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.     

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.     

Formation of proline metabolites in chick embryo bone: Interference with the measurement of free hydroxyproline by ion-exchange chromatography

Metabolites of -[¹⁴C]proline were found in the trichloroacetic acid-soluble fraction of 16-day-old chick embryo frontal bones. In several ion-exchange procedures these metabolites interfered with the analysis of hydroxyproline derived from the metabolic breakdown of collagen. The major metabolite was identified as glutamic acid by its chromatographic and crystallization properties. It was eluted from AG50 cation-exchange resin with 1.0 HCL in the hydroxyproline region, but was separated from hydroxyproline on a DC-6A column in the amino acid analyzer. Another metabolite was identified as aspartic acid. It was not separated from hydroxyproline on either AG50 using 1 HCL for elution or on DC-6A using 0.1 sodium citrate, pH 3.25, for elution, but adequate separation was obtained by elution with 0.2 sodium citrate buffer at pH 2.91. Formation of these metabolites was not related either to protein synthesis or proline hydroxylation. Therefore, it is possible to analyze for hydroxyproline accurately by using a separate unhydroxylated sample to correct for the presence of the metabolites. The formation of glutamic acid suggested that proline oxidase activity might be present in bone tissue, but none was detected using a sensitive radioisotopic assay. Although the amount of radioactivity found in the metabolites was 36% of the amount of [¹⁴C]proline incorporated into protein, no radioactive glutamic or aspartic acid was present in protein hydrolyzates. This observation suggests that the metabolites did not enter the major amino acid pool used for protein synthesis.     

The appearance of free hydroxyproline as the major product of degradation of newly synthesized collagen in cell culture

Embryonic lung fibroblasts and rabbit vascular smooth muscle cells have the ability to degrade newly synthesized collagen. Analysis of 24-h pulse media from cultures given [¹⁴C]proline demonstrates that greater than 90% of the degraded collagen is represented by free hydroxyproline rather than the peptide-bound imino acid. The addition of cycloheximide or α-α-dipyridyl to the culture medium during the pulse period severely diminished the formation of the free hydroxyproline demonstrating its enzymatic and protein (collagen) origin. It is proposed that assessment of free hydroxyproline formation may allow us to distinguish between intracellular and extracellular collagen degradation.     

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%.     

Oestradiol inhibits collagen breakdown in the involuting rat uterus

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100mug of oestradiol-17beta/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[(14)C]-proline by the administration of [(14)C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[(14)C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [(14)C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.     

Comparative analysis of the structure and thermal stability of sea urchin peristome and rat tail tendon collagen

We have purified collagen from two distinct sources; the vertebrate, rat tail tendon and an invertebrate, sea urchin adult tissue, the peristome. The collagenous nature of the purification products was confirmed by amino acid compositional analysis. Both preparations had high contents of glycine and proline residues and hydroxyproline was also present. The total pyrrolidine (proline+hydroxyproline) content decreased from 17.9 mole% in rat tail collagen to 12.9 mole% in peristome collagen. Distinctly different circular dichroic spectra were measured for these collagens. Analyses of spectra, measured as a function of temperature, revealed distinct thermal denaturation profiles. The melting temperature for rat tail collagen was 38.5 degrees C, while the corresponding value for peristome collagen was significantly lower at 27 degrees C. A similar thermal denaturation profile was obtained for rat tail collagen in digestion experiments using a 41-kDa gelatinase activity, isolated from sea urchin eggs. These results identify structural differences between a typical, vertebrate type I fibrillar collagen and an echinoderm collagen which serves as a constituent of a mutable connective tissue. These differences may relate to the functional roles played by collagen in these distinctly different tissues.     

A sensitive method for the separation and quantitation of (14C)proline and (14C)hydroxyproline from the collagen of rat uterus

A specific and sensitive method is described for the isolation and quantitation of [¹⁴C]proline and [¹⁴C]hydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a protease-free bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment with collagenase. There is a consistent recovery of [¹⁴C]proline and [¹⁴C]hydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [¹⁴C]proline into collagen of the rat uterus and to quantitate its conversion to [¹⁴C]hydroxyproline. Coupled with the colorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.     

Determination of radioactivity of proline and hydroxyproline in tissue hydrolysates after the elimination of interference by primary amino acids by deamination

A simple method for the determination of radioactivity of proline and hydroxyproline, particularly of small amounts, in hydrolysates of tissues is described. Specificity is assured by eliminating primary amino acids from the hydrolysates by deamination and then extraction before separation of proline from hydroxyproline by paper chromatography. Six to eight tissue samples may be compared simultaneously. The efficiency and reproducibility are good, as indicated by the use of labeled l-proline, labeled dl-hydroxyproline, a hydrolysate of a protein in which the amino acids (and proline) were labeled, and hydrolysates of tissues cultured in media containing radioactive l-proline. The method is particularly useful when ion-exchange column chromatography of amino acids is not in routine use.     

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.     

Quantitative study of tissue collagen metabolism

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.     

The in vivo inhibition of collagen synthesis and the reduction of prolyl hydroxylase activity by 3,4-dehydroproline.

Various proline analogs and iron chelators were tested for their effect on collagen formation which occurs in the uterus of the immature rat following the administration of estradiol-17β. dl-3,4-Dehydroproline, l-α-azetidine-2-carboxylic acid and l-pyroglutamic acid reduced the estradiol-17β stimulated formation of hydroxyproline which occurs in the uterus following administration of the hormone while l-thiazolidine-4-carboxylic acid was without effect on this response. The activity of the d- and l-isomers of 3,4-dehydroproline was compared with the racemic mixture; the l-isomer was twice as active as the latter, while the d-isomer was only half as active. l-3,4-Dehydroproline was approximately four times as potent as l-α-azetidine-2-carboxylic acid, the second most active analog of those tested. dl-3,4-Dehydroproline inhibited the incorporation of l-[¹⁴C]proline into the proline and hydroxyproline of uterine collagen; it also inhibited the incorporation of [¹⁴C]glycine into collagen while having less effect on the incorporation of these amino acids into noncollagen protein. These results indicate dl-3,4-dehydroproline is a fairly specific and potent inhibitor of collagen formation in vivo.These observations indicate that dl-3,4-dehydroproline reduces the hydroxylation of prolyl residues in collagen. Presumably, this occurs in part due to the incorporation of the analog into the collagen molecule in place of proline. It is probably also related to a reduction of prolyl hydroxylase activity which can be demonstrated in the tissues of animals treated with 3,4-dehydroproline. A significant reduction of prolyl hydroxylase activity was shown to persist in the uterus, lung, and heart for approximately 24 h following a single intraperitoneal dose of dl-3,4-dehydroproline (200 mg/kg).     

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.     

Rice callus growth, proline content and activity of proline and IAA oxidase under the influence of hydroxyproline and NaCl

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR₁₁) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD₅₀ concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth, proline content and activity of proline and IAA oxidases of the cultured tissues were determined at the end of 0, 2, 4, and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing hydroxyproline and NaCl showed increased dry weight and proline content as compared to NaCl stressed calli. The levels of proline and IAA oxidases were also low in the hydroxyproline resistant calli.     

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.     

16, 16 Dimethyl prostaglandin E2 decreases the formation of collagen in fibrotic rat liver slices

The effect of 16, 16 dimethyl prostaglandin E₂ (DMPG) on fibrogenesis was studied in slices from normal and fibrotic rat liver. Rats received a cirrhogenic diet for seven months; supplemented controls received a diet with the deficient nutrients restored. Slices from fibrotic livers incorporated more ¹⁴C-proline and produced more ¹⁴C-hydroxyproline in TCA precipitable proteins than slices from control livers. DMPG (10^−10M) decreased the incorporation of labeled proline and the synthesis of labeled hydroxyproline in slices from fibrotic livers to the same extent, suggesting that DMPG did not affect the hydroxylation of proline per se. The magnitude of the DMPG induced decrease in labeled proline incorporation correlated with the hydroxyproline content in the liver (i.e. with increasing fibrosis there was a greater effect of DMPG; while in control rat liver slices, DMPG had no effect). DMPG did not change the size of the proline pool, its specific activity, or the activity of proline oxidase. We conclude that under these conditions of enhanced fibrogenesis, DMPG decreases the formation of collagen in vitro, possibly by lowering the incorporation of proline into collagen precursors. This may explain, at least in part, the inhibition of fibrogenesis by DMPG in vivo.     

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.     

Effects of hydroxyproline on the growth and cell-wall protein metabolism of excised root segments of Pisum sativum

Summary Hydroxyproline, in the presence of sucrose, enhanced the extension growth of excised 2–4 mm pea root segments in aseptic media. About 90% of protein-bound hydroxyproline in the pea root segments was confined to the cell-wall fraction where it occurred as trans-4-hydroxy-l-proline. The amounts of wall-bound hydroxyproline increased dramatically towards the cessation of extension growth, but when the segments were cultured in trans-hydroxyproline, this increase was considerably less.Externally supplied cis and trans-hydroxyproline inhibited the formation of protein-bound [¹⁴C]hydroxyproline from [¹⁴C]proline without affecting the total amount of [¹⁴C]proline incorporated into proteins. Studies with -dipyridyl showed that, although some of the externally supplied trans-[¹⁴C]hydroxyproline was incorporated directly into cell-wall proteins, most of it was first converted into proline which was then incorporated into proteins and subsequently reconverted, in part, into hydroxyproline. The effect of externally supplied hydroxyproline is discussed in relation to protein-bound proline hydroxylation.     

Urine glycyl-L-proline increase and skin trophicity

Summary Glycyl-L-proline (gly-pro) is an end product of collagen metabolism that is further cleaved by prolidase (EC 3.4.13.9); the resulting proline molecules are recycled into collagen or other proteins. We postulated a relationship between defective gly-pro hydrolysis, increased collagen degradation and skin destruction. This relationship was tested using HPLC to measure the gly-pro in urine. 24 hour urine samples were collected from 27 old people (86 ± 6 years old), of whom 15 were suffering from skin pressure sores of the sacrum or calcaneus. The urine from patients with pressure sores contained significantly more gly-pro than the urine from the control. A cut-off at 7mol/ mmol creatinine gave the test a positive predictive value of 70%. Collagen breakdown was also increased as indicated by the increase of hydroxyproline (hyp) in the urine. But this breakdown seemed to stop at the gly-pro step.     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.