Monocytes Release Pro-Cathepsin D to Drive Blood-to-Brain Transcytosis in Diabetes.

BACKGROUND: Microvascular complications are the major outcome of type 2 diabetes progression, and the underlying mechanism remains to be determined. METHODS: High-throughput RNA sequencing was performed using human monocyte samples from controls and diabetes. The transgenic mice expressing human CTSD (cathepsin D) in the monocytes was constructed using CD68 promoter. In vivo 2-photon imaging, behavioral tests, immunofluorescence, transmission electron microscopy, Western blot analysis, vascular leakage assay, and single-cell RNA sequencing were performed to clarify the phenotype and elucidate the molecular mechanism. RESULTS: Monocytes expressed high-level CTSD in patients with type 2 diabetes. The transgenic mice expressing human CTSD in the monocytes showed increased brain microvascular permeability resembling the diabetic microvascular phenotype, accompanied by cognitive deficit. Mechanistically, the monocytes release nonenzymatic pro-CTSD to upregulate caveolin expression in brain endothelium triggering caveolae-mediated transcytosis, without affecting the paracellular route of brain microvasculature. The circulating pro-CTSD activated the caveolae-mediated transcytosis in brain endothelial cells via its binding with low-density LRP1 (lipoprotein receptor-related protein 1). Importantly, genetic ablation of CTSD in the monocytes exhibited a protective effect against the diabetes-enhanced brain microvascular transcytosis and the diabetes-induced cognitive impairment. CONCLUSIONS: These findings uncover the novel role of circulatory pro-CTSD from monocytes in the pathogenesis of cerebral microvascular lesions in diabetes. The circulatory pro-CTSD is a potential target for the intervention of microvascular complications in diabetes.

Lpp of Escherichia coli K1 inhibits host ROS production to counteract neutrophil-mediated elimination.

Escherichia coli (E. coli) is the most common Gram-negative bacterial organism causing neonatal meningitis. The pathogenesis of E. coli meningitis, especially how E. coli escape the host immune defenses, remains to be clarified. Here we show that deletion of bacterial Lpp encoding lipoprotein significantly reduces the pathogenicity of E. coli K1 to induce high-degree of bacteremia necessary for meningitis. The Lpp-deleted E. coli K1 is found to be susceptible to the intracellular bactericidal activity of neutrophils, without affecting the release of neutrophil extracellular traps. The production of reactive oxygen species (ROS), representing the primary antimicrobial mechanism in neutrophils, is significantly increased in response to Lpp-deleted E. coli. We find this enhanced ROS response is associated with the membrane translocation of NADPH oxidase p47phox and p67phox in neutrophils. Then we constructed p47phox knockout mice and we found the incidence of bacteremia and meningitis in neonatal mice induced by Lpp-deleted E. coli is significantly recovered by p47phox knockout. Proteomic profile analysis show that Lpp deficiency induces upregulation of flagellar protein FliC in E. coli. We further demonstrate that FliC is required for the ROS induction in neutrophils by Lpp-deleted E. coli. Taken together, these data uncover the novel role of Lpp in facilitating intracellular survival of E. coli K1 within neutrophils. It can be inferred that Lpp of E. coli K1 is able to suppress FliC expression to restrain the activation of NADPH oxidase in neutrophils resulting in diminished bactericidal activity, thus protecting E. coli K1 from the elimination by neutrophils.

Dynamin-2 mediates clathrin-dependent endocytosis for amyloid-ß internalization in brain microvascular endothelial cells.

Dynamin is recognized as a crucial regulator for membrane fission and has three isoforms in mammals. But the expression patterns of dynamin isoforms and their roles in non-neuronal cells are incompletely understood. In this study, the expression profiles of dynamin isoforms and their roles in endocytosis was investigated in brain endothelial cells. We found that Dyn2 was expressed at highest levels, whereas the expression of Dyn1 and Dyn3 were far less than Dyn2. Live-cell imaging was used to investigate the effects of siRNA-mediated knockdown of individual dynamin isoforms on transferrin uptake, and we found that Dyn2, but not Dyn1 or Dyn3, is required for the endocytosis in brain endothelial cells. Results of dextran uptake assay showed that dynamin isoforms are not involved in the clathrin-independent fluid-phase internalization of brain endothelial cells, suggesting the specificity of the role of Dyn2 in clathrin-dependent endocytosis. Immunofluorescence and electron microscopy analysis showed that Dyn2 co-localizes with clathrin and acts at the late stage of vesicle fission in the process of endocytosis. Further results showed that Dyn2 is necessary for the basolateral-to-apical internalization of amyloid-ß into brain endothelial cells. We concluded that Dyn2, but not Dyn1 or Dyn3, mediates the clathrin-dependent endocytosis for amyloid-ß internalization particularly from basolateral to apical side into brain endothelial cells.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli.

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

BAG3 Suppresses Loading of Ago2 to IL6 mRNA in Pancreatic Ductal Adenocarcinoma.

Pancreatic stellate cells (PSCs) are a subset of pancreatic cancer-associated fibroblasts, which play a critical role in pancreatic fibrosis, a characteristic feature of pancreatic cancer. The interplay between PSCs and pancreatic cancer cells is vital for promotion of tumor progression and metastasis. BAG3 is correlated with poor prognostics in patients with pancreatic ductal adenocarcinoma (PDAC), however, the exact mechanisms remain largely unknown. In this study, we demonstrated that BAG3 downregulation decreased IL6 release by PDACs, and IL6 reduction was, at least partially, responsible for suppression of PSCs activation by PDACs with BAG3 downmodulation. Importantly, BAG3 expression positively correlated with fibrosis in pancreatic cancer tissue. With regard to the underlying mechanism, we demonstrated that BAG3 knockdown facilitated recruitment of Agonaute 2 (Ago2) to IL6 mRNA, resulting in destabilization of IL6 mRNA. In addition, the current study demonstrated that phosphorylation at Serine (Ser) 387 site was required for recruitment of Ago2-containing miRISC to IL6 mRNA and BAG3 knockdown facilitated Ago2 loading to IL6 mRNA via increasing its phosphorylation at Ser 387. This study shed new light on the tumor-promoting role of BAG3 in PDAC tumors, suggesting BAG3 might represent an interesting therapeutic opportunity to PDAC patients.

BAG3 regulates stability of IL-8 mRNA via interplay between HuR and miR-4312 in PDACs.

Bcl-2 associated athanogene 3 (BAG3) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), and its high expression appears to be a poor prognostic factor for patients with PDAC. In this study, we show that BAG3 knockdown significantly decreases migration and invasion of PDACs via reduction of interleukine-8 (IL-8) production. BAG3 knockdown regulates IL-8 expression at the posttranscriptional levels via interplay between recruitment of RNA-binding protein HuR and miR-4312. HuR binds to the cis-elements located in the 3'-untranslational region (UTR) of the IL-8 transcript to stabilize it, whereas miR-4312-containing miRNA-induced silencing complex (miRISC) is recruited to the adjacent seed element to destabilize it. The binding of HuR prevents the recruitment of Argonaute (Ago2), overriding miR-4312-mediated translation inhibition of IL-8. BAG3 knockdown decreases cytoplasmic distribution of HuR via increasing its phosphorylation at Ser202, therefore compromising its recruitment while promoting recruitment of miR-4312 containing miRISC to IL-8 transcript. Furthermore, our data indicate that only phosphorylated Ago2 at Ser387 interacts with IL-8 transcript. BAG3 knockdown increases phosphorylation of Ago2 at Ser387, thereby further promoting loading of miR-4312 containing miRISC to IL-8 transcript. Taken together, we propose that BAG3 promotes invasion by stabilizing IL-8 transcript via HuR recruitment, and subsequently suppressing the loading of miR-4312 containing miRISC in PDACs. Our results reveal a novel pathway linking BAG3 expression to enhanced PDAC metastasis, thus making BAG3 a potential target for intervention in pancreatic cancer.

BAG3 directly stabilizes Hexokinase 2 mRNA and promotes aerobic glycolysis in pancreatic cancer cells.

Aerobic glycolysis, a phenomenon known historically as the Warburg effect, is one of the hallmarks of cancer cells. In this study, we characterized the role of BAG3 in aerobic glycolysis of pancreatic ductal adenocarcinoma (PDAC) and its molecular mechanisms. Our data show that aberrant expression of BAG3 significantly contributes to the reprogramming of glucose metabolism in PDAC cells. Mechanistically, BAG3 increased Hexokinase 2 (HK2) expression, the first key enzyme involved in glycolysis, at the posttranscriptional level. BAG3 interacted with HK2 mRNA, and the degree of BAG3 expression altered recruitment of the RNA-binding proteins Roquin and IMP3 to the HK2 mRNA. BAG3 knockdown destabilized HK2 mRNA via promotion of Roquin recruitment, whereas BAG3 overexpression stabilized HK2 mRNA via promotion of IMP3 recruitment. Collectively, our results show that BAG3 promotes reprogramming of glucose metabolism via interaction with HK2 mRNA in PDAC cells, suggesting that BAG3 may be a potential target in the aerobic glycolysis pathway for developing novel anticancer agents.

BAG3 promotes stem cell-like phenotype in breast cancer by upregulation of CXCR4 via interaction with its transcript.

BAG3 is an evolutionarily conserved co-chaperone expressed at high levels and has a prosurvival role in many tumor types. The current study reported that BAG3 was induced under specific floating culture conditions that enrich breast cancer stem cell (BCSC)-like cells in spheres. Ectopic BAG3 overexpression increased CD44+/CD24- CSC subpopulations, first-generation and second-generation mammosphere formation, indicating that BAG3 promotes CSC self-renewal and maintenance in breast cancer. We further demonstrated that mechanically, BAG3 upregulated CXCR4 expression at the post-transcriptional level. Further studies showed that BAG3 interacted with CXCR4 mRNA and promoted its expression via its coding and 3'-untranslational regions. BAG3 was also found to be positively correlated with CXCR4 expression and unfavorable prognosis in patients with breast cancer. Taken together, our data demonstrate that BAG3 promotes BCSC-like phenotype through CXCR4 via interaction with its transcript. Therefore, this study establishes BAG3 as a potential adverse prognostic factor and a therapeutic target of breast cancer.

Induction of epithelial-mesenchymal transition (EMT) by Beclin 1 knockdown via posttranscriptional upregulation of ZEB1 in thyroid cancer cells.

Beclin 1 has emerged as a haploinsufficient tumor suppression gene in a variety of human carcinomas. In order to clarify the role of Beclin 1 in thyroid cancer, Beclin 1 was knockdown in thyroid cancer cell lines. The current study demonstrated that knockdown of Beclin 1 resulted in morphological and molecular changes of thyroid cancer cells consistent with epithelial-mesenchymal transition (EMT), a morphogenetic procedure during which cells lose their epithelial characteristics and acquire mesenchymal properties concomitantly with gene expression reprogramming. In addition, the current study presented evidence demonstrating that Beclin 1 knockdown triggered this prometastatic process via stabilization of the EMT inducer ZEB1 mRNA through upregulation of AU-binding factor 1 (AUF1), which is recruited to the 3'-untranslated region (UTR) of the ZEB1 mRNA and decreases its degradation. We also found a negative correlation of Beclin 1 with AUF1 or ZEB1 in thyroid cancer tissues. These results indicated that at least some tumor suppressor functions of Beclin 1 were mediated through posttranscriptional regulation of ZEB1 via AUF1 in thyroid cancers.

Involvement of Nrf2 in proteasome inhibition-mediated induction of ORP150 in thyroid cancer cells.

Oxygen-regulated protein 150 (ORP150) is an inducible ER chaperone by numerous cellular insults and sustains cellular viability. We have previously reported that ORP150 is differentially induced in a panel thyroid cancer cells and represents as an unwanted molecular consequence during exposure to proteasome inhibition. However, the molecular basis for induction of ORP150 by proteasome inhibitors in thyroid cancer cells remains unclear. In the current study, we found that -421/-307 and -243/+53 regions at the ORP150 gene were responsible for its transactivation by MG132 in thyroid cancer cells. Nrf2 directly transactivated the ORP150 gene by direct binding with the -421/-307 region. Nrf2 also indirectly activated OPR150 transcription via facilitating recruitment of ATF4 to the -243/+53 region. Collectively, this study highlights the molecular mechanism by which proteasome inhibition stimulates ORP150 expression via Nrf2 in thyroid cancer cells.