Collagen metabolism in mouse lung after X irradiation

Collagen and total protein synthesis rates have been determined in the lungs of CBA mice irradiated with single doses of X rays between 8 and 16 Gy. Mice were injected with [3H]proline accompanied by a large dose of unlabeled proline, and synthesis rates were measured at 2-month intervals from 8 to 31 weeks after irradiation. At 2 months after radiation treatment, collagen and total protein synthesis rates were significantly depressed but they had recovered by 4 months. By 6 months collagen synthesis rates had increased above control in a dose-dependent manner, so that in the 14-Gy dose group the fractional synthesis rate for collagen was 4.6 times higher than in control mice as measured by incorporation of [3H]proline. However, a significant net accumulation of collagen was seen only in the lungs of the highest dose group at 31 weeks, as indicated by total hydroxyproline measurements. There was a slight increase in the ratio of types I and III collagen. Late radiation damage in the CBA mouse lung is characterized by increased collagen metabolism, which may or may not lead to a net accumulation of collagen.     

Stability of rat skin collagen during recovery from under-nutrition.

The growth of young rats was arrested for 6 weeks from 48 h after receiving an injection of L-[5-3H]proline. The 3H in the hydroxyproline of the newly synthesized skin collagen remained steady during under-nutrition and did not decrease during the subsequent recovery period. It was concluded that in this animal model the renewed growth did not induce degradation of the pre-existing collagen fibres.     

Inhibition of cell metabolism by a smokeless tobacco extract: tissue and species specificity.

Smokeless tobacco contains a nonnicotine inhibitor of posttranslational modification of collagen (hydroxylation of [3H]proline) by cultured chick embryo tibias and osteoblasts. This study was undertaken to determine whether a methanol extract of smokeless tobacco (STE) containing the inhibitor has similar effects on collagen-producing cells and tissues other than bone. Its effects on DNA synthesis and cell proliferation (incorporation of [3H]thymidine) were also determined. Frontal bone, aorta, and cartilage were incubated for 2 days in medium containing STE. Glycolysis (lactate production) was stimulated by 80% in cartilage, but was not affected in the other tissues; medium alkaline phosphatase activity was unaffected. In frontal bone and cartilage, [3H] hydroxyproline content was decreased 88% and 57%, respectively, and [3H]proline content was decreased 68% and 37%, respectively; neither was affected in the aorta. Confluent cultures of collagen-producing mouse fibroblasts or primary osteoblasts obtained from chick embryo calvarias were incubated for 2 days in medium containing increasing concentrations of STE. Glycolysis and DNA synthesis were not affected. Cell proliferation was unaffected in fibroblasts, but was inhibited (34%) at the highest STE concentration in osteoblasts. AIPase activity was not detectable in fibroblast medium, but was decreased up to 72% in osteoblast medium. Inhibition of collagen synthesis by STE was concentration related in both cell types. At the highest concentration, [3H] hydroxyproline and [3H]proline contents in the cell layers were decreased to the following respective values: fibroblasts 56% and 45% and osteoblasts 50% and 29%, respectively. When incubation with STE was discontinued for 1 day, recovery did not occur. These findings suggest that inhibition of collagen synthesis by STE is not specific for bone, that collagen-producing cells are directly affected, and that recovery is not immediate. This inhibitor could contribute to the periodontal disease often seen in users of smokeless tobacco. Its identification and removal would produce a safer product.     

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.     

Influence of bleomycin on the metabolism of dermal collagen in albino rats

The metabolism of collagen in male rats by treatment with bleomycin was studied following the injection of [3H]proline and the determination of specific and total activity of [3H]hydroxyproline in skin collagen fractions and urine. In the case of the bleomycin-treated animals, there was found to be an increase in the neutral salt soluble collagen content with no change in insoluble collagen content as compared to the control group. The specific and total radioactivity of [3H]hydroxyproline in soluble and insoluble collagen fractions was also increased. Examination of [3H]hydroxyproline activity in soluble and insoluble collagen showed that the conversion of soluble to insoluble collagen was improved by the bleomycin-treated group. It was found that this was accompanied by a decrease in urinary excretion of total hydroxyproline and [3H]hydroxyproline during the first 12 hr after the administration of [3H]proline. Therefore, the results of the present investigation clearly indicate that the maturation of soluble to insoluble collagen is promoted and accompanied by a decrease in the catabolism of soluble collagen in the bleomycin-treated animals. In addition, administration of bleomycin increased the synthesis of collagen.     

Glucose deficiency reduces collagen synthesis in breast cancer MCF7 cells

We decided to study the effect of glucose deprivation on collagen metabolism in MCF7 cells. The incorporation of [³H]‐proline into collagenase‐sensitive and hydroxyproline‐containing proteins was used as an index of collagen synthesis, whereas pulse—chase technique was employed to evaluate the degradation of newly synthesized proteins. The MCF7 cells incubated in high glucose medium synthesized detectable amounts of collagenous proteins. Most of them were found in the cell layer. The shortage of glucose resulted in about 30% reduction in collagen synthesis. The pulse—chase experiments demonstrated that proportionally less collagen was degraded in cultures incubated in low‐glucose than in high‐glucose media.     

Proline recycling during collagen metabolism as determined by concurrent 18O2- and 3H-labeling

In a previous study where rat skin collagen was labeled with ¹⁸O in the hydroxyl group of the collagen hydroxyproline we noticed that the decay rate of this label was much faster than had been observed when the skin collagen hydroxyproline was labeled with ³H in the prolyl ring. In this study a rat was labeled concurrently with [¹⁸O₂] and [³H] proline and the rate of decline of both labels was determined in rat skin collagen hydroxyproline. After correction for growth dilution of the skin collagen the [¹⁸O] hydroxyproline was found to have a half-life of 27 days while the [³H] hydroxyproline had a half-life of 53 days. The decay rate of the [¹⁸O] hydroxyproline represents the true turnover rate of collagen since there is no possibility of recycling this label. Hence, the difference between this and the [³H] hydroxyproline decay rate is due to recycling of l-[³H] proline into new collagen. The efficiency of recycling of proline from catabolized collagen into new collagen was about 93%.     

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.     

Biosynthesis of skin collagens in normal and diabetic mice.

Synthesis of collagens in vitro was studied on minced mouse skins incubated with [3H]-proline in organ-culture conditions. A comparative study was carried out on genetically diabetic mice (KK strain) and control mice (Swiss strain). After incubation, neutral-salt-soluble and acid-soluble collagens were extracted. The insoluble dermis was digested by pepsin and type I and type III collagens separated by differential precipitation in neutral salt solutions. Type I and Type III collagens were characterized by ion-exchange and molecular-sieve chromatography, amino acid analysis and by the characterization of CNBr peptides. In diabetic-mouse skin, the relative proportion of type III collagen was significantly higher than in control-mouse skin. The incorporation of radioactively labelled proline into hydroxyproline of type III collagen was significantly faster in diabetic-mouse skin than in control-mouse skin.No significant modifications in the total collagen content of the skin or of their rates of synthesis were observed between the two strains. Alteration in the ratio of type III to type I collagen in the diabetic-mouse skin can be interpreted as a sign of alteration of the regulation of collagen biosynthesis and may be related to the structural alterations observed in the diabetic intercellular matrix.     

Collagen biosynthesis by human skin fibroblasts. III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. the results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased the amount of [3H]hydroxyproline synthesized. Ascorbic acid, however, did not increase the synthesis of 3H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and secretion of procollagen.     

Effect of beta-D-xylosides on collagen synthesis in cultured fibroblasts.

Cultured normal human skin fibroblasts were incubated with [¹⁴C]proline in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose. Formation of non-dialyzable hydroxyproline was used as a measure of collagen synthesis. Although total [¹⁴C]proline incorporation was similar in the two cultures, [¹⁴C]hydroxyproline formation was significantly decreased in the β-xyloside-treated cultures. Increasing the period of incubation increased the radioactivity of the insoluble collagen fraction in untreated fibroblasts, however, in β-xyloside-treated cultures no such increase was observed. In contrast to the decreased production of collagen, growth of cells in the presence of the β-xyloside induced the synthesis of high levels of soluble glycosaminoglycans as measured by ³⁵SO₄ incorporation into isolated polysaccharide.     

Response of rat connective tissues to streptozotocin-diabetes. Tissue-specific effects on collagen metabolism

We assessed the effect of streptozotocin-diabetes on in vivo collagen metabolism in skin, aorta and intestine by injecting [³H]proline into rats, 20 days after administering the diabetogen, streptozotocin. One day after [³H]proline injection, diabetic and control animals were killed, their tissues analyzed for both ³H-labeled and unlabeled hydroxyproline and results expressed per entire tissue. Thereby, the effect of diabetes on net collagen synthesis and tissue collagen mass, respectively, was evaluated.Diabetes resulted in a lower content of [³H]collagen in skin and aorta, suggesting decreased net collagen synthesis. This decrease in net synthesis was accompanied by a decrease of collagen mass in skin, whereas aortic collagen mass was unaffected. Consequently, an acceleration of collagen degradation in skin is postulated to have accompanied the expected depression of collagen synthesis; alterations of the physiochemical properties of skin from diabetic rats support this interpretation. For intestine, both net collagen synthesis and mass increased in diabetic rats, reflecting increased collagen synthesis—possibly associated with polyphagy.In conclusion, with regard to collagen metabolism, representative connective tissues respond differently to experimental diabetes, and we suggest that this insight will be useful in future studies aimed at understanding the pathophysiology of connective tissues affected by diabetes.     

Collagen biosynthesis by human skin fibroblasts III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. The results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased tha amount of [³H]hydroxyproline syntehsized. Ascorbic acid, however, did not increase the synthesis of ³H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and selection of procollagen.     

Metabolism of glomerular basement membrane in short- and long-term streptozotocin diabetic rats

The synthesis of glomerular basement membrane (GBM) total protein and collagen was assessed by two methods in vivo in normal and streptozotocin diabetic rats 4-6 weeks and 42-44 weeks after onset of hyperglycaemia, using L-[2, 3, 3H] proline as a radioactive precursor. The incorporation of tritiated proline into GBM hydroxyproline was used as a measure of collagen synthesis and that into proline as total protein synthesis. The basement membrane fractions from both short- and long-term diabetic rats attained much higher proline and hydroxyproline specific activities compared to normal GBM proline and hydroxyproline specific activities. Early insulin therapy with normalization of blood sugar levels in short-term (4-6 weeks) diabetic rats returned the abnormal increases in GBM total protein and collagen synthesis to normal. By contrast, poor glycaemic control with insulin did not prevent the increases in GBM protein synthesis. The results of the present study suggest that overall enhancement of GBM protein synthesis occurs in both short- and long-term streptozotocin diabetes. Early insulin therapy with normalization of blood sugar levels prevents this increase in GBM protein synthesis. Poor glycaemic control had no effect on abnormal GBM protein synthesis. This may be of potential significance in view of preventing chronic diabetic microvascular complications such as nephropathy.     

Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 1802 labelling.

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1. After the administration of l-[G-(3)H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.     

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 58% and 39%, respectively, by the addition of exogenous proteoglycan at a concentration of 10 mg/ml growth media. The incorporation of L-[3H]proline into acid-insoluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity on the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-translational modification of collagen and proteoglycan.     

The embryonic rat parietal yolk sac. The role of the parietal endoderm in the biosynthesis of basement membrane collagen and glycoprotein in vitro.

Basement membrane biosynthesis in vitro was studied in a rapidly growing embryonic tissue, the rat parietal yolk sac. This tissue consists of a thick, nonvascular basement membrane (Reichert's membrane) separating two cellular layers (parietal endoderm and trophoblast). Morphologically, Reichert's membrane appeared similar to other basement membranes. Previous analysis of the amino acid and carbohydrate composition of acellular Reichert's membrane showed it to be typical of basement membranes isolated from other tissues and species. Analysis of [14-C]proline incorporation and hydroxy [14-C]proline synthesis during the third quarter ogestation in vitro showed that basement membrane collagen synthesis in the parietal yolk sac was maximal around the 14th day of gestation. At this time, basement membrane collagen represented nearly 10% of the newly synthesized protein. The collagen synthesized in this system was characteristic of basement membrane collagen in that about 11% of the total hydroxy [14-C]proline was present as the 3-isomer. In addition, after incubation in the presence of [14-C]lysine, 83 to 94% of the hydroxy[14-C]lysine was glycosylated, with the predominant form being glucosylgalactosylhydroxy[14-C]lysine. When the parietal endoderm and trophoblast were incubated separately with [14-C]proline, it was determined that the former was solely responsible for the synthesis of basement membrane collagen since essentially all of the 4-hydroxy[14-C]proline was associated with this cell type. Autoradiographic experiments with [3-H]glucosamine also served to localize the synthesis of noncollagen basement membrane glycoprotein components to the parietal endoderm. As with the results reported for basement membrane collagen secretion in embryonic chick lens cells, there appeared to be approximately a 60-min delay between the incorporation of [14-C]proline into protein and the secretion of collagen as measured by the appearance of 4-hydroxy[14-C]proline in the culture medium. Experiments utilizing [3H]glucosamine to monitor glycoprotein synthesis did not show a delay between the incorporation of [3H]glucosamine and the secretion of nondialyzable 3-H into the medium. The results obtained using the parietal yolk sac system to study basement membrane biosynthesis were compared to those previously obtained using the kidney glomerular and embryonic chick lens systems. It was concluded that the parietal yolk sac system is superior for a number of reasons: (a) the extracellular matrix appeared to contain only basement membrane components; there was no contamination by acid mucopolysaccharides or other types of collagen; (b) only a single cell type appeared to be responsible for the synthesis of basement membrane components; and (c) a relatively large percentage of the newly synthesized protein was basement membrane collagen.     

The biosynthesis of basement-membrane collagen by isolated rat glomeruli.

A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of [14C]proline into non-diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non-diffusible 4-hydroxy[14C]proline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5% of total protein synthesis in glomeruli. When isolated glomeruli were incubated with [14C]proline, it was found that approximately 16% of the hydroxyproline present in the collagenous component occurred as the 3-isomer. When glomeruli were incubated with [14C]lysine over 90% of the hydroxy[14C]lysine synthesised was glycosylated and most of the glycosylated hydroxy[14C]lysine was present as glucosyl-galactosyl-hydroxy[14C]lysine. The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the 14C-labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C-labelled protein on an agarose column equilibrated and eluted with buffer containing 0.1% (w/v) sodium dodecyl sulphate. The initial form of [14C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140 000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with [14C]proline for 2 h and chased with unlabelled proline for 4 h there was a time-dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co-chromatograph with tendon pro-alpha chains (molecular weight approx. 120 000).