Georges Bellon

Isotopic biogeochemistry (13C,15N) of fossil vertebrate collagen: application to the study of a past food web including Neandertal man

Abstract TheC/Nratio and amino acid composition of organic matter extracted from fossil mammal bones from the Paleolithic site at Marillac (Charentes, France) shown that this organic matter comes from collagen.δ 13 Candδ 15 Nvalues of known-diet fossil species demonstrate that these values have been preserved through fossilization processes, and that these fossil mammals can be used as ecological references to determine the Neandertal position in the past food web. Initial Neandertalδ 13 Candδ 15 N values suggest that he was mostly carnivorous.

Effect of diet, physiology and climate on carbon and nitrogen stable isotopes of collagen in a late pleistocene anthropic palaeoecosystem: Marillac, Charente, France

Abstract In fossil mammal bones and teeth from Marillac (40,000–45,000 years Br), collagen has been found to be preserved. This collagen presents a similar amino acid composition to modern collagen. An isotopic difference between collagen of herbivorous and carnivorous species is clearly documented in this paper and it is shown that the decrease of the amount of collagen in bones and teeth through diagenesis has not altered the original isotopic signal. It has been possible to demonstrate the carnivorous diet of Neanderthals. In addition, more subtle isotopicdifferences have been documented between the different species within each trophic group consumed. These differences can be linked to physiology, or to particular kinds of food of each animal species. Another isotopic signal due to nursing effects has also been observed. A young mammal suckling its mothers milk is one trophic level higher than an adult of the same species. Consequently, the collagen of deciduous teeth records an isotopic signal from the food consumed before weaning. Also, in species where most of the tooth growth occurs before weaning time, the collagen of permanent teeth records, in part, information about the milk diet. Thus the 15 N composition of such teeth is different from the isotopic composition of bones in the same individual. The variation of the 15 N composition of bone collagen along the stratigraphical sequence of the site shows that this isotopic signal may also be used for the palaeoclimatic reconstruction; for example, evidence of an arid period is seen in the layer #7 in Marillac.

Gelatinase B is involved in the in vitro wound repair of human respiratory epithelium.

Following epithelial injury, extracellular matrix undergoes imposing remodelings. We examined the contribution of matrix metalloproteinases, gelatinases A and B, in an in vitro wound repair model of human respiratory epithelium. Confluent human surface respiratory epithelial (HSRE) cells cultured from dissociated surface cells of human nasal polyps were chemically injured. Over the next 3 to 5 days, cells migrated onto the injured area to repair the circular wound. Repair kinetics of these wounds was monitored until wound closure occurred. Gelatinolytic activities were analysed in culture supernates and in cell protein extracts derived from repairing migratory and non repairing stationary cells. Small amounts of gelatinase A were expressed by HSRE cells, and variations of this gelatinase remained very weak for the time of the wound repair. In contrast, gelatinase B was upregulated during the wound repair process, with a maximum peak observed at wound closure. A marked gelatinase B activation occurred only in cells involved in the repair process. Gelatinase B was localized in some migratory basal cells, recognized by an anti‐cytokeratin 14 antibody and located around the wound. We could not detect any gelatinase A in repairing or in stationary HSRE cells. Addition of the 6‐6B monoclonal antibody, known to inhibit gelatinase B activation, to the culture medium during the repair process resulted in a dose‐dependent decrease of the wound repair speed. These results suggest that gelatinase B, produced by epithelial cells, actively contributes to the wound repair process of the respiratory epithelium.

LRP-1 silencing prevents malignant cell invasion despite increased pericellular proteolytic activities

ABSTRACT The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.

Analysis of the ex vivo specificity of human gelatinases A and B towards skin collagen and elastic fibers by computerized morphometry.

Cutaneous aging and chronic exposure to UV irradiation leads to alterations in the appearance and biochemical composition of the skin. Members of the MMP family have been involved in the destruction of the extracellular matrix. Among them, gelatinases A and B were found to display elastolytic activity, in vitro. In this study, we first determined the ex vivo elastolytic potential of both endopeptidases, using human skin tissue sections and computerized morphometric analyses, and compared it with those of neutrophil elastase. In such conditions, gelatinase B (50 nM) induced 50% elastolysis. The percentage of elastic fibers degraded by gelatinase A (10-100 nM) never exceeded 10%. Elastolysis by gelatinase B and leukocyte elastase was characterized by a decrease in fiber length and an increase in the average diameter of the fibers. In addition, gelatinase B exhibited fibrillin-degrading activities. On the contrary, gelatinase A (50 nM) elicited up to 50% hydrolysis of collagen fibers, preferentially degrading type III collagen fibers. Gelatinase B did not promote any collagen degrading activity. Our data suggested that in vivo gelatinases could disrupt most extracellular matrix structures of human skin. Gelatinase B and to a much lesser extent, gelatinase A would degrade components of the elastic fibers network while gelatinase A, but not gelatinase B, would alter mostly collagen fibers and also degrade constituents of the dermo-epidermal junction.

Glutamine increases collagen gene transcription in cultured human fibroblasts

We have previously shown that glutamine stimulates the synthesis of collagen in human dermal confluent fibroblast cultures (Bellon, G. et al. [1987] Biochim. Biophys. Acta, 930, 39-47). In this paper, we examine the effects of glutamine on collagen gene expression. A dose-dependent effect of glutamine on collagen synthesis was demonstrated from 0 to 0.25 mM followed by a plateau up to 10 mM glutamine. Depending on the cell population, collagen synthesis was increased by 1.3-to 2.3-fold. The mean increase in collagen and non-collagen protein synthesis was 63% and 18% respectively. Steady-state levels of alpha 1(I) and alpha 1(III) mRNAs, were measured by hybridizing total RNA to specific cDNA probes at high stringency. Glutamine increased the steady-state level of collagen alpha 1(I) and alpha 1(III) mRNAs in a dose-dependent manner. At 0.15 mM glutamine, collagen mRNAs were increased by 1.7-and 2.3-fold respectively. Nuclear run-off experiments at this concentration of glutamine indicated that the transcriptional activity was increased by 3.4-fold for the pro alpha 1(I) collagen gene. The effect of glutamine on gene transcription was also supported by the measurement of pro alpha 1(I) collagen mRNA half-life since glutamine did not affect its stability. Protein synthesis seemed to be required for the glutamine-dependent induction of collagen gene expression since cycloheximide suppressed the activation. The effect of glutamine appeared specific because analogues and/or derivatives of glutamine, such as acivicin, 6-diazo-5-oxo-L-norleucine, homoglutamine, ammonium chloride and glutamate did not replace glutamine. The influence of amino acid transport systems through plasma membrane was assessed by the use of 2(methylamino)-isobutyric acid and beta 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. The glutamine-dependent induction of collagen gene expression was found to be independent of transport system A but dependent on transport system L whose inhibition induced a decrease in pro alpha 1(I) collagen gene transcription by an unknown mechanism. Thus, glutamine, at physiological concentrations, indirectly regulates collagen gene expression.

Chronic UVB- and all-trans retinoic-acid-induced qualitative and quantitative changes in hairless mouse skin

Histochemical and ultrastructural studies have already demonstrated that chronic exposure to UV radiation induces profound alterations in all structural elements of the skin and that topical all-trans retinoic acid (tRA) can substantially correct much of the tissue damage. However, previous biochemical studies on dermal components of the extracellular matrix have led to contradictory results, particularly with regard to the effect of chronic UV exposure. The aim of our study was to investigate changes in collagen content and other dermal modifications induced by tRA in irradiated and non-irradiated hairless mouse skin. Hairless mice were exposed to increasing doses of UVB for 10 weeks (the cumulative total dose was 4.6 J cm-2). After the UV irradiation period the animals were treated with 0.05% tRA or with ethanol-polyethylene glycol vehicle alone three times a week for up to 10 weeks. Non-irradiated animals underwent the same treatments. The main clinical and histological changes induced by UVB exposure were erythema, wrinkling, keratosis and epidermal thickening. Following UVB exposure, tRA treatment did not improve the clinical aspect but increased the width of the dermal repair zone. Fibronectin, laminin and type I and VI collagens were detected by indirect immunofluorescence techniques in this zone. Type I and III collagens were quantitated in skin fragments after cyanogen bromide digestion and polyacrylamide gel electrophoresis. Under our experimental conditions, UVB irradiation alone induced neither changes in total collagen nor in type I and III collagen levels. tRA treatment of irradiated skin significantly increased both type I and III collagen levels by factors of 1.33 and 1.88 respectively. The ratio of type III to types I + III increased significantly. Topical tRA also increased collagen type levels in non-irradiated hairless mouse skin. Type I collagen increased proportionally to type III. This study leads to the conclusion that topical tRA exerts direct or indirect effects on collagen metabolism in irradiated as well as non-irradiated hairless mouse skin.

Elastase-type activity associated with high density lipoproteins in human serum

Abstract Elastase-type activities were found associated with lipoprotein fractions in human serum. A metallo-protease hydrolysing Suc(Ala) 3 PNA and soluble elastin peptides was isolated from apoHDL by Sepharose-elastin chromatography. Polyacrylamide gel electrophoresis and immunoelectrophoresis indicated that this elastase-type activity was associated with apoA 1 .

Elastin-derived peptides: matrikines critical for glioblastoma cell aggressiveness in a 3-D system.

In the most common primary brain tumors, malignant glioma cells invade the extracellular matrix (ECM) and proliferate rapidly in the cerebral tissue, which is mainly composed of hyaluronan (HA) along with the elastin present in the basement membrane of blood vessels. To determine the role of ECM components in the invasive capacity of glioma cell lines, we developed a 3‐D cell‐culture system, based on a hydrogel in which HA can be coreticulated with kappa‐elastin (HA‐κE). Using this system, the invasiveness of cells from four glioma cell lines was dramatically increased by the presence of κE and a related, specific peptide (VGVAPG)3. In addition, MMP‐2 secretion increased and MMP‐12 synthesis occurred. Extracellular injections of κE or (VGVAPG)3 provoked a pronounced and dose‐dependent increase in [Ca2+]i. κE significantly enhanced the expression of the genes encoding elastin‐receptor and tropoelastin. We propose the existence of a positive feedback loop in which degradation of elastin generates fragments that stimulate synthesis of tropoelastin followed by further degradation as well as migration and proliferation of the very cells responsible for degradation. All steps in this ECM‐based loop could be blocked by the addition of either of the EBP antagonists, lactose, and V‐14 peptide, suggesting that the loop itself should be considered as a new therapeutic target.

Role of the elastin receptor complex (S-Gal/Cath-A/Neu-1) in skin repair and regeneration.

Impaired elastic fiber assembly constitutes one major problem in skin wound healing. Recent data indicate that a ternary complex involving a splicing form of β‐galactosidase associated with cathepsin‐A and neuraminidase‐1 directs the transport of tropoelastin to the fibroblast plasma membrane and participates in the deposition of the elastin precursor onto a microfibrillar scaffold. In addition, this elastin receptor complex is ubiquitously expressed and also acts as a true receptor for elastin‐derived peptides produced during the initial stage of wound repair following elastase‐mediated proteolysis action. Among the peptides generated, those having a x.G.x.x.P.G. motif upregulate (i) keratinocyte migration, (ii) endothelial cell angiogenic phenotype, (iii) fibroblast proliferation, and (iv) induction of the expression of matrix metalloproteinases, type I collagen, and tropoelastin. All of these properties could accelerate the different stages of wound repair. Elastin‐derived peptides from a chemical or a proteolytic digest of insoluble elastin alone or linked to the collagen scaffold significantly improve skin wound healing and dermal regeneration in vivo in several animal models. Such a beneficial influence has been recently extended to the treatment of burn patients. In this respect, recent investigations have focused on the design of elastin‐derived peptides or elastin‐building blocks, as obtained from peptide chemistry or by genetic engineering, to elaborate biocompatible elastin peptides, which are considered as ideal biomaterials for “catalyzing” skin repair and regeneration following injury.