Antiestrogens specifically up-regulate bone morphogenetic protein-4 promoter activity in human osteoblastic cells

Bone morphogenetic protein-4 (BMP-4) plays an important role in the onset of endochondral bone formation in humans, and a reduction in BMP-4 expression has been associated with a variety of bone diseases. Here we describe, by transient transfection assays in bone cells, that the human BMP-4 promoter recently characterized in our laboratory can be stimulated specifically by antiestrogens but not by estrogens or other steroid hormones. This activity is dependent on the presence of the estrogen receptor (ER)-alpha, although the promoter lacks a consensus estrogen-responsive element. No activity was observed in the presence of ERbeta, but synergy was observed when both ER subtypes were cotransfected. The observed stimulation of BMP-4 promoter activity by antiestrogens appeared bone cell specific and was reversed upon addition of estrogens. Since antiestrogens are known to be effective in hormone replacement therapies for postmenopausal women, this observation may help to develop new strategies for treatment and prevention of osteoporosis.     

Role of the N-terminus of epidermal growth factor in ErbB-2/ErbB-3 binding studied by phage display

Epidermal growth factor (EGF) binds with high affinity to the EGF receptor, also known as ErbB-1, but upon replacement of the N-terminal linear region by neuregulin (NRG) 1 or transforming growth factor (TGF) alpha sequences it gains in addition high affinity for ErbB-2/ErbB-3 heterodimers. However, these chimeras weakly bind to ErbB-3 alone. To further dissect the ligand binding selectivity of the ErbB network, we have applied the phage display technique to examine the role of the linear N-terminal region in EGF for interaction with ErbB-2/ErbB-3 heterodimers. A library of EGF variants was constructed in which residues 2, 3, and 4 were randomly mutated, followed by selection for binding to intact MDA-MB-453 cells that overexpress ErbB-2 and ErbB-3 but lack ErbB-1. Analysis of the selected phage EGF variants revealed clones with high binding affinity to ErbB-2/ErbB-3 while maintaining high affinity to ErbB-1. In these variants, Trp (or alternatively His) was almost exclusively present at position 2, while specific combinations of hydrophobic, basic, and small residues were found at positions 3 and 4. The mitogenic activity of the phage EGF variants corresponded with their relative binding affinity. Two of the selected EGF variants, EGF/WVS and EGF/WRS, were further characterized as recombinant proteins. In contrast to previously characterized chimeras of EGF with NRG-1 or TGF-alpha, these variants did not only show high binding affinity for ErbB-2/ErbB-3 heterodimers but also for ErbB-3 alone. These data show that the linear N-terminal region of EGF-like growth factors is directly involved in binding to ErbB-3.     

Termination of diapause in the Cinnabar moth (Tyria jacobaeae) by chilling

The effect of chilling time on the termination of pupal diapause was studied in the Cinnabar moth, Tyria jacobaeae L., Lepidoptera: Arctiidae. Chilling followed by a temperature of 20°C was effective in terminating diapause. Chilling for 120 days immediately after pupation yielded the highest percentage emergence and resulted in the shortest total pupal development time (162 days). A more natural treatment, a warm period before cold treatment, did not result in a higher percentage of emergence or in a shorter total pupal development time.During hibernation this species experiences two successive processes: diapause development during the first three to four months of cold, followed by a period of quiescence. Application of high temperatures in this latter period immediately starts morphogenesis. The impact of a relative humidity of 70% during the pupal stage for complete wing unfolding is demonstrated. The underlying mechanism of diapause is discussed.

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Résumé L'effet sur la levée de la diapause de différents régimes de température infligés aux chrysalides est étudié chez Tyria jacobaeae L. (Lepidoptera: Arctiidae). Une mise au froid suivie de hautes températures lève efficacement la diapause. Un refroidissement pendant 120 jours immédiatement après la nymphose entraîne le plus grand pourcentage d'éclosions et la durée totale du développement des chrysalides la plus courte (162.3 j). Un traitement plus naturel, une période chaude avant la période froide, n'entraîne pas un plus grand pourcentage d'éclosions ni une durée plus courte de développement.Il est proposé que durant l'hibernation cette espèce passe par deux processus successifs: un diapause durant les premiers trois ou quatre mois de froid, suivie d'une période de quiescence. Le transfert à des hautes températures pendant cette derpériode déclanche immédiatement la morphogénèse. L'influence d'un taux élevé d'humidité relative durant le stade chrysalide sur le déploiement complet des ailes est démontré. Le méchanisme de diapause sous-jacent est discuté.

     

Myeloid-Related Protein-14 Contributes to Protective Immunity in Gram-Negative Pneumonia Derived Sepsis

Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis. Myeloid related protein 8 (MRP8, S100A8) and MRP14 (S100A9) are the most abundant cytoplasmic proteins in neutrophils. They can form MRP8/14 heterodimers that are released upon cell stress stimuli. MRP8/14 reportedly exerts antimicrobial activity, but in acute fulminant sepsis models MRP8/14 has been found to contribute to organ damage and death. We here determined the role of MRP8/14 in K. pneumoniae sepsis originating from the lungs, using an established model characterized by gradual growth of bacteria with subsequent dissemination. Infection resulted in gradually increasing MRP8/14 levels in lungs and plasma. Mrp14 deficient (mrp14^−/−) mice, unable to form MRP8/14 heterodimers, showed enhanced bacterial dissemination accompanied by increased organ damage and a reduced survival. Mrp14^−/− macrophages were reduced in their capacity to phagocytose Klebsiella. In addition, recombinant MRP8/14 heterodimers, but not MRP8 or MRP14 alone, prevented growth of Klebsiella in vitro through chelation of divalent cations. Neutrophil extracellular traps (NETs) prepared from wildtype but not from mrp14^−/− neutrophils inhibited Klebsiella growth; in accordance, the capacity of human NETs to kill Klebsiella was strongly impaired by an anti-MRP14 antibody or the addition of zinc. These results identify MRP8/14 as key player in protective innate immunity during Klebsiella pneumonia.     

Role of the prostanoid FP receptor in action potential generation and phenotypic transformation of NRK fibroblasts

By using an shRNA approach to knockdown the expression of the prostaglandin (PG)-F(2alpha) receptor (FP-R), the role of PGF(2alpha) in the process of phenotypic transformation of normal rat kidney (NRK) fibroblasts has been studied. Our data show that PGF(2alpha) up-regulates Cox-2 expression both at the mRNA and protein level, indicating that activation of FP-R in NRK fibroblasts induces a positive feedback loop in the production PGF(2alpha). Knockdown of FP-R expression fully impaired the ability of PGF(2alpha) to induce a calcium response and subsequent depolarization in NRK cells. However, these cells could still undergo phenotypic transformation when treated with a combination of EGF and retinoic acid, but in contrast to the wild-type cells, this process was not accompanied by a membrane depolarization to -20 mV. Knockdown of FP-R expression also impaired the spontaneous firing of calcium action potentials by density-arrested NRK cells. These data show that a membrane depolarization is not a prerequisite for the acquisition of a transformed phenotype. Furthermore, our data provide the first direct evidence that activity of PGF(2alpha) by putative pacemaker cells underlies the generation of calcium action potentials in NRK monolayers.     

Membrane regulation of the Na+,K+-ATPase during the neuroblastoma cell cycle: correlation with protein lateral mobility

The pumping activity of the plasma membrane-bound Na+,K+-ATPase shows considerable variation during the cell cycle of mouse neuroblastoma Neuro-2A cells. Addition of external ATP at millimolar concentrations, which selectively enhances the plasma membrane permeability of Neuro-2A cells for sodium ions, stimulates the Na+,K+-ATPase pumping activity at all phases of the cell cycle from a factor of 1.05 in mitosis up to 2.2 in G1 phase. Determination of the number of Na+,K+-ATPase copies per cell by direct 3H-ouabain binding studies in the presence of external ATP shows a gradual increase in the number of pump sites on passing from mitosis to the late S/G2-phase by approximately a factor of 2. From these data the pumping activity per copy of Na+,K+-ATPase, optimally stimulated with respect to its various substrate ions, has been determined during the various phases of the cell cycle. This optimally stimulated pumping activity per enzyme copy, which is a reflection of the physicochemical state of the plasma membrane, is high in mitosis, almost twofold lower in early G1 phase, and increases gradually again during the other phases of the cell cycle. This shows that the observed regulation of Na+,K+-ATPase activity during the cell cycle is caused by a combination of three independent factors--namely variation in intracellular substrate availability (Na+), changes in number of enzyme copies per cell, and modulation of the plasma membrane environment of the protein molecules. The modulation of the optimal pumping activity per enzyme copy shows a good correlation (rho = 0.96) with the known modulation of protein lateral mobility during the cell cycle, such that a high protein lateral mobility correlates with a low enzyme activity. It is concluded that changes in plasma membrane properties take place during the Neuro-2A cell cycle that result in changes in the rate of protein lateral diffusion and Na+,K+-ATPase activity in directly correlated way.     

Effect of external ATP on the plasma membrane permeability and (Na+ +K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na+ +K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na+ +K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2 X 10(-6)M. Neuro-2A cells contain (3.5 +/- 0.7) X 10(5) ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7 +/- 0.4) X 10(-20) mol K+/min per copy of (Na+ +K+)-ATPase at room temperature.     

Family tree analysis of a transformed cell line and the transition probability model for the cell cycle

Variation in intermitotic time between individual cells in culture can be ascribed to the occurrence of random transitions in the cell cycle. We have analysed a family tree of mouse neuroblastoma cells, and observed that variation in difference in intermitotic time between sister cells is smaller than between cousin cells, and this difference is again smaller than between second-cousin and unrelated cells. This observation is incompatible with all transition probability models presented so far. We propose a model for the cell cycle with a single random transition, but with the additional assumption that the (two) system parameters may show variability within the population such that the closer cells are in their relation to each other, the closer their values of the system parameters will be. This model describes correctly the behaviour of the family tree of the cell line and in addition is able to explain why differences in intermitotic time between sister cells are exponentially distributed, while intermitotic times themselves are more or less normally distributed. Methods have been described to quantify the various system parameters.     

Identification of the type-B receptor for platelet-derived growth factor in human embryonal carcinoma cells

The human embryonal carcinoma (EC) cell line Tera 2 clone 13 (T2/13) can be induced to differentiate in vitro into neuroectodermal cell types with retinoic acid. Undifferentiated cells are characterized by rapid proliferation, whereas differentiated cells show a prolonged generation time, have a limited life span, and possess new cell-surface markers. In the present study we establish that both differentiated and undifferentiated T2/13 cells express the type-B platelet-derived growth factor (PDGF) receptor mRNA and bind PDGF-BB with high affinity. Differentiation causes a three-fold increase in receptor number per cell and leaves the affinity of the receptors unaffected. These data are the first to describe expression of this receptor in EC cells. The biosynthesis and degradation of this receptor were studied in undifferentiated as well as in differentiated T2/13 cells using an anti-type-B receptor antibody. These experiments revealed that high concentrations of recombinant PDGF-AA did not accelerate receptor metabolism in both cell types. In contrast, human PDGF or recombinant PDGF-BB added to the culture dishes readily increased receptor degradation. These results demonstrate that T2/13 cells express functional type-B PDGF receptors and suggest that cells responsive to PDGF might be present during mammalian development before the onset of mesoderm formation.     

Osmotic behaviour of Acholeplasma laidlawii B cells with membrane lipids in liquid-crystalline and gel state.

The osmotic behaviour of Acholeplasma laidlawii B cells was investigated with combined spectrophotometric and enzymatic measurements. The conclusion could be drawn that this osmotic behaviour depends largely on the physical state of the membrane lipids. When part of the membrane lipids is in the liquid-crystalline phase the cell is able to swell and behaves as a good osmometer. However, when the membrane lipid is in the gel phase, the cell is unable to swell and the change in absorbance of the cell suspension is then completely due to lysis.