Autophagy: a barrier or an adaptive response to cancer

Macroautophagy or autophagy is a degradative pathway terminating in the lysosomal compartment after the formation of a cytoplasmic vacuole that engulfs macromolecules and organelles. The recent discovery of the molecular controls of autophagy that are common to eukaryotic cells from yeast to human suggests that the role of autophagy in cell functioning is far beyond its nonselective degradative capacity. The involvement of proteins with properties of tumor suppressor and oncogenic properties at different steps of the pathway implies that autophagy must be considered in tumor progression. Autophagy as a stress response mechanism protects cancer cells from low nutrient supply or therapeutic insults. Autophagy is also involved in the elimination of cancer cells by triggering a non-apoptotic cell death program, suggesting a negative role in tumor development. These two aspects of autophagy will be discussed in this review.     

An assessment of ADAMs in bone cells: absence of TACE activity prevents osteoclast recruitment and the formation of the marrow cavity in developing long bones

ADAMs (A Disintegrin And Metalloprotease domain) are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Since such events are critical for bone resorption and osteoclast recruitment, we investigated whether they require ADAMs. We report here which ADAMs we have identified in bone cells, as well as our analysis of the generation, migration and resorptive activity of osteoclasts in developing metatarsals of mouse embryos lacking catalytically active ADAM 17 [TNFalpha converting enzyme (TACE)]. The absence of TACE activity still allowed the generation of cells showing an osteoclastic phenotype, but prevented their migration into the core of the diaphysis and the subsequent formation of marrow cavity. This suggests a role of TACE in the recruitment of osteoclasts to future resorption sites.     

Probing the role of divalent metal ions in a bacterial psychrophilic metalloprotease: binding studies of an enzyme in the crystalline state by x-ray crystallography

The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated in Antarctica belongs to the clan of metzincins, for which a zinc ion is essential for catalytic activity. Binding studies in the crystalline state have been performed by X-ray crystallography in order to improve the understanding of the role of the zinc and calcium ions bound to this protease. Cocrystallization and soaking experiments with EDTA in a concentration range from 1 to 85 mM have resulted in five three-dimensional structures with a distinct number of metal ions occupying the ion-binding sites. Evolution of the structural changes observed in the vicinity of each cation-binding site has been studied as a function of the concentration of EDTA, as well as of time, in the presence of the chelator. Among others, we have found that the catalytic zinc ion was the first ion to be chelated, ahead of a weakly bound calcium ion (Ca 700) exclusive to the psychrophilic enzyme. Upon removal of the catalytic zinc ion, the side chains of the active-site residues His-173, His-179 and Tyr-209 shifted approximately 4, 1.0, and 1.6 A, respectively. Our studies confirm and also explain the sensitivity of PAP toward moderate EDTA concentrations and propose distinct roles for the calcium ions. A new crystal form of native PAP validates our previous predictions regarding the adaptation of this enzyme to cold environments as well as the proteolytic domain calcium ion being exclusive for PAP independent of crystallization conditions.     

Prenatal nicotine affects catecholamine gene expression in newborn rat carotid body and petrosal ganglion

Nicotine exposure modifiesthe expression of catecholamine and opioid neurotransmitter systemsinvolved in attenuation of hypoxic chemosensitivity. We used insitu hybridization histochemistry to determine the effect of prenataland early postnatal nicotine exposure on tyrosine hydroxylase (TH),dopamine -hydroxylase (DH), preproenkephalin (PPE), andD₂-dopamine receptor mRNA levels in the rat carotid bodyand petrosal ganglion during postnatal development. In the carotidbody, nicotine increased TH mRNA expression in animals at 0 and 3 postnatal days (both, P < 0.05 vs. control) withoutaffecting TH mRNA levels at 6 and 15 days. At 15 postnatal days, DHmRNA levels were increased in the carotid body of nicotine-exposed animals. Dopamine D₂-receptor mRNA levels in the carotidbody increased with postnatal age but were unaffected by nicotineexposure. PPE was not expressed in the carotid body at any of the agesstudied in control or treated animals. In the petrosal ganglion,nicotine increased the number of ganglion cells expressing TH mRNA inanimals at 3 days (P < 0.01 vs. control). DH mRNAexpression was not induced nor was PPE mRNA expression increased in thepetrosal ganglion in treated animals. Prenatal nicotine exposureupregulates mRNAs involved in the synthesis of two inhibitoryneuromodulators, dopamine and norepinephrine, in peripheral arterialchemoreceptors, which may contribute to abnormalities incardiorespiratory control observed in nicotine exposed animals.

     

Common origin and evolution of glycosyltransferases using Dol-P-monosaccharides as donor substrate

On the basis of the analysis of 64 glycosyltransferases from 14 species we propose that several successive duplications of a common ancestral gene, followed by divergent evolution, have generated the mannosyltransferases and the glucosyltransferases involved in asparagine-linked glycosylation (ALG) and phosphatidyl-inositol glycan anchor (PIG or GPI), which use lipid-related donor and acceptor substrates. Long and short conserved peptide motifs were found in all enzymes. Conserved and identical amino acid positions were found for the alpha 2/6- and the alpha 3/4-mannosyltransferases and for the alpha 2/3-glucosyltransferases, suggesting unique ancestors for these three superfamilies. The three members of the alpha 2-mannosyltransferase family (ALG9, PIG-B, and SMP3) and the two members of the alpha 3-glucosyltransferase family (ALG6 and ALG8) shared 11 and 30 identical amino acid positions, respectively, suggesting that these enzymes have also originated by duplication and divergent evolution. This model predicts a common genetic origin for ALG and PIG enzymes using dolichyl-phospho-monosaccharide (Dol-P-monosaccharide) donors, which might be related to similar spatial orientation of the hydroxyl acceptors. On the basis of the multiple sequence analysis and the prediction of transmembrane topology we propose that the endoplasmic reticulum glycosyltransferases using Dol-P-monosaccharides as donor substrate have a multispan transmembrane topology with a first large luminal conserved loop containing the long motif and a small cytosolic conserved loop containing the short motif, different from the classical type II glycosyltransferases, which are anchored in the Golgi by a single transmembrane domain.     

A conserved interaction between beta1 integrin/PAT-3 and Nck-interacting kinase/MIG-15 that mediates commissural axon navigation in C. elegans

BACKGROUND: Integrins are heterodimeric (alphabeta) transmembrane receptors for extracellular matrix (ECM) ligands. Through interactions with molecular partners at cell junctions, they provide a connection between the ECM and the cytoskeleton and regulate many aspects of cell behavior. A number of integrin-associated molecules have been identified; however, in many cases, their function and role in the animal remain to be clarified. RESULTS: We have identified the Nck-interacting kinase (NIK), a member of the STE20/germinal center kinase (GCK) family, as a partner for the beta1A integrin cytoplasmic domain. We find that NIK is expressed in the nervous system and other tissues in mouse embryos and colocalizes with actin and beta1 integrin in cellular protrusions in transfected cells. To demonstrate the functional significance of this interaction, we used Caenorhabditis elegans, since it has only one beta (PAT-3) integrin chain, two alpha (INA-1 and PAT-2) integrin chains, and a well-conserved NIK ortholog (MIG-15). Using three methods, we show that reducing mig-15 activity results in premature branching of commissures. A significant aggravation of this defect is observed when mig-15 activity is compromised in a weak ina-1 background. Neuronal-specific RNA interference against mig-15 or pat-3 leads to similar axonal defects, thus showing that both mig-15 and pat-3 act cell autonomously in neurons. Finally, we show a genetic interaction between mig-15, ina-1, and genes that encode Rac GTPases. CONCLUSIONS: Using several models, we provide the first evidence that the kinase NIK and integrins interact in vitro and in vivo. This interaction is required for proper axonal navigation in C. elegans.     

Inflammatory microcrystals alter the functional phenotype of human osteoblast-like cells in vitro: synergism with IL-1 to overexpress cyclooxygenase-2

Chronic crystal-associated arthropathies such as gout and pseudogout can lead to local bone destruction. Because osteoblasts, which orchestrate bone remodeling via soluble factors and cell-to-cell interactions, have been described in contact with microcrystals, particularly in uratic foci of gout, we hypothesized that microcrystals of monosodium urate monohydrate (MSUM) and of calcium pyrophosphate dihydrate (CPPD) could alter osteoblastic functions. MSUM and CPPD adhered to human osteoblastic cells (hOB) in vitro and were partly phagocytized as shown by scanning electron microscopy. MSUM and CPPD dose-dependently stimulated the production of PGE(2) in hOB as assessed by enzyme immunoassay, a response that was synergistically enhanced in the presence of IL-1. The mechanism of this synergism was, at least in part, at the level of the expression of cyclooxygenase-2 as evaluated by immunoblot analysis. MSUM and CPPD also stimulated the expression of IL-6 and IL-8 and reduced the 1,25-dihydroxyvitamin D(3)-induced activity of alkaline phosphatase and osteocalcin in hOB (with no synergism with IL-1). MSUM- or CPPD-stimulated expression of IL-6 in hOB pretreated with the selective cyclooxygenase-2 inhibitor NS-398 was increased, unlike that induced by IL-1 alone which was partially reduced. MSUM-, CPPD- or IL-1-induced expression of IL-8 was unchanged by pretreating hOB with NS-398. These results suggest that inflammatory microcrystals alter the normal phenotype of hOB, redirecting them toward reduced bone formation and amplified osteoblast-mediated bone resorption, abnormalities that could play a role in the bone destruction associated with chronic crystal-induced arthritis.     

New N-pyridinyl(methyl)-indole-2- and 3-(alkyl)carboxamides and derivatives acting as systemic and topical inflammation inhibitors

A series of novel N-substituted-(indol-2-yl)carboxamides (12-18) and (indol-3-alkyl)carboxamides (25-31) were synthesized and evaluated as inhibitors of the inflammation process. Pharmacomodulation at the level of the amidic nitrogen by incorporation of the previously described pharmacophoric moieties 6-aminolutidine, beta-picolylamine, 4-aminopyridine and piperazine was investigated; only two compounds (12) and (31) exhibited significant (approximately 40%) inhibitory effect in the carrageenan-induced rat paw edema after oral administration of a dose of 0.1 mM kg(-1). Replacement of the indole core by indazole failed to increase activity. Incorporation of an alkyl chain spacer led to more efficient compounds (46-52) especially in the indolepropanamide sub-series. Determination of the efficiency of the most active compounds on topical inflammation, by measuring reduction of ear thickness in the acute tetradecanoyl phorbol acetate (TPA)-induced mouse ear swelling assay, confirmed the high potency of propanamides (49) and (51) after oral administration: ID50 = 0.041 +/- 0.013 and 0.042 +/- 0.016 mM kg(-1) respectively. The less toxic propanamide (51) exerted a high level of inhibitory activity after topical application of 2 x 100 microg/ear: 78 +/- 2%.