Akt-dependent Skp2 mRNA translation is required for exiting contact inhibition, oncogenesis, and adipogenesis

The requirement of Akt for cell proliferation and oncogenesis is mammalian target of rapamycin complex 1 (mTORC1) dependent. SV40 large T expression in Akt-deficient cells restores cell proliferation rate, but is insufficient for exiting contact inhibition and oncogene-induced anchorage-independent growth, because of a failure to promote Skp2 mRNA translation. Skp2 mRNA and protein are induced upon exiting contact inhibition, which enables entry into mitosis. While Skp2 mRNA is induced in Akt-deficient cells, it is not translated, preventing entry into mitosis. Restoring Skp2 expression in Akt-deficient cells is sufficient to restore exit from contact inhibition and oncogenesis. Skp2 mRNA translation is dependent on mTORC1 and the eukaryotic translation initiation factor 4E (eIF4E). Thus, the requirement of Akt for exiting contact inhibition is mediated by the induction of Skp2 mRNA translation in eIF4E-dependent mechanism. These results provide a new insight into the role of the Akt/mTORC1/eIF4E axis in tumourigenesis. Akt-dependent Skp2 mRNA translation is also required for mitotic clonal expansion (MCE)--the earliest event in adipogenesis. Skp2 re-expression in Akt-deficient preadipocytes, which are impaired in adipogenesis, is sufficient to restore adipogenesis. These results uncover the mechanism by which Akt mediates adipogenesis.     

Bronchioalveolar stem cells increase after mesenchymal stromal cell treatment in a mouse model of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) remains a major complication of prematurity resulting in significant morbidity and mortality. The pathology of BPD is multifactorial and leads to alveolar simplification and distal lung injury. Previous studies have shown a beneficial effect of systemic treatment with bone marrow-derived mesenchymal stromal cells (MSCs) and MSC-conditioned media (MSC-CM) leading to amelioration of the lung parenchymal and vascular injury in vivo in the hyperoxia murine model of BPD. It is possible that the beneficial response from the MSCs is at least in part due to activation of endogenous lung epithelial stem cells. Bronchioalveolar stem cells (BASCs) are an adult lung stem cell population capable of self-renewal and differentiation in culture, and BASCs proliferate in response to bronchiolar and alveolar lung injury in vivo. Systemic treatment of neonatal hyperoxia-exposed mice with MSCs or MSC-CM led to a significant increase in BASCs compared with untreated controls. Treatment of BASCs with MSC-CM in culture showed an increase in growth efficiency, indicating a direct effect of MSCs on BASCs. Lineage tracing data in bleomycin-treated adult mice showed that Clara cell secretory protein-expressing cells including BASCs are capable of contributing to alveolar repair after lung injury. MSCs and MSC-derived factors may stimulate BASCs to play a role in the repair of alveolar lung injury found in BPD and in the restoration of distal lung cell epithelia. This work highlights the potential important role of endogenous lung stem cells in the repair of chronic lung diseases.     

Protective effect of curcumin on pulmonary and cardiovascular effects induced by repeated exposure to diesel exhaust particles in mice

Particulate air pollution has been associated with increased risk of cardiopulmonary diseases. However, the underlying mechanisms are not fully understood. We have previously demonstrated that single dose exposure to diesel exhaust particle (DEP) causes lung inflammation and peripheral thrombotic events. Here, we exposed mice with repeated doses of DEP (15 μg/animal) every 2(nd) day for 6 days (a total of 4 exposures), and measured several cardiopulmonary endpoints 48 h after the end of the treatments. Moreover, the potential protective effect of curcumin (the yellow pigment isolated from turmeric) on DEP-induced cardiopulmonary toxicity was assessed. DEP exposure increased macrophage and neutrophil numbers, tumor necrosis factor α (TNF α) in the bronchoalveolar lavage (BAL) fluid, and enhanced airway resistance to methacoline measured invasively using Flexivent. DEP also significantly increased plasma C-reactive protein (CRP) and TNF α concentrations, systolic blood pressure (SBP) as well as the pial arteriolar thrombosis. It also significantly enhanced the plasma D-dimer and plasminogen activator inhibitor-1 (PAI-1). Pretreatment with curcumin by oral gavage (45 mg/kg) 1 h before exposure to DEP significantly prevented the influx of inflammatory cells and the increase of TNF α in BAL, and the increased airway resistance caused by DEP. Likewise, curcumin prevented the increase of SBP, CRP, TNF α, D-dimer and PAI-1. The thrombosis was partially but significantly mitigated. In conclusion, repeated exposure to DEP induced lung and systemic inflammation characterized by TNFα release, increased SBP, and accelerated coagulation. Our findings indicate that curcumin is a potent anti-inflammatory agent that prevents the release of TNFα and protects against the pulmonary and cardiovascular effects of DEP.     

NMR based metabonomics study of NPY Y5 receptor activation in BT-549, a human breast carcinoma cell line

Overexpression of neuropeptide Y (NPY) and its receptors has been found in various cancers. In our previous study, we demonstrated expression of NPY Y5 receptor (Y5R) in various breast cancer cell lines along with Y1 receptor. In Y5R expressing BT-549 cells, NPY induced cell proliferation that was blocked by Y5R-selective antagonist CGP1683A (CGP). Here, NMR-based metabonomics was used to monitor the metabolic profile of BT-549 cells in the presence of NPY and CGP to assess the effect of Y5R activation and inhibition during NPY-induced cell proliferation. To study changes in intra and extra cellular metabolites in response to various treatments, 1D ¹H-NMR spectra of both hydrophilic cell extracts and growth medium were recorded from BT-549 with three treatments: (1) NPY, (2) CGP, and (3) CGP followed by NPY (CGP/NPY). Principal component analysis and statistical significance analysis indicated changes in intracellular concentrations of seven metabolites in hydrophilic cell extracts with NPY treatment: decreases in lactate, succinate, myo-inositol, and creatine, and increases in acetate, glutamate, and aspartate. A significant increase in intracellular lactate level and attenuation of other metabolites to baseline was detected in CGP/NPY group. Also, significant decreases in lactate and increases in pyruvate were observed in growth medium from NPY treated cells. Based on the metabonomics analysis, Y5R activation induces cell proliferation by increasing the rate of glycolysis, glutaminolysis, and TCA cycle. Inhibition of Y5R by CGP counteracts NPY-induced changes in cellular metabolites. These changes may play a role in cell proliferation and migration by NPY through Y5R activation.     

Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans

Autophagy plays key roles both in host defense against bacterial infection and in tumor biology. Helicobacter pylori (H. pylori) infection causes chronic gastritis and is the single most important risk factor for the development of gastric cancer in humans. Its vacuolating cytotoxin (VacA) promotes gastric colonization and is associated with more severe disease. Acute exposure to VacA initially triggers host autophagy to mitigate the effects of the toxin in epithelial cells. Recently, we demonstrated that chronic exposure to VacA leads to the formation of defective autophagosomes that lack CTSD/cathepsin D and have reduced catalytic activity. Disrupted autophagy results in accumulation of reactive oxygen species and SQSTM1/p62 both in vitro and in vivo in biopsy samples from patients infected with VacA (+) but not VacA (-) strains. We also determined that the Crohn disease susceptibility polymorphism in the essential autophagy gene ATG16L1 increases susceptibility to H. pylori infection. Furthermore, peripheral blood monocytes from individuals with the ATG16L1 risk variant show impaired autophagic responses to VacA exposure. This is the first study to identify both a host autophagy susceptibility gene for H. pylori infection and to define the mechanism by which the autophagy pathway is affected following H. pylori infection. Collectively, these findings highlight the synergistic effects of host and bacterial autophagy factors on H. pylori pathogenesis and the potential for subsequent cancer susceptibility.     

Neuraminidase-1 is required for the normal assembly of elastic fibers

The assembly of elastic fibers in tissues that undergo repeated cycles of extension and recoil, such as the lungs and blood vessels, is dependent on the proper interaction and alignment of tropoelastin with a microfibrillar scaffold. Here, we describe in vivo histopathological effects of neuraminidase-1 (Neu1) deficiency on elastin assembly in the lungs and aorta of mice. These mice exhibited a tight-skin phenotype very similar to the Tsk mouse. Normal septation of Neu1-null mice did not occur in neonatal mice, resulting in enlarged alveoli that were maintained in adults. The abnormal development of elastic fibers was remarkable under electron microscopy and confirmed by the overlapping distribution of elastin, fibrillin-1, fibrillin-2, and fibulin-5 (Fib-5) by the light microscopy immunostainings. Fib-5 fibers appeared diffuse and unorganized around the alveolar walls and the apex of developing secondary septal crests. Fibrillin-2 deposition was also abnormal in neonatal and adult lungs. Dispersion of myofibroblasts appeared abnormal in developing lungs of Neu1-null mice, with a random distribution of myofibroblast around the alveolar walls, rather than concentrating at sites of elastin synthesis. The elastic lamellae in the aorta of the Neu1-null mice were thinner and separated by hypertrophic smooth muscle cells that were surrounded by an excess of the sialic acid-containing moieties. The concentration of elastin, as measure by desmosine levels, was significantly reduced in the aorta of Neu1-null mice. Message levels for tropoelastin and Fib-5 were normal, suggesting the elastic fiber defects in Neu1-null mice result from impaired extracellular assembly.     

Overexpression of PPK-1, the Caenorhabditis elegans Type I PIP kinase, inhibits growth cone collapse in the developing nervous system and causes axonal degeneration in adults

Growth cones are dynamic membrane structures that migrate to target tissue by rearranging their cytoskeleton in response to environmental cues. The lipid phosphatidylinositol (4,5) bisphosphate (PIP₂) resides on the plasma membrane of all eukaryotic cells and is thought to be required for actin cytoskeleton rearrangements. Thus PIP₂ is likely to play a role during neuron development, but this has never been tested in vivo. In this study, we have characterized the PIP₂ synthesizing enzyme Type I PIP kinase (ppk-1) in Caenorhabditis elegans. PPK-1 is strongly expressed in the nervous system, and can localize to the plasma membrane. We show that PPK-1 purified from C. elegans can generate PIP₂in vitro and that overexpression of the kinase causes an increase in PIP₂ levels in vivo. In developing neurons, PPK-1 overexpression leads to growth cones that become stalled, produce ectopic membrane projections, and branched axons. Once neurons are established, PPK-1 overexpression results in progressive membrane overgrowth and degeneration during adulthood. These data suggest that overexpression of the Type I PIP kinase inhibits growth cone collapse, and that regulation of PIP₂ levels in established neurons may be important to maintain structural integrity and prevent neuronal degeneration.     

Purification and characterization of phycoferritin from the blue-green alga, Arthrospira (Spirulina) platensis

Phycoferritin from the nutritionally important blue-green alga Arthrospira platensis has been isolated, by application of conventional biochemical techniques. The molecular mass, yield, iron and total neutral carbohydrate contents of the purified protein were 470 kDa, 0.044 mg g⁻¹ of Arthrospira, 1.4 and 20%, respectively. The iron content was much lower when compared to bacterial and mammalian ferritins. The P: Fe ratio of Arthrospira phycoferritin was 1: 3.5, a value akin to bacterioferritins. Native gel-electrophoresis revealed the presence of isoforms. Subunit analysis by SDS-PAGE and Western blotting showed a protein subunit with an apparent molecular mass of 18 kDa. Oligomeric forms of the protein subunit were also present. The phycoferritin exhibited cross-reactivity with anti-pea seed ferritin suggesting phylogenetic relationship with that of higher plants. Carbohydrate analysis of phycoferritin by GC-MS revealed the presence of sugars such as galactose, glucose and mannose similar to that of mammalian ferritins. Interestingly, the analysis also revealed sugars such as rhamnose, xylose and talose, which has not been reported in the structure of ferritins. Except for very low histidine content in phycoferritin, the rest of the amino acid composition resembled to ferritins of other species. UV-visible spectral analysis of the phycoferritin revealed the presence of haem groups, a property characteristic of bacterioferritins. The fluorescence intensity of phycoferritin was higher than equine spleen ferritin. Circular dichroic spectra revealed a lower degree of helicity.