Collagen Precursors Research Articles

Cellular regulation of collagen biosynthesis.

THE synthesis of collagen involves several enzymatic modifications of the polypeptide precursor of collagen, protocollagen1–4. First, certain proline and lysine residues in protocollagen are hydroxylated in similar enzymatic reactions involving molecular 02, Fe2+, α-ketoglutarate and ascorbic acid5–8. Much of the hydroxylation occurs after completed polypeptide chains have been released from the polyribosomal complexes9–11. Some hydroxylysine residues are then glycosylated by the stepwise addition of glucose and galactose, to form disaccharide appendages on the polypeptide chain12 and this glycosylation may be essential for the secretion of collagen into the extracellular matrix13,14. Although there are conflicting reports concerning the regulatory role of the hydroxylating enzymes15–20, no information is available on the genetic controls of collagen biosynthesis.

Effect of hydrocortisone acetate, fluocinolone acetonide, fluclorolone acetonide, betamethasone-17-valerate and fluprednyliden-21-acetate on collagen biosynthesis

Abstract The effect of hydrocortisone acetate, fluocinolone acetonide, fluclorolone acetonide, betamethasone-17-valerate and fluprednyliden-21-acetate on collagen biosynthesis was studied both in vivo and in vitro . In experiments in vivo , test substances and [ 14 C]proline were injected on the chorioallantoic membrane of 11-day-old chick embryos. In experiments in vivo , chick embryo tibiae were incubated in a medium containing the corticosteroids to be tested and [ 14 C]proline. In both types of experiments, the formation of [ 14 C]hydroxyproline was taken as a measure of the rate of collagen biosynthesis. In addition, the effect of these corticosteroids on the activity of protocollagen proline hydroxylase was tested. [ 14 C]hydroxyproline formation in vivo was inhibited by all the corticosteroids tested, and no clear difference in the degree of inhibition could be observed between the various corticosteroids. in vitro , betamethasone-17-valerate inhibited hydroxyproline formation more than the other substances tested. The concentration of corticosteroids required to inhibit collagen formation was considerably higher in vivo than in vitro . The corticosteroids tested did not affect the activity of protocollagen proline hydroxylase in the bones. In all these experiments, total protein synthesis, measured as incorporation of total 14 C-radioactivity into the bones, was inhibited by corticosteroids to the same extent as hydroxyproline formation. The results seem to suggest that corticosteroids inhibit collagen biosynthesis by inhibiting the formation of polypeptide precursors of collagen.

Studies on protocollagen: Identification of a precursor of proto α1

In the presence of α,α-dipyridyl chick calvaria accumulate a hydroxyproline and hydroxylysine deficient collagen, protocollagen. In addition to components with the properties of proto α1 and α2 chains, a component was identified that has the properties of nonhydroxylated α1 from a precursor of collagen and is therefore designated protopro α1.

Evidence for procollagen, a biosynthetic precursors of collagen.

Incubation of rat calvaria for short times in the presence of a labeled amino acid revealed the existence of a collagen fraction (procollagen) that functions as a biosynthetic precursor of collagen. Procollagen contains an alpha1-like chain (pre-alpha1) that elutes earlier from CM-cellulose than does rat-bone alpha1 and has a molecular weight, estimated by acrylamide gel electrophoresis, of 120,000. A time-dependent conversion of pre-alpha1 to alpha1 was demonstrated by incubation of calvaria for periods varying from 9 to 60 min and by a pulse-chase experiment. Limited cleavage of procollagen with pepsin resulted in a molecule with a chain resembling alpha1 in chromatographic properties, molecular weight, and relative hydroxyproline and proline contents. Thus, conversion of procollagen to collagen is likely to occur in vivo by a proteolytic mechanism. The additional peptide sequences in procollagen may serve to initiate chain association in triple-helix formation, to facilitate molecular transport, and to inhibit intracellular fibrogenesis.

Stimulation of Tissue Regeneration by Pulsed Plane-Wave Ultrasound

Plane-wave ultrasound at certain dosages can cause a statistically significant increase in the rate at which tissue re- generates to replace that lost after injury. The experimental model used in this study is the replacement of tissue in the pinna of the ear of a rabbit after the removal of tissue of area 1 cm2 through the full thickness of the ear. The regenerate consists of skin and its derivatives and elastic cartilage. The method of irradiation and the effects of a range of dosages on the rate of tissue replacement are described. Experimental evidence indicates that the growth rate increases found are not due to thermal effects caused by ultrasound; it is thought that a mechanical factor, streaming, may be involved. Electron microscopic studies show changes in the irradiated tissue that may be interpreted as indicating an increase in protein synthesis and temporary interference with the polymerization of collagen precursors. Autoradiographic investigation of 3H-thymidine uptake shows that the rate of synthesis of DNA in the regenerating tissue is increased by treatment with ultrasound. ISTROUL-CTIOX HERE have been several reports that ultrasound at certain dosages can increase enzyme activity (l), (2) and thus generally increase metabolic ac- tivity. In plants this effect stimulates seed germination and the growth rate of young seedlings (3), (4). This stimulation of plant growth suggested the use of ultra- sound as a possible stimulant of tissue growth in animals. The control of regenerative growth, i.e., the replacement of lost tissue, is clinically important in the treatment of injuries and in post-operative care. It is possible to stimulate growth by systemic means, for example, by the repeated injection of anabolic hormones (5), but such treatment affects other sites as well as the regenerate and can produce undesirable side effects. The direct

Collagen and mucopolysaccharide biosynthesis in mass cultures and clones of chick corneal fibroblasts in vitro

Abstract Collagen and mucopolysaccharide biosynthesis by cell populations of the chick cornea have been studied in vitro. Stromal fibroblasts from 14-day embryos were grown as mass cultures and clones; corneal epithelium was isolated as intact sheets of cells from embryos of the same age. Cultures were incubated with radioactive precursors of collagen or mucopolysaccharides. Corneal epithelium hydroxylated a portion of the administered proline and therefore appeared to synthesize collagen. Mass cultures of stromal fibroblasts were incubated with the labeled precursors at various times during culture cycles. Low- and highly sulfated chondroitin sulfate, keratosulfate, and collagen, end products of differentiation in the cornea in vivo, were synthesized during both logarithmic and saturation phases of growth. Changes in the rate of sulfate incorporation into individual polysaccharides during the culture cycle did not parallel those of glucosamine incorporation, suggesting a change in the degree of sulfation of the polysaccharides synthesized as the cell density of cultures increased. Analysis of 68 clones of stromal fibroblasts, each double-labeled with proline-3, 4-3H + Na235SO4 or proline-14C (uniformly labeled) + glucosamine-6-3H, showed that in direct proportion to their size 49 of these clones could make all three corneal polysaccharides and collagen. In all but one of the remaining smaller clones, all three polysaccharides were detected. The clonal analysis therefore suggested that each cell of this tissue-specific population of fibroblasts is specialized for the production of more than one end product of differentiation.

Unterschiede in der Reifung des Kollagens zwischen Rücken- und Schwanzhaut von Mäusen im Hinblick auf die unterschiedliche Tumoranfälligkeit dieser Hautanteile

1. 1. Après le traitement de la peau de la souris avec une substance cancérogène, on a observé la formation de cancers dans la région dorsale, et non sur la queue. Considérant le rôle du collagène dermique sur la croissance et sur la différenciation de l'épiderme, on a étudié, chez la souris, les différences entre la maturation du collagène de la peau de la queue et celle de la région dorsale. 2. 2. Dans la queue, la plus grande partie du collagène récemment synthétisé s'agrège sous une forme extractible par la cystéamine. Cette forme favorise la formation du réseau covalent; celle-ci se passe plus rapidement et atteint un degré plus élevé que dans la peau dorsale du même animal. Dans la région dorsale par contre, un pourcentage relativement élevé du collagène récemment synthétisé a pu être retrouvé plus tard sous une forme acidosoluble. 3. 3. Les résultats soutiennent l'hypothèse que le collagène acido-soluble représente une voie à part dans le processus de la maturation du collagène. 4. 4. L'impossibilité d'induire des cancers dans la peau de la queue pourrait s'expliquer d'un côté par une plus grande vitesse d'agrégation et de formation de fibrilles, et de l'autre côté par un ralentissement de l'agression par les collagénases. Ces deux facteurs empêchent la perte de collagène qui a été observée à la suite du badigeonnage du dos par le méthylcholanthrène. 1. 1. Painting with carcinogenic hydrocarbons produces carcinomas in the back skin of mice, but is ineffective in tail skin. Since dermal collagen is thought to be involved in the control of epidermal proliferation and differentiation, it was investigated whether there are differences between back and tail skin with respect to the maturation of collagen. 2. 2. Newly synthesized collagen of tail skin is composed predominantly of a cysteaminextractable form which appears to be favourable for further cross linkage. Cross linkage in tail skin collagen proceeds faster and to a greater extent than in collagen of the back skin of the same animal. By contrast, a relatively high percentage of newly synthesized collagen of back skin is later found in an acid-soluble form. 3. 3. The results support the hypothesis that the acid-soluble form of collagen is not a precursor of the insoluble collagen. 4. 4. The faster rate of collagen aggregation and fibril formation in tail skin is thought to result in a highly cross linked form of collagen more resistant to degradation by collagenases. This process obviously prevents a loss of collagen analogous to that observed in back skin after methylcholanthrene application. This might possibly be the reason for the inability to induce carcinomas in tail skin. 1. 1. Durch Behandlung mit Cancerogen können in der Rücken-, nicht aber in der Schwanzhaut von Mäusen Carcinome erzielt werden. Im Hinblick auf die Rolle des Kollagens der Dermis bei der Steuerung von Wachstum und Differenzierung der Epidermis wurden die Unterschiede in der Kollagenreifung zwischen Schwanz- und Rückenhaut von Mäusen untersucht. 2. 2. Frisch synthetisiertes Kollagen aggregiert in der Schwanzhaut zum Großteil in einer mit Cysteaminlösung extrahierbaren Form, welche die weitere kovalente Vernetzung sehr zu begünstigen scheint, da diese sowohl rascher vonstatten geht als auch einen höheren Grad erreicht als in der Rückenhaut der gleichen Tiere. Demgegenüber wird in der Rückenhaut ein verhältnismäßig großer Prozentsatz der frisch synthetisierten Kollagens später als säurelösliches Kollagen gefunden. 3. 3. Die Ergebnisse stützen die Ansicht, daß das säurelösliche Kollagen einen Seitenweg in der Kollagenreifung darstellt. 4. 4. Die Nichtinduzierbarkeit von Carcinomen in der Schwanzhaut könnte damit zusammenhängen, daß dort mit einer rascheren Kollagenaggregation und Fibrillenbildung eine durch die höhere Vernetzung bedingte langsamere Auflösung durch Kollagenasen einhergeht; beide Faktoren verhindern einen Kollagenverlust, wie er in der Rückenhaut bei Einwirkung von Methylcholanthren zu beobachten ist.

Development of protocollagen proline hydroxylase activity in the chick embryo

The activity of protocollagen proline hydroxylase, the enzyme hydroxylating proline residues in protocollagen, the proline-rich and hydroxyproline-deficient polypeptide precursor of collagen, was determined in the chick embryo during embryonic development. In whole chick embryos, the activity of this enzyme increased until the 14th day of embryonic development and thereafter declined. The activity of protocollagen proline hydroxylase was found to be present in all tissues tested, the highest activities being observed in bone and skin. These tissues showed the same type of development curve for this enzyme as whole embryos. In contrast, in the other tissues the enzyme activity did not change markedly during embryonic development. The changes in protocollagen proline hydroxylase activity correlated well with the changes observed in collagen synthesis in the developing chick embryo and thus support the earlier conclusions that the activity of this enzyme may be an indicator of collagen synthesis. Administration of ferrogluconate onto the chorioallantoic membrane led to a marked increase in protocollagen proline hydroxylase activity in whole embryo homogenates, whereas administration of ascorbate caused only a slight increase in activity.

Protocollagen Proline Hydroxylase in Normal Liver and in Hepatic Fibrosis

Previous observations indicated that the hydroxyproline in collagen is synthesized by the hydroxylation of proline which has been incorporated into a large polypeptide precursor of collagen called protocollagen, and that the hydroxylation of protocollagen is one of the terminal reactions in the intracellular synthesis of collagen. Significant levels of protocollagen proline hydroxylase activity were demonstrated both in normal rat liver and in a single specimen of human liver. The enzymatic activity in rat and human liver was similar to protocollagen proline hydroxylase isolated from chick embryos in that it was inhibited by poly-L-proline II, and it required ascorbate, α-ketoglutarate, and ferrous iron. When hepatic fibrosis was produced in rats with carbon tetrachloride or with a choline-deficient diet, liver levels of protocollagen proline hydroxylase increased over 4-fold. In all of the experiments the increase in protocollagen proline hydroxylase activity occurred more promptly and it was more marked than the increase in collagen hydroxyproline.

Effect of tetracycline on collagen biosynthesis in cultured embryonic bones

Abstract The biosynthesis of collagen was studied with 14 C-proline in organ cultures of embryonic ulnae. The rate of conversion of the incorporated 14 C-proline to 14 C-hydroxyproline in cultured ulnae was optimal up to 6 days, provided that the medium contained ascorbate and was replaced daily. When the medium was not replaced daily, the rate of 14 C-proline incorporation was reduced to about 60 per cent, and only a small part of the incorporated 14 C-proline was converted to 14 C-hydroxyproline. In these conditions, a considerable part of the 14 C-proline was found to be in the form of protocollagen, the proline-rich and hydroxyproline-deficient polypeptide precursor of collagen. The addition of 10, 30 or 100 μg ml tetracycline to the culture medium reduced the rate of incorporation of 14 C-proline into protein. The rate of 14 C-hydroxyproline synthesis was reduced even more, and thus a decrease was observed in the conversion of the incorporated 14 C-proline to 14 C-hydroxyproline. Similar changes were observed when embryonic ulnae from pregnant mice receiving tetracycline were cultured in a medium not containing tetracycline. In experiments with purified protocollagen hydroxylase, tetracycline was found to inhibit the hydroxylation of 14 C-proline-labelled protocollagen, the degree of inhibition being dependent on the concentration of ferrous iron in the reaction mixture. Cultivation of the ulnae with tetracycline had no effect on the total amount of protocollagen hydroxylase activity in the bones when the determination was made in the presence of excess of ferrous iron. The results suggest that tetracycline inhibits collagen synthesis by inhibiting both the incorporation of proline into protocollagen and the conversion of protocollagen to collagen. The latter inhibition is probably due to an interaction of tetracycline with ferrous iron, which is required in the hydroxylation of proline by protocollagen hydroxylase.

Anticollagen antibodies following thermal trauma.

SummaryIt was shown by a latex agglutination technique that rats, following thermal injury, are stimulated to produce antibodies to the acid-extractable fraction of collagen in skin. These antibodies do not agglutinate particles coated with the salt-extractable collagen but can be absorbed by this precursor of acid-extractable mature collagen. The anticollagen antibody in rats also reacts with bovine gelatin. An antibody-containing fraction of serum from burned rats was isolated by gel filtration and appears to be a 16.5 S20 macroglobulin by ultracentrifugation and is inactivated by mercaptane reduction. Antibodies to rat collagen and bovine gelatin were also demonstrated in the serum of normal humans. In burned humans the agglutinins disappeared following an extensive burn, presumably by reaction with circulating antigen, and reappeared during convalescence.

Serological and immunofluorescent study of soluble collagen in tissue culture of fibroblasts.

SummaryTo localize the synthesis of collagen precursor within fibroblasts, antibodies against purified neutral salt-soluble collagen fraction of chicken leg tendon were induced in adult rabbits. The antibodies against this presumed precursor of insoluble mature collagen had, (a) a high titer against specific antigen on complement fixation (1:1600), and on hemagglutination (1:15,000); (b) a single precipitin band in agar double diffusion and in immunoelectrophoresis; and (c) high specificity as to species and collagen fraction in cross reactions with citrate-soluble and insoluble collagen of chicken tendon, with neutral salt-soluble collagen of bovine sclera, and with an acetic extract of ichthyocol. Neutral salt-soluble collagen was localized by immunofluorescence in the cytoplasm, and processes of mature cultured fibroblasts, but not in undifferentiated cells.

Kinetics of incorporation of C 14-proline into mesogleal protocollagen and collagen of the sea anemone Aiptasia

1. Methods are described for labeling and purifying mesogleal collagen from the sea anemone Aiptasia sp. These techniques were applied to the study of the kinetics of the in vivo formation of mesogleal collagen. 2. Free C14-proline, injected into the coelenteron, was incorporated into bound C14-proline and bound C14-hydroxyproline of a protein(s) which becomes soluble on autoclaving. 3. The autoclave soluble material, in addition to containing collagen, is thought to contain an unhydroxylated collagen precursor. Within a few hours after the C14-proline was administered, the ratio of C14-proline to C14-hydroxyproline was 20/1; the proline/hydroxyproline ratio of unlabeled material was about 6/1.

Hydroxyprolinemia III. The origin of free hydroxyproline in hydroxyprolinemia. Collagen turnover. Evidence for a biosynthetic pathway in man

1. 1. A patient with hydroxyproline oxidase deficiency offered a unique opportunity to investigate the origin of plasma and urine hydroxyproline, since most of the free hydroxyproline produced in this patient is accumulated in blood and excreted unchanged in the urine. Dietary origin had been ruled out previously. 2. 2. The hydroxyproline-containing peptides in the urine were not different from normal. There was no evidence of abnormal collagen breakdown. Results indicate that the free hydroxyproline derives from a normal collagen turnover of about 2 g/day. 3. 3. Trial of a scorbutic diet unexpectedly resulted in a striking increase in excretion of free and peptide-bound hydroxyproline in the urine, and a further increase in plasma hydroxyproline concentration. This is in contrast to the decreased urinary hydroxyproline observed in scorbutic guinea pigs. Administration of ascorbic acid after the period of depletion resulted in further increase in excretion of free hydroxyproline. This suggests that ascorbic acid depletion in man increases turnover of mature collagen or of partially hydroxylated collagen precursors. 4. 4. When labeled glyoxylate was administered, a significant though small amount of urinary free hydroxyproline became labeled, indicating a slight degree of biosynthesis from glyoxylate in man, as has been described in rat livers.

Decarboxylation of alpha-ketoglutarate coupled to collagen proline hydroxylase.

The collagen proline hyd.roxylase system (also referred to as protocollagen hydroxylase), which converts selected proline residues to hydroxyproline in polypeptide precursors of collagen, has now been purified from a variety of animal tissues.1-4 M\/Iost purified preparations have an absolute requiremenlt for Fe++ and for a reducing agent, which can be ascorbic acid, other enediols, or tetrahydropteridines. An additional requirement discovered by Hutton et al.1' is a-ketoglutarate. This keto acid cannot be replaced by pyruvate, oxaloacetate, or related compounds and is an absolute requirement for proline hydroxylation. Removal of the keto acid by incubation with crystalline glutamic dehydrogenase, NH4+, and reduced nicotinamide-adeniine dinucleotide (NADH) leads to total inactivation of crude homogenates; activity is restored by the addition of aketoglutarate. Earlier attempts in this laboratory to determine whether a-ketoglutarate was consumed during the course of the hydroxylation reaction were inconclusive because the chick-embryo enzyme preparations used contained other enzymes that metabolized a-ketogluftarate.1 More recently, we have been able to obtain preparations from fetal, rat; skin that are far more active with respect to proline hydroxylation and that do not contain competing enzymes. With these preparations, it has now been possible to show a substrate-dependent and stoichiometric decarboxylation of a--ketoglutarate coupled to the hydroxylation of peptidyl proline residues. Ml/aterials and M11ethods.-Both H(3,4-Hl3-Pro-Gly-Pro),OH (mol wt 4100; spee. act. 0.9 mec/nmg) and poly-L-proline 11 (mol wt 9000) were gifts from Drs. J. Kurtz and A. 13erger, Weizmanari I nstitute of S ciences, Rehovoth, Israel. We obtaiiied a-ketoglutarate5-C14, (9.33 me/mnmole) from Nuelear-Chicago Corp., and a-ketoglutarate-U-C14 (3.2 me/mmole) an-d a-ketoglutarate-1-C14 (11 me/mmole) from Calbiochem, Inc. These were diluted with nonradioactive a-ketogluitarate before use. Crystalline bovine liver L-glutamic acid dehydrogenase) crystalline bovine liver catalase, and NADH were all products of Sigma Chemical Company. NCS reagent (for trapping C02) was obtained from Nuclear-Chicago Corp., and crystalline bovine serum albumin from Pentex Corp. Collagen proline hydroxylase was prepared from skins of newborn rats. Approximately 300 gm of tissue were homnogenized in 2 vol of 0.25 M. sucrose containing 0.01 mM ethylenediaini,1etet-aacetate (E})TA) aid O.l OJmM dithiothreitol, and the homogenate was centrifuged at 27,000 X g. All solutions used in subsequent purificatioin contained the same coneentrations of ditliothieitol. TFwo vol of saturated ammonium sulfate at pH 7.0 were added to the supelrnatant fraetion, and after centrifugation, the precipitate was dissolved in sodium cacodylate buffer, pH 7.0, and treated with 0.64 mg streptomycin sulfate per mg protein to remove nucleic acids. After centrifugation for 1 hr at 68,000 X g, the supernatanit fluid was treated with nieutral ammonium sulfate, and the fraction that precipitated between 17%( and 39% saturation (12 and 27 gm/100 ml) was dissolved in 0.05 M cacodylate, pH 7.0, containing 0.2 11f NaCl. After dialysis against 4 liters of the same solution, the en-zyme (800 mg of protein) was charged onto an O-(diethyl-

Denatured Collagen from the Cuticle of Ascaris lumbricoides as a Substrate for Protocollagen Proline Hydroxylase

Abstract Recent studies have shown that the hydroxyproline and hydroxylysine found in the collagen of vertebrates are synthesized by the hydroxylation of proline and lysine, which have been incorporated into a large polypeptide precursor of collagen called protocollagen. Because the collagen isolated from the cuticle of Ascaris lumbricoides contains less hydroxyproline and hydroxylysine than any other known collagen, it was of interest to determine whether this collagen could serve as a substrate for the synthesis of hydroxyproline by protocollagen proline hydroxylase from a vertebrate source. Essentially no synthesis of hydroxyproline was observed when the hydroxylase from chick embryos was incubated with native cuticle collagen solubilized by either 0.5 m NaCl extraction or pepsin treatment. Both of these large aggregate forms of the collagen, however, served as substrates for hydroxyproline synthesis after they were denatured by heating at 100° for 10 min. A subunit form of the collagen with a lower specific optical rotation than the large aggregate preparations served as a substrate without prior heat treatment, but the rate of hydroxyproline synthesis was increased by boiling the preparation. Comparison of the relative substrate activities and specific optical rotations of various forms of the cuticle collagen indicated an inverse relationship between the rate of hydroxyproline synthesis and the apparent degree of helicity. Previous reports by others have suggested that the subunit structure of collagen from Ascaris cuticle may involve a helical conformation different from that found in other collagens, and it is possible that an unusual conformation of the polypeptide chain or chains of the native cuticle collagen may explain its inability to serve as a substrate for protocollagen proline hydroxylase.

The fibroblast and wound repair.

SummaryThis review of connective tissue repair has attempted to place into historical perspective information obtained by newer approaches. The literature review is incomplete, as it was unfortunately necessary to leave many interesting studies out of the discussion. Emphasis has been placed upon what is known of the inflammatory response, the fine structure of the connective tissue cells in healing wounds and with correlated chemical findings in these tissues.An optimal inflammatory response appears to be an important, rapid, non‐specific stimulus for fibroplasia. It is not clear how inflammation exerts this effect. The inflammatory cells and their enzymes markedly alter the extracellular matrix of injured tissue. The matrix of connective tissue may itself participate in the control of its own synthesis and degradation. It is possible that modification of this environment by injury and/or inflammation with ensuing matrix alteration may provide a stimulus for cell migration and protein synthesis. The converse may also be true, that is, a given level of matrix concentration may have an inhibitory effect upon the connective tissue cells. The inter‐relationships between the connective tissue matrix and the cells, and the possibilities of feedback mechanisms playing a role in maintaining a balance between these two are important areas for future investigation. In this regard, additional questions may be asked concerning the role of the fibroblast in remodelling and degradation of connective tissue. It is not yet clear how important a balance between collagenolytic enzymes and the solubility states, or stability, of collagen are in each connective tissue. It will be interesting to determine which cells make collagenolytic and/or proteolytic enzymes upon appropriate stimulus.It is possible to distinguish between the fibroblast and the monocyte, or potential macrophage with the electron microscope. The rough endoplasmic reticulum with its large numbers of attached ribosomes is extensively developed in the fibroblast in contrast to the monocyte. The endoplasmic reticulum sequesters collagen precursors and other secretory proteins for transport either directly to the extracellular space, as appears to be the case for collagen, or to the Golgi complex as is the case for other exportable proteins. Collagen precursors are secreted into the environment and are not shed from within the cell surface.A number of cytoplasmic alterations have been described for fibroblasts and other cells during various pathological states. The significance of these alterations is not clear. It will be important to distinguish between specific and non‐specific responses to injury, if these alterations are to aid us in understanding the various cellular responses.The source of the fibroblasts in granulation tissue appears to be mesenchymal cells from adjacent tissues rather than blood‐borne precursors. Although contact inhibition can be demonstrated in vitro, it is not clear how important this phenomenon is in vivo, nor are the reasons for the ability of some tissues to heal by regeneration rather than by scar tissue formation understood.These and many other questions remain to be answered. The healing wound is multifaceted and presents the opportunity for systematic investigation into the problems of cell proliferation, cell and matrix interactions, and protein synthesis in vivo and it also can help to further our understanding of the ubiquitous fibroblast and its complex extracellular matrix.

Collagen formation in developing molar teeth of rats

In terms of collagen production, the odontoblast can be divided into two chief functional parts: a synthetic part (the cell body), and a part engaged in the disposition of the product (the odontoblastic process). The Golgi apparatus, mitochondria, granular endoplasmic reticulum, and free ribosomes are within the cell body, but they are not present in the odontoblastic process. The cell membrane of the odontoblastic process remains intact during fibrogenesis. The outer surface of the cell membrane is closely associated with collagen fibers, but no continuity can be detected between collagen fibers and comparable elements within the cell. Although the odontoblastic process contains a number of formed organelles, on the basis of the morphological and autoradiographic data, none of these are regarded as being, or containing, the collagen precursor. On the other hand, the collagen precursor is regarded as being within the cytoplasmic matrix of the odontoblastic process. A mechanism of fibrogenesis, in predominantly collagen producing cells, is proposed. This takes into account the inherent property of collagen “molecules” to aggregate into fibers in an appropriate cell free medium, and the observation that fibrogenesis appears to occur in relation to the outer surface of the cell membrane.

Hydroxylation of proline after the release of proline-rich polypeptides from ribosomal complexes during uninhibited collagen biosynthesis

Abstract Pulse-labeling techniques were used to study the hydroxylation of proline in isolated embryonic cartilage which synthesizes collagen at a rapid rate in vitro . In cartilage incubated with [ 14 C]proline for short periods of time, the synthesis of [ 14 C]hydroxyproline consistently lagged behind the incorporation of 14 C into protein. The ratio of [ 14 C]hydroxyproline to total 14 C in non-dialyzable fractions was less than 1 % after incubation with [ 14 C]proline for 1 min, and it gradually approached a maximal value of 20 to 25 % in 10 to 30 min. Incubation of extracts of pulse-labeled tibiae with purified protocollagen hydroxylase suggested that a large part of the [ 14 C]proline incorporated in 1 min was in polypeptides resembling collagen. Gel-filtration chromatography of extracts of pulse-labeled cartilage indicated that after short incubation periods with [ 14 C]proline, significant amounts of [ 14 C]proline were incorporated into molecules which were of about the same size as the complete polypeptide chains of α-collagen, but which did not contain significant amounts of [ 14 C]hydroxyproline. Analysis of the elution patterns of 14 C made it possible to estimate the time required to synthesize complete polypeptides de novo by a technique which apparently has not been used previously. The synthesis time calculated with this technique was about 1 min. Since maximal hydroxylation of collagen polypeptides in the system required over 10 min, the results indicated that cells synthesizing collagen under relatively normal conditions contain a significant intracellular pool of complete polypeptide precursors of collagen which are incompletely hydroxylated. A few proline residues may be hydroxylated while polypeptides are still attached to ribosomes, but the results suggested that most of the synthesis of collagen hydroxyproline occurs after completed polypeptides are released from ribosomal complexes.

Intracellular pool of unhydroxylated polypeptide precursors of collagen.

The hydroxyproline and hydroxylysine in collagen are synthesized by an apparently unique pathway in which proline and lysine are hydroxylated after they are incorporated into a large polypeptide precursor of collagen called protocollagen. When the hydroxylation of protocollagen in isolated tissues is intermittently interrupted, hydroxylation can occur after complete polypeptides are released from ribosomal complexes. Cartilage from chick embryos was incubated with the iron chelator alpha,alpha'-dipyridyl for 2 hours to inhibit protocollagen hydroxylase, and then the inhibition was reversed by transferring the tissues to medium containing ferrous iron and no alpha,alpha'-dipyridyl. "Pulse labeling" of the tissues during these two periods indicated that both the accumulated protocollagen and the polypeptides synthesized after reversal of the inhibition were hydroxylated at the same rate. Even when no measures are taken to inhibit the hydroxylation of protocollagen, most of the hydroxyproline in collagen is probably synthesized after complete protocollagen polypeptides are released from ribosomes.