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    1995, U. Seitzer- Microgravity and hypergravity effects on collagen biosynthesis of human dermal fibroblasts

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    1995, U. Seitzer - Microgravity and Hypergravity Effects On Collagen Biosynthesis of Human Dermal Fibroblasts

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    jeffrey Eellis

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    © All Rights Reserved
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    Cell Tissue Res (1995) 282:513-517 Cella Tissue ch © Springer-Verlag 1995. Microgravity and hypergravity effects on collagen biosynthesis of human dermal fibroblasts Ulrike Seitver!, Michael Bodo’, Peter K. Miller!, Yahya Agil!, Boris Batge® "Institut fir Medizinische Molekularbiologie, Medizinische Universitat zu Libeck, Ratreburger Allee 160, D-23538 Lalbeck, Germany Department of Biology 0322, University of Califor san Diego, 9500 Gilman Drive, La Jolla, CA 92098, USA Kink fur Innere Medizin, Medizinische Universitit 2u Lbeck, Ratzeburger Allee 160, D-23538 Lubeck, Germany Received: 13 February 1995 / Accepted: 12 July 1995 Abstract. Astronauts experiencing long periods of space flight suffer from severe loss of bone tissue, particularly in those bones that carry the body weight under normal, gravity. It is assumed that the lack of mechanical load decreases connective tissue biosynthesis in bone-form- ing cells. To test this assumption, quantitative and quali- tative aspects of collagen synthesis under microgravity, normal gravity, and hypergravity conditions were inves. tigated by incubating human fibroblast cultures with [H]-proline for 4, 7, 10, and 20 h during the Spacelab D2-mission in 1993. Quantitative analysis revealed an increase of collagen synthesis under microgravity condi- tions, being up to 143% higher than in 1g controls. In, contrast, hypergravity samples showed a decrease in col- lagen synthesis with increasing g, being at the 13% level at 10 g. ‘The relative proportion of collagen in total syn- thesized protein showed a slight decrease with increas- ing g. The secretion of collagen by the cells, proline hy- droxylation of individual collagen d-chains, and the rel- ative proportions of synthesized collagens I, III, and V ‘were not affected under any of the applied conditions, Key words: Microgravity ~ Collagen — Bone ~ Dermal fibroblasts — Human Introduction Astronauts and Jaboratory animals experiencing long pe- riods of space flight suffer from severe loss of bone tis sue, Closer inspection reveals that the biosynthesis of bone minerals and proteins is diminished (Russel and Simmons 1985). Bone formation and remodeling are stringently controlled processes, and a variety of hor- monal factors influence calcium uptake/release, and pro- tein synthesis and catabolism. Changing blood levels of several key hormones have been found in astronauts, ‘Our research was supported financially by Dara GmbH Bona (grant. no. O1QV’ 8866), the Deutsche Forschungsgemeinschaft (SFR A1/367) and BMFT grant, no. 01 KM 9303/8 Correspondence to: U. Seiteer (Planel_and Oser 1984). On the other hand, it appears that only the weight-bearing bones are affected by mi crogravity (Wronski and Morey 1983). Hormonal chang- es, however, would be expected to cause the same symp- toms in all bones. A decrease in mechanical strain appar ently results in a decrease of connective tissue synthesis by bone-forming cells. To test this hypothesis, a cell cul- ture experiment has been conceived to elucidate the rele vant biochemical mechanisms with respect to collagen synthesis under microgravity. As experiments with intact organisms are difficult to interpret in terms of molecular mechanisms, the investigation of the effect of weight- lessness on isolated connective tissue cells is an impor- tant complementation to existing results of animal exper- iments. Human dermal fibroblasts have been chosen, rather than osteoblasts as an experimental model system, because they can easily be isolated in large quantity and maintained in culture; they are thus more suitable for the experimental requirements under the microgravity con- ditions in the Spacelab facility. They have a biosynthetic profile similar to that of bone-forming cells, both ost blasts and fibroblasts being of mesenchymal origin and producing predominantly collagen type 1 in vivo. Colla- gen type Lis the most abundant protein of bones and oth- fer connective tissues and is required for bone mineral- ization and stability. Small amounts of collagens type III and V are also produced in cell cultures of fibroblasts. In the present experiment, we have investigated the effect of microgravity, normal gravity, and hypergravity on quantitative and qualitative aspects of collagen synthesis by incubating the cells with [H]-protine, which is post- translationally modified to hydroxyproline as a unique constituent of all collagen chains. Incubation should al- low investigation of the collagen synthesis rate, the per- cent collagen of total protein synthesized, the secretory behavior of the cells, the relative proportions of collagen 1, IIL, and V synthesized, and the degree of proline hy- droxylation of individual collagen a-chains, by the de- termination of the amount of [SH)-proline and (*H|-hy- droxyproline incorporated. The determination of the rel- ative amount of collagen synthesized with respect to to- sia tal protein synthesis is a parameter that registers the specificity of an effect on collagen synthesis. The com- position of the collagen types synthesized indicates the quality of the matrix formed (van der Rest and Garrone 1991), and the degree of hydroxylation of proline plays a major role in the stability of individual collagen mole- cules (Piez 1984). ‘This experimental design thus allows specific infor- mation concerning matrix synthesis as a prerequisite for orderly matrix formation to be obtained. Materials and methods Cell culture Haman dem fibroblasts, obtained from human volunteers aged 28-15, were seeded in the twelln passage a 10 eelsen? onto 8% 24.6 mm ‘Thermanox coverslips (NUNC, Roskilde, Denmark) in Sovel plats and grown to contlaeny for 48 f in‘Dulbecco's ‘noid Eagles medium (DMEND supplemented with 10% fetal Calf seam (FCS), 10°1U. peaiilin, 100 mg suepromyein, 2 mM [egutamine, and 0.25 mM Ne-ascorhate (Bioehrom, Bein, Ger tary) Cells were cultured ina humidified stmosphere with 5% CO, ai 37°C. For incubation, the coafluent cell coverslips. were trabfered t0 the ineuhaton chambers (see bslow) comsining 26a incubation medium (DMEM, 1.2 mM Paminoproprionitle, {25 mM Ne-scorate, 10° LU. penicillin, 1% FCS). CH) proline (NEN-Dupont, Dreiich, Germany) wa ket in a separate compat ment in the incubation chamber G7 MBqml (1-proline in 0.26 ml incubation medium). The samples were maintained at 37°C for 31h inching the launch ofthe shut hen tersfered othe respective 37°C incubators microgravity and 1g round reference) and 37°C centrifuges (in-flight 1g reference to assess the effect of exmic radiation on collagen synthesis, 18 ground, and 6 ¢ fd 0 g hyperfage. and fet to adj for 6h. Hypereavity cond ‘tions were achieved in a specially constructed hyperfuge incubator (Diport AG, Uster, Switzeriand), The incubations were started by theradiion ofthe radioactive ace solution and two samples each sere taken at 4,7, 10, and 20 h, The incobtion was stopped by Separating the ell fom the medvm and freezing the samples “20°C until analysis. The env experiment. including al applied trviy conditions and samples taken, wos performed in parle With 22 delay on ground samples stlizing ieneal conditions ‘wth respect th cells, medium and incubation medium wed Fig. 1. A Confluent monolayer of human dermal fibroblasts on a ‘Theemanox coverslip prepared for incubation. B Cell monolayer after incubation, space Flight, and freezing to ~20°C: the cells are still attached to the coverslip, demonstrating that they must have been viable atthe time ofthe incubation, C Fluorography of (H- Hardware. Incubation with radioactive media was performed in safety culture chambers that permitted the addition and removal of radioactive liquids to the cells without exposure of the operator to radioactivity hazards, Analysis of collagen types ‘Samples were digested with 0.1 mg/ml pepsin (Boehringer, Mann- heim, Germany) in 0.05% acetic acid adjusted to pH. 1 for 16 h at 4°C. Aer addition of carrier solution containing collagen 1, Hl, and V, extracts were concentrated and washed with Microsep ul- rafilration units with a cut-off of 10 kDa (Filtron, Karlscuhe, Germany) to cemove unbouad radioactivity. After direct resuspen= sion in sample buffer for electrophoresis, the d-chains were sepa- rated by SDS-PAGE (polyacrylamide gel electrophoresis) under conditions of delayed reduction (Sykes ct al. 1976). After separa- tion and visualization of individual bands with Coomassie blue, the separated chains were cut out, and the activity per a-chain was determined in a liquid scintillation counter. The relative propor- tions of individual a-chains were expressed as a percent of the t0- tal counts of each sample, Hydroxyproline per u-chain was deter- ‘mined by amino acid analysis (see below). Quantitative collagen analysis ‘Medium and cells were analyzed separately. The samples were concentrated and washed by ultrafiltration as described above, re- suspended in 6 N HCL hydrolyzed (110°C. 24 b), and dried in a desiceator, After resuspension in amino-acid sample-dilution buff et (Beckman, Munich, Germany) and the determination of the PH}-proline ‘and [PH}-tydroxyproline amounts by amino-acid analysis using ion exchange chromatography with a solid phase scintillation detector, the percent collagen of the total protein was calculated (Wiestner et al. 1979). Quantitative collagen synthesis ‘was expressed as hydroxyprotine counts present per cell, and the secretion factor was caleulated as. [H]-hydroxyprolinegntun/ (PHL nydroxyprotinegasn + PHT hydroxyproline) Results Microscopic evaluation of the cells on the coverslips of, all incubated samples revealed that the cells were still at- collagen a-chain

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