Glycoprotein, elastin, and collagen secretion by rat smooth muscle cells.

Abstract

Smooth muscle cells from rat heart secreted extracellular matrix components at high rates for many generations in culture. The matrix proteins remained anchored to the culture dish and were characterized after removal of cellular material with sodium dodecyl sulfate. Sequential enzyme digestion demonstrated the presence of at least three components, including glycoprotein(s), elastin, and collagen. Prolonged extraction of the matrix with detergent under reducing conditions solubilized a fucosylated glycoprotein having an apparent molecular weight of 250,000 and two other proteins with molecular weights of 72,000 and 45,000, respectively. Sublines derived from discrete colonies of smooth muscle cells synthesized all of the matrix components, and the proportion of collagen secreted by some sublines increased with time in culture. The biosynthesis of a mixed extracellular matrix and the relationships among the component proteins were therefore studied in one system producing milligram quantities of material.The extracellular matrix proteins are of great importance to the functioning of an organism. Present culture models for their biosynthesis are not altogether satisfactory because most cells tend to lose their differentiated properties when they are removed from their normal environment (1)-for example, 3T3 and 3T6 cells, which are often used for studies of collagen biosynthesis, have largely lost their ability to synthesize this protein (2, 3). However, it has recently been shown that primary avian tendon cells synthesize physiological amounts of collagen under the correct culture conditions (1).The current interest in the proteins comprising the extracellular matrix (4-7) involves not only collagen and elastin but also the high molecular weight glycoproteins which probably play a major role in the organization and properties of the matrix. The microfibrillar protein of the elastic fiber has an apparent molecular weight of 270,000 (8) and has been suggested to be involved in the organization of the fiber (7). Fibronectin (LETS protein) has also been shown to be a predominantly matrix protein (9), to bind to collagen (10), and possibly to act as a bridge in the binding of cells to collagen (11). Our understanding of the biosynthesis, processing, and interactions of these proteins has been retarded not only by the lack of culture systems that produce them rapidly in physiological amounts but also by the lack of quantitative data on the matrix components. There is little information in the literature as to the actual quantities of material secreted by cultured cells, and no studies have been reported on the biochemical analysis of a mixed matrix produced in vitro.In this report we describe a culture system in which large amounts of an extracellular matrix are formed in a relatively short time. Smooth muscle cells cultured from rat heart form a multilayered structure containing milligram quantities of connective tissue proteins in a crosslinked, insoluble form within 2 weeks, even after many generations in ...

Metastatic behavior of human melanoma cell lines in nude mice correlates with urokinase-type plasminogen activator, its type-1 inhibitor, and urokinase-mediated matrix degradation.

“…These results suggest that plasmin is involved in the matrix degradation . As the extracellular matrices were produced by smooth muscle cells without ad- dition of ascorbic acid to the culture medium they contain little or no collagen (10,29,30) . The matrix degradation was mediated virtually only by t-PA in three of the cell lines (M14, IF6, and 530) while in two cell lines (BLM and MV3) it was mediated virtually only by u-PA (Fig .…”

Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats.

“…Cells were plated into tissue culture flasks (T35, 10 6 cells/flask). Primary cultures were routinely passaged for use in experimental regimes, and phenotypic characterization was performed as described by Jones et al 20 For the present study, we used smooth muscle cells from the 3rd-14th passages, during which the investigated parameters remained steady. The large number of experiments performed necessitated use of different pairs of isolates at differing passage numbers, but for each single experiment the cells within any pair were used at a matched passage number.…”

“…In total, 18 pairs of isolates were made, and for any given pair the areas from which the tissue was obtained were carefully matched for length and location. Procedures used were as described previously by Jones et al 20 and Chamley-Campbell et al 21 with minor modifications. After decapitation of animals, thoracic or abdominal aorta vessel sections (from the ventricular origin to the branching of the renal arteries) were rapidly removed and washed in phosphate-buffered saline (4° C) that contained 200 units/ml of both penicillin and streptomycin.…”

Morphological and metastatic murine melanoma variants: motility, adhesiveness, cell surface and in vivo properties

“…To produce extracellular matrix (ECM) cells were grown to confluency in 3.5 cm petri dishes and ascorbic acid added for the following 5 days. ECM was prepared according to the method of Jones et al (1979 (Avnur & Geiger, 1981 Cells were grown in cMEM + 0.1 I Ci ml-1 3H TdR for 48 h and then harvested with 0.02% EDTA/PBS, washed, adjusted to 2.5 x 105 cellsml-l cMEM and 2ml was added to each well of a 3.5 cm 6 well tissue culture plate and incubated at 37°C. Each plate consisted of triplicates of each cell line.…”

“…To produce extracellular matrix (ECM) cells were grown to confluency in 3.5 cm petri dishes and ascorbic acid added for the following 5 days. ECM was prepared according to the method of Jones et al (1979 (Avnur & Geiger, 1981). Coverslips were then washed in PBS pH 7.3, placed in 3.5 cm petri dishes containing medium and 104 cells were added and incubated in a 5% CO2 humidifled atmosphere.…”