Ribosomal-processing cysteine protease homolog modulates Streptococcus mutans glucan production and interkingdom interactions.

Glucan-dependent biofilm formation is a crucial process in the establishment of Streptococcus mutans as a cariogenic oral microbe. The process of glucan formation has been investigated in great detail, with glycosyltransferases GtfB, GtfC, and GtfD shown to be indispensable for the synthesis of glucans from sucrose. Glucan production can be visualized during biofilm formation through fluorescent labeling, and its abundance, as well as the effect of glucans on general biofilm architecture, is a common phenotype to study S. mutans virulence regulation. Here, we describe an entirely new phenotype associated with glucan production, caused by a mutation in the open reading frame SMU_848, which is located in an operon encoding ribosome-associated proteins. This mutation led to the excess production and accumulation of glucan-containing droplets on the surface of biofilms formed on agar plates after prolonged incubation. While not characterized in S. mutans, SMU_848 shows homology to the phage-related ribosomal protease Prp, essential in cleaving off the N-terminal extension of ribosomal protein L27 for functional ribosome assembly in Staphylococcus aureus. We present a further characterization of SMU_848/Prp, demonstrating that the deletion of this gene leads to significant changes in S. mutans gtfBC expression. Surprisingly, it also profoundly impacts the interkingdom interaction between S. mutans and Candida albicans, a relevant dual-species interaction implicated in severe early childhood caries. The presented data support a potential broader role for SMU_848/Prp, possibly extending its functionality beyond the ribosomal network to influence important ecological processes. IMPORTANCE: Streptococcus mutans is an important member of the oral biofilm and is implicated in the initiation of caries. One of the main virulence mechanisms is the glucan-dependent formation of biofilms. We identified a new player in the regulation of glucan production, SMU_848, which is part of an operon that also encodes for ribosomal proteins L27 and L21. A mutation in SMU_848, which encodes a phage-related ribosomal protease Prp, leads to a significant accumulation of glucan-containing droplets on S. mutans biofilms, a previously unknown phenotype. Further investigations expanded our knowledge about the role of SMU_848 beyond its role in glucan production, including significant involvement in interkingdom interactions, thus potentially playing a global role in the virulence regulation of S. mutans.

Risk factors for venous thromboembolism in a single pediatric intensive care unit in China.

BACKGROUND: Analyses of extensive, nationally representative databases indicate a rising prevalence of venous thromboembolism (VTE) among critically ill children. However, the majority of studies on childhood VTE have primarily concentrated on Caucasian populations in the United States and European countries. There is a lack of epidemiological studies on VTE in Chinese children. METHODS: We conducted a retrospective cohort study of data from the Pediatric Intensive Care (PIC) database. Data were obtained and extracted by using Structured Query Language (SQL) and the administrative platform pgAdmin4 for PostgreSQL. Bivariate analyses were conducted in which categorical variables were analyzed by a chi-square test and continuous variables were analyzed by a Student's t-test. Separate multivariable logistic regressions were employed to investigate the associations between VTE and sociodemographic factors as well as clinical factors. RESULTS: Our study included 12,881 pediatric patients from the PIC database, spanning the years 2010 to 2018. The incidence rate of pediatric VTE was 0.19% (24/12,881). The venous thrombotic locations were deep venous thrombosis extremities (n = 18), superior vena cava (n = 1), cerebral sinovenous (n = 1), and other deep venous thrombosis (n = 4). Univariate analysis showed that age, weight, shock, sepsis, cancer and vasopressor receipt were statistically significant risk factors for pediatric VTE (all p ≤ 0.05). After multivariable logistic regression analysis, only shock (aOR: 6.77, 95%CI: 1.33-34.73, p = 0.019) and admission for sepsis (aOR: 6.09, 95%CI: 1.76-21.09, p = 0.004) were statistically significant associated with pediatric VTE. CONCLUSIONS: In conclusion, data obtained from the Pediatric Intensive Care (PIC) database revealed a prevalence of VTE in pediatric patients of 0.19%. The most common location for venous thrombi was deep venous thrombosis (DVT) in the extremities. We identified that shock and sepsis were statistically significant factors associated with pediatric VTE.

Effect of Epidermoid Cysts on the Efficacy of Intralesional Corticosteroid Therapy for Hypertrophic Scars and Keloids: A Prospective Pilot Study.

BACKGROUND: Patients with hypertrophic scars (HSs) or keloids occasionally have epidermoid cysts (ECs), and the effect of ECs on the effectiveness of intralesional corticosteroids (ILCs) treatment in these patients has not been reported. OBJECTIVE: This study aims to evaluate the influence of ECs on the outcomes of ILCs treatment in patients with HSs or keloids. MATERIALS AND METHODS: This prospective study included 572 patients with keloids ( n = 461) or HSs ( n = 111). Patients received intralesional triamcinolone acetonide injection (0.05 mL/injection) at a concentration of 40 mg/mL and every 28 days for 4 sessions, with a 1-year follow-up. RESULTS: A higher incidence of ECs was observed in keloid patients (16.92%) compared with HSs patients (7.21%). Keloid patients with ECs were older ( p = .008) and had a longer disease duration ( p = .0148), higher Vancouver scar scale (VSS) scores ( p = .04), and greater thickness ( p = .006). Keloid patients with ECs showed less improvement in VSS scores ( p < .0001) and thickness ( p < .0001) after ILCs treatment, with a higher recurrence rate ( p < .0001). The overall complication rate in keloid patients with ECs after ILCs treatment was 49.51%. CONCLUSION: Epidermoid cysts under keloids were associated with a poor response to ILCs therapy. Therefore, it is recommended to incorporate ultrasonography as a routine examination for keloid patients to aid in better decision making in clinical practice.

Maintenance of cathepsin D-dependent autophagy-lysosomal function protects against cardiac ischemia/reperfusion injury.

Cardiac ischemia/reperfusion(I/R) induced-cardiac vascular endothelial injury is an important pathological process that appears in the early stage of cardiac I/R injury. The autophagy-lysosomal pathway is essential for the maintenance of cellular homeostasis. However, in cardiac I/R injury, the role of the autophagy-lysosomal pathway is controversial. The present study aimed to use oxygen-glucose deprivation/oxygen-glucose resupply(OGD/OGR) in human coronary artery endothelial cells(HCAECs) with I/R injury to assess the role of the autophagy-lysosomal pathway in I/R-induced endothelial injury. The results revealed lysosomal dysfunction and impaired autophagic flux in endothelial cells exposed to OGD/OGR. Meanwhile, our data showed that the levels of cathepsin D(CTSD) decreased time-dependently. Knockdown of CTSD caused lysosomal dysfunction and impaired autophagic flux. Conversely, restoration of CTSD levels protected HCAECs against OGD/OGR induced-defects in autophagy-lysosomal function and cellular damage. Our findings indicated that I/R induced-impaired autophagic flux, rather than excessive autophagic initiation, mediates endothelial cells injury. The maintenance of autophagy-lysosomal function is critical to protect endothelial cells against I/R injury, and CTSD is a key regulator. Thus, strategies focused on restoring CTSD function are potentially novel treatments for cardiac reperfusion injury.

Isolation and Identification of Novel Highly Pathogenic Avian Influenza Virus (H5N8) Subclade 2.3.4.4b from Geese in Northeastern China.

H5N8, a highly pathogenic avian influenza, has become a new zoonotic threat in recent years. As of December 28, 2021, at least 3,206 H5N8 cases had been reported in wild birds and poultry worldwide. In January 2021, a novel virus strain named A/goose/China/1/2021 was isolated during an H5N8 goose influenza outbreak in northeastern China. The PB2, PB1, HA, and M genes of A/goose/China/1/2021 were highly identical to those of H5N8 strains emerging in Kazakhstan and Russia in Central Asia from August to September 2020, while the remaining four genes had the closest homology to those of H5N8 viruses isolated in South Korea in East Asia from November to December 2020. We thus speculate that A/goose/China/1/2021 is likely a reassortant virus that formed in the 2020 to 2021 influenza season and that the migratory birds via the two migration routes of Central Asia and East Asia-Australia may have played an essential role in the genetic reassortment of this virus. The phylogenetic analysis indicated that the HA genes of H5N8 viruses belonging to group II of subclade 2.3.4.4b, including A/goose/China/1/2021, may be derived from strains in Central Asia. Given the complex global spread of H5N8 virus, our study highlights the necessity to strengthen the function of the global surveillance network for H5N8 virus and to accelerate the pace of vaccine development to confront the current challenges posed by H5N8 virus of subclade 2.3.4.4. IMPORTANCE H5N8, a highly pathogenic avian influenza, not only has an impact on public health, but also has a huge negative impact on animal health, food safety, safety, and even on the local and international economy. The migratory wild birds play a vital role in the intercontinental transmission of H5N8 virus. It is urgent that we should strengthen the function of the global surveillance network for H5N8 virus and accelerate the pace of vaccine development to confront the current challenges posed by H5N8 virus of subclade 2.3.4.4.

Defect-Rich W/Mo-Doped V2O5 Microspheres as a Catalytic Host To Boost Sulfur Redox Kinetics for Lithium-Sulfur Batteries.

It is very important to develop ideal electrocatalysts to accelerate the sulfur redox kinetics in both the discharging and charging processes for high-performance lithium-sulfur batteries. Herein, defect-rich cation-doped V2O5 yolk-shell microspheres are reported as a catalytic host of sulfur. The doping of W or Mo cations induces no impurities, broadens the lattice spacing of V2O5, and enriches the oxygen vacancy defects. Thus, the doped V2O5 host affords sufficient active sites for chemically anchoring polysulfides and promising catalytic effect on the mutual conversion between different sulfur intermediates. As a result, the S/W-V2O5 cathode delivers a discharging capacity of 1143.3 mA g-1 at an initial rate of 0.3 C and 681.8 mA g-1 at 5 C. Even under a sulfur loading of up to 5.5 mg cm-2 and a minimal electrolyte/sulfur ratio of 6 µL mg-1, the S/W-V2O5 cathode could still achieve good sulfur utilization and dependable cycle stability. Thus, this work offers an electrocatalytic host based on the cation doping strategy to greatly enhance the sulfur redox kinetics for high-performance Li-S batteries.

Circulating expression and clinical significance of LncRNA ANRIL in diabetic kidney disease.

BACKGROUND: Long noncoding RNA ANRIL has been found to be involved in the pathogenesis of diabetic kidney disease (DKD) and is expected to be a new target for prevention of DKD. However, the circulating expression and clinical significance of ANRIL in DKD patients is uncertain. This study aims to explore this issue. METHODS: The study consisted of 20 healthy controls, 22 T2DM patients (normalbuminuria) and 66 DKD patients (grouped as follows: microalbuminuria, n = 23; macroalbuminuria, n = 22 and renal dysfunction, n = 21). The expressions of ANRIL in peripheral whole blood of all participants were measured by RT-qPCR. RESULTS: The expression of ANRIL was significantly up-regulated in DKD patients (microalbuminuria, macroalbuminuria and renal dysfunction groups) than that in healthy control group. ANRIL was also over-expressed in macroalbuminuria and renal dysfunction groups in comparison with normalbuminuria group. ANRIL expression was positively correlated with Scr, BUN, CysC, urine ß2-MG and urine α1-MG; while negatively correlated with eGFR in DKD patients. In addition, ANRIL was the risk factor for DKD with OR value of 1.681. The AUC of ANRIL in identifying DKD was 0.922, and the sensitivity and specificity of DKD diagnosis were 83.3% and 90.5%, respectively. CONCLUSION: Our results indicated that highly expressed ANRIL in peripheral blood is associated with progression of DKD. Circulating ANRIL is an independent risk factor of DKD and has a highly predictive value in identifying DKD.

Activation of Autophagy Induces Monocrotaline-Induced Pulmonary Arterial Hypertension by FOXM1-Mediated FAK Phosphorylation.

PURPOSE: It has been shown that activation of autophagy promotes the development of pulmonary arterial hypertension (PAH). Meanwhile, forkhead box M1 (FOXM1) has been found to induce autophagy in several types of cancer. However, it is still unclear whether FOXM1 mediates autophagy activation in PAH, and detailed mechanisms responsible for these processes are indefinite. METHOD: PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-smooth muscle actin (α-SMA) staining, and Ki67 staining were performed to evaluate the development of PAH. The protein levels of FOXM1, phospho-focal adhesion kinase (p-FAK), FAK, and LC3B were determined by immunoblotting or immunohistochemistry. RESULTS: FOXM1 protein level and FAK activity were significantly increased in MCT-induced PAH rats, this was accompanied with the activation of autophagy. Pharmacological inhibition of FOXM1 or FAK suppressed MCT-induced autophagy activation, decreased RVSP, RVHI and %MT in MCT-induced PAH rats, and inhibited the proliferation of pulmonary arterial smooth muscle cells and pulmonary vessel muscularization in MCT-induced PAH rats. CONCLUSION: FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.

Pembrolizumab associated Stevens-Johnson syndrome with porokeratosis in a patient in the setting of primary hepatocellular carcinoma.

Pembrolizumab is a humanised therapeutic antibody against the PD-1 receptor. It has been used in various advanced cancer immunotherapies. Here, we report an extremely rare case of a 32-year-old man who developed Stevens-Johnson syndrome (SJS) with porokeratosis simultaneously during pembrolizumab treatment for primary hepatocellular carcinoma (T3N1M1).

Rac1/ROCK-driven membrane dynamics promote natural killer cell cytotoxicity via granzyme-induced necroptosis.

BACKGROUND: Natural killer (NK) cells play an important role in cancer immunosurveillance and therapy. However, the target selectivity of NK cell activity is still poorly understood. RESULTS: Here, we used live-cell reporters to unravel differential epithelial cancer target killing by primary human NK cells. We found highly variable fractions of killing by distinct NK cell cytotoxic modes that were not determined by NK ligand expression. Rather, epithelial plasma membrane dynamics driven by ROCK-mediated blebs and/or Rac1-mediated lamellipodia promoted necrotic mode in preference to the apoptotic mode of killing. Inhibition of granzyme B and key necroptosis regulators RIP1, RIP3, and MLKL significantly attenuated the necrotic killing, revealing a novel NK cell cytotoxic pathway by granzyme-induced necroptosis that conferred target selectivity. CONCLUSIONS: Our results not only elucidate a new NK cell effector mechanism but also suggest that tissue microenvironment and oncogenic signaling pathways that promote membrane dynamics, e.g., Rac1 and Rho/ROCK, could be exploited to enhance proinflammatory NK cell killing.

Activation of AMPK inhibits Galectin-3-induced pulmonary artery smooth muscle cells proliferation by upregulating hippo signaling effector YAP.

Galectin-3(Gal-3) is an effective regulator in the pathological process of pulmonary arterial hypertension (PAH). However, the detailed mechanisms underlying Gal-3 contribution to PAH are not yet entirely clear. The aim of the present study was to explore these issues. Proliferation of rat pulmonary arterial smooth muscle cells (PASMCs) was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Small interfering RNA (siRNA) was applied to silence the expression of yes-associated protein (YAP) and Forkhead box M1 (FOXM1). The protein expression and phosphorylation were measured by immunoblotting. The subcellular location of YAP was determined using immunoblotting and immunofluorescence. Gal-3-stimulated PASMCs proliferation in a time- and dose-dependent manner, this was accompanied with, YAP upregulation, dephosphorylation, and nucleus translocation. Gal-3 further increased FOXM1 and cyclinD1 expression via YAP activation. Interfering YAP/FOXM1 axis suppressed Gal-3-induced PASMCs proliferation. Activation of AMPK also inhibited Gal-3-triggered cells proliferation by targeting YAP/FOXM1/cyclinD1 pathway. Gal-3 induced PASMCs proliferation by regulating YAP/FOXM1/cyclinD1 signaling cascade, and activation of AMPK targeted on this axis and suppressed Gal-3-stimulated PASMCs proliferation. Our study provides novel therapeutic targets for prevention and treatment of PAH.

DAP1 negatively regulates autophagy induced by cardamonin in SKOV3 cells.

Autophagy is closely related to the formation and development of multiple human tumors including ovarian cancer. As a major regulator of this process, the role of mTOR (mammalian target of rapamycin) has been well proven. Cardamonin, a kind of flavonoid from plants, has effects on induction of autophagy and thus antiproliferation of cancer cells. However, the detailed mechanism remains unclear. DAP1 (death-associated protein 1) is a proline-rich protein, which is involved in the regulation of cellular growth and programmed cell death including autophagy and apoptosis. The aim of this study was to investigate whether DAP1 is involved in proliferation inhibition and autophagy induced by cardamonin in tumor cells. Using online bioinformatics tools, we found that DAP1 expression is closely related to the survival of patients with ovarian cancer. Our study showed that autophagy induced by cardamonin was associated with mTOR inhibition, and DAP1 was involved in this process. Silence of DAP1 decreased cell proliferation but enhanced the antiproliferative effect of cardamonin in SKOV3 cells. The level of autophagy was elevated by DAP1 silencing in SKOV3 cells. Notably, cardamonin showed higher autophagy flux in the DAP1 small interfering RNA group. Taken together, our results implied that DAP1 negatively regulates autophagy induced by cardamonin, and it may be a potential target for ovarian cancer therapy.

Requirement of Rab21 in LPS-induced TLR4 signaling and pro-inflammatory responses in macrophages and monocytes.

Lipopolysaccharide (LPS) induces macrophage/monocyte activation and pro-inflammatory cytokines production by activating Toll-like receptor 4 (TLR-4) signaling. Rab GTPase 21 (Rab21) is a member of the Rab GTPase subfamily. In the present study, we show that LPS induced TLR4 and Rab21 association and endosomal translocation in murine bone marrow-derived macrophages (BMDMs) and primary human peripheral blood mononuclear cells (PBMCs). In BMDMs, shRNA-mediated stable knockdown of Rab21 inhibited LPS-induced expression and production of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α). Conversely, forced overexpression of Rab21 by an adenovirus construct potentiated LPS-induced IL-1ß, IL-6 and TNF-α production in BMDMs. Further studies show that LPS-induced TLR4 endosomal traffic and downstream c-Jun and NFκB (nuclear factor-kappa B) activation were significantly inhibited by Rab21 shRNA, but intensified with Rab21 overexpression in BMDMs. Finally, in the primary human PBMCs, siRNA-induced knockdown of Rab21 significantly inhibited LPS-induced IL-1ß, IL-6 and TNF-α production. Taken together, we suggest that Rab21 regulates LPS-induced pro-inflammatory responses by promoting TLR4 endosomal traffic and downstream signaling activation.

S1P induces pulmonary artery smooth muscle cell proliferation by activating calcineurin/NFAT/OPN signaling pathway.

The upregulation of osteopontin(OPN) has been found to contribute to the proliferation of pulmonary artery smooth muscle cells(PASMCs), and activation of PPARγ has been shown to suppress OPN expression in THP-1 cells. However, the molecular mechanisms underlying the upregulation of OPN expression and PPARγ agonist modulation of OPN expression in PASMCs remain largely unclear. Here we found that S1P stimulated PASMCs proliferation and up-regulated OPN expression in rat PASMCs, which was accompanied with the activation of phospholipase C(PLC), calcineurin and translocation of NFATc3 to nucleus. Further study showed that inhibition of PLC by U73122, suppression of calcineurin activity by cyclosporine A(CsA) or knockdown of NFATc3 using small interfering RNA suppressed S1P-induced OPN up-regulation. Activation of PPARγ by pioglitazone suppressed S1P-induced activation of calcineurin/NFATc3 signaling pathway and followed OPN up-regulation. Taken together, our study indicates that S1P stimulates OPN expression by activation of PLC/calcineurin/NFATc3 signaling pathway, and activation of PPARγ suppresses calcineurin/NFATc3-mediated OPN expression in PASMCs.

COP9 signalosome subunit 6 mediates PDGF -induced pulmonary arterial smooth muscle cells proliferation.

Up-regulation of mammalian COP9 signalosome subunit 6 (CSN6) and consequent reduction of SCF ubiquitin ligase substrate receptor ß-transduction repeat-containing protein (ß-TrCP) have been shown to be associated with cancer cells proliferation. However, it is unclear whether CSN6 and ß-TrCP are also involved in PDGF-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation. This study aims to address this issue and further explore its potential mechanisms. Our results indicated that PDGF phosphorylated Akt, stimulated PASMCs proliferation; while inhibition of PDGF receptor (PDGFR) by imatinib prevented these effects. PDGF further up-regulated CSN6 protein expression, this was accompanied with ß-TrCP reduction and increase of Cdc25A. Inhibition of PDGFR/PI3K/Akt signaling pathway reversed PDGF-induced such changes and cell proliferation. Prior transfection of CSN6 siRNA blocked PDGF-induced ß-TrCP down-regulation, Cdc25A up-regulation and cell proliferation. Furthermore, pre-treatment of cells with MG-132 also abolished PDGF-induced ß-TrCP reduction, Cdc25A elevation and cell proliferation. In addition, pre-depletion of Cdc25A by siRNA transfection suppressed PDGF-induced PASMCs proliferation. Taken together, our study indicates that up-regulation of CSN6 by PDGFR/PI3K/Akt signaling pathway decreases ß-TrCP by increasing its ubiquitinated degradation, and thereby increases the expression of Cdc25A, which promotes PDGF-induced PASMCs proliferation.

Activation of peroxisome proliferation-activated receptor-γ inhibits transforming growth factor-ß1-induced airway smooth muscle cell proliferation by suppressing Smad-miR-21 signaling.

The aims of the current study were to examine the signaling mechanisms for transforming growth factor-ß1 (TGF-ß1)-induced rat airway smooth muscle cell (ASMC) proliferation and to determine the effect of activation of peroxisome proliferation-activated receptor-γ (PPAR-γ) on TGF-ß1-induced rat ASMC proliferation and its underlying mechanisms. TGF-ß1 upregulated microRNA 21 (miR-21) expression by activating Smad2/3, and this in turn downregulated forkhead box O1 (FOXO1) mRNA expression. In addition, TGF-ß1-Smad-miR-21 signaling also downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and thus de-repressed the PI3K-Akt pathway. Depletion of PTEN reduced the nuclear FOXO1 protein level without affecting its mRNA level. Inhibition of the PI3K-Akt pathway or proteasome function reversed PTEN knockdown-induced nuclear FOXO1 protein reduction. Our study further showed that loss of FOXO1 increased cyclin D1 expression, leading to rat ASMC proliferation. Preincubation of rat ASMCs with pioglitazone, a PPAR-γ activator, blocked TGF-ß1-induced activation of Smad2/3 and its downstream targets changes of miR-21, PTEN, Akt, FOXO1, and cyclin D1, resulting in the inhibition of rat ASMC proliferation. Our study suggests that the activation of PPAR-γ inhibits rat ASMC proliferation by suppressing Smad-miR-21 signaling and therefore has a potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.

Sphingosine-1-phosphate induces airway smooth muscle cell proliferation, migration, and contraction by modulating Hippo signaling effector YAP.

Sphingosine-1-phosphate (S1P), a bioactive lipid, has been shown to be elevated in the airways of individuals with asthma and modulates the airway smooth muscle cell (ASMC) functions, yet its underlying molecular mechanisms are not completely understood. The aim of the present study is to address this issue. S1P induced yes-associated protein (YAP) dephosphorylation and nuclear localization via the S1PR2/3/Rho-associated protein kinase (ROCK) pathway, and this in turn increased forkhead box M1 (FOXM1) and cyclin D1 expression leading to ASMC proliferation, migration, and contraction. Pretreatment of cells with S1PR2 antagonist JTE013, S1PR3 antagonist CAY10444, or ROCK inhibitor Y27632 blocked S1P-induced alterations of YAP, FOXM1, cyclin D1, and ASMC proliferation, migration, and contraction. In addition, prior silencing of YAP or FOXM1 with siRNA reversed the effect of S1P on ASMC functions. Taken together, our study indicates that S1P stimulates ASMC proliferation, migration, and contraction by binding to S1PR2/3 and modulating ROCK/YAP/FOXM1 axis and suggests that targeting this pathway might have potential value in the management of asthma.

SphK1/S1P Mediates PDGF-Induced Pulmonary Arterial Smooth Muscle Cell Proliferation via miR-21/BMPRII/Id1 Signaling Pathway.

BACKGROUND/AIMS: The underlying molecular mechanisms involved in sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) mediation of platelet-derived growth factor (PDGF)-induced pulmonary arterial smooth muscle cell (PASMC) proliferation are still unclear, and the present study aims to address this issue. METHODS: Small interfering RNA (siRNA) and microRNA inhibitor transfection was performed to block the expression of SphK1, bone morphogenetic protein receptor II (BMPRII) and microRNA-21 (miR-21). Gene expression levels of SphK1, BMPRII and inhibitor of DNA binding 1 (Id1) were detected by immunoblotting, miR-21 expression level was examined with qRT-PCR, and S1P production was measured by ELISA. Additionally, PASMC proliferation was determined by BrdU incorporation assay. RESULTS: Our results indicated that PDGF increased the expression of SphK1 protein and S1P production, up-regulated miR-21 expression, reduced BMPRII and Id1 expression, and promoted PASMCs proliferation. Pre-silencing of SphK1 with siRNA reversed PDGF-induced S1P production, miR-21 up-regulation, BMPRII and Id1 down-regulation, as well as PASMC proliferation. Pre-inhibition of miR-21 also blocked BMPRII and Id1 down-regulation as well as PASMC proliferation caused by PDGF. Knockdown of BMPRII down-regulated Id1 expression in PASMCs. We further found that inhibition of PI3K/Akt and ERK signaling pathways, particularly ERK cascade, suppressed PDGF-induced above changes. CONCLUSION: Our study indicates that SphK1/S1P pathway plays an important role in PDGF-induced PASMC proliferation via miR-21/BMPRII/Id1 axis and targeting against SphK1/S1P axis might be a novel strategy in the prevention and treatment of pulmonary arterial hypertension (PAH).

Anti-inflammatory Effects of Cardamonin in Ovarian Cancer Cells Are Mediated via mTOR Suppression.

Cardamonin exhibits a variety of pharmacological activities including anti-inflammatory and antitumor, which are correlated with the inhibition of nuclear factor-kappaB and the mammalian target of rapamycin, respectively. However, whether the anti-inflammatory effects of cardamonin are mediated by the mammalian target of rapamycin remains unknown. In this study, ovarian cancer SKOV3 cells were cultured with lipopolysaccharide to induce inflammation, and the inhibitory effects and underlying molecular mechanisms of cardamonin were investigated using specific inhibitors of the mammalian target of rapamycin and the nuclear factor-kappaB pathway (rapamycin and pyrrolidine dithiocarbamate, respectively). Our results indicated that cardamonin inhibited the viability of normal and lipopolysaccharide-pretreated SKOV3 cells in a concentration-dependent manner. In accordance with rapamycin, the activation of the mammalian target of rapamycin and its downstream target, ribosomal protein S6 kinase 1, was inhibited by cardamonin, while pyrrolidine dithiocarbamate substantially blocked nuclear factor-kappaB activation and mildly inhibited the phosphorylation of the mammalian target of rapamycin and ribosomal protein S6 kinase 1. Pretreated with pyrrolidine dithiocarbamate, the effect of cardamonin on the mammalian target of rapamycin signalling was not affected, but the expression of inflammatory factors was further reduced. In cells pretreated with rapamycin, the inhibitory effects of cardamonin were completely suppressed with regards to the phosphorylation of the mammalian target of rapamycin, ribosomal protein S6 kinase 1, TNF-α, and interleukin-6, and nuclear factor-kappaB p65 protein expression was decreased. In conclusion, our findings indicate that the anti-inflammatory effects of cardamonin are correlated with mammalian target of rapamycin inhibition.

Glycolysis inhibition via mTOR suppression is a key step in cardamonin-induced autophagy in SKOV3 cells.

BACKGROUND: Autophagy occurs in cells that undergoing nutrient deprivation. Glycolysis rapidly supplies energy for the proliferation of cancer cells. Cardamonin inhibits proliferation and enhances autophagy by mTORC1 suppression in ovarian cancer cells. Here, we investigate the relationship between cardamonin-triggered autophagy and glycolysis inhibition via mTORC1 suppression. METHODS: Treated with indicated compounds, ATP content and the activity of hexokinase (HK) and lactate dehydrogenase (LDH) were analyzed by the assay kits. Autophagy was detected by monodansylcadaverin (MDC) staining. The relationship between cardamonin-triggered autophagy and glycolysis inhibition via mTORC1 suppression was analyzed by Western blot. RESULTS: We found that cardamonin inhibited the lactate secretion, ATP production, and the activity of HK and LDH. The results demonstrated that cardamonin enhanced autophagy in SKOV3 cells, as indicated by acidic compartments accumulation, microtubule-associated protein 1 Light Chain 3-II (LC3-II) and lysosome associated membrane protein 1 up-regulation. Our results showed that the activation of mTORC1 signaling and the expression HK2 were reduced by cardamonin; whereas the phosphorylation of AMPK (AMP-activated protein kinase) was increased. We also confirmed that the AMPK inhibitor, Compound C, reversed cardamonin-induced upregulation of LC3-II. CONCLUSION: These results suggest that cardamonin-induced autophagy is associated with inhibition on glycolysis by down-regulating the activity of mTORC1 in ovarian cancer cells.