Influence of various extracellular matrix components on the behavior of cultured chick embryo dermal cells

Dermal cells isolated from the back of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III or IV collagen, fibronectin and several glycosaminoglycans: hyaluronate, chondroitin-4, chondroitin-6, dermatan or heparan sulfate). The effect of these substrates on cell behavior was compared with that of culture dish polystyrene. Three parameters of cell behavior were examined: cell proliferation and patterning, spreading (cell surface) and locomotion (velocity and directionality). Data were collected by sequential microphotography and analyzed by computer assisted morphometry. Types I and III collagen, hyaluronate and heparan sulfate had a slowing down effect on cell proliferation and patterning. The inhibitory effect of type I collagen was also detected in mixtures with glycosaminoglycans. The other components had no effect. While the smallest spreading was observed on fibronectin substrate, the largest was recorded on chondroitin-6 sulfate and heparan sulfate. The slowest velocity of locomotion was measured on fibronectin, types I and IV collagen and a mixture of type I collagen and chondroitin-6 sulfate. The fastest speed was recorded on chondroitin-4 sulfate. These effects are discussed in view of our knowledge of the role of the dermis in the development of skin and cutaneous appendages, and in the light of the morphogenetically related microheterogeneous distribution of collagens, fibronectin and various glycosaminoglycans in the developing skin.     

The effect of embryo extract on the types of collagen synthesized by cultured chick chondrocytes.

Growth of embryonic chick chondrocytes in dialyzed embryo extract results in both a change in morphology of the cells toward that of a fibroblast and a change in the type of collagen synthesized from the cartilage-specific Type II collagen (chain composition [α1(II)]₃) to a mixture of Type I collagen (chain composition [α1(I)]₂α2) and the Type I trimer (chain composition [α1(I)]₃). Analyses after 6 days of growth in embryo extract show that the synthesis of only Type I collagen and the Type I trimer can be detected. However, on subculturing the cells to a low density and allowing a period of growth without embryo extract, colonies of chondrocytes reappear and the synthesis of Type II collagen apparently resumes. It is suggested that the observed changes represent a “modulation” in cell behavior, this being expressed not only by the morphological changes but also by changes in cell-specific protein synthesis as demonstrated by the changes in the type of collagen synthesized.     

Production of components of extracellular matrix by cultured rat Sertoli cells

Sertoli cells from rats aged 15, 20, and 25 days were cultured in plastic dishes and extracted with Triton X-100 (0.1 percent w/v) or sodium deoxycholate (2 percent w/v). Residues left after extraction were found to contain three proteins characteristic of extracellular matrix (fibronectin, collagen IV, and laminin). These proteins were identified by four methods: indirect immunofluorescence, co-migration with standard proteins on electrophoresis in polyacrylamide gels, immunoblotting (Western blots), and immunoprecipitation after incubating the Sertoli cells with [35S]methionine. In addition, fibronectin was identified by immunoelectron microscopy with a second antibody conjugated to colloidal gold. In the same cell residues, heparan sulfate was tentatively identified by the first of these methods. The cells used in these studies were shown, by electron microscopy, to be essentially pure cultures of Sertoli cells (greater than 95% pure). Since 100 percent of the cells examined showed positive and specific immunofluorescent staining with well-characterized antibodies to the four components of the extracellular matrix, and since studies with colloidal gold revealed the presence of fibronectin closely associated with and inside cells identified by electron microscopy as Sertoli cells, it must be concluded that Sertoli cells synthesize these four proteins and presumably heparan sulfate. Evidently, cultured Sertoli cells can synthesize and secrete some of the components of an extracellular matrix.     

Activity of matrix metalloproteinase-9 against native collagen types I and III

Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain. MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III. Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37 degrees C and 25 degrees C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that it was MMP-9-derived. Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters/one-quarter locus was the site of initial attack. Digestion of type III collagen generated a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage. These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.     

Differential effect of hypertonic initiation block on the synthesis of collagen chains by cultured chick embryo cells

The major type of collagen synthesized by fibroblasts or bone cells, type I collagen, consists of two chains normally found in a 2:1 ratio designated alpha 1(I)2 alpha 2(I) or more simply alpha 1(I)2 alpha 2. I have analyzed the relative synthesis of type I chains in these cells under conditions which reduce the initiation of protein synthesis. It was found that in bone cells, which make a large amount of collagen, the alpha 1(I):alpha 2 ratio is unaltered whereas in fibroblasts, which make smaller amounts of collagen, the alpha 2 chain is particularly sensitive to these same conditions. Examination of the collagen secreted into the medium, under these same conditions, also revealed an altered chain ratio from cells making low amounts of collagen.     

Immunolocalization of collagen types I and III, tenascin, and fibronectin in intramembranous bone.

Structural components of the organic bone matrix were located by immunohistochemical techniques in fresh-frozen sections of normal and dysplastic bone. Fine and coarse birefringent fibers were identified as separate and distinctive features in the extracellular matrix by antibodies raised against human collagen Type III. The glycoprotein tenascin was located on a proportion of the fibers in a characteristic beaded pattern, which was absent in dysplastic bone. The fibers originated in the periosteum or in the fibrous stroma of the marrow cavity and were oriented with regard to both the spatial and the lamellar organization of the bone. The disposition and composition of the fibers suggests that they form a preliminary framework on which intramembranous bone modeling proceeds, and that the specific location of tenascin on the fibers in normal developing membrane bone may be important in determining the alignment of the bone tissue. Epitopes recognized by the collagen Type I and fibronectin antibodies were demonstrated throughout the mineralized matrix, but their incorporation into the collagen "Type III" fibers was evident only outside the mineralized matrix.     

Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis

The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.     

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [³H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K_m = 370 μM and V_max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na⁺ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (K_m = 12 μM; V_max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N⁶-benzyladenosine. In decreasing order of potency, N⁶-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.     

Analysis of extracellular matrix proteins produced by cultured cells

The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.     

Studies of fibronectin synthesized by cultured chick hepatocytes

We have adapted a chick embryo liver cell system for studying the synthesis of proteins secreted by hepatocytes. In primary liver cell cultures maintained for several days in arginine-deficient medium containing ornithine (0.7 mM) and carbamyl phosphate (1 mM), only hepatocytes demonstrated normal morphological and biosynthetic characteristics, indicating that they possessed a functional ornithine cycle as a source of arginine production. Non-parenchymal liver cells, such as fibroblasts, which lack the ornithine cycle were excluded. Hepatocytes in arginine-deficient or arginine-containing medium synthesized fibronectin (Fn) over several days at a constant rate of 3 micrograms +/- 1 microgram/mg cell protein per day, with fibronectin representing approximately 3% of the total secreted hepatocyte proteins during any culture period after the first 24 h. Pulse-chase experiments indicated that Fn synthesis and secretion was relatively rapid (t1/2 = 45 min) and represented approximately 95% of the intracellularly labelled Fn. This Fn is secreted predominantly as a 450 kD dimer with a subunit size that is indistinguishable from the plasma form as assessed by one-dimensional electrophoretic analysis. Continuous exposure of hepatocytes to insulin caused a moderate decrease (26%) in Fn synthesis, whereas there was no effect of short-term exposure. In contrast, dexamethasone stimulated Fn production 2-3-fold, consistent with its known ability to stimulate hepatocyte production of acute phase proteins. Under these conditions, electrophoretic analyses showed that an increased quantity of intact hepatocyte Fn was produced having the same molecular size of plasma Fn.     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

Study of some enzyme activities in cultured chick embryo brain nerve cells treated by chick embryo brain extracts

Brain extracts from 8-day-old chick embryos have been shown to influence morphological development of dissociated brain cells from 7-day-old chick embryos in culture. Stimulatory, effects on size of the neuronal somas and on growth of long processes were observed by adding the cytosol of the brain extract or the dialysate of the cytosol. These morphological changes parallel modifications of various enzyme activities according to the age of the cultures. Adenyl cyclase, (Na⁺, K⁺)- and Mg²⁺-ATPase, 5-nucleotidase, choline acetyltransferase, and acetylcholinesterase activities were studied between 5 and 14 days of culture. Adenyl cyclase activity was strongly stimulated at 8 days by both extracts. (Na⁺, K⁺)-and Mg²⁺-ATPase activities were stimulated in 8-day-old cultures only by the dialysate. 5-Nucleotidase activity was stimulated in 8-day-old cultures by the dialysate and in 11-day-old cultures by both extracts. Choline acetyltransferase activity was stimulated by the cytosol in 8-day-old cultures and by the dialysate in 11-day-old cultures. The total acetylcholinesterase activity was higher in 8-, 11-, and 14-day-old cultures treated with the cytosol. When the cells were treated with the dialysate, the activity was only higher in 14-day-old cultures. We also found that following the addition of brain extracts, the specific activity of the enzymes we studied was enhanced and became close to the values found in vivo during embryogenesis. Thus in parallel to the morphological modifications observed in nerve cell cultures treated by embryo brain extracts, biochemical variations especially involved in synaptogenesis and membrane development could be measured.     

Denervation does not change the ratio of collagen I and collagen III mRNA in the extracellular matrix of muscle

Denervation or inactivity is known to decrease the mass and alter the phenotype of muscle and the mechanics of tendon. It has been proposed that a shift in the collagen of the extracellular matrix (ECM) of the muscle, increasing type III and decreasing type I collagen, may be partially responsible for the observed changes. We directly investigated this hypothesis using quantitative real-time PCR on muscles and tendons that had been denervated for 5 wk. Five weeks of denervation resulted in a 2.91-fold increase in collagen concentration but no change in the content of collagen in the muscle, whereas in the tendon there was no change in either the concentration or content of collagen. The expression of collagen I, collagen III, and lysyl oxidase mRNA in the ECM of muscle decreased (76 +/- 1.6%, 73 +/- 2.3%, and 83 +/- 3.2%, respectively) after 5 wk of denervation. Staining with picrosirius red confirmed the earlier observation of a change in staining color from red to green. Taken with the observed equivalent decreases in collagen I and III mRNA, this suggests that there was a change in orientation of the ECM of muscle becoming more aligned with the axis of the muscle fibers and no change in collagen type. The change in collagen orientation may serve to protect the smaller muscle fibers from damage by increasing the stiffness of the ECM and may partly explain why the region of the tendon closest to the muscle becomes stiffer after inactivity.     

Immunofluorescent localization of collagen types I, II, and III in the embryonic chick eye.

The appearance and distribution of type I, II, and III collagens in the developing chick eye were studied by specific antibodies and indirect immunofluorescence. At stage 19, only type I collagen was detected in the primary corneal stroma, in the vitreous body, and along the lens surface. At later stages, type I collagen was located in the primary and secondary corneal stroma and in the fibrous sclera, but not around the lens. Type II collagen was first observed at stage 20 in the primary corneal stroma, neural retina, and vitreous body. It was particularly prominent at the interface of the neural retina and vitreous body and, from stage 30 on, in the cartilaginous sclera. The primary corneal stroma consisted of a mixture of type I and II collagens between stages 20 and 27. Invasion of the primary corneal stroma by mesenchyme and subsequent deposition of fibroblast-derived collagen corresponded with a pronounced increase of type I collagen, throughout the entire stroma, and of type II collagen, in the subepithelial region. Type II collagen was also found in Bowman's and Descemet's membranes. A transient appearance of type III collagen was observed in the corneal epithelial cells, but not in the stroma (stages 20–30). The fully developed cornea contained both type I and II collagens, but no type III collagen. Type III collagen was prominent in the fibrous sclera, iris, nictitating membrane, and eyelids.     

Heterogeneous distribution of the precursor of type I and type III collagen and fibronectin in the rough endoplasmic reticulum of palatal mesenchymal cells of the mouse embryo cultured in ascorbate-depleted medium

Summary In order to examine the intracellular distribution of precursors of type I and type III collagen and fibronectin in the palatal mesenchymal (MEPM) cells of the mouse embryo cultured under ascorbate-deficient conditions, immuno-electron-microscopic studies were carried out by use of affinity purified antibodies for these proteins. MEPM cells were obtained from the palatal shelves of 14-day-old mouse fetuses and cultured for 3–7 days in medium, either with or without 50 ng/dish/day ascorbic acid. Results obtained were as follows: (1) Although the rough endoplasmic reticulum (rER) of MEPM cells cultured for 5 days in ascorbate-supplemented medium was flattened, that in cells cultured in ascorbate-deficient medium had a distended or vesicular appearance. (2) Vesicular or distended rER showed heterogeneous staining for both type I and type III collagen, namely, some parts of rER showed positive staining for both types of collagen, while others showed negative staining. (3) Both type I and type III collagen showed codistribution in the same vesicular rER. (4) Vesicular rER showed negative or very faint labelling for fibronectin. These results may suggest regional differences in the function of rER.