Waltraud Dessau

Origin of collagen types I, III, and V in cultures of avian skeletal muscle

In this study we present biochemical and immunological evidence that avian myoblasts synthesize types I, III, and V (AB2-) collagen. Clonal and mass cultures of embryonic quail breast muscle were radioactively labeled, and the collagen from cell layer and culture medium was pepsin treated and analyzed by sodium dodecyl sulfate-gel electrophoresis. Types I, III, and V collagen are found in cell layer and medium, but most of the type V collagen is retained in the cell layer. Time course experiments revealed that types I and V collagen are produced from the first day on in culture, while type III collagen synthesis increased from less than 5% of the total collagen to about 30% on the third day in mass cultures. Immunofluorescence analysis of the collagens produced in myogenic clones revealed that only mononucleated cells of spindle-shaped or stellate morphology stained for types I and III collagen, while type V collagen was found in mononucleated cells as well as in myotubes. In myogenic clones stained without fixation, an extensive meshwork of extracellular collagen fibers was observed. This suggests that not only fibroblasts, but also myoblasts participate in the deposition of extracellular connective tissue fibers in skeletal muscle.

SYNTHESIS OF COLLAGEN BY HUMAN FIBROBLASTS AND THEIR SV40 TRANSFORMANTS

Abstract Synthesis of collagen was studied in human fibroblasts (WI26, WI38) and their SV40 transformants. Viral transformation decreased the amount of collagen synthesized by a factor of 8 during a 24 h pulse and affected the rate of conversion of procollagen to collagen. No change was observed in the proportions of type I and type III collagen, the degree of hydroxylation of α-chains of the newly synthesized collagen remained the same. The collagen of viral transformants contained substantial amounts of collagen molecules which were composed of α1(I)-chains only. Immunofluorescence analysis using specific antibodies for type I collagen and fibronectin showed less deposition of extracellular fibrils in the transformed cell layers than in the normal cells.

Similarity of antigelatin factor and cold insoluble globulin.

Antigelatin factor, a protein capable of complexing denatured collagen, was separated from human serum by adsorption onto immobilized collagen. Antiserum raised against the material binding to denatured collagen permitted the development of a radioassay for the determination of antigelatin factor in which the complex of antigelatin factor and denatured 125I-labeled collagen is precipitated with this antiserum. Further purification of antigelatin factor was achieved by chromatography on DEAE-cellulose yielding an electrophoretically homogeneous protein. Its migration rate in dodecyl sulfate-polyacrylamide gel electrophoresis was identical with that of cold insoluble globulin (molecular weight approx. 440 000) prepared from human plasma by a published procedure amended by DEAE-cellulose chromatography. Reduction of disulfide bonds yielded subunits of molecular weight approx. 220 000, indistinguishable from those of cold insoluble globulin. The amino acid composition of both proteins was very similar. Immunological identity of both proteins was demonstrated by gel diffusion against monospecific anti-cold insoluble globulin antiserum. Closely related binding curves were obtained if denatured 125I-labeled collagen was reacted with increasing amounts of either cold insoluble globulin or antigelatin factor and the complexes formed were precipitated with anti-cold insoluble globulin antiserum. In addition, antigelatin factor and cold insoluble globulin mediated the fixation of denatured 125I-labeled collagen to trypsinized macrophages in the same way. Therefore, it is concluded that antigelatin factor and cold insoluble globulin are identical or very closely related proteins.

Collagen types synthesized by isolated calvarium cells

Abstract Three populations of cells were isolated from embryonic chick calvaria by three sequential incubations with a collagenase-trypsin solution. Population 1 contained presumptive ‘osteoprogenitor cells’ of fibroblast-like cell shape. A compact, polygonal cell type, called ‘osteoblast-like’ cells, was predominant in the populations 2 and 3. Incorporation of 45 Ca 2+ into acid-soluble material was measured for additional characterization of the cells. All calvarium cells synthesized type I collagen and a small amount of type V (A,B) collagen. Type II collagen was not detected in any culture. Type III collagen was synthesized by a few cells of the first population but not by cells of subsequent harvests. The overall level of collagen synthesis was higher in calvarium cells, particularly in the populations 2 and 3, as compared with tendon fibroblasts. The qualitative pattern of collagen synthesis remained virtually unchanged during 6–8 weeks in primary cultures and was not influenced by subculturing either.

Rates of Synthesis of Basement Membrane Proteins by Differentiating Teratocarcinoma Stem Cells and Their Modulation by Hormones

The embryonal carcinoma mouse cell line F-9 was used as a convenient model for a quantitative study of the production of the basement membrane proteins laminin and type IV collagen. Both proteins could be identified in the culture medium and cell layer by radioimmuno assays, metabolic labeling and immunofluorescence. More than 95% of the material is secreted into the medium. Lack of ascorbic acid inhibits secretion of type IV collagen but not of laminin. Induction of differentiation into endoderm-like cells by retinoic acid consistently caused after a lag period of 2-3 days a 5-10 fold increase in the production of basement membrane proteins but not of total protein. Dibutyryl cyclic AMP further potentiated this specific effect particularly with respect to type IV collagen synthesis. Insulin, epidermal growth factor and nerve growth factor produced only moderate increases (10-60%) in the amount of laminin and type IV collagen. Effects of these hormones were only observed with certain doses and were quite variable between different experiments.

Biochemical and immunological studies of fibroblasts derived from a patient with Ehlers-Danlos Syndrome Type IV

SummaryFibroblasts derived from a skin biopsy of a patient with the Ehlers-Danlos syndrome (EDS) type IV were cultured in monolayer. The amount of collagen synthesized during a 24-h pulse was not different from that found with normal fibroblasts. Chromatographic procedures and immunofluorescence staining showed a normal synthesis of type I procollagen and collagen but a deficiency in synthesis of type III procollagen and collagen. This could be corroborated by radioimmuno assays showing a reduction in type III procollagen by about 90%. The secretion and degradation of collagens was not altered. The results demonstrate that the molecular defect in this particular patient is due to an impairment of the mechanism controlling the gene expression for type III procollagen.ZusammenfassungVon einem Patienten mit den klinischen Symptomen eines Ehlers-Danlos-Syndroms Typ IV wurden Fibroblastenkulturen gezüchtet. Diese synthetisierten dieselbe Menge an Kollagen wie Kontroll-Fibroblasten und zeigten auch ein identisches Sekretions-und Degradations-Verhalten. Biochemische und immunochemische Methoden wiesen jedoch eine signifikante Reduktion des Anteils von Typ III-Kollagen auf etwa 10% des Normalwertes auf.