Stimulation of rat uterine collagen synthesis by relaxin

Immature, ovariectomized, estrogen-primed rats respond to the administration of porcine relaxin by an increase in the incorporation of labeled amino acids ([14C]leucine, [14C]phenylalanine, [3H]proline) into uterine proteins in vitro. The maximum response occurs about 12 hr after a single injection of 0.1 mg relaxin in benzopurpurine 4B solution; subsequently, the relaxin effect declines but is still apparent after 24 hr. Smaller, but still significant increases in incorporation rates can be induced by relaxin in the absence of estrogen priming. Uterine collagen synthesis, as indicated by the incorporation of [3H]proline and its conversion to hydroxyproline, appears to be a primary target of the relaxin stimulus, since the effect of relaxin upon proline incorporation into uterine collagen is significantly greater than its effect upon labeling of noncollagen protein.     

Collagen studies in late pregnant relaxin null mice

The relaxin knockout (rlx -/-) mouse was used to assess the effect, during pregnancy, of relaxin with regard to water, collagen content, growth, and morphology of the nipple (N), vagina (V), uterus, cervix (C), pubic symphysis (PS), and mammary gland (MG). The results presented here indicate that during pregnancy, relaxin increases the growth of the N, C, V, and PS. Large increases in water content in the PS (20%) occurred in pregnant (Day 18.5) wild-type (rlx +/+) mice but not in rlx -/- animals. This indicates that in the PS, relaxin might increase the concentration of a water-retaining extracellular matrix component (hyaluronate). In the pregnant rlx +/+ mouse, collagen content decreased significantly in the N and V but not in other tissues. There were no significant changes in the rlx -/- mouse. This contrasts with findings in the rat, in which relaxin has been found to cause decreases in collagen concentrations in the V, C, and PS. Histological analysis showed that the collagen stain was more condensed in the tissues (V, C, PS, N, and MG) of rlx -/- mice than in those of rlx +/+ mice. This phenomenon indicates that the failure of collagen degradation and lack of growth in the N underlie the inability of the rlx -/- mice to feed their young, as reported previously. Vaginal and cervical luminal epithelia, which proliferated markedly in the rlx +/+ pregnant mice, remained relatively atrophic in the rlx -/- mice. As proliferation and differentiation of uterine and vaginal epithelia are thought to be induced by a paracrine stromal factor that acts upon estrogen stimulation, our results indicate that relaxin may be this paracrine factor.     

Progesterone inhibits the uterotrophic effect of relaxin in immature rats

Injection of progesterone for 3 days before treatment with relaxin inhibited the trophic effect of the peptide in both estrogen-primed and unprimed uteri. The depression in collagen concentration and increase in apparent rate of proline incorporation into collagen induced by relaxin alone were also eliminated, indicating a fundamental blockade of the effect of relaxin in this experimental design as well as a close association of changes in collagen concentration with tissue hypertrophy. The effect of relaxin on incorporation of proline into soluble protein was not blocked by progesterone, however, suggesting a separate mechanism for this anabolic effect of relaxin.     

Structure of a porcine relaxin inactive on the mouse pubic ligament

In addition to B31 (CM-a) and B28 (CM-B) relaxins, acid-acetone extracts of ovaries of pregnant sows contain a third major relaxin species (relaxin C). The major components of relaxin C possess about half the activity of CM-a or CM-B in the guinea pig palpation assay, but are completely inactive in the mouse pubic ligament assay. Its uterotrophic and protein anabolic effects in ovariectomized, estrogen-primed mice, however, are comparable to those of CM-B. Sequence analysis indicates that the two major components of relaxin C, like the other porcine relaxins, consist of two polypeptide chains linked by disulfide bonds. The shorter (A) chains are identical to the A chains of the other porcine relaxins, except for the absence of the N-terminal arginine residue. The B chains display microheterogeneity; the B sequences of the two predominant species are the same as those of the other porcine relaxins through B25, but terminate at valine residue B25 or serine residue B26, respectively.     

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1)

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.     

Trophic effects of relaxin on reproductive tissue: role of the IGF system.

Although the growth promoting actions of relaxin on the reproductive tract have been well documented, the means by which relaxin stimulates reproductive tissue growth has not been identified. This report is an overview of studies from our laboratory investigating the role of the insulin-like growth factor (IGF) system in relaxin-induced growth of ovarian and uterine tissues. In the pig ovary, concentrations of relaxin that promote both theca and granulosa cell (GC) DNA synthesis in vitro also significantly (P < 0.05) increased GC IGF-I secretion. When IGF-I activity was blocked in the presence of an IGF-I antibody, the trophic effects of relaxin on GC [3H]thymidine incorporation into DNA were inhibited. However, there was no effect of relaxin on GC IGF binding proteins or IGF-I receptor. In the uterus, in vivo relaxin administration to prepubertal pigs resulted in the stimulation of growth and increases in uterine luminal IGF-I, IGF-II, and IGF binding proteins-2 and -3 secretion (P < 0.05). Thus, the trophic effects of relaxin on ovarian granulosa cells and the uterus involve tissue-specific changes in the IGF system. Additional studies are necessary to better understand the contribution of relaxin to follicular growth and uterine accommodation. These include characterization of the relaxin receptor and post-receptor binding events, as well as the potential impact of relaxin on other growth factor systems and how these systems interact to ultimately drive reproductive tissue growth.     

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).     

Inhibition of postpartum uterine involution in the rat by relaxin

The possible contribution of relaxin to the support of uterine accommodation during late gestation by retarding tissue lysis was examined using the involuting postpartum uteri of unilaterally pregnant rats. In otherwise intact animals, twice-daily administration of 0.1 mg of relaxin (porcine fraction B) significantly retarded the regression of both gravid and, to a greater extent, nongravid tissue during the first 4 days postpartum, and collagenolysis was similarly delayed. Immediate postpartum ovariectomy had little effect on the uterus, although 5 micrograms estradiol benzoate daily suppressed uterine involution in the gravid tissue to about 50% and was even more effective in the nongravid uterus. Relaxin alone had little effect on the gravid uterus following ovariectomy, but augmented estrogen to the extent that less than half of the tissue and its collagen were lost during 4 days. The effect on nongravid tissue was even more striking in that the combination of estrogen and relaxin prevented any degradation of tissue in general or of collagen. Although we have reported that relaxin can stimulate uterine collagen synthesis as well as uterine growth, the magnitude of its postpartum effect in the presence of estrogen suggests a stabilizing or anticatabolic effect upon the uterus.     

Effects of the hormone relaxin on the metabolism of the glycosaminoglycans in the mouse symphysis pubis

The influence of the peptide hormone relaxin on the glycosaminoglycan (GAG) metabolism was investigated in the pubic ligament of the symphysis pubis and in serum of the virgin mouse. Fresh weight DNA and GAG content per 1 ligament is significantly increased, the level of water soluble protein is not affected. A shift in the electrophoretic GAG pattern by an increasing amount of hyaluronic acid and a decreasing amount of chondroitin sulfate and dermatan sulfate can be observed. Concerning GAG-splitting enzymes (N-acetylglucosaminidase, arylsulfatase, beta-glucuronidase) the N-acetylglucosaminidase reveals a significant increase of its activity in the interpubic ligament and in the serum. The data demonstrate that relaxin treatment induces some changes in the GAG metabolism.     

Relaxin and beta-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by beta-estradiol. We postulated that relaxin and beta-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, beta-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered beta-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 microg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. Beta-estradiol, relaxin, or beta-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or beta-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by beta-estradiol, relaxin, or beta-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.     

Preparation of biologically active 32P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain

Relaxin is a member of the insulin family of polypeptide hormones and is known to exert its biological effects on various parts of the mammalian reproductive system. Biologically active human relaxin has been chemically synthesized based on the nucleotide sequence obtained from an ovarian cDNA clone. In the present study synthetic human relaxin was radiolabled by phosphorylation with cAMP-dependent protein kinase and [gamma-32P]ATP to a specific activity of 5000 Ci/mmol. The phosphorylated relaxin was purified on cation exchange high performance liquid chromatography and was shown to co-migrate with relaxin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed a single phosphorylated site on the B chain of relaxin. The 32P-relaxin was able to bind to a goat anti-relaxin antibody, and this binding could be displaced by unlabeled relaxin in a concentration-dependent manner. A comparison of the concentration responses of cellular cAMP production stimulated by relaxin and phosphorylated relaxin in a primary human uterine cell line showed that phosphorylation did not affect the in vitro biological efficacy of relaxin. This made it suitable for in situ autoradiographic localization of relaxin binding sites in rat uterine, cervical, and brain tissue sections. Displacement of the binding of 100 pM 32P-relaxin by 100, 10, and 3 nM unlabeled relaxin, but not by 100 nM insulin, insulin-like growth factor-I, and an insulin-like growth factor-I analog, demonstrated the high affinity and specificity of such binding. We conclude that 32P-labeled human relaxin is biologically and immunologically active and that this novel probe binds reversibly and with high affinity to classical (e.g. uterus) and unpredicted (e.g. brain) tissues.     

H3 relaxin demonstrates antifibrotic properties via the RXFP1 receptor

Human gene 3 (H3) relaxin is the most recently discovered member of the relaxin peptide family and can potentially bind all of the defined relaxin family peptide receptors (RXFP1-4). While its effects as a neuromodulator are being increasingly studied through its primary receptor, RXFP3, its actions via other RXFPs are poorly understood. Hence, we specifically determined the antifibrotic effects and mechanisms of action of H3 relaxin via the RXFP1 receptor using primary rat ventricular fibroblasts in vitro, which naturally express RXFP1, but not RXFP3, and a mouse model of fibrotic cardiomyopathy in vivo. Transforming growth factor β1 (TGF-β1) administration to ventricular fibroblasts significantly increased Smad2 phosphorylation, myofibroblast differentiation, and collagen deposition (all p < 0.05 vs untreated controls), while having no marked effect on matrix metalloproteinase (MMP) 9, MMP-13, tissue inhibitor of metalloproteinase (TIMP) 1, or TIMP-2 expression over 72 h. H3 relaxin (at 100 and 250 ng/mL) almost completely abrogated the TGF-β1-stimulated collagen deposition over 72 h, and its effects at 100 ng/mL were equivalent to that of the same dose of H2 relaxin. Furthermore, H3 relaxin (100 ng/mL) significantly inhibited TGF-β1-stimulated cardiac myofibroblast differentiation and TIMP-1 and TIMP-2 expression to an equivalent extent as H2 relaxin (100 ng/mL), while also inhibiting Smad2 phosphorylation to approximately half the extent of H2 relaxin (all p < 0.05 vs TGF-β1). Lower doses of H3 (50 ng/mL) and H2 (50 ng/mL) relaxin additively inhibited TGF-β1-stimulated collagen deposition in vitro, while H3 relaxin was also found to reverse left ventricular collagen overexpression in the model of fibrotic cardiomyopathy in vivo. These combined findings demonstrate that H3 relaxin exerts antifibrotic actions via RXFP1 and may enhance the collagen-inhibitory effects of H2 relaxin.     

The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts

The rate of collagen synthesis relative to the rate of synthesis of noncollagen protein was determined in several lines of cultured fibroblasts using an assay which measures [¹⁴C]proline incorporation into the polypeptide chains of collagen. In this assay procedure, collagen is degraded by protease-free collagenase regardless of whether proline and lysine residues are hydroxylated, thus separating the process of polypeptide synthesis from hydroxylation. It was found that the relative rate of collagen synthesis in L-929 cells was approximately 0.8–1% at all stages of growth. There was no significant increase in the relative rate of collagen synthesis in stationary phase compared to log phase cells in the lines Balb 3T3, 3T6, 3T12, and Swiss mouse 3T6. In all cases, the absolute incorporation of [¹⁴C]proline into both collagen and noncollagen proteins expressed as radioactivity incorporated per milligram of cellular protein, was 2–10 times higher in log phase cells, depending on the line examined.     

Changes of large molecular weight hyaluronan and versican in the mouse pubic symphysis through pregnancy

During pregnancy, the mouse pubic symphysis undergoes expansion and remodeling resulting in formation of a flexible and elastic interpubic ligament allowing passage of a term fetus. In the current study, we sought to identify and characterize components of the extracellular matrix that likely play an important role in elongation and flexibility of the interpubic ligament during parturition. Mouse pubic symphyses and interpubic ligaments collected at time points during pregnancy and postpartum were utilized to evaluate collagen type, collagen content, processing and solubility, matricellular protein, and proteoglycan expression and quantitative assessment of all glycosaminoglycans. These studies revealed increased gene expression for hyaluronan synthase 1, hyaluronan synthase 2, and versican on Gestation Day 18 as well as a decline in protein expression for the versican-degrading protease a disintegrin-like and metalloprotease with thrombospondin type 1 (ADAMTS1) motif. These findings suggest that the primary mediators of increased elongation and flexibility of the interpubic ligament at term result from increased synthesis and reduced metabolism of viscoelasticity-promoting molecules such as high molecular weight hyaluronan and versican.     

A0-phenylalanyl] relaxin (porcine): an active intermediate

A [phenylalanylA0] relaxin has been isolated as a byproduct during large scale porcine relaxin preparations, using ion exchange chromatography on CM-cellulose at pH 7.8 followed by high performance liquid chromatography on reversed phase columns. The elongation at the N terminus of the A-chain has been demonstrated by amino acid and sequence analyses of the isolated and carboxymethylated relaxin-A-chain. The phenylalanyl relaxin and B29 relaxin are indistinguishable by circular dichroism spectroscopy, in mouse pubic ligament assay, and radioimmunoassay. The occurrence of phenylalanyl relaxin may be caused by an incomplete conversion of prorelaxin to relaxin.     

The correlation between specific protein synthesis and tumoricidal function in murine peritoneal macrophages

Macrophages from the lipopolysaccharide (LPS)-responsive C3H/HeN mouse strain and the closely related LPS-nonresponsive C3H/HeJ strain were compared for tumoricidal activation and protein synthetic changes following in vivo and in vitro stimulation, utilizing two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Peritoneal macrophages elicited from C3H/HeN mice with heat-killed Propionibacterium acnes exhibited tumoricidal activity in a 16-hr cytolytic assay and expressed cytoplasmic levels of a 23.5-kDa protein during 48 hr of culture. The inability to detect persistent expression of p23.5 in P. acnes-stimulated C3H/HeJ macrophages correlated with the cytolytic impotence of those cells in the 16-hr chromium release assay. C3H/HeN macrophage populations lacking tumoricidal capacity could be rendered lytic, as could P. acnes-elicited C3H/HeJ macrophages, following in vitro stimulation with bacterial lipopolysaccharide. Concomitant with the LPS-induced expression of new functional activity was the appearance of augmented levels of several macrophage-specific proteins, including p23.5. This effect was dependent upon the lipid A moiety of LPS as the effects of LPS could be blocked by inclusion of polymyxin B sulfate in the culture medium. However, neither tumoricidal function nor protein modulation could be readily induced in C3H/HeJ proteose peptone-elicited or resident macrophages. These results identify biochemical responses to stimuli which may be requisite to acquisition or execution of cytolytic activity.     

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.     

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.     

The effect of oxygen partial pressure on protein synthesis and collagen hydroxylation by mature periodontal tissues maintained in organ cultures

Mature periodontal tissues from adult-mouse first mandibular molars were cultured in a continuous-flow organ-culture system which allowed the regulation of both ascorbic acid concentration and pO(2) (oxygen partial pressure). Protein synthesis was measured by analysing the incorporation of [(3)H]proline into collagenous and non-collagenous proteins during the last 24h of a 2-day culture. At low pO(2) [16.0kPa (approx. 120mmHg)] approx. 60% of protein-incorporated [(3)H]proline was found in collagenous proteins. However, it was evident that this collagen was considerably underhydroxylated. At high pO(2) [56.0kPa (approx. 420mmHg)], both the amount of collagen deposited in the tissues and the degree of hydroxylation were increased considerably. In contrast, no significant effect on non-collagenous protein was observed. Tissues cultured at low pO(2) for the first 48h were unable to respond to a subsequent increase in pO(2) during the last 24h. Analysis of pepsin-solubilized collagen alpha-chains labelled with [(14)C]glycine demonstrated the synthesis of both type-I and type-III collagens by explants cultured for 48h at high pO(2). Type-III collagen comprised 20-30% of the radioactivity in alpha-chains in both the periodontal ligament and the tissues of the alveolar process. The pattern of protein synthesis in the alveolar tissues at high pO(2) was similar to that observed in these tissues in vivo. However, in the cultured periodontal ligament the proportions of non-collagenous proteins and type-III collagens were increased in comparison with the tissue in vivo.     

Procollagen synthesis and processing in periodontal ligament in vivo and in vitro. A comparative study using slab-gel fluorography.

A combination of dodecylsulphate/polyacrylamide gel electrophoresis and fluorography has been used to quantify the synthesis of type I and type III collagens by periodontal ligament in situ and periodontal-ligament fibroblasts in vitro. The separation of 14C-labelled collagen alpha chains was achieved by introducing an interrupted reduction step, and the total radioactivity in the alpha-chain bands related to the fluorographic response by a series of standard curves. From these curves an accurate assessment of the relative amounts of type I and III collagen synthesized could be made. The same system also allowed the synthesis and processing of the respective procollagens to be analyzed. For the study in vivo, 200-g male rats were injected with 2 mCi [14C]glycine and killed 0.5-6 h later. Periodontal ligament was dissected from the mandibular molars and the newly-synthesized collagens extracted with 0.45 M sodium chloride. In the study in vitro, confluent monkey periodontal-ligament fibroblasts were cultured in the presence of [14C]proline and [14C]glycine. Analysis of labelled collagens showed a rapid conversion of type I procollagen to collagen but type III collagen was recovered as a procollagen intermediate both in vitro and in vivo. Analysis of duplicate samples after pepsin digestion showed type III collagen synthesis to comprise 15% of the total collagen synthesized in vivo and 20% in early subcultures in vitro. However, the proportion of type III synthesized by the fibroblasts decreased on subculturing. The data demonstrate that fibroblasts in vitro retain the basic characteristics of collagen synthesis and procollagen processing found in vivo, but the overall phenotypic expression of the cells is not stable in culture.