Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells.

Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [3H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [3H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.     

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

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

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.     

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

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

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

Ionizing radiation induces early, sustained increases in collagen biosynthesis: a 48-week study in mouse skin and skin fibroblast cultures

Groups of 10 CF1 female mice, irradiated to the thorax with a dual-head 137Cs gamma-RAY source, received single doses of 0, 5, 10, 15, or 25 Gy. One to forty-eight weeks later collagen synthesis was measured in minced skin specimens incubated in medium containing [3H]proline and then assayed for radioactive hydroxyproline. A progressive, generally dose-dependent increase in collagen biosynthesis, up to 50% above control sites, was found 1, 4, and 12 weeks after radiation exposure. These changes showed further small fluctuations at 12-36 weeks, increasing again at the 48-week interval. At the same times throughout the study fibroblasts were cultured from skin explants. Following the second subculture, these cells were also incubated in medium containing [3H]proline, and collagen synthesis was again determined by [3H]hydroxyproline assay. At all radiation dose levels studied, collagen production increased threefold by 12 weeks postradiation and remained elevated for the 48-week duration of the study. In vitro radiation dose response differences were not observed.     

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.     

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.     

Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures

A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [14C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [14C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.     

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.     

Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture

A method is presented for isolating osteoblasts from newborn mouse calvaria without the use of digestive enzymes. The procedure is based on the ability of osteoblasts to migrate from bone onto small glass fragments (Jones, S.J., and A. Boyde, 1977, Cell Tissue Res., 184:179- 193). The isolated cells were cultured for up to 14 d in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml of ascorbic acid. 7-d cultures were incubated for 24 h with [3H]proline. High levels of collagen synthesis relative to total protein were found, as measured by collagenase digestion of medium and cell layer proteins. Analysis of pepsin-digested proteins from the same cultures by SDS PAGE showed that type I collagen was predominantly produced with small amounts of type III and V (alpha 1 chains) collagens. Osteoblasts grown in the presence of beta-glycerophosphate were able to initiate mineral deposition in culture. Electron microscopic analysis of the cultures revealed the presence of needle- shaped apatite-like crystals associated with collagen fibrils and vesicles in the extracellular space. Mouse skin fibroblasts cultured under identical conditions failed to initiate mineralization. Electron histochemical studies revealed the presence of alkaline phosphatase activity, associated with osteoblast membranes, matrix vesicles and on or near collagen fibrils. Thus these isolated osteoblasts retained in culture their unique property of initiating mineralization and therefore represent a model of value for studying the mineralization process in vitro.     

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.     

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.     

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.     

Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts

Human fibroblasts when induced to make nonhelical , defective collagen have mechanisms for degrading up to 30% of their newly synthesized collagen intracellularly prior to secretion. To determine if at least a portion of the degradation of defective collagen occurs by lysosomes, extracts of cultured HFL-1 fibroblasts were examined for proteinases capable of degrading denatured type I [3H]procollagen. The majority of the proteolytic activity against denatured [3H]-procollagen had a pH optimum of 3.5-4; it was stimulated by dithiothreitol and inhibited 95% by leupeptin, 10% by pepstatin, and 98% by leupeptin and pepstatin together. Extracts of purified lysosomes from the fibroblasts were active in degrading denatured [3H]procollagen and were completely inhibited by leupeptin and pepstatin. To demonstrate directly that human lung fibroblasts can translocate a portion of their defective collagen to lysosomes, cultured cells were incubated with cis-4-hydroxyproline and labeled with [14C]proline to cause the cells to make nonhelical [14C]procollagen. About 3% of the total intracellular hydroxy[14C]proline was found in lysosomes. If, however, the cells were also treated with NH4Cl, an inhibitor of lysosomal function, 18% of the intracellular hydroxy[14C]proline was found in lysosomes. These results demonstrate that cultured human lung fibroblasts induced to make defective collagen are capable of shunting a portion of such collagen to their lysosomes for intracellular degradation.     

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.     

Vitamin A and bone formation. Effect of an excess of retinol on bone collagen synthesis in vitro.

The influence of an excess of retinol on bone formation was studied by using cultures of embryonic-chick calvaria. Retinol decreased collagen synthesis in a dose-dependent manner, non-collagenous protein synthesis being relatively unaffected. Collagen synthesis was significantly inhibited after 24 h of culture with retinol and was progressively decreased, compared with control cultures containing no retinol, as the period of culture was increased. The effect of retinol on collagen synthesis could be reversed by incubation of calvaria for further periods in retinol-free medium. Incorporation of [3H]thymidine and [3H]uridine into DNA and RNA respectively was not altered by culturing calvaria with retinol for 22 h. These latter findings, and the selectivity for collagen synthesis, all suggested that the effect observed was not a cell-toxicity phenomenon. The effect of retinol on collagen synthesis by chick calvarial osteoblasts was probably direct and not mediated by osteoclasts, since a negligible number of the latter cells is present in chick calvaria. In cultures of neonatal murine calvaria, which contain many osteoclasts, retinol similarly inhibited synthesis of collagen, but not of non-collagenous protein; the concentrations of retinol necessary to produce the response were similar to those required to stimulate bone resorption in vitro.     

Effect of L-3,4-dehydroproline on collagen synthesis and prolyl hydroxylase activity in mammalian cell cultures.

The incorporation of DL-3,4-dehydro[14C]proline into collagen and total protein of 3T3 cells occurred at approximately one-fifth the rate observed for L-[14C]proline. Addition of L-3,4-dehydroproline to the culture medium inhibited markedly the incorporation of [14C]glycine and L-[3H]lysine into the collagen of 3T3 cells, but there was only slight inhibition of the incorporation of the radiolabeled amino acids into total cellular proteins, indicating that the action of L-3,4-dehydroproline is specific for collagen. When 1 mM L-3,4-dehydroproline was added to the culture medium the [14C]hydroxyproline content was reduced 40% in the cell layer and 70% in the medium. The D isomer of 3,4-dehydroproline did not inhibit [14C]hydroxyproline formation. These findings indicate that L-3,4-dehydroline reduced the hydroxylation of the susceptible prolyl residues in the collagen molecule and the secretion of collagen from the cell. The reduction in the hydroxyproline content is probably related in part to a reduction in the activity of prolyl hydroxylase; when various mammalian cell cultures were exposed to 0.2 mM L-3,4-dehydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroxylase was not affected. Under these conditions, cell growth and lactic dehydrogenase required protein synthesis. Removal of L-3,4-dehydroproline from the growth medium resulted in a time-dependent increase in the specific activity of prolyl hydroxylase.     

Collagen degradation in I-cells is normal

There is evidence that lysosomal proteases mediate the intracellular degradation of structurally abnormal collagen. I-Cell disease (Mucolipidosis II) is characterized by marked deficiency of many lysosomal hydrolases, including the collagenolytic enzyme cathepsin B. The experiments reported here tested the hypothesis that degradation of abnormal collagen would be severely impaired in I-cells. Skin fibroblasts from 3 patients with I-cell disease were incubated with and without cis-hydroxyproline, a proline analog that causes structural abnormalities in collagen, and [14C]proline. The amount of [14C]hydroxyproline in a low molecular weight fraction relative to total [14C]hydroxyproline was used as a measure of intracellular collagen degradation. Levels of degradation were significantly higher in I-cells exposed to cis-hydroxyproline than in cells incubated without the analog. Similar data were obtained for normal human fetal lung fibroblasts incubated under the same conditions. Degradation of [125I]-epidermal growth factor was used to assess the functionality of the lysosomal pathway for protein degradation, and it was much lower in I-cells than in normal cells. It can be concluded that a completely functional complement of lysosomal enzymes is not necessary for structurally abnormal collagen to be degraded intracellularly; the data suggest that a nonlysosomal pathway exists.