Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts

Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not beta-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled alpha 2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment.     

Coated vesicles purified from chick tendon fibroblasts contain newly synthesized type I procollagen

Coated vesicles were purified from embryonic chick tendon fibroblasts pulsed with [3H]proline. They were morphologically and biochemically similar to coated vesicles purified from other sources. Furthermore, they contained newly synthesized Type I procollagen which was protected from bacterial collagenase digestion unless detergent was present. The procollagen remained associated with coated vesicles during immune precipitation and agarose gel electrophoresis. Data from pulse-chase experiments demonstrated that the specific activity of the coated vesicle preparations was approx. 5-fold higher at the 10 min chase point than at either the 0 or 40 min chase points. These data are consistent with the hypothesis that coated vesicles are intermediates in the intracellular transport of newly synthesized Type I procollagen in chick tendon fibroblasts.     

Generalized inhibition of cell-free translation by the amino-terminal propeptide of chick type I procollagen

Fragments of the amino-terminal propeptide of procollagen have been shown to inhibit the synthesis of procollagen in cultured cells and in a reticulocyte lysate cell-free system (for review see Timpl, R. and Glanville, R.W. (1981) Clin. Orth. Rel. Res. 158, 224-242). In this report, we show that the full-length amino-terminal propeptide of chick pro alpha1(I) chains inhibits the translation of chick tendon mRNA and rat brain mRNA in a reticulocyte lysate cell-free system. The synthesis of procollagen and non-collagenous proteins was equally affected. Inhibition was dose-dependent up to 10 microM. A similar pattern of inhibition was observed for the collagenase-resistant fragment, col 1(I).     

Giantin is required for intracellular N-terminal processing of type I procollagen

Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.     

Surface-induced aggregation of type I procollagen

We have examined the state of aggregation of type I procollagen in the concentration range 5 to 800 micrograms/ml. Electron microscopy typically indicates a high proportion of aggregated material (greater than 50%), when a range of preparative techniques are used. Aggregates of in-register molecules (segment-long-spacing-like aggregates) are frequently observed, often with units of in-register molecules connected via the C-terminal propeptides. In contrast, studies using gel-filtration chromatography and density-gradient ultracentrifugation demonstrate only limited aggregation in solution (less than 5%) even at 800 micrograms/ml. The aggregated material is mainly dimeric and probably not segment-long-spacing-like. We conclude that aggregation of procollagen is strongly favoured by adsorption to a surface when samples are prepared for electron microscopy. The possible relevance of these observations to the fate of procollagen secreted by cells in vivo is discussed.     

D-periodic assemblies of type I procollagen

The solubility limit of purified chick type I procollagen, incubated at 37 degrees C in phosphate-buffered saline, was found to be in the range 1 to 1.5 mg/ml. At higher concentrations large aggregates formed. These comprised: (1) D-periodic assemblies; (2) narrow filaments with no apparent periodicity; and (3) segment-long-spacing-like aggregates. The D-periodic assemblies, which predominated at high concentrations, were separated from the other types of aggregate and found to be ribbon-like. Ribbons were uniform in thickness (approximately 8 nm) and up to 1 micron wide. Staining patterns showed features similar to those in native-type collagen fibrils. Immunolabelling indicated that the carboxyl-terminal propeptide domains were close to the carboxyl-terminal gap-overlap junction, and that the amino-terminal propeptide domains were folded over into the amino-terminal side of the overlap zone. Both propeptide domains appeared to be located on the surface of the assemblies. These observations show that intact propeptide domains hinder, but do not prevent, the formation of D-periodic assemblies. The presence of the propeptide domains on the surface of a growing assembly could restrict its lateral growth and limit its final thickness.     

Assembly and processing of procollagen type III in chick embryo blood vessels

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.     

NADPH oxidase activity is associated with cardiac osteopontin and pro-collagen type I expression in uremia

Abstract Cardiovascular disease is a frequent complication inducing mortality in chronic kidney disease (CKD) patients, which can be determined by both traditional risk factors and non-traditional risk factors such as malnutrition and oxidative stress. This study aimed to investigate the role of oxidative stress in uremia-induced cardiopathy in an experimental CKD model. CKD was induced in Sprague-Dawley rats by a 4-week diet supplemented in adenine, calcium and phosphorous and depleted in proteins. CKD was associated with a 3-fold increase in superoxide anion production from the NADPH oxidase in the left ventricle, but the maximal activity of mitochondrial respiratory chain complexes was not different. Although manganese mitochondrial SOD activity decreased, total SOD activity was not affected and catalase or GPx activities were increased, strengthening the major role of NADPH oxidase in superoxide anion output. Superoxide anion output was associated with enhanced expression of osteopontin (×7.7) and accumulation of pro-collagen type I (×3.7). To conclude, the increased activity of NADPH oxidase during CKD is associated with protein modifications which could activate a pathway leading to cardiac remodelling.     

Sulfoxide stimulation of chondrogenesis in limb mesenchyme is accompanied by an increase in type II collagen enhancer activity

We have utilized a modification of the limb bud mesenchyme micromass culture system to screen compounds that might stimulate chondrogenesis. Two compounds in the sulfoxide family (methylphenylsulfoxide and p-chlorophenyl methyl sulfoxide) were stimulatory at 10(-2) M and 10(-3) M, respectively; whereas other sulfoxides and organic solvents were not active at these concentrations. In addition, specific growth factors (basic FGF, IGF-I, IGF-II) were not chondroinductive at concentrations that are active in other cell systems. Both sulfoxide compounds stimulated cartilage nodule formation, [35S]sulfate incorporation, and activity of the regulatory sequences of the collagen II gene. In contrast, transforming growth factor beta-1 (10 ng/ml) stimulated sulfate incorporation but produced only a diffuse deposition of cartilage matrix and reduced the ability of the cells to utilize the regulatory sequences of the collagen II gene. The sulfoxides appear to promote the differentiation of limb bud cells to chondrocytes and thus exhibit chondroinductive activity.     

Pseudomonas invasion of type I pneumocytes is dependent on the expression and phosphorylation of caveolin-2

Pseudomonas aeruginosa is a major cause of pneumonia in patients with cystic fibrosis and other immuncompromising conditions. Here we showed that P. aeruginosa invades type I pneumocytes via a lipid raft-mediated mechanism. P. aeruginosa invasion of rat primary type I-like pneumocytes as well as a murine lung epithelial cell line 12 (MLE-12) is inhibited by drugs that remove membrane cholesterol and disrupt lipid rafts. Confocal microscopy demonstrated co-localization of intracellular P. aeruginosa with lipid raft components including caveolin-1 and -2. We generated caveolin-1 and -2 knockdowns in MLE-12 cells by using RNA interference techniques. Decreased expression of caveolin-2 significantly impaired the ability of P. aeruginosa to invade MLE-12 cells. In addition, the lipid raft-dependent tyrosine phosphorylation of caveolin-2 appeared to be a critical regulator of P. aeruginosa invasion.     

Quantitation of type II procollagen mRNA levels during chick limb cartilage differentiation

A single-stranded DNA probe complementary to chicken type II procollagen mRNA has been used to quantitate levels of that mRNA present in chicken limb mesenchyme during cartilage differentiation. Excess labeled probe prepared from a cDNA template cloned in M13mp9 was hybridized to completion to increasing amounts of total RNA and assayed by protection from S1 nuclease digestion. Estimates of the absolute levels of type II procollagen RNA were determined using the M13mp9 template containing the coding strand as a standard. RNA complementary to the probe increased from 20 copies per diploid genome in stage 24 limb to approximately 2000 copies per diploid genome in stage 24 limb mesenchyme which had differentiated to cartilage in culture. Similar levels were found in cartilage from stage 31 limb. Sternal cartilage from 17-day embryos contained approximately 10,000 copies per diploid genome suggesting that the level of expression of this gene is different in limb growth cartilage compared with sternal cartilage. Low but detectable levels of RNA complementary to the probe were observed in limb at stages 20-24. Since a large fraction of the type II procollagen RNA in these early limbs is associated with polysomes, the type II procollagen gene appears to be expressed at a low level prior to phenotypic differentiation and prior to the accumulation of immunologically detectable levels of type II collagen.     

Transformation of cultured epithelial cells by ethylnitrosourea: Altered expression of type I procollagen chains

Cultured epithelial rodent cells were transformed in vitro using ethylnitrosourea as a carcinogen either alone or in combination with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The frequency of transformation in the absence of TPA was 5·10^?4 at 10 μg/ml ethylnitrosourea. Growth of ethylnitrosourea-treated cells in TPA-substituted medium increased the transformation frequency 8-fold. Colonies of transformed cells were isolated from soft agar and analyzed for the production of pericellular matrix glycoproteins. The ethylnitrosourea-transformed cells retained pericellular matrix structures, typical of the nontransformed control cells. Parent cells produced into their culture media fibronectin and procollagen types I and III as their major pericellular glycoproteins. The ethylnitrosourea-transformed cells synthesized and secreted altered procollagen polypeptides. The procollagen of ethylnitrosourea-transformed cells apparently consisted mainly of homotrimeric proα1 molecules, with smaller amounts of basement membrane procollagen-like chains. Fibronectin synthesis or secretion was not affected by ethylnitrosourea-induced transformation, but the production of fibronectin was enhanced in the transformed cultures treated with TPA. Also, the deposition of procollagen and fibronectin into the pericellular matrix was not affected by ethylnitrosourea-transformation. Very similar changes had previously been observed in murine sarcoma virus-transformed cells. The change of procollagen type I thus appears to be a correlate of malignant transformation of cultured epithelial cells. The results indicate that ethylnitrosourea can induce malignant transformation of epithelial cells in culture and modify production and deposition of pericellular glycoproteins.     

Translation of chick calvarial procollagen messenger RNA'S by a messenger RNA dependent reticulocyte lysate.

A method was developed for the extraction of RNA from chick embryo calvaria which should be generally applicable to other connective tissues. Total RNA prepared by this method was translated by a mRNA-dependent reticulocyte lysate into discrete pro alpha chains. Several criteria were used to identify these translation products, including (1) preferential labeling with [3H]proline, (2) appropriate migration on sodium dodecyl sulfate-polyacrylamide gels, (3) selective sensitivity to collagenase digestion, and (4) specific precipitability by two different antisera against procollagen. Data from the immunoprecipitation experiments indicated that the majority of the pro alpha chains contained the carboxy-terminal antigenic determinants. These results demonstrate that this translation system can be used as an assay for intact procollagen mRNAs and as a source of in vitro synthesized pro alpha chains for future structural analysis.     

Glucocorticoids decrease the synthesis of type I procollagen mRNAs

Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression.