Metazoan stress granule assembly is mediated by P-eIF2α-dependent and -independent mechanisms

Stress granules (SGs) are cytoplasmic bodies wherein translationally silenced mRNAs are recruited for triage in response to environmental stress. We report that Drosophila cells form SGs in response to arsenite and heat shock. Drosophila SGs, like mammalian SGs, are distinct from but adjacent to processing bodies (PBs, sites of mRNA silencing and decay), require polysome disassembly, and are in dynamic equilibrium with polysomes. We further examine the role of the two Drosophila eIF2α kinases, PEK and GCN2, in regulating SG formation in response to heat and arsenite stress. While arsenite-induced SGs are dependent upon eIF2α phosphorylation, primarily via PEK, heat-induced SGs are phospho-eIF2α-independent. In contrast, heat-induced SGs require eIF2α phosphorylation in mammalian cells, as non-phosphorylatable eIF2α Ser51Ala mutant murine embryonic fibroblasts do not form SGs even after severe heat shock. These results suggest that mammals evolved alternative mechanisms for dealing with thermal stress.     

NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.     

PPARγ-dependent pathway in the growth-inhibitory effects of K562 cells by carotenoids in combination with rosiglitazone

Background

Carotenoids have been found to play roles in the prevention and therapy of some cancers which PPARγ was also discovered to be involved in. The present studies were directed to determine the inhibitory effects of carotenoids in combination with rosiglitazone, a synthetic PPARγ agonist, on K562 cell proliferation and elucidate the contribution of PPARγ-dependent pathway to cell proliferation suppression.

Methods

The effects of carotenoid and rosiglitazone combination on K562 cell proliferation were evaluated by trypan blue dye exclusion assay and MTT assay. When PPARγ has been inhibited by GW9662 and siRNA, cycle-related regulator expression in K562 cells treated with carotenoid and rosiglitazone combination was analyzed by Western blotting.

Results

Rosiglitazone inhibited K562 cell proliferation and augmented the inhibitory effects of carotenoids on the cell proliferation greatly. Specific PPARγ inhibition attenuated the cell growth suppression induced by carotenoid and rosiglitazone combination. GW9662 pre-treatment attenuated the enhanced up-regulation of PPARγ expression caused by the combination treatment. Moreover, GW9662 and PPARγ siRNA also significantly attenuated the up-regulation of p21 and down-regulation of cyclin D1 caused by carotenoids and rosiglitazone.

Conclusions

PPARγ signaling pathway, via stimulating p21 and inhibiting cyclin D1, may play an important role in the anti-proliferative effects of carotenoid and rosiglitazone combination on K562 cells.

General significance

Carotenoids in combination with rosiglitazone are hopeful to provide attractive dietary or supplementation-based and pharmaceutical strategies to treat cancer diseases.     

α-Galactosylceramide-induced airway eosinophilia is mediated through the activation of NKT cells

Invariant NKT (iNKT) cells bridge innate and adaptive immune responses, resulting in the expansion of Ag-specific B and T cell responses. α-Galactosylceramide (α-GalCer), the most studied glycolipid that activates iNKT cells, has been proposed to be an effective adjuvant against infections and tumors. We found that the activation of iNKT cells by intranasal injection of α-GalCer induced airway eosinophilia in naive mice. Eosinophils, which mediate tissue damage and dysfunction by secreting mediators, play important roles in the pathogenesis of allergic diseases. In this study, we investigated the mechanism of how eosinophils are recruited to the lung by α-GalCer. Our results demonstrated that α-GalCer-induced eosinophil inflammation was mediated through iNKT cells. These cells secreted IL-5 to recruit eosinophils directly to the lung and/or secreted IL-4 and IL-13 to recruit eosinophils indirectly by inducing lung epithelial cells, endothelial cells, and fibroblast to secrete the eosinophil chemoattractant eotaxin. In addition, in the OVA-alum murine model of allergic asthma, α-GalCer administration in OVA-immunized mice also increased airway eosinophilia after challenge. Given our findings, intranasal administration of α-GalCer induced airway eosinophilic inflammation in both naive and allergic mice. Hence, it remains to be determined whether the activation of iNKT cells would be applicable in therapeutics for human diseases.     

Synthetic lethality of PARP inhibition in BRCA-network disrupted tumor cells is associated with interferon pathway activation and enhanced by interferon-γ

Tumor suppressor genes BRCA1 and BRCA2 function in a complex gene network that regulates homologous recombination and DNA double-strand break repair. Disruption of the BRCA-network through gene mutation, deletion, or RNAi-mediated silencing can sensitize cells to small molecule inhibitors of poly (ADP-ribose) polymerase (PARPi). Here, we demonstrate that BRCA-network disruption in the presence of PARPi leads to the selective induction and enhancement of interferon pathway and apoptotic gene expression in cultured tumor cells. In addition, we report PARPi cytotoxicity in BRCA1-deficient tumor cells is enhanced >10-fold when combined with interferon-γ. These findings establish a link between synthetic lethality of PARPi in BRCA-network disrupted cells and interferon pathway activation triggered by genetic instability.     

Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway

Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-β1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-β1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-β1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.     

Cigarette smoke-induced accumulation of lung dendritic cells is interleukin-1α-dependent in mice

Background

Evidence suggests that dendritic cells accumulate in the lungs of COPD patients and correlate with disease severity. We investigated the importance of IL-1R1 and its ligands IL-1α and β to dendritic cell accumulation and maturation in response to cigarette smoke exposure.

Methods

Mice were exposed to cigarette smoke using a whole body smoke exposure system. IL-1R1-, TLR4-, and IL-1α-deficient mice, as well as anti-IL-1α and anti-IL-1β blocking antibodies were used to study the importance of IL-1R1 and TLR4 to dendritic cell accumulation and activation.

Results

Acute and chronic cigarette smoke exposure led to increased frequency of lung dendritic cells. Accumulation and activation of dendritic cells was IL-1R1/IL-1α dependent, but TLR4- and IL-1β-independent. Corroborating the cellular data, expression of CCL20, a potent dendritic cells chemoattractant, was IL-1R1/IL-1α-dependent. Studies using IL-1R1 bone marrow-chimeric mice revealed the importance of IL-1R1 signaling on lung structural cells for CCL20 expression. Consistent with the importance of dendritic cells in T cell activation, we observed decreased CD4⁺ and CD8⁺ T cell activation in cigarette smoke-exposed IL-1R1-deficient mice.

Conclusion

Our findings convey the importance of IL-1R1/IL-1α to the recruitment and activation of dendritic cells in response to cigarette smoke exposure.     

Autocrine modulation of glucose transporter SGLT2 by IL-6 and TNF-α in LLC-PK1 cells

We determined in cultured kidney epithelial cells (LLC-PK(1)) the effects of high glucose, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) on mRNA and protein expression of the renal glucose transporters SGLT1 and SGLT2. Cultured monolayers were incubated with similar concentrations of IL-6 and TNF-α to those produced by LLC-PK(1) in the presence of 20 mM glucose. Confluent monolayers with either 5 (controls, C) or 20 mM glucose (high glucose, HG) were incubated in the presence of 5 mM glucose, 20 mM glucose, 10 pg/ml IL-6, or TNF-α alone or in combination. Separate groups with IL-6 and TNF-α were incubated with antibodies to their respective receptors. HG induced an increased SGLT1 mRNA at 48 h (p<0.05 vs. C) and protein expression in 120 h (p<0.05 vs. C). HG also induced an increased SGLT2 mRNA at 72 and 96 h (P<0.05 vs. C) and SGLT2 protein expression at 120 h (p<0.05 vs. C). In C, 10 pg/ml IL-6 or TNF-α did not modify SGLT1 mRNA (n.s vs. in the absence of cytokines). In contrast, cytokines induced an increased expression of SGLT1 protein at 120 h (p<0.05 vs. in the absence of cytokines), and SGLT2 mRNA and protein were increased at 96 and 120 h, respectively (p<0.05 vs. in absence of cytokines). No changes were observed when cells were incubated with cytokines and HG (n.s vs. C). In conclusion, this study showed that SGLT2 increased in the presence of IL-6 and TNF-α, indicating an autocrine modulation of the expression of this transporter by cytokines.     

Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.     

The PGC-1α-dependent pathway of mitochondrial biogenesis is upregulated in type I endometrial cancer

PGC-1α-dependent pathway of mitochondrial biogenesis was investigated for the first time in type I endometrial cancer and in normal endometrium. In cancer endometrial tissue the citrate synthase activity, the mitochondrial DNA content and the TFAM level were found doubled compared to control endometrial tissue. Moreover, a 1.6- and 1.8-fold increase, respectively, of NRF-1 and PGG-1α expression was found. This study demonstrates, for the first time, that the increased mitochondrial biogenesis in type I endometrial cancer is associated to the upregulation of PGC-1α signalling pathway.     

Induction of growth cessation by acacetin via β-catenin pathway and apoptosis by apoptosis inducing factor activation in colorectal carcinoma cells

Molecular Biology Reports - Acacetin, a bioflavanoid, contains anti-inflammatory and anti-cancer activities as shown in different experimental models. However, its anticancer potential and...     

Overexpression of tissue factor pathway inhibitor in CHO-K1 cells results in increased activation of NF-κB and apoptosis mediated by a caspase-3 independent pathway

There is now circumstantial evidence that tissue factor pathway inhibitor (TFPI) is not only a major anticoagulant, but also has proapoptotic properties. The current study was designed to address the role of TFPI on signalling pathways and apoptosis. The non-TFPI expressing cell line CHO-K1 was stably transfected with pcDNA3.1/V5-His-TOPO-TFPI and control cells were established by transfecting the CHO-K1 cells with pcDNA3.1/V5-His-TOPO. Sodium butyrate (NaBut) has been shown to induce the expression of recombinant proteins. Here we have used NaBut to increase the expression of TFPI as assessed by qRT-PCR and ELISA. Compared to the control cells, TFPI induced apoptosis in a concentration dependent manner as measured by a cell death detection assay. Independent of caspase-3 activation an increased cleavage of PARP was detected in the TFPI expressing cells. This was accompanied by downregulation of Bcl-XL, elevated levels of Bax, and increased translocation of the apoptosis initiating factor. Increased DNA binding activity of NF-κB was revealed by electrophoretic mobility shift assay when the TFPI level was elevated by NaBut together with an increased translocation of the NF-κB subunit p65. The results indicate that TFPI affected the apoptotic activity through a process independent of caspase-3, and was also able to increase the activation of the NF- κB pathway.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-α

Cytosolic phospholipase A₂-α (cPLA₂) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA₂ is dependent on a rise in cytosolic Ca²⁺ concentration, membrane association via the Ca²⁺-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA₂ via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP₂), including the role of a “pleckstrin homology (PH)-like” region of cPLA₂ (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate + the Ca²⁺ ionophore, A23187, or epidermal growth factor + A23187, expression of the PH domain of phospholipase C-δ1 (which sequesters membrane PIP₂) attenuated cPLA₂ activity. Stimulated cPLA₂ activity was also attenuated by the expression of cPLA₂ 135-366, or cPLA₂ 2-366, and expression of a PIP₂-specific 5′-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP₂, with high affinity, only cPLA₂ 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA₂s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA₂ activity can be modulated by sequestration or depletion of cellular PIP₂, although the interaction of cPLA₂ with membrane PIP₂ appears to be indirect, or of weak affinity.