrotic kidney.     

间质表皮转化因子MET信号通路及其在肿瘤中的研究进展

间质表皮转化因子(Mesenchymal to epithelial transition factor,MET)蛋白作为一种受体酪氨酸激酶,通常存在于上皮细胞中,被HGF等配体激活后,能够参与调控细胞的增殖、凋亡、迁移侵袭和细胞形态等多种生物学功能。随着研究的深入,MET已被证实在多种恶性肿瘤中异常表达或基因扩增,其与肿瘤患者的预后有着密切的关系。因此,针对MET的抑制剂研究发展比较迅速,且其良好的抗肿瘤效果也得到了证实。本文结合目前本实验室的研究,对MET的结构、功能及其抑制剂研究的现状等进行了综述,为今后的研究者提供一个阶段性的数据资料。     

Deglycosylation of epithelial cell adhesion molecule affects epithelial to mesenchymal transition in breast cancer cells

The transmembrane glycoprotein epithelial cell adhesion molecule (EpCAM) is overexpressed in most epithelial cancers including breast cancer, where it plays an important role in cancer progression. Previous study has demonstrated that knockdown of EpCAM inhibits breast cancer cell growth and metastasis via inhibition of the Ras/Raf/ERK signaling pathway and matrix metallopeptidase-9 (MMP-9). Although glycosylation is believed to be associated with the function of EpCAM, the contribution of N-glycosylation to this function remains unclear. We constructed the N-glycosylation mutation plasmid of EpCAM and used it to treat breast cancer cells. Loss of N-glycosylation at all three sites EpCAM had no effect on its level of expression or membrane localization. However, mutation at glycosylation sites significantly reduced the ability of EpCAM to promote epithelial to mesenchymal transition in breast cancer. N-glycosylation mutation of EpCAM led to decrease phosphorylation of Raf, ERK, and Akt, and inhibited the Ras/Raf/ERK and PI3K/Akt signaling pathways. Furthermore, we demonstrated that N-glycosylation mutation of EpCAM-mediated invasion and metastasis of breast carcinoma cells required the downregulation of MMP-9 via inhibition of these two signaling pathways. Our results identified the characteristics and function of EpCAM glycosylation. These data could illuminate molecular regulation of EpCAM by glycosylation and promote our understanding of the application of glycosylated EpCAM as a target for breast cancer therapy.