Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.

A growing body of evidence demonstrates that autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of atherosclerosis and has become a potential therapeutic target. Here we tested the effect of two inhibitors of phosphatidylinositol 3-kinase, 3-methyladenine (3-MA) and 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), commonly used as inhibitors of autophagy, in atherosclerosis in apolipoprotein E-/- mice. Systemic application of 3-MA but not LY294002 markedly reduced the size of atherosclerotic plaque and increased the stability of lesions in high-fat diet-fed mice as compared with controls. Furthermore, 3-MA had multiple atheroprotective effects, including modulating macrophage autophagy and foam cell formation and altering the immune microenvironment. Long-term treatment with 3-MA promoted oxidized low-density lipoprotein (oxLDL)-induced macrophage autophagy and suppressed foam cell formation and cell viability in vitro. Furthermore, systemic application of 3-MA promoted lipid droplet breakdown and decreased apoptosis, most likely associated with autophagy. 3-MA treatment strikingly enhanced the expression of immune-negative molecules such as interleukin 10 (IL-10), transforming growth factor β and IL-35, as well as forkhead box P3 (Foxp3), the specific transcriptional factor for regulatory T cells, but did not affect the level of proinflammatory cytokines in the arterial wall. We provide strong evidence for the potential therapeutic benefit of 3-MA in inhibiting atherosclerosis development and improving plaque stability.

Interleukin (IL)-35 is a newly identified immune negative molecule which is secreted by CD4(+)Foxp3(+) T regulatory cells (Tregs) and contributes to their suppressive capacity. Early data have shown that IL-35 inhibits development of several autoimmune diseases. However, the role of IL-35 in atherosclerosis, a lipid-driven chronic inflammatory disease in arterial wall, remains to be investigated. Here, we found that IL-35 was involved in atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. ApoE(-/-) mice with established atherosclerotic lesion displayed a lower level of IL-35 compared to age-matched wild type C57BL/6 mice without plaque. However, IL-35 expression increased significantly in ApoE(-/-) mice with attenuated plaque. More importantly, we found that modulation of ER stress treated by chemical chaperone, 4-Phenyl butyric acid (PBA) in vivo, mainly upregulated immune negative regulating molecule IL-35, as well as IL-10 and Foxp3, accompanied by increased Tregs. However, no obvious impact on pro-inflammatory molecules such as TNF-α, IFN-γ, IL-17 and IL-23 was observed, which provides new insight into the benefit of ER stress recovery from attenuated plaque. Our results suggest that IL-35 might have a potential value for atherosclerotic therapy.

Programmed cell death-4 (PDCD4), a selective protein translation inhibitor, has shown proinflammatory effect in some inflammatory diseases, but its roles in obesity remain unestablished. This study aims to investigate the effects of PDCD4 on obesity, inflammation, and insulin resistance. Surprisingly, high-fat diet (HFD)-fed PDCD4-deficient (PDCD4(-/-)) mice exhibited an absolutely lean phenotype together with improved insulin sensitivity. Compared with wild-type obese mice, HFD-fed PDCD4(-/-) mice showed higher energy expenditure, lower epididymal fat weight, and reduced macrophage infiltration inflammatory cytokine secretion in white adipose tissue (WAT). Alleviated hepatic steatosis along with decreased plasma levels of triglyceride and cholesterol was also observed in these mice. Importantly, PDCD4 appeared to disturb lipid metabolism via inhibiting the expression of liver X receptor (LXR)-α, a master modulator of lipid homeostasis, which was elevated in HFD-fed PDCD4(-/-) mice accompanied by upregulation of its target genes and relieved endoplasmic reticulum stress in WAT. These data demonstrate that PDCD4 deficiency protects mice against diet-induced obesity, WAT inflammation, and insulin resistance through restoring the expression of LXR-α, thereby proposing PDCD4 as a potential target for treating obesity-associated diseases.

Tumor invasion and metastasis are the major reasons for leading death of patients with hepatocellular carcinoma (HCC). Therefore, to identify molecules that can suppress invasion and metastasis of tumor will provide novel targets for HCC therapies. Tumor necrosis factor (TNF)-alpha-induced protein 8-like 2, TIPE2, is a novel immune negative molecule and an inhibitor of the oncogenic Ras in mice but its function in human is unclear. Our previous research has shown that TIPE2 is downregulated in human primary HCC compared with the paired adjacent non-tumor tissues.

Brain-derived neurotrophic factor (BDNF) is a growth factor that plays vital roles in the neuron survival, growth, and neuroplasticity. Alteration to BDNF expression is associated with major depressive disorder. However, the BDNF translational machinery in depression remains unknown. Herein, we pointed that Pdcd4, a suppressor oncogene, acted as an endogenous inhibitor for the translation of BDNF, and selectively repressed the translation of BDNF splice variant IIc mRNA in an eIF4A-dependent manner. Chronic restraint stress (CRS) up-regulated Pdcd4 expression in hippocampus via decreasing mTORC1-mediated proteasomes degradation pathway, which resulted in the reduction of BDNF protein expression. Moreover, over-expression of Pdcd4 in the hippocampus triggered spontaneous depression-like behaviors under the non-stressed conditions in mice, while systemic or neuron-specific knockout of Pdcd4 reverses CRS-induced depression-like behaviors. Importantly, administration of Pdcd4 siRNA or an interfering peptide that interrupts the Pdcd4-eIF4A complex substantially promoted BDNF expression and rescued the behavioral disorders which were caused by CRS. Overall, we have discovered a previously unrecognized role of Pdcd4 in controlling BDNF mRNA translation, and provided a new method that boosting BDNF expression through blocking the function of Pdcd4 in depression, indicating that Pdcd4 might be a new potential target for depressive disorder therapy.

Herein, fifteen new compounds containing coumarin, 1,2,3-triazole and benzoyl- substituted arylamine moieties were designed, synthesized and tested in vitro for their anticancer activity. The results showed that all tested compounds had moderate antiproliferative activity against MDA-MB-231, a human breast cancer cell line, under both normoxic and hypoxic conditions. Furthermore, the 4-substituted coumarin linked with benzoyl 3,4-dimethoxyaniline through 1,2,3-triazole (compound 5e) displayed the most prominent antiproliferative activities with an IC50 value of 0.03 μM, about 5000 times stronger than 4-hydroxycoumarin (IC50 > 100 μM) and 20 times stronger than doxorubicin (IC50 = 0.60 μM). Meanwhile, almost all compounds revealed general enhancement of proliferation-inhibiting activity under hypoxia, contrasted with normoxia. A docking analysis showed that compound 5e had potential to inhibit carbonic anhydrase IX (CA IX).

Programmed cell death 4 (Pdcd4) was found to be related to apoptosis upon first discovery. It was later found to play the role of tumor suppressor gene in a variety of tumors by inhibiting transcription and translation. Recently, it has been proposed that it may play an important role in some inflammatory diseases and in the immune response. In our previous study, deficiency of Pdcd4 was found to attenuate the formation of atherosclerotic plaques. This might be because deficiency of Pdcd4 may increase IL-10 expression and lipoautophagy by macrophages and attenuate the formation of foam cells. However, the effect of Pdcd4 on the subsets of T cells in hyperlipidemic mice still remained unclear. In the present study, results showed that Pdcd4 deficiency decreased the percentage of CD8+ T cells and increased that of regulatory T cells (Tregs) under hyperlipidemic conditions both in vitro and in vivo, which may be due to the reduced expression of co-stimulatory molecules CD28 and CD137, and the enhancive expression of co-inhibitory molecules CTLA-4. These results indicated that endogenous Pdcd4 promotes immune response mediated by T cells through regulation of the co-stimulatory molecules expression, which may contribute to the development of advanced atherosclerotic plaques. The current work provides new data to understand the role of Pdcd4 in different T cell subsets under hyperlipidemic microenvironment.

Obesity and insulin resistance greatly increase the risk of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). We have previously discovered that whole-body and adipocyte-specific Ip6k1deletion protects mice from high-fat-diet-induced obesity and insulin resistance due to improved adipocyte thermogenesis and insulin signaling. Here, we aimed to determine the impact of hepatocyte-specific and whole-body Ip6k1 deletion (HKO and Ip6k1-KO or KO) on liver metabolism and NAFLD/NASH.

Depression is one of the most common psychiatric disorders. Recently, studies demonstrate that antidepressants generating BDNF not only maintain synaptic signal transmission but also repress neuroinflammatory cytokines such as IL-6 and IL-1β. Therefore, promoting BDNF expression provides a strategy for the treatment of depression. Our recent research has indicated that programmed cell death 4 (Pdcd4) is a new target for antidepressant treatment by facilitating BDNF. Herein, we modified Pdcd4 specific small interfering RNA (siPdcd4) with the rabies virus glycoprotein peptide (RVG/siPdcd4) which enables it cross the blood-brain barrier (BBB). We found that RVG/siPdcd4 complex was selectively delivered to neurons and microglia and silenced the expression of Pdcd4, thereby up-regulating the level of BDNF and down-regulating IL-6 and IL-1β expression. More importantly, RVG/siPdcd4 injection attenuated synaptic plasticity impairment and protected mice from CRS-induced depressive behavior. These findings suggest that RVG/siPdcd4 complex is a potential therapeutic medicine for depression.

Mitochondria are unavoidably subject to organellar stress resulting from exposure to a range of reactive molecular species. Consequently, cells operate a poorly understood quality control programme of mitophagy to facilitate elimination of dysfunctional mitochondria. Here, we used a model stressor, deferiprone (DFP), to investigate the molecular basis for stress-induced mitophagy. We show that mitochondrial fission 1 protein (Fis1) is required for DFP-induced mitophagy and that Fis1 is SUMOylated at K149, an amino acid residue critical for Fis1 mitochondrial localization. We find that DFP treatment leads to the stabilization of the SUMO protease SENP3, which is mediated by downregulation of the E3 ubiquitin (Ub) ligase CHIP. SENP3 is responsible for Fis1 deSUMOylation and depletion of SENP3 abolishes DFP-induced mitophagy. Furthermore, preventing Fis1 SUMOylation by conservative K149R mutation enhances Fis1 mitochondrial localization. Critically, expressing a Fis1 K149R mutant restores DFP-induced mitophagy in SENP3-depleted cells. Thus, we propose a model in which SENP3-mediated deSUMOylation facilitates Fis1 mitochondrial localization to underpin stress-induced mitophagy.

Monoamine oxidase A (MAOA) is an important mitochondria-bound enzyme that catalyzes the oxidative deamination of monoamine neurotransmitters. Accumulating evidence suggests a significant association of increased MAOA expression and advanced high-grade prostate cancer (PCa) progression and metastasis. Herein, a series of novel conjugates combining the MAOA inhibitor isoniazid (INH) and tumor-targeting near-infrared (NIR) heptamethine cyanine dyes were designed and synthesized. The synthesized compounds G1⁻G13 were evaluated in vitro for their cytotoxicity against PC-3 cells using the MTT assay, and molecular docking studies were performed. Results showed that most tested compounds exhibited improved antitumor efficacy compared with INH. Moreover, conjugates G10 and G11 showed potent anticancer activity with IC50 values (0.85 and 0.4 μM respectively) comparable to that of doxorubicin (DOX). This may be attributable to the preferential accumulation of these conjugates in tumor cells. G10, G11, and G12 also demonstrated moderate MAOA inhibitory activities. This result and the results of molecular docking studies were consistent with their cytotoxicity activities. Taken together, these data suggest that a combination of the MAOA inhibitor INH with tumor-targeting heptamethine cyanine dyes may prove to be a highly promising tool for the treatment of advanced prostate cancer.

Estrogen receptor alpha (ERα), a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

Metastasis of hepatocellular carcinoma (HCC) can be facilitated by TNF-α, a prototypical inflammatory cytokine in the HCC microenvironment. A20 is a negative regulator of NF-κB signaling pathway. In the present study we ask whether A20 plays a role in HCC metastasis. We found that A20 expression was downregulated in the invasive cells of microvascular invasions (MVI) compared with the noninvasive cells in 89 tissue samples from patients with HCC by immunochemistry methods. Overexpression of A20 in HCC cell lines inhibited their motility induced by TNF-α. Furthermore, the overexpression of A20 inhibited epithelial-mesenchymal transition (EMT), FAK activation and RAC1 activity. By contrast, knockdown of A20 in one HCC cell line results in the converse. In addition, the overexpression of A20 restrained the formation of MVI in HCC xenograft in nude mice treated with TNF-α. All the results suggested that A20 functioned as a negative regulator in motility of HCC cells induced by TNF-α.

Mechanical strain plays a critical role in the proliferation, differentiation and maturation of bone cells. As mechanical receptor cells, osteoblasts perceive and respond to stress force, such as those associated with compression, strain and shear stress. However, the underlying molecular mechanisms of this process remain unclear. Using a four-point bending device, mouse MC3T3-E1 cells was exposed to mechanical tensile strain. Cell proliferation was determined to be most efficient when stimulated once a day by mechanical strain at a frequency of 0.5 Hz and intensities of 2500 µε with once a day, and a periodicity of 1 h/day for 3 days. The applied mechanical strain resulted in the altered expression of 1992 genes, 41 of which are involved in the mitogen-activated protein kinase (MAPK) signaling pathway. Activation of ERK by mechanical strain promoted cell proliferation and inactivation of ERK by PD98059 suppressed proliferation, confirming that ERK plays an important role in the response to mechanical strain. Furthermore, the membrane-associated receptors integrin β1 and integrin β5 were determined to regulate ERK activity and the proliferation of mechanical strain-treated MC3T3-E1 cells in opposite ways. The knockdown of integrin β1 led to the inhibition of ERK activity and cell proliferation, whereas the knockdown of integrin β5 led to the enhancement of both processes. This study proposes a novel mechanism by which mechanical strain regulates bone growth and remodeling.

Non-small cell lung cancer (NSCLC) is one of the leading causes of all cancer-related deaths worldwide. Despite extensive efforts to improve the diagnosis and treatment of this neoplasm, limited progress has been made. Tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2 or TNFAIP8L2) is a newly introduced negative immune regulator, which also controls tumorigenesis. However, the role of TIPE2 in angiogenesis is unknown. In the present study, we investigated the expression and roles of TIPE2 in NSCLC. TIPE2 upregulation in human NSCLC tissues was negatively associated with the primary tumor size, lymph node metastasis, and advanced clinical stage, which can be used to predict lymph node metastasis. Moreover, overexpression of TIPE2 not only inhibited the colony formation, migration, and invasion of NSCLC cells but also indirectly suppressed the proliferation, migration, and tube formation of vascular endothelial cells. Furthermore, TIPE2 suppressed tumor invasiveness and angiogenesis via inhibiting the activation of Rac1 and subsequently weakening its downstream effects, including F-actin polymerization and VEGF expression. Collectively, these results indicate that TIPE2 plays a key role in NSCLC metastasis, suggesting that forced TIPE2 expression might be a novel strategy for the treatment of NSCLC.

A series of selenium-containing miconazole derivatives were identified as potent antifungal drugs in our previous study. Representative compound A03 (MIC = 0.01 μg/mL against C.alb. 5314) proved efficacious in inhibiting the growth of fungal pathogens. However, further study showed lead compound A03 exhibited potential hemolysis, significant cytotoxic effect and unfavorable metabolic stability and was therefore modified to overcome these drawbacks. In this article, the further optimization of selenium-containing miconazole derivatives resulted in the discovery of similarly potent compound B17 (MIC = 0.02 μg/mL against C.alb. 5314), exhibiting a superior pharmacological profile with decreased rate of metabolism, cytotoxic effect and hemolysis. Furthermore, compound B17 showed fungicidal activity against Candida albicans and significant effects on the treatment of resistant Candida albicans infections. Meanwhile, compound B17 not only could reduce the ergosterol biosynthesis pathway by inhibiting CYP51, but also inhibited biofilm formation. More importantly, compound B17 also shows promising in vivo efficacy after intraperitoneal injection and the PK study of compound B17 was evaluated. In addition, molecular docking studies provide a model for the interaction between the compound B17 and the CYP51 protein. Overall, we believe that these selenium-containing miconazole compounds can be further developed for the potential treatment of fungal infections.

Dysregulation of the mitochondrial fission machinery has been linked to cell death following ischemia. Fission is largely dependent on recruitment of Dynamin-related protein 1 (Drp1) to the receptor Mitochondrial fission factor (Mff) located on the mitochondrial outer membrane (MOM). Drp1 is a target for SUMOylation and its deSUMOylation, mediated by the SUMO protease SENP3, enhances the Drp1-Mff interaction to promote cell death in an oxygen/glucose deprivation (OGD) model of ischemia. Another interacting partner for Drp1 is the Bcl-2 family member Bcl-x L , an important protein in cell death and survival pathways. Here we demonstrate that preventing Drp1 SUMOylation by mutating its SUMO target lysines enhances the Drp1-Bcl-x L interaction in vivo and in vitro. Moreover, SENP3-mediated deSUMOylation of Drp1 promotes the Drp1-Bcl-x L interaction. Our data suggest that Mff primes Drp1 binding to Bcl-x L at the mitochondria and that Mff and Bcl-x L can interact directly, independent of Drp1, through their transmembrane domains. Importantly, SENP3 loss in cells subjected to OGD correlates with reduced Drp1-Bcl-x L interaction, whilst recovery of SENP3 levels in cells subjected to reoxygenation following OGD correlates with increased Drp1-Bcl-x L interaction. Expressing a Bcl-x L mutant with defective Drp1 binding reduces OGD plus reoxygenation-evoked cell death. Taken together, our results indicate that SENP3-mediated deSUMOlyation promotes an Mff-primed Drp1-Bcl-x L interaction that contributes to cell death following ischemia.

Insulin resistance is a pathological state often associated with obesity, representing a major risk factor for type 2 diabetes. Limited mechanism-based strategies exist to alleviate insulin resistance. Here, using single-cell transcriptomics, we identify a small, critically important, but previously unexamined cell population, p21Cip1 highly expressing (p21high) cells, which accumulate in adipose tissue with obesity. By leveraging a p21-Cre mouse model, we demonstrate that intermittent clearance of p21high cells can both prevent and alleviate insulin resistance in obese mice. Exclusive inactivation of the NF-κB pathway within p21high cells, without killing them, attenuates insulin resistance. Moreover, fat transplantation experiments establish that p21high cells within fat are sufficient to cause insulin resistance in vivo. Importantly, a senolytic cocktail, dasatinib plus quercetin, eliminates p21high cells in human fat ex vivo and mitigates insulin resistance following xenotransplantation into immuno-deficient mice. Our findings lay the foundation for pursuing the targeting of p21high cells as a new therapy to alleviate insulin resistance.

Examining associations between viral genomic loads of enteroviruses and clinical severity is important for promoting and improving development of antiviral drugs related to hand, foot and mouth disease (HFMD).