Collagenase-sensitive Protein Research Articles

Estrogen regulation of alpha 1(I)-procollagen messenger ribonucleic acid in the rat uterus.

A cDNA library, prepared from mRNA isolated from the uteri of 3-day estradiol-stimulated immature rats, was constructed in pBR322. From this library an estrogen-regulated clone, pERU3, was isolated. This clone contained sequences complementary to uterine mRNA that migrated during gel electrophoresis as a double band of about 5.0 and 5.8 kilobases. Little of this mRNA was seen in several other tissues examined. An increase in the amount of this RNA in uterus was seen 2 h after estradiol treatment, with maximum hybridization occurring, in different experiments, between 18 and 36 h, followed by a decline. Hybridization of the cDNA insert of the pERU3 plasmid with known probes indicated that it coded for alpha 1(I)-procollagen. This conclusion was supported by in vitro translation experiments in which the hybrid-selected mRNA complementary to pERU3 DNA was shown to code for a collagenase-sensitive protein with a size corresponding to that of alpha 1(I)-procollagen. This system, therefore, provides an additional tool for the study of the estrogen regulation of gene expression in the uterus.

Colchicine does not Provide a Sustained Blockage of Collagen and Plasma Protein Secretion by Rat Hepatocytes

Colchicine has been reported to disrupt microtubules and thereby inhibit collagen secretion. Because of this “Canti-collagen” activity, colchicine has been suggested for use in the treatment of hepatic fibrosis. Using biochemical and immunohistochemical techniques, our laboratory has identified the hepatocyte as one possible source of collagen in the liver. The present study examined the direct effect of colchicine on collagen secretion by hepatocytes in culture. Parenchymal cells were isolated from the livers of adult rats four days following a two-thirds hepatectomy. Total collagen and the fraction secreted into the medium were quantitated as incorporation of [3H]proline into bacterial collagenase-sensitive protein. Treatment of the hepatocytes with 100[,M colchicine (2-3 hours) resulted in a substantial inhibition of collagen secretion. However, upon longer exposure of the hepatocytes to the drug (24 hours and 8 days), the inhibitory effect on collagen secretion was abolished. The anti-protein secretion activity of the colchicine in the conditioned medium removed from the hepatocytes was still present as verified by a 2.5 hour fibroblast-collagen secretion bioassay. The secretion of the plasma proteins albumin, fibrinogen and the third component of complement was not altered by the presence of colchicine. We conclude that the hepatocyte is a highly efficient secretory cell, and is not entirely dependent upon microtubules as organelles for protein secretion. Therefore, to the extent that hepatocytes may contribute to hepatic fibrosis, the therapeutic use of colchicine to block collagen secretion might be expected to have only limited effectiveness.

Electrophoretic characterisation of human parotid saliva protein fractions isolated by preparative isoelectric focusing

AbstractHuman parotid saliva proteins fractionated by preparative isoelectric focusing into 3 main fractions have been characterised electrophoretically and immunochemically. The anodic proteins constitute a polymorphic family of collagenase and trypsin sensitive, immunochemically related, phosphoproteins. Many of these contain a common polypeptide of Mr 23 000 either alone or in combination with other lower (Mr 21 000) or higher (Mr 25 000) molecular weight polypeptides. Parotid amylase was substantially purified by this technique and an antiserum against these fractions reacted monospecifically with amylase in a number of systems. The cathodic proteins are all collagenase sensitive proteins with Mr values of 12 500 to 77 000.

Dermal wound repair: Role of collagen matrix implants and synthetic polymer dressings

The effects of two different polymeric wound dressings and a new collagen matrix (CM) implant on the healing and scarring of full-thickness excision wounds were studied in swine. The synthetic polymers comprised an occlusive O 2-impermeable hydrocolloid dressing (HCD) and an occlusive O 2-permeable Polyurethane film (PUF). The CM implant consisted of an acellular collagen sponge fabricated from purified bovine tendon type I collagen. Wounds were evaluated for granulation tissue-production capacity by measuring 14C proline incorporation into collagenase-sensitive protein. Epidermal resurfacing and wound contraction were measured by computerized morphometric image analysis of wounds made on a tattooed grid. In comparison with air-exposed wounds, the relative collagen synthetic capacity was greater in the granulation tissue of wounds treated with HCD, PUF, or CM with occlusion. Both HCD and PUF accelerated by 40% the epidermal resurfacing over the granulating wound bed. Wound contraction was significantly reduced by CM but was not altered by the occlusive dressings.

Pathogenesis of Tumor Desmoplasia. II. Collagens Synthesized By Line 1 And Line 10 Guinea Pig Carcinoma Cells And By Syngeneic Fibroblasts In Vitro<xref ref-type="fn" rid="FN2">2</xref>

For the investigation of the pathogenesis of desmoplasia, the capacities to synthesize collagen in vitro of 2 bile duct carcinomas (lines 1 and 10) of Sewall-Wright inbred strain 2 guinea pigs and of syngeneic dermal fibroblasts were studied. Line 10 cells synthesized collagen type IV as judged by sensitivity to bacterial collagenase, by immunoprecipitation, by migration of pro alpha (IV) chains and pepsin-resistant fragments on sodium dodecyl sulfate-polyacrylamide gels, and by immunofluorescence. Line 1 cells also synthesized small amounts of collagenase-sensitive protein. Neither line 1 nor line 10 cells synthesized detectable collagen type I, III, or V. Only about 1% of [14C]proline incorporated by tumor cells was found in collagenase-sensitive protein. In contrast, dermal fibroblasts synthesized 4 and 128 times as much collagenase-sensitive protein as line 10 and line 1 cells, respectively, amounting to 20% of total protein synthesized. Fibroblasts produced mostly collagen types I and III, in a ratio of 7:1, and smaller amounts of collagen type V. Thus lines 1 and 10 carcinoma cells produce primarily basement membrane collagen, whereas interstitial collagens, abundant in desmoplastic tumor stroma, are fibroblast products.

Growth, synthesis and regional specialization of the embryonic chicken lens capsule

The growth, synthesis and regional specialization of the lens capsule has been studied in chicken embryos and compared to adult chickens and mammals. During the final 15 days of embryonic development the surface area of the capsule increased 11-fold. This represents the minimum estimate of capsule growth, since it also increased in thickness during this period. Autoradiographic examination of [ 3H]proline- or 35SO 4-labeled, detergent-cleaned, flat-mounted capsules suggested that all lens cells participated in capsule synthesis. This was supported by the observation that both lens epithelial and fiber cells incorporated [ 35S]-methionine into collagenase-sensitive proteins with molecular weights similar to type IV collagen. Histochemical staining of detergent-cleaned capsule explants (DCCEs) revealed regional differences in the carbohydrate composition of the capsule. Differences in collagenous proteins were also seen between the anterior and posterior regions of the capsule of the embryonic lenses. Following SDS-PAGE and silver staining a total of six collagenase-sensitive proteins were seen with molecular weights between 150 and 180 K. Three of these polypeptides were common to both anterior and posterior capsules, one was found only in the anterior capsule and the other two were found predominantly in the posterior capsule. No regional differences were seen in the collagenous proteins of capsules from adult chickens, rats or hamsters. The possibility is discussed that the unique pattern of collagenous polypeptides seen in embryonic capsules is related to the rapid growth of the embryonic lens.

Collagen Synthesis in Cultured Human Skin Fibroblasts: Effect of Ascorbic Acid and Its Analogs

In confluent human skin fibroblasts maintained in 0.5% serum-supplemented medium. L-ascorbate specifically stimulated the rate of incorporation of labeled proline into total collagenase-sensitive protein, without changing the specific activity of the intracellular free proline. This influence of ascorbate reached a maximum at 30 microM and continued for at least 4 days, resulting in a 4-fold increase. The ascorbate effect occurred in cells at both confluent and subconfluent densities and was evident at all serum concentrations from 0.5-20%. The effect was independent of duration of the radioactive pulse between 2-6 h. D-Ascorbate, D-isoascorbate, and L-dehydroascorbate also stimulated collagen synthesis but at considerably higher concentrations, i.e., 250-300 microM. The stimulation of collagen synthesis by ascorbate and its analogs was accompanied by a decline in prolyl hydroxylase activity and a rise in lysyl hydroxylase activity; again L-ascorbate was found to be most effective. Dimethyltetrahydropterine and L-lactate failed to produce these effects.

Detection and partial characterization of collagen synthesis stimulating activities in rat mammary adenocarcinomas.

Rat mammary epithelium and adenocarcinomas derived from it synthesize type IV collagen, a structural protein of basement membranes. In cultures of cells, net production of collagen is stimulated 2-fold more than total cell protein by epidermal growth factor. Mammary adenocarcinoma cells also respond to epidermal growth factor but to a much reduced extent. This difference in growth factor responsiveness appears to be due to the production of collagen synthesis stimulating factors by the mammary tumor cells. Such factors have been partially purified and shown to differentially stimulate the incorporation of proline into collagenase-sensitive protein by 2.5-10-fold in normal rat mammary epithelium, normal rat kidney, and mouse 3T3 cells. The tumor factors do not stimulate net collagen production in cultures of tumor cells from which the factors are derived, suggesting that tumor cells produce sufficient stimulatory factors for optimal synthesis of collagen. Pulse-chase studies indicate that the tumor factors stimulate collagen synthesis rather than block collagen turnover. The activities in the extract have been partially purified by gel filtration, ion exchange column chromatography, and isoelectric focusing. The major species has a molecular weight of about 68,000 and a pI of 5.9. A smaller peak of activity with a molecular weight of 6,000 is also present. Since collagen synthesis appears to be necessary for the growth of mammary adenocarcinomas in vivo, production of these collagen synthesis stimulating factors may be important for tumor growth.

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro: I. Characterization of the chondrocyte-specific phenotypes

We have maintained chick embryo chondrocytes in culture for more than 2 months, passaging the floating cells in the absence of ascorbic acid. Throughout the culture period some of the cells attached to the dish, assuming an epithelial-like morphology and subsequently giving rise to new floating cells. The interconversion of the two cell populations was highest in primaries and decreased with the aging of the culture. Cartilage cells synthesized pro-α 1 (II) collagen and sulphated proteoglycans in vitro; compared with floaters, the epithelial-like cells secreted relatively large amounts of fibronectin. When ascorbic acid was added to the medium, all cells attached, maintaining their rounded shape; in this condition the pro-α 1 (II) collagen was matured and collagen fibres were detectable outside the cells. Other specific proteins synthesized by the chondrocytes in culture were also identified. One of these, a 64 K collagenase-sensitive protein, was not related to the type II collagen and may represent a new collagen type.

Structural components and characteristics of Reichert's membrane, an extra-embryonic basement membrane.

This paper presents an analysis of mouse and rat Reichert's membrane, a thick basement membrane formed between the trophoblast and parietal endoderm cells of early mammalian embryos. When analyzed by polyacrylamide gel electrophoresis. Reichert's membranes from rat and mouse conceptuses appear simple and consist primarily of collagen and 4 noncollagenous glycoproteins of approximate Mr = 415,000, 245,000, 170,000, and 50,000. The proteins at 415,000 and 245,000 are similar in molecular weight to laminin and are immunoprecipitated by anti-laminin antiserum. The protein at 170,000 is co-precipitated with laminin and represents a novel form of a laminin-like protein, whereas the protein at 50,000 is unrelated to laminin. Using metabolic labeling experiments, it is shown that parietal endoderm cells synthesize these components and incorporate them into the matrix in organ cultures but do not degrade Reichert's membrane in the conditions used over a 6-day culture period. Evidence is presented that the high molecular weight collagenase-sensitive proteins (greater than 420,000) observed on polyacrylamide gel electrophoresis are due to lysyl oxidase-derived cross-links between Reichert's membrane components.

Effects of inhibition of basement membrane collagen deposition on rat mammary gland development

Mammary ducts and alveoli in culture produce collagen exclusively of the type IV variety. In these culturescis-hydroxyproline selectively blocks the accumulation of collagenase-sensitive protein compared to total cell protein. Associated with this is an inhibition of cell growth. A mammary cell line which does not synthesize collagen was insensitive to the proline analog.In vivo, cis-hydroxyproline was found to cause an involution-like structural alteration of the mammary glands of animals given perphenazine to induce mammary proliferation. Thecis-hydroxyproline was nontoxic, caused no observed change in hormonal status of the animals, and produced no observed changes in the unstimulated mammary gland. The results are interpreted as indicating that the mammary epithelial cell is dependent on the integrity of the basement membrane for viability. A model for the role of basement membrane collagen metabolism in normal mammary physiology is presented.

Changes in the Growth and Metabolism of Cells Cultured from Normal, Sclerotic and Rheumatoid Connective Tissue Brought about by D-Penicillamine and by Sodium Salicylate

D-penicillamine and sodium salicylate were equally effective in suppressing the proliferation of fibroblasts from normal and scleroderma skin and rheumatoid synovial cells. The effects were concentration-dependent, beginning at around 100 microgram/ml, and proliferation was almost halted at 1600 microgram/ml. Individual cell strains of each type showed differences in drug susceptibility but there was no consistent difference between cells from normal and abnormal tissues. Acid mucopolysaccharide secretion was more clearly inhibited in scleroderma fibroblasts than in synovial cells, and penicillamine produced greater inhibition than salicylate, beginning at 10 microgram/ml and reaching almost 50% at 500 microgram/ml. Confluent cultures of scleroderma fibroblasts showed 23% less incorporation of 3H-proline into collagenase-sensitive protein in the presence of 1600 microgram/ml penicillamine, without significant effect on other protein synthesis; sodium salicylate had no effect. These data suggest that D-penicillamine may affect connective tissue metabolism in other ways than by interfering with collagen synthesis or cross-linking.

De novo protein synthesis by human chondrosarcoma in cell and organ culture: evidence of unusually high collagen production by a neoplastic tissue.

Protein synthesis by human chondrosarcoma tissue and normal articular cartilage was studied in organ and primary monolayer cell culture systems. Protein synthesis by cell cultures was evaluated at 2.7 and 21 days after plating. When compared to normal, incorporation of 3H-proline and 35S-methionine into proteins was elevated in chondrosarcoma samples under both culture conditions. Hydroxyproline analyses of tissue hydrolysates indicated that chondrosarcoma samples contained significantly less collagen than normal articular cartilage, yet were incorporating significantly greater amounts of 3H-proline into 3H-hydroxyproline. Collagen production by cell cultures was assessed by digestion of samples with purified clostridial collagenase. Chondrosarcoma cells produced more collagenase-sensitive protein than normal cells at all intervals. SDS polyacrylamide gel analyses of all preparations showed two collagenase-sensitive proteins with apparent molecular weights of 165,000 and 175,000. Decreased synthesis of another major protein, apparent molecular weight 210-220,000 was noted in chondrosarcoma preparations in both culture systems.

The identification of collagen messenger RNA.

RNA isolated from calvaria of 16- to 18- day-old chick embryos, assayed in rabbit reticulocyte lysates, programs the synthesis of a collagenase-sensitive protein with the molecular weight of collagen pro-alpha-chains. When RNA labeled with [(3)H]uridine for 2 hr and chased for 1 or 2 hr was electrophoresed on aqueous polyacrylamide gels, most of the radioactivity not in 28S or 18S rRNA migrated with an apparent molecular weight of about 1,800,000. After oligo(dT)-cellulose chromotography and analysis in 99% formamide gels, this nonribosomal, rapidly labeled calvaria RNA species migrates at 28S-30S and thus has a molecular weight of at least 1,600,000. Both the ability to program the synthesis of collagenase-sensitive protein in reticulocyte lysates and the presence of a single prominent rapidly labeled 30S peak in acrylamide gels strongly support the deduction that there is only one major mRNA species in calvaria and that this species is collagen messenger RNA.