Compartmentalization of proline pools and apparent rates of collagen and non-collagen protein synthesis in arterial smooth muscle cells in culture.

Rates of collagen and non-collagen protein synthesis in rabbit arterial smooth muscle cells (SMC) were determined by using the specific (radio)activity of [3H]proline in the extracellular, intracellular, and prolyl-tRNA pools. The intracellular free proline specific activity was only 25% of the extracellular value in cultures incubated for 12 h in 0.25 mM-proline. The specific activity of prolyl-tRNA was less than 10% of the extracellular specific activity. Increasing the extracellular proline concentration 10-fold (to 2.5 mM), while keeping the extracellular specific activity of proline constant, resulted in equilibration of the specific activities of intracellular and extracellular free proline, but the specific activity of prolyl-tRNA remained at less than 10% of the extracellular specific activity. Therefore, calculated rates of collagen and non-collagen protein synthesis were greatly underestimated using the intracellular or extracellular specific activity of proline. SMC were also incubated with 0.1 mM-[14C]ornithine in 0.25 nM or 2.5 mM non-labelled proline to examine synthesis de novo of proline and prolyl-tRNA from ornithine. In SMC cultures containing 0.25 mM unlabelled proline, the specific activity of intracellular ornithine was approx. 45% of the extracellular specific activity, due to the production of unlabelled ornithine. The specific activity of ornithine-derived intracellular free proline in SMC incubated with 2.5 mM-proline was significantly lower than in SMC incubated in 0.25 mM-proline, due to the influx of unlabelled proline. However, a corresponding difference in the specific activity of [14C]prolyl-tRNA between SMC in 0.25 mM- or 2.5 mM-proline was not observed. Ornithine-derived [14C]proline was incorporated into proteins in a manner different from that of exogenously added radiolabelled proline. A much higher proportion of the proline synthesized de novo was channelled into collagen synthesis relative to total protein synthesis. Together, these results show that intracellular proline pools are highly compartmentalized in arterial SMC. They also suggest that proline synthesized from ornithine may enter a prolyl-tRNA pool separate from that of proline entering from the extracellular medium.     

Rates of collagen synthesis in lung, skin and muscle obtained in vivo by a simplified method using [3H]proline.

Methods for measurement of rates of collagen synthesis in vivo have thus far been technically difficult and often subject to quite large errors. In this paper a simplified method is described for obtaining synthesis rates of collagen and non-collagen proteins, for tissues of rabbits. This involves an intravenous injection of [3H]proline, administered with a large dose of unlabelled proline, and measurement of the specific radioactivity of proline and hydroxyproline in body tissues up to 3 h later. The specific radioactivity of [3H]proline in plasma and the tissue free pools rises rapidly to a plateau value which is maintained for at least 2 h, when the specific radioactivity of the type I collagen precursors, isolated from the skin, was similar to that of the plasma and tissue-free pool. Furthermore, over this period, the increase in the specific radioactivity of proline in collagen and non-collagen protein was linear with respect to time. These results suggest that the large dose of proline floods the precursor pools for protein synthesis, and that this effect can be maintained for quite long periods of time. Such kinetics greatly simplified the method for obtaining collagen synthesis rates in vivo, which were calculated for lung, heart, skin and skeletal muscle, and shown to be quite rapid, ranging between about 3 and 10%/day. The lung was a particularly metabolically active tissue, with synthesis rates of about 10%/day for collagen and 35%/day for total non-collagen proteins, indicating rapid turnover of both intracellular and extracellular proteins of this tissue.     

Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro.

Analysis of pepsin-resistant proteins produced in culture by monkey aortic smooth muscle cells (SMC) indicates the synthesis of types I and III collagen. As determined by carboxymethylcellulose chromatography and disc gel electrophoresis, SMC cultures synthesize more type III collagen than monkey skin fibroblast cultures; aortic adventitial cell cultures (a mixture of SMC and fibroblasts) synthesize an intermediate amount of type III collagen. Both types I and III procollagens can also be isolated from the culture medium of SMC and skin fibroblasts. The procollagens were separated by diethylaminoethylcellulose (DEAE-cellulose) chromatography in identified by electrophoresis and after cleavage with pepsin and cyanogen bromide. Quantitation of the procollagen by DEAE-cellulose chromatography suggests that 68% of the SMC procollagens and less than 10% of the skin fibroblast procollagens are type III. On the other hand, estimation of the proportions of collagen types secreted by cells, employing pepsin digestion of cell culture medium at 15 degrees C, leads to an underestimation of the amount of type III collagen relative to type I. SMC and fibroblasts may differ in their ability to convert type I procollagen to collagen ad indicated by the observation that skin fibroblast culture medium contains both pN and pC collagen intermediates after 24 h, while cultures of SMC essentially lack the pC collagen intermediates.     

A specific decrease in collagen synthesis in acutely fasted, vitamin C-supplemented, guinea pigs

Weight loss often results from various experimental conditions including scurvy in guinea pigs, where we showed that decreased collagen synthesis was directly related to weight loss, rather than to defective proline hydroxylation (Chojkier, M., Spanheimer, R., and Peterkofsky, B. (1983) J. Clin. Invest. 72, 826-835). In the study described here, this effect was reproduced by acutely fasting normal guinea pigs receiving vitamin C, as determined by measuring collagen and non-collagen protein production after labeling tissues in vitro with [3H]proline. Collagen production (dpm/microgram of DNA) decreased soon after initiating fasting and by 96 h it had reached levels 8-12% of control values. Effects on non-collagen protein were much less severe, so that the percentage of collagen synthesis relative to total protein synthesis was 20-25% of control values after a 96-h fast. These effects were not due to changes in the specific radioactivity of free proline. Refeeding reversed the effects on non-collagen protein production within 24 h, but collagen production did not return to normal until 96 h. The effect of fasting on collagen production was independent of age, sex, ascorbate status, species of animal, and type of connective tissue and also was seen with in vivo labeling. Pulse-chase experiments and analysis of labeled and pre-existing proteins by gel electrophoresis showed no evidence of increased collagen degradation as a result of fasting. Procollagen mRNA was decreased in tissues of fasted animals as determined by cell-free translation and dot-blot hybridization with cDNA probes. In contrast, there was no decrease in translatable mRNAs for non-collagen proteins. These results suggest that loss of nutritional factors other than vitamin C lead to a rapid, specific decrease in collagen synthesis mainly through modulation of mRNA levels.     

Rapid intracellular degradation of newly synthesized collagen by bone cells Effect of dichloromethylenebisphosphonate

Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesizes collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [³H]proline into collagenase-digestible proteins or by the conversion of [³H]proline into [³H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation.     

A microassay to quantitate collagen synthesis by cells in culture

A method to quantitate collagen synthesis, total protein synthesis, and DNA in 24-well culture plates is presented. Collagen-producing cells such as human intestinal smooth muscle cells and dermal fibroblasts were pulse-labeled with [3H]proline. After incubation, the plates were heated to 90 degrees C to stop isotope incorporation and sonicated to lyse the cells and an aliquot was removed for DNA quantitation. Carrier protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was removed by repeated precipitations. After incubation with purified bacterial collagenase, both the soluble 3H-labeled collagen-derived peptides and the remaining insoluble 3H-labeled noncollagen protein were quantified. Results were expressed as the amount of radioactivity incorporated into collagen and noncollagen protein per nanogram DNA and also as the percentage of collagen synthesis per total protein synthesized. The advantage of this technique over previous attempts to scale down the assay is that the entire assay for DNA, collagen, and non-collagen protein can be carried out in the same well without any transfer of material. This technique also provides a significant savings of culture medium, serum, growth factors, and cell material.     

Collagen synthesis in explants from rat intestine

Collagen is the major structural component of the intestinal wall and its metabolism is of special interest for intestinal strength. We describe collagen synthesis in short-term (3 h) incubations of rat intestinal tissue, as measured in terms of incorporation of [3H]proline in collagenase-digestible protein and percentage relative collagen synthesis. In this time span, incorporation of [3H]proline in collagen increases linearly with time and tissue weight. Addition of unlabeled proline during incubation, in excess of the 0.1 microM [3H]proline always present, strongly increases both total protein and collagen synthesis, suggesting that proline transport is rate limiting. Further experiments have been performed in the presence of labeled proline alone and with the addition of 0.35 mM unlabeled proline. Collagen synthesis is significantly higher in colon than in ileum, comprising 0.37 and 0.21%, respectively, of total protein synthesis. Also, collagen synthesis decreases considerably with age, both in ileum and colon. The results presented here demonstrate that rat intestinal explants synthesize measurable amounts of collagen in vitro and that the system used is able to detect changes in in vivo synthetic capability such as those induced by ageing.     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor

Pinocytosis was measured in monkey aortic smooth muscle cells (SMC), bovine aortic endothelial cells, and Swiss 3T3 cells in culture as cellular uptake of [U-(14)C]sucrose and horseradish peroxidase (HRP) from the tissue culture medium. Monkey arterial SMC and Swiss 3T3 cells were maintained in a quiescent state of growth at low cells density in medium containing 5 percent monkey plasma-derived serum (PDS). Replacement of PDS with 5 percent monkey whole blood serum (WBS) from the same donor, or addition to PDS of partially purified platelet-derived growth factor(s) (PF), resulted in a marked stimulation of pinocytosis as well as of cellular proliferation. In SMC, enhancement of the rate of pinocytosis occurred 4-6 h after exposure to WBS or PF, and the rate was up to twofold higher than the rate in medium containing PDS. In contrast, [(3)H]thymidine uptake by SMC did not increase until 12-16 h after exposure to PF. In endothelial cells the presence of PF or WBS did not enhance either the rate of pinocytosis or the rate of proliferation over that in PDS. Thus, endothelial cells did not become quiescent at subconfluent densities in PDS but maintained rates of proliferation and pinocytosis that were equivalent to those in WBS. By autoradiography, the fraction of labeled nuclei in SMC cultures 24 h after change of medium increased from 0.061 +/- 0.004 in quiescent cultures to 0.313 +/- 0.028 after exposure to WBS or PF. In contrast, labeling indices of endothelial cells were similar for cultures grown in PDS, WBS, or PF at any single time point after change of medium. These findings suggest that the rate of pinocytosis maybe be coupled in some fashion to growth regulation, which may be mediated in part by specific growth factors, such as that derived from the thrombocyte.     

Extracellular matrix metabolism by chondrocytes: VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[³H]proline into hydroxyproline and [³H]acetate into glycosaminoglycans, was shown to be depressed by 59% and 39%, respectively, by the addition of exogenous proteoglycan at a concetration of 10 mg/ml growth media. The incorporation of L-[³H]proline into acid-in-soluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity of the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-traslational modification of collagen and proteoglycan.     

High-performance liquid chromatographic quantitation of collagen biosynthesis in explant cultures

The capacity of lung explant cultures to synthesize collagen can be estimated by determining the content of [³H]hydroxyproline in protein following incubation with [³H]proline. The technique requires acid hydrolysis followed by quantitative separation of hydroxyproline from proline for scintillation counting and is often restricted to methods that can accommodate large samples because of relatively low specific radioactivity. A method which is useful for such samples, providing rapid separation of nonderivatized amino acids by ion-exchange HPLC, is described here. The HPLC system employs an HPX-87C cation-exchange column in 10 mm calcium acetate, pH 5.5, at 85°C. Under isocratic conditions hydroxyproline is completely resolved from proline with quantitative recovery of the ³H cpm applied to the column. Large amounts of material, equivalent to at least 150 mg wet wt of lung, can be applied without affecting resolution or recovery, and samples can be injected at intervals as short as 40 min. This method was used to study collagen biosynthesis in a model of pulmonary fibrosis induced in rabbits by the tumor-promoting agent, phorbol myristate acetate (PMA), and provides information concerning total protein synthesis as well as production of collagen. The data show a doubling in the rate of collagen production in lung explants prepared from animals treated with PMA compared with explants from control animals.     

Studies on the effect of l-3,4-dehydroproline on collagen synthesis by chick embryo polysemes

Various proline analogs have been tested in vitro for their ability to inhibit the enzymatic aminoacylation of tRNA by proline. Of these, l-3,4-dehydroproline is the most potent inhibitor. This inhibition is competitive; the K_i is 100 μm. It was shown that l-3,4-dehydroproline can serve as substrate in the aminoacylation reaction. However, the incorporation of radioactivity from l-3,4-[¹⁴C]dehydroprolyl-tRNA into protein occurs at one-fifth the rate observed for l-prolyl-tRNA. The addition of l-3,4-dehydroproline in vitro inhibits the synthesis of collagen to a greater extent than non-collagen protein.     

Synthesis of procollagen by odontogenic cells of rabbit tooth germ

Studies were performed to determine whether cultured odontogenic cells from rabbit tooth germ (RP cell) could synthesize dentine-like collagen. When cells were cultured with [¹⁴C]proline, 33% of the total incorporated proteins present were collagenous. Cultured RP cells were labelled with [¹⁴C]proline in the presence of β-aminopropionitrile. The resulting fractions, on analysis by CM-cellulose chromatography, contained three radioactive protein peaks, α1(I), [α1(III)]₃, α2. From the radioactive measurements, RP cells synthesized a significant amount of type III collagen, comparable to type I collagen.DEAE-cellulose chromatography was used to separate collagen molecules from collagen precursors. The results showed that 60% of total collagen precursor was type III precursor and the remainder was type I precursor.CM-cellulose chromatography of CNBr peptides of collagen from culture medium and cell extract revealed the presence of type I and type III collagen. Thus, the RP cell, which is a diploid cell, is unique in the predominance of type III collagen in culture, differing thereby from the character of collagen in vivo.     

The synthesis and secretion of collagen by cultured sea urchin micromeres

Circumstantial evidence in several previous studies has suggested that sea urchin embryo micromeres, the source of primary mesenchyme cells which produce the embryonic skeleton, contribute to the extracellular matrix of the embryo by synthesizing collagen. A direct test of this possibility was carried out by culturing isolated micromeres of the sea urchin Stronglyocentrotus purpuratus in artificial sea water containing 4% (v/v) horse serum. Under these conditions the micromeres divide and differentiate to produce spicules with the same timing as intact embryos. Collagen synthesis was determined by labeling cultures with [3H]proline or [35S]methionine and the medium and cell layer were assayed for collagen. The results indicate that by the second day in culture micromeres synthesize and secrete a collagenase-sensitive protein doublet with a molecular weight of about 210 kDa. Densitometry indicates a 2:1 ratio of the respective bands in the doublet which is characteristic of Type I collagen. The doublet is insensitive to digestion with pepsin. This differential sensitivity is characteristic of collagen. Over 90% of the collagen synthesized by micromeres is soluble in the seawater culture medium. On days 2-4 in culture, collagen accounts for 5% of the total protein synthesized and secreted. Additional collagenase-sensitive bands are noted at 145 and 51 kDa. The relationship of the described collagen metabolism to previously characterized collagen gene expression in sea urchin embryos is discussed.     

Expression of the heterogenous nuclear ribonucleoprotein complex K protein and the prolyl-4-hydroxylase alpha-subunit in atherosclerotic arterial smooth muscle cells.

Smooth muscle cells (SMC) play a major role in the formation of atherosclerotic lesions found on major blood vessels. SMC proliferation, migration, and protein synthesis promote the progression of the early lesion, the fatty streak, into a complex myointimal fibrous plaque. To investigate altered gene expression in SMC during atherogenesis, we characterized differences between SMC from normal rabbits, rabbits fed a 2% cholesterol diet, and Watanabe Heritable Hyperlipidemic rabbits (WHHL). We detected and isolated a 501 bp cDNA fragment representing the A isoform of heterogenous nuclear ribonucleoprotein complex K (hnRNP-K) and a 281 bp cDNA fragment representing the prolyl-4-hydroxylase alpha-subunit (alphaPH) mRNAs. hn-RNP-K was upregulated in SMC from cholesterol-fed rabbits isolated in primary culture, as well as in SMC medial tissue from both the cholesterol-fed and WHHL rabbits. alphaPH was upregulated in SMC from the cholesterol-fed rabbits isolated in primary culture and in the tissue from WHHL rabbits. These data demonstrate genes consistent with increased proliferation and collagen production are upregulated in SMC during atherogenesis and may shed new light on gene expression changes and corresponding phenotype changes in SMC during atherogenesis.     

Activation of collagen synthesis in primary culture of rat liver parenchymal cells (hepatocytes)

An increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high-molecular-weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (alpha A and alpha B) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. Immuno-electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day-8 hepatocytes in primary culture.     

Influence of serum cholesterol on atherogenesis and intimal hyperplasia after angioplasty: inhibition by amlodipine

The objectives of the present study were to determine whether serum hypercholesterolemia (HC) promotes the development of spontaneous and angioplasty-induced lesions and whether amlodipine inhibits these lesions and cellular processes underlying their genesis. Rabbits were fed normal, 0.5%, or 2% cholesterol diets for 9 wk, which resulted in the development of increasing HC. After week one, balloon dilation of the abdominal aorta was performed while the thoracic aorta was not disturbed and monitored for the development of spontaneous lesions. Lesion size increased with the degree of HC and was accompanied by increased collagen synthesis and smooth muscle cell (SMC) proliferation at each site. Amlodipine (5 mg/kg p.o.) inhibited lesion size by 50% (P < 0.01) at both sites in cholesterol-fed animals but not at angioplasty sites in animals on a normal diet. Local collagen synthesis was inhibited at both sites by amlodipine in the diet animals. The increase in HC was accompanied by a 1.7-fold increase in basal Ca2+ uptake in SMCs in the thoracic aorta, which was not altered by amlodipine, nifedipine, Ni2+, or La3+, revealing an uninhibitable calcium leak during atherogenesis. In culture, cholesterol enrichment increased SMC proliferation, collagen synthesis, and the secretion of a soluble SMC mitogen, which were inhibited by amlodipine (10(-9) M). Finally, in SMC membranes, amlodipine uniquely restored the cholesterol-expanded membrane bilayer width without any effect on membrane fluidity. This study establishes a causal role between serum HC and the development of spontaneous and angioplasty-induced lesions and the ability of amlodipine to disrupt this action by a novel remodelling action on the SMC membrane.     

Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation, and maturation

Collagen metabolism was studied in the regenerating forelimbs of adult newts (Notophthalmus viridescens) with respect to the pattern of accumulation relative to total protein accretion, maturation, and rate of biosynthesis. Measurements of collagen and noncollagen protein in regenerating limbs at various stages indicate that a preferential enrichment in collagen occurs at two periods correlating with (1) the onset of differentiation and chondrogenesis and (2) the initial period of elongation and outgrowth following morphogenesis. The maturation of collagen was determined by measuring the distribution of collagen into acetic acid soluble and insoluble forms. Soluble collagen increased to 30% during the differentiative period, remained at a high level during digit-formation, and decreased progressively following morphogenesis.Tracer studies were performed to determine whether the net accumulation of collagen resulted from a preferential increase in collagen biosynthesis. Separation of collagen and noncollagen proteins labeled in vivo with [³H]proline was performed enzymatically using purified clostridial collagenase. Rates of incorporation of proline into collagen relative to noncollagen proteins did not vary significantly during regeneration, although a threefold increase in incorporation rates into both species occurs at the onset of differentiation. Collagen synthesis constitutes 7–11% of the total protein synthesis in the regenerate. Estimates of variations in the absolute rates of protein synthesis, based on endogenous levels of proline, indicate that the highest rates of protein synthesis occur during morphogenesis. The relationship between protein content and relative rates of synthesis suggests that the net accumulation is governed by variations in rates of degradation. The relationship between collagen content and solubility also suggests that the rate of insolubilization plays a role in the net accumulation of collagen.     

Effects of transforming growth factor-β on collagen gene expression and collagen synthesis level in mineralizing cultures of osteoblast-like cell line,MC3T3-E1

• 1.1. High levels of type I collagen mRNA and [³H]proline incorporation into collagenase digestable protein by MC3T3-E1 cells were detected during the first 7 days of culture, after which they declined.
• 2.2. Type I collagen gene expression was stimulated by TGF-β in the early culture stage when the collagen gene expression was fully functioning.
• 3.3. However, these stimulatory effects disappeared at the differentiation stages. Although collagen gene expression was stimulated by TGF-β (2.0 ng/ml) in early culture, collagen synthesis in medium was not.
• 4.4. This study shows that collagen synthesis and collagen gene expression were affected by the state of differentiation in MC3T3-E1 cells and that the rate of stimulation by TGF-β in collagen gene expression decreased over time in culture.