Collagen/β1 integrin signaling up-regulates the ABCC1/MRP-1 transporter in an ERK/MAPK-dependent manner

The mechanisms by which β1 integrins regulate chemoresistance of cancer cells are still poorly understood. In this study, we report that collagen/β1 integrin signaling inhibits doxorubicin-induced apoptosis of Jurkat and HSB2 leukemic T-cells by up-regulating the expression and function of the ATP-binding cassette C 1 (ABCC1) transporter, also known as multidrug resistance-associated protein 1. We find that collagen but not fibronectin reduces intracellular doxorubicin content and up-regulates the expression levels of ABCC1. Inhibition and knockdown studies show that up-regulation of ABCC1 is necessary for collagen-mediated reduction of intracellular doxorubicin content and collagen-mediated inhibition of doxorubicin-induced apoptosis. We also demonstrate that activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway is involved in collagen-induced reduction of intracellular doxorubicin accumulation, collagen-induced up-regulation of ABCC1 expression levels, and collagen-mediated cell survival. Finally, collagen-mediated up-regulation of ABCC1 expression and function also requires actin polymerization. Taken together, our results indicate for the first time that collagen/β1 integrin/ERK signaling up-regulates the expression and function of ABCC1 and suggest that its activation could represent an important pathway in cancer chemoresistance. Thus simultaneous targeting of collagen/β1 integrin and ABCC1 may be more efficient in preventing drug resistance than targeting each pathway alone.     

Role of CSN5/JAB1 in Wnt/β-catenin activation in colorectal cancer cells

CSN5/JAB1 is a critical subunit of the COP9 signalosome (CSN) and is overexpressed in many human cancers, but little is known about the role of CSN5 in colorectal cancer (CRC). To explore the functional role of CSN5 in colorectal tumorigenesis, we applied siRNA technology to silence CSN5 in HeLa, SW480, HCT116, HT29, and CaCo2 cells. CSN5 knock-down led to reduced β-catenin and phospho-bcatenin levels and this was paralleled by reduced CRC cell proliferation and reduced apoptosis rates, whereas the short-term β-catenin protein stability was enhanced by CSN5 knock-down in SW480 cells. Together, these data implicate the CSN in the pathogenesis of CRC via regulation of the Wnt/β-catenin pathway     

Enterovirus 71 induces integrin β1/EGFR-Rac1-dependent oxidative stress in SK-N-SH cells: role of HO-1/CO in viral replication

Oxidative stress became emerged as a key player in the development and progression of many pathological conditions including virus-induced encephalitis. Heme oxygenase-1 (HO-1) plays a crucial role in defending the body against oxidant-induced injury during inflammatory processes. Therefore, we investigated the induction of HO-1 level in host cells, which may exert a beneficial effect to minimize viral replication in SK-N-SH cells. In this study, we found that enterovirus 71 (EV71) induced the generation of reactive oxygen species (ROS) and activation of NADPH oxidase. EV71-induced ROS generation was mediated through activation of integrin β1, an epidermal growth factor receptor (EGFR), Rac1 and NADPH oxidase which revealed by using selective pharmacological inhibitors or transfection with respective siRNAs. In addition, the reduction of viral load was observed with NADPH oxidase inhibitors (apocynin and diphenyleneiodonium chloride), ROS scavenger (N-acetylcysteine), and transfection with p47(phox) siRNA in Western blot and real-time PCR analyses. Consistently, overexpression of HO-1 attenuated EV71-induced NADPH oxidase/ROS generation and EV71 replication which were abrogated by pretreatment with an HO-1 inhibitor, zinc protoporphyrin IX (ZnPP IX). Moreover, metabolite of HO-1, carbon monoxide (CO), also diminished ROS formation and EV71 replication which were reversed by pretreatment with a CO scavenger (hemoglobin) and a cyclic GMP-dependent protein kinase (PKG) inhibitor (KT5823). These findings suggest that up-regulation of HO-1 exerts as a host cellular defense mechanism against EV71 infection in SK-N-SH cells.     

A mitotic role for BRCA1/BARD1 in tumor suppression?

The tumor-suppressor protein BRCA1 is thought to act by preserving genomic integrity. In this issue of Cell, Joukov et al. demonstrate that the BRCA1/BARD1 heterodimer participates in mitotic spindle assembly, a process conducted by the GTPase Ran. Loss of this mitotic function might contribute to tumorigenesis.     

The Netrin-4/Laminin γ1/Neogenin-1 complex mediates migration in SK-N-SH neuroblastoma cells

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It arises during development of the sympathetic nervous system. Netrin-4 (NTN4), a laminin-related protein, has been proposed as a key factor to target NB metastasis, although there is controversy about its function. Here, we show that NTN4 is broadly expressed in tumor, stroma and blood vessels of NB patient samples. Furthermore, NTN4 was shown to act as a cell adhesion molecule required for the migration induced by Neogenin-1 (NEO1) in SK-N-SH neuroblastoma cells. Therefore, we propose that NTN4, by forming a ternary complex with Laminin γ1 (LMγ1) and NEO1, acts as an essential extracellular matrix component, which induces the migration of SK-N-SH cells.     

Kinetics of β-phase formation in 1-O-alkylglycerols

The rate of β-phase formation in the ether lipids 1-O-alkylglycerols have been investigated at various temperatures. The concentrations of the phases vs. time in 1-O-hexadecylglycerol (C₁₆G) were measured using automatic X-ray powder diffraction peak area measurements. In 1-O-decylglycerol (C₁₆G) the rate was estimated using the heat evolved during the transition. At least two factors are important for the low transition rate. At higher temperatures the rate appears to be limited by a low probability of β crystallite formation (nucleation). As the temperature is decreased, crystallite formation probably increases. A second factor involves an activation of the metastable lattice. The activation process results in a lower rate at decreased temperatures. The two factors together give the highest transition rate at the α ? sub-α-phase transition temperature.     

ADD1/SREBP1c activates the PGC1-α promoter in brown adipocytes

Cold adaptation elicits a paradoxical simultaneous induction of fatty acid synthesis and β-oxidation in brown adipose tissue. We show here that cold exposure coordinately induced liver X receptor α (LXRα), adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein-1c (SREBP1c) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) in brown and inguinal white adipose tissues, but not in epididymal white adipose tissue. Using in vitro models of white and brown adipocytes we demonstrate that β-adrenergic stimulation induced expression of LXRα, ADD1/SREBP1c and PGC1α in cells with a brown-like adipose phenotype. We demonstrate that ADD1/SREBP1c is a powerful inducer of PGC1α expression via a conserved E box in the proximal promoter and that β-adrenergic stimulation led to recruitment of ADD1/SREBP1c to this E box. The ability of ADD1/SREBP1c to activate the PGC1α promoter exhibited a striking cell type dependency, suggesting that additional cell type-restricted factors contribute to ADD1/SREBP1c-mediated activation. In conclusion, our data demonstrate a novel role of ADD1/SREBP1c as a regulator of PGC1α expression in brown adipose tissue.     

Ypt1/Rab1 regulates Hrr25/CK1δ kinase activity in ER–Golgi traffic and macroautophagy

ER-derived COPII-coated vesicles are conventionally targeted to the Golgi. However, during cell stress these vesicles also become a membrane source for autophagosomes, distinct organelles that target cellular components for degradation. How the itinerary of COPII vesicles is coordinated on these pathways remains unknown. Phosphorylation of the COPII coat by casein kinase 1 (CK1), Hrr25, contributes to the directional delivery of ER-derived vesicles to the Golgi. CK1 family members are thought to be constitutively active kinases that are regulated through their subcellular localization. Instead, we show here that the Rab GTPase Ypt1/Rab1 binds and activates Hrr25/CK1δ to spatially regulate its kinase activity. Consistent with a role for COPII vesicles and Hrr25 in membrane traffic and autophagosome biogenesis, hrr25 mutants were defective in ER–Golgi traffic and macroautophagy. These studies are likely to serve as a paradigm for how CK1 kinases act in membrane traffic.     

Lipoteichoic acid restrains macrophage senescence via β-catenin/FOXO1/REDD1 pathway in age-related osteoporosis

Osteoporosis and its related fractures are common causes of morbidity and mortality in older adults, but its underlying molecular and cellular mechanisms remain largely unknown. In this study, we found that lipoteichoic acid (LTA) treatment could ameliorate age-related bone degeneration and attenuate intramedullary macrophage senescence. FOXO1 signaling, which was downregulated and deactivated in aging macrophages, played a key role in the process. Blocking FOXO1 signaling caused decreased REDD1 expression and increased phosphorylation level of mTOR, a major driver of aging, as well as aggravated bone loss and deteriorated macrophage senescence. Moreover, LTA elevated FOXO1 signaling through β-catenin pathway while β-catenin inhibition significantly suppressed FOXO1 signaling, promoted senescence-related protein expression, and accelerated bone degeneration and macrophage senescence. Our findings indicated that β-catenin/FOXO1/REDD1 signaling plays a physiologically significant role that protecting macrophages from senescence during aging.     

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2 in mice

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor α (TNFα), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ET(A)/ET(B) receptor antagonist bosentan, and selective ET(A) or ET(B) receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFα and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c(+) markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ET(A)- and ET(B)-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2-dependent mechanism.     

Effects of daintain/AIF-1 on β cell dysfunction in INS-1 cells

We investigated the effects of daintain/AIF-1, a novel inflammatory cytokine, on INS-1β cells. Cells incubated with daintain/AIF-1 showed decreased cell viability and glucose-stimulated insulin secretion, as well as upregulated apoptosis and NO production. These deleterious effects of daintain/AIF-1 indicate that daintain/AIF-1 plays important roles in the dysfunction of pancreatic β cells in type-1 diabetes.     

IL-1α/IL-1R1 expression in chronic obstructive pulmonary disease and mechanistic relevance to smoke-induced neutrophilia in mice

Background

Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. Despite this, the cellular and molecular mechanisms that contribute to COPD pathogenesis are still poorly understood.

Methodology and Principal Findings

The objective of this study was to assess IL-1 α and β expression in COPD patients and to investigate their respective roles in perpetuating cigarette smoke-induced inflammation. Functional studies were pursued in smoke-exposed mice using gene-deficient animals, as well as blocking antibodies for IL-1α and β. Here, we demonstrate an underappreciated role for IL-1α expression in COPD. While a strong correlation existed between IL-1α and β levels in patients during stable disease and periods of exacerbation, neutrophilic inflammation was shown to be IL-1α-dependent, and IL-1β- and caspase-1-independent in a murine model of cigarette smoke exposure. As IL-1α was predominantly expressed by hematopoietic cells in COPD patients and in mice exposed to cigarette smoke, studies pursued in bone marrow chimeric mice demonstrated that the crosstalk between IL-1α+ hematopoietic cells and the IL-1R1+ epithelial cells regulates smoke-induced inflammation. IL-1α/IL-1R1-dependent activation of the airway epithelium also led to exacerbated inflammatory responses in H1N1 influenza virus infected smoke-exposed mice, a previously reported model of COPD exacerbation.

Conclusions and Significance

This study provides compelling evidence that IL-1α is central to the initiation of smoke-induced neutrophilic inflammation and suggests that IL-1α/IL-1R1 targeted therapies may be relevant for limiting inflammation and exacerbations in COPD.     

Critical Role of S1PR1 and Integrin β4 in HGF/c-Met-mediated Increases in Vascular Integrity

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met (1). We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.     

Effects of Daintain/AIF-1 on β Cell Dysfunction in INS-1 Cells

We investigated the effects of daintain/AIF-1, a novel inflammatory cytokine, on INS-1β cells. Cells incubated with daintain/AIF-1 showed decreased cell viability and glucose-stimulated insulin secretion, as well as upregulated apoptosis and NO production. These deleterious effects of daintain/AIF-1 indicate that daintain/AIF-1 plays important roles in the dysfunction of pancreatic β cells in type-1 diabetes.     

DGKζ is involved in LPS-activated phagocytosis through IQGAP1/Rac1 pathway

Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ(-/-) mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.     

OxLDL up-regulates Niemann-Pick type C1 expression through ERK1/2/COX-2/PPARα-signaling pathway in macrophages

The Niemann-Pick type C1 (NPC1) is located mainly in the membranes of the late endosome/lysosome and controls the intracellular cholesterol trafficking from the late endosome/lysosome to the plasma membrane. It has been reported that oxidized low-density lipoprotein (oxLDL) can up-regulate NPC1 expression. However, the detailed mechanisms are not fully understood. In this study, we investigated the effect of oxLDL stimulation on NPC1 expression in THP-1 macrophages. Our results showed that oxLDL up-regulated NPC1 expression at both mRNA and protein levels in a dose-dependent and time-dependent manner. In addition, oxLDL also induced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Treatment with oxLDL significantly increased cyclooxygenase-2 (COX-2) mRNA and protein expression in the macrophages, and these increases were suppressed by the ERK1/2 inhibitor PD98059 or ERK1/2 small interfering RNA (siRNA) treatment. OxLDL up-regulated the expression of peroxisome proliferator-activated receptor α (PPARα) at the mRNA and protein levels, which could be abolished by COX-2 siRNA or COX-2 inhibitor NS398 treatment in these macrophages. OxLDL dramatically elevated cellular cholesterol efflux, which was abrogated by inhibiting ERK1/2 and/or COX-2. In addition, oxLDL-induced NPC1 expression and cellular cholesterol efflux were reversed by PPARα siRNA or GW6471, an antagonist of PPARα. Taken together, these results provide the evidence that oxLDL can up-regulate the expression of the NPC1 through ERK1/2/COX-2/PPARα-signaling pathway in macrophages.     

Reversal effects of pantoprazole on multidrug resistance in human gastric adenocarcinoma cells by down-regulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway in vitro and in vivo

To investigate reversal effects of pantoprazole (PPZ) on multidrug resistance (MDR) in human gastric adenocarcinoma cells in vivo and in vitro. Human gastric adenocarcinoma cell SGC7901 was cultured in RPMI‐1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5% CO₂ atmosphere at 37°C. Adriamycin (ADR)‐resistant cells were cultured with addition of 0.8 µg/ml of ADR maintaining MDR phenotype. ADR was used to calculate ADR releasing index; CCK‐8 Assay was performed to evaluate the cytotoxicity of anti‐tumor drugs; BCECF‐AM pH‐sensitive fluorescent probe was used to measure intracellular pH (pHi) value of cells, whereas pH value of medium was considered as extracellular pH (pHe) value; Western blotting and immunofluorescent staining analyses were employed to determine protein expressions and intracellular distributions of vacuolar H⁺‐ATPases (V‐ATPases), mTOR, HIF‐1α, P‐glycoprotein (P‐gp), and multidrug resistant protein 1 (MRP1); SGC7901 and SGC7901/ADR cells were inoculated in athymic nude mice. Thereafter, effects of ADR with or without PPZ pretreatment were compared by determining the tumor size and weight, apoptotic cells in tumor tissues were detected by TUNEL assay. At concentrations greater than 20 µg/ml, PPZ pretreatment reduced ADR releasing index and significantly enhanced intracellular ADR concentration of SGC7901 (P < 0.01). Similarly, PPZ pretreatment significantly decreased ADR releasing index of SGC7901/ADR dose‐dependently (P < 0.01). PPZ pretreatment also decreased cell viabilities of SGG7901 and SGC7901/ADR dose‐dependently. After 24‐h PPZ pretreatment, administration of chemotherapeutic agents demonstrated maximal cytotoxic effects on SGC7901 and SGC7901/ADR cells (P < 0.05). The resistance index in PPZ pretreatment group was significantly lower than that in non‐PPZ pretreatment group (3.71 vs. 14.80). PPZ at concentration >10 µg/ml significantly decreased pHi in SGC7901 and SGC7901/ADR cells and diminished or reversed transmembrane pH gradient (P < 0.05). PPZ pretreatment also significantly inhibited protein expressions of V‐ATPases, mTOR, HIF‐1α, P‐gp, and MRP1, and alter intracellular expressions in parent and ADR‐resistant cells (P < 0.05). In vivo experiments further confirmed that PPZ pretreatment could enhance anti‐tumor effects of ADR on xenografted tumor of nude mice and also improve the apoptotic index in xenografted tumor tissues. PPZ pretreatment enhances the cytotoxic effects of anti‐tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR by downregulating the V‐ATPases/mTOR/HIF‐1α/P‐gp and MRP1 signaling pathway. J. Cell. Biochem. 113: 2474–2487, 2012. © 2012 Wiley Periodicals, Inc.