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Intracellular Degradation of Newly Synthesized Collagen
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THE .JOURNAL OF INVESTIGATIVE DERMATOLOGY, 79:778-828, 1982
Copyright ©
1982
Vol. 79,
by The Williams & Wilkins Co.
Intracellular Degradation of Newly Synthesized Collagen STEPHEN 1. RENNARD, M.D., LARUE E. STIER, M.S., AND RONALD G. CRYSTAL, M.D. Pulmonary Branch, National Heart, Lung, and Blood Institute Bethesda, Maryland,
The intracellular degradation of newly synthesized collagen is a cellular pathway that accounts for the destruction of 10-60% of collagen synthesized by a vari ety of cell types prior to secretion. This pathway can serve in a regulatory role to limit the secretion of defec tive molecules, and, in response to some extracellular mediators, regulates the amount and type of collagens secreted. In addition, this pathway may contribute to the pathogenesis of a variety of conditions affecting the extracellular matrix including fibrosis, diabetes mellitus, and scurvy.
Collagen is the most abundant extracellular structural protein of higher organisms. Although the process by which cells pro duce collagen has been described in considerable detail, the mechanisms which regulate the production of this macromole cule are not fully understood. Recent evidence indicates that in addition to tra �scriptional and translational controls, collagen producing cells can modulate collagen production by degrading a portion of newly synthesized collagen prior to secretion. In this context, this review will describe: (1) the pathway of intracellular degradation of collagen including the experimental evidence that demonstrates this pathway in various cells and tissues; (2) the role of intracellular degradation as an important mechanism by which cells regulate both the quality and the quantity of collagen produced; and (3) the relationship between the intracellular degradation of newly synthesized collagen and some disease states. THE PATHWAY OF INTRACELLULAR DEGRADATION OF COLLAGEN: TECHNICAL CONSIDERATIONS Certain aspects of collagen biosynthesis provide important means for quantifying the intracellular degradation of newly synthesized collagen (see reference 1 for review). Specifically, the modification of prolyl and lysyl residues in the nascent polypeptide of the newly synthesized procollagen chain serve as specific markers for collagen (Figure). In this regard, the hydroxylation of proline and lysine are considered to be rela tively specific for collagen in most tissues. Thus, although it is known that hydroxylation of prolyl residues does occur in elastin [2] and in the collagenous regions of the C1q component of complement [3] and acetylcholinesterase [4], quantitatively, the vast amount of hydroxyproline is found in collagen. Indeed, the hydroxyproline content of tissues is generally utilized as a measure of collagen content, and the error due to hydroxypro line from other sources has been estimated to be less than 5% [5]. Importantly, since hydroxyproline and hydroxylysine are fonned only after the synthesis of the polypeptide chain of the collagen molecule [1], the presence of small peptide fragments containing these modified amino acids can be utilized as a marker of collagen degradation. The analysis of such low mo lecular weight hydroxylated derivatives forms the basis for studies regarding intracellular degradation of collagen.
Reprint requests to: Stephen l. Rennard, Pulmonary Branch, Na tional Heart, Lung. and Blood Institute, Bethesda, MD 20205.
U.S.A.
Historical Aspects
A large number of early studies demonstrated that, following the addition of labeled proline to cells or tissues in vitro or to animals in vi vo, there was rapid formation of low molecular weight peptides containing hydroxyproline (Table). Although at the time the significance of these low molecular weight hydroxyproline containing molecules was unclear, it is now recognized that they represented rapid degradation of newly synthesized collagenous chains. Moreover, although the major ity of these studies were not designed to address the question of the site of the rapid degradation of newly synthesized colla gen, as will be described below, considerable experimental evidence exists in these reports in support of an intracellular site for this process. Experimental Proof of an Intracellular Site for the Degradation of Newly Synthesized Collagen
The evidence indicating that rapid degradation of newly synthesized collagen occurs at an intracellular location is now clearly established. Although early investigators considered the possibility that the rapid formation of low molecular weight hydroxyproline containing species indicated intracellular deg radation of newly synthesized collagen [6-12], it was the studies of Bienkowski and colleagues that definitively demonstrated the intracellular location for the degradation of newly synthe sized collagen [13,14]. Utilizing cultured lung fibroblasts [1:3], these studies demonstrated that, following the addition of ra diolabeled proline to culture medium, low molecular weight hydroxyproline was found within 8 minutes in the cells but not in the culture medium. Since collagen synthesis, processing, and secretion requires about :30 minutes, the early presence of hydroxyproline in low molecular weight form dearly indicated collagen degradation prior to secretion. In addition, these workers performed several important con trol experiments. The possibility that the low molecular weight hydroxyproline containing species represented partially com pleted collagen chains which had undergone hydroxylation while still bound to the ribosome was exluded by two experi ments: (1) The majority of hydroxyproline was found to be in a relatively homogeneous population of very small fragments (partially completed nascent chains would likely be present as a range of variably sized fragments); and (2) timed experiments showed that the small hydroxyproline containing species were not converted to complete collagen chains. The possibility that phagocytosis of extracellular collagen contributed to the for mation of low molecular weight hydroxyproline was excluded by studies demonstrating that exogenous collagen added to cultures could be recovered intact. The possibility that some unusual metabolic pathway might be producing labeled hy droxyproline de novo was excluded since the amount of degra dation observed in these studies (10-:30%) was about the same whether hydroxyproline or hydroxylysine was utilized as a marker. Lastly, extracellular degradation was demonstrated to be minimal since (1) the test cells produced no active collagen ase, (2) the addition of extracellular protease inhibitors had no effect on the collagen degradation observed, and (:3) exogenous collagen added to these cultures was recovered intact. Taken together, these observations indicated that a portion of collagen molecules are degraded rapidly after synthesis and 778
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RENNARD ET AL
788
fetal mouse calvaria [7], an d guinea pig granulomata [16]. The rapid formation of low molecular weight hydroxyproline has also been demonstrated in cells derived from tendon (Berg RA, personal communication), lens [8], gingiva [17], liver [18], and skin [9,17,19,20], as well as from lung [13]. The Widespread Nature of Intracellular Degradation:
vivo •
Translation (Synthesis)
r
•
Intracellular
Degradation
Fragments
•
Modification of Amino Acids
•
Addition of Carbohydrate
Assembly of
Procollagen Molecules • Se cre t io n •
Cleavage of � P rocollage n "
�__ " �
Extensions
Co :2 ., u
�
)( UJ
Schematic outline of the pathway of collagen production; major intracellular and extracellular steps are indicated. The posttranslational modifications to proline and lysine residues occur prior to intracellular
in
Studies
A variety of in vivo studies also offer evidence in support of the widespread nature of intracellular degradation of newly synthesized collagen (Table). The experimental design of most of these studies involves the injection of radiolabeled proline into live animals and evaluating the specific activity and the time course of hydroxyproline excreted in the urine. These kinetic studies suggest that significant amounts of urinary hy droxyproline originate from a pool of collagen with a half life of no more than several hours in man [21] or in rat [10]. In addition, labeled hydroxyproline is detectab le in the urine soon after the labeled proline is administered in monkey [22] and guinea pig [to], in agreement with the concept that a pool of rapidly degraded collagen exists in vivo. Moreover, the specific activity of the excreted hydroxyproline in these studies is very high suggesting that little degradation of mature, unlabeled collagen is taking place and that this rapid degradation is selective for newly synthesized collagen chains. Further in vivo evidence in support of intracellular degrada tion in specific tissues has been presented by Schneir and colleagues who have detected low molecular weight hydroxy proline in skin, aorta, and intestine after the injection of labeled proline into rats [23,24]. Two lines of evidence strongly suggest that his in vivo collagen degradation was due to an intracellular process. First, the high specific activity of the low molecular
degradation; low molecular weight fragments containing these hydrox ylated aminoacids can be utilized as indicators of intracellular degra
TABLE 1. Rapid degradation of newly synthesized collagen
dation.
excluded the possibility that the low molecular weight hydrox yproline represents the degradation of secreted collagen either extracellularly or foll owing phagocytosis. Widespread Nature
of Intracellular Degradation: in
Experimental Design
In vitro
Skin (emCalvaria (embryo)
Studies
Mandible
been
,
,
Percentage of newly synthesized collagen degraded
Reference
16
Chick
bryo)
vitro
The rapid degradation of newly synthesized collagen has demonstrated in a number of in vitro studies (Table) . Although the early studies which demonstrated the rapid deg radation of newly synthesized collagen and the rapid formation of hydroxyproline following the addition of radioactive proline to various experimental systems were not specific ally designed to demonstrate the intracellular degradation of newly synthe sized collagen they do present evidence which supports the concept that rapid, likely intracellular, degradation of collagen occurs in many tissues. For example, Daughaday and Mariz reported the presence of low molecular weight hydroxyproline within 2 hr after the addition of labeled proline to rat cartilage in culture [6]. They showed that this material accumulated at a linear rate for the duration of the labeling period, and, in addition, demonstrated that an extracellular pool of collagen could not be the source of the low molecular weight hydroxy proline. Taken together, these data are similar to that of Bien kowski, Baum, and Crystal for lung fibroblast cultures [13] and suggest that in rat cartilage there is also rapid intracellular degradation of a portion of newly synthesized collagen. Similar evidence in support of the intracellular degradation of newly synthesized collagen also exists for other tissues and cells (Table). Low molecular weight hydroxyproline has been observed within 15 min following the addition of labeled proline to cultures of fetal chick skin [9] and to intact chick em bryos [15]. Furthermore, low molecular weight hydroxyproline accu lumates at a constant rate in c u l tures of fetal chick skin [9]
Species
Tissue
Rat
25-30
Mouse
20-30
7
40
46
15-25
6
Chick
Cartilage
Rat
Lens
Chick
Granuloma
Guinea pig
Lung
Human Rabbit Hamster
Embryo
Chick
26
4,47
8
16 20-40
14
6-24
51
10
15
(whole) Cells
In vitro
Fibroblast skin
In vivo
30-32
Human
17,48
30
9, 19
Calf
30
Mouse
Chick
Lens
Chick
Lung
Human
Gingiva
Mouse
30
17
3T6
Mouse
20,60
38
Hepatocyte
Rat
Whole ani-
Rat
mal
34
8
10,30
13, 35,39
37
55
45 90
37 18
10,11,56
Guinea pig
57
Monkey
22
Man Skin
Rat Guinea pig
Intestine
Rat
Aorta
Rat
h 8 h 10 9h
58,21 23, 24 25
2:3 23
not available. Values probably represent an underestimate; see text for discussion.
" Rapid degradation detected but quantitative data h
20 17,53
Tendon
July 1982
INTRACELLULAR
DEGRADATION
OF NEWL Y
SYNTHESIZED
COLLAGEN
798
weight hydroxyproline peptides indicated a maximal amount of degradation soon after synthesis. Second, although an increase in the percentage of rapid degradation was present in skin of diabetic rats, in mixing experiments in vitro, diabetic skin did not cause the degradation of newly synthesized collagen pro duced by normal skin, thus excluding an extracellular mecha nism for collagen degradation.
suprising. However, the available evidence indicates that the intracellular degradation of collagen is a widespread process which can occur simultaneously with extracellular collagen degradation. It is likely that these 2 pathways for the destruc tion of collagen molecules play very different roles in the metabolism of collagen.
Quantitive Studies of Intracellular Degradation
Collagen
In general, all quantitative studies of intracellular degrada tion in normal cells for which data are available are quite similar, demonstrating 10-40% of newly synthesized collagen being degraded (Table). The hepatocyte appears to be an exception to this rule; hepatocyes in culture degrade 90% of newly synthesized collagen [18]. It is not known whether this high level of intracellular degradation found in hepatocyte cultures reflects a special property of these cells, a general property of epithelial cells, or if this result depends on the specific conditions utilized in this study. Interestingly, in spite of technical limitations of making such measurements in vivo, the available quantitative data for in vivo estimates of intracellular collagen degradation agree well with the in vitro estimates. For example, Schneir et al [23,24] have estimated intracellular degradation to be 8% in skin and 9% in intestine based on a 4 hr labeling period, in good agree ment with Barnes et al [25] who observed 9% of hydroxyproline to be present in low molecular weight form in guinea pig skin in vivo. While these values are somewhat lower than those obtained by in vitro methods, both of these investigators pres ent evidence that the low molecular weight species are cleared rapidly from tissues. This is consistent with the urinary excre tion data and suggests that the estimates of 8-9% represent a lower limit for the rapid degradation of newly synthesized collagen.
Recent studies indicate that collagen is not the only secreted protein subject to intracellular degradation. Insulin [27,28], parathyroid hormone [29-31], and prolactin [32] all appear to be subject to intracellular degradation within the cell prior to secretion. Interestingly, in the case of these hormones, the degradation appears to be selective for recently synthesized molecules that are resident within an intracellular storage pool and appears to regulate the amount available for secretion in response to a variety of physiologic stimuli.
Intracellular Degradation Compared to Extracellular Degradation
It is quite clear that the intracellular degradation of newly synthesized collagen and the extracellular degradation of col lagen by specific neutral proteases are distinct processes. How ever, in some tissues, intracellular and extracellular degradation of collagen appear to take place simultaneously. For example, in gingival fibroblasts [17], in intact chick lens [8], and in cultured lens cells [8], labeled proline is rapidly converted to low molecular weight hydroxyproline suggesting collagen deg radation. However, the addition of extracellular protease inhib itors results in a decrease in the amount degraded, indicating extracellular collagen degradation. Importantly, however, some collagen degradation occurs even in the presence of these extra cellular protease inhibitors. Thus, although Grant, Kefalides, and Prockop found that the addition of serum completely blocked the degradation of labeled extracellular collagen added to cultured lens cells, the degradation of newly synthesized collagen still constituted greater than 20% of collagen synthesis, and therefore must represent an intracellular process [8]. Sim ilarly, Roszkowski and Sauk observed that extracellular pro tease inhibitors could block much, but not all, of the degrada tion of newly synthesized collagen in gingival cell cultures [17]. Studies of the specific activity of the hydroxyproline found in low molecular weight fragments under these conditions in dicated that the most recently synthesized collagen was being degraded while older, extracellular collagen, if degraded at all, was degraded at a much lower rate. Thus, extracellular protease inhibitors could decrease extracellular collagen degradation while the simultaneous intracellular degradation of newly syn thesized collagen was unaffected. That many cell types can produce proteases capable of de grading collagen is well known [26], and that some extracellular degradation of collagen might occur in some systems in which intracellular degradation is simultaneously occurring is not
Intracellular Degradation of Secreted Proteins Other Than
ROLE OF INTRACELLULAR DEGRADATION OF NEWLY SYNTHESIZED COLLAGEN IN THE CELLULAR CONTROL OF COLLAGEN PRODUCTION Current concepts suggest that the intracellular degradation of newly synthesized collagen serves two distinct roles in mod ulating collagen production. First, intracellular collagen degra dation provides a mechanism for the destruction of defective molecules before their secretion, thus preventing the incorpo ration of defective molecules into the extracellular matrix. Second, intracellular degradation provides one means to regu late the amount of collagen and the relative amounts of collagen types produced by cells in response to certain extracellular mediators. Degradation of Defective Molecules
The formation of the triple helical structure of the collagen molecule requires severe constraints on the amino acid com position of the component polypeptide chains [1,33]. Since collagen is a large molecule with nearly 1,000 amino acid residues per chain, even a very small percentage of errors would result in the production of significant amounts of abnormal molecules. For example, every third amino acid must be a glycine to allow the chains to properly fold into a triple helix [1,33]; substitution of a glycine residue with another amino acid results in a molecule that can not assume a normal conforma tion. In addition, the hydroxyprolyl residues in the collagen chain are also required for a stable triple helical structure [1,33]. Thus, it is possible that intracellular degradation of newly synthesized collagen could function as a cellular "quality control" mechanism to rapidly destroy any abnormal molecules that might be synthesized. Support for the hypothesis that intracellular degradation selectively disposes of abnormal collagen comes from 3 lines of evidence. First, incubation of cell" with the analogues of proline such as cis-hydroxyproline or azetidine result in the incorporation of these analogues into collagen molecules that can not assume a stable triple helix [34]. In both situations, there is an increase in the amount of collagen degraded within the cell [13,35]. Second, when hydroxylation of proline is blocked, cells pro duce abnormal, underhydroxylated collagen; these circum stances are associated with an increase in the proportion of newly synthesized collagen which is rapidly degraded [36-38]. For example, in the absence of ascorbate, lung fibroblasts degrade up to 60% of newly synthesized collagen [36], a 4- to 6fold increase in baseline intracellular collagen degradation. In terestingly, some of the abnormal collagen chains produced by these cells are still secreted, suggesting that the mechanism of intracellular degradation may have a limited capacity, i.e., in the absence of ascorbate, abnormal molecules can still "overflow" into the extracellular space. If such "overflow" oc-
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curs in cell types which also secrete proteases capable of extra cellular collagen degradation, this may account for the increase in extracellular degradation of collagen sometimes observed when cells produce abnormal collagen. Third, lung fibroblasts underhydroxylate collagen during the log phase of growth; this condition is associated with a nearly 3-fold increase in the proportion of newly synthesized collagen degraded intracellularly over that observed when cells achieve confluency [39]. Taken together, these experiments clearly support the con cept that abnormal collagen molecules can be detected within the cell and shunted into a degradative pathway. Intracellular Site of Degradation of Defective Collagen Molecules
Studies with lung fibroblasts have demonstrated that the increase in intracellular degradation that occurs in association with the formation of abnormal collagen chains is due to a lysosomal process [35,40]. For example, inhibitors of lysosomal proteolytic activity block the increased intracellular degrada tion that occurs when cells make defective collagen. Moreover, the decrease in production of low molecular weight hydroxy proline containing fragments in the presence of inhibitors of lysosomal function is accompanied by an accumulation of hy droxyproline containing material within lysosomes [35]. Al though one study of ascorbate deficient rat skin fibroblasts showed no decrease in the percentage of intracellular degrada tion with lysosomal inhibitors [17], in this case, only the me dium and an acetic acid extract of the cell layers was analyzed, and therefore, intralysosomal material was not fully accounted for, i.e., the effect of lysosomal inhibitors may have been un derestimated. Further support of the localization of intracellular degrada tion to a lysosomal site derives from studies of Karim, Cournil, and Leblond [41] which demonstrate material within lysosom9s that was antigenically cross reactive with procollagen. This material appears to reach lysosomes by way of the endoplasmic reticulum and the Goigi apparatus where collagen containing microvesicular bodies are formed. Interestingly, such microves icular bodies have also been implicated in mediating the lyso somal destruction of other intracellular components [42]. Although the available evidence supports the concept that a lysosomal process is involved in the increased intracellular degradation of newly synthesized collagen that occurs with the production of defective molecules, lysosomal inhibitors do not inhibit all intracellular degradation [17,35,40]. In the )Jresence of these agents approximately 10% of newly synthesized colla gen molecules are still degraded. Thus, in some circumstances, intracellular extralysosomal processes may be important for the intracellular degradation of collagen. Intracellular Degradation of Newly Synthesized Collagen as a Mechanism for Control of Collagen Production
Intracellular degradation of newly synthesized collagen is one means by which cells regulate collagen production in response to exogenous mediators. For example, intracellular degradation is important in the response of lung and skin fibroblasts to stimuli which affect the cAMP system [43]. Agents which elevate cAMP in these cells also cause an increase in the intracellular degradation of newly synthesized collagen with a corresponding decrease in the amount of collagen :-;ecreted. Moreover, the effect of elevated levels of cAMP appears to be selective in increasing the degradation of type I collagen, i.e., the production of type III collagen by these cells appears to be unaffected by cAMP levels [44]. In this context, since certain properties of the extracellular matrix may critically depend on the ratio of collagen types present, intracellular degradation may play an important role in regulating matrix composition [45]. Although intracellular degradation of collagen has been noted
in bone [7,46] (Kream BE and Raisz LG, personal communi cation) and cartilage [6], tissues where collagen production is sensitive to hormonal control, the relative role of intracellular degradation in the control of collagen production in these sites is just beginning to be understood. In cultured bone, for exam ple, intracellular degradation appears to be less important than other mechanisms for the control of collagen production [47]. Thus, the quantitative importance of intracellular degradation in the control of collagen synthesis is likely to be different in various tissues. Control of Intracellular Degradation
Several lines of evidence suggest that widely differing cellular events can lead to the intracellular degradation of newly syn thesized collagen. First, agents which increase cAMP within cells do not affect the degree of prolyl hydroxylation [43]; i.e., the effects of cAMP on collagen degradation are not mediated through changes in the conformation of the collagen molecule. Second, under circumstances where collagen is underhydroxy lated, production of both types I and III collagen is reduced [36] (Rennard SI, Berg RA, and Crystal RG, unpublished observations); in contrast, the cAMP mediated effect is specific in decreasing the amount of type I [44]. Thus the cell can increase intracellular degradation of newly synthesized collagen in a different fashion in response to different stimuli. Third, intracellular degradation has also been observed to be increased in skin fibroblasts following a phagocytic stimulus [48] although it is unknown whether this event affects either intracellular cAMP or the degree of hydroxylation of collagen. Together, these results strongly suggest that more than one cellular pathway leads to the intracellular degradation of newly synthe sized collagen. INTRACELLULAR DEGRADATION OF NEWLY SYNTHESIZED COLLAGEN AND DISEASE STATES Very few disease states have been studied with regard to a pathogenic role for intracellular degradation of newly synthe sized collagen. Although it is possible that abnormal intracel lular degradation of collagen on a congenital basis may contrib ute to one of the primary heritable collagen disorders, this phenomenon has only been studied in skin fibroblasts from patients with osteogenesis imperfecta, a group of inherited disorders characterized by the underproduction of type I col lagen. In these cells, intracellular degradation of collagen is normal and does not appear to contribute to the disease process [19]. In acquired diseases, however, current evidence suggests a role for intracellular degradation in 3 distinct disease states: fibrosis, diabetes mellitus, and scurvy. Fibrosis is a state characterized by an abnormal accumulation of connective tissue and can result from a wide variety of causes. Several separate lines of evidence suggest a role for intracellular degradation in certain types of fibrosis. 1. Fibrosis has been associated with ,B-blocking drugs such as propanolol or practolol, and may be due to a pharmacologic interruption in the control of intracellular degradation of col lagen [49]. As detailed above, ,B-agonists, by increasing intra cellular cAMP levels, induce an increase in the intracellular degradation of collagen and reduce collagen production by certain fibroblasts [43,44]. ,B-blocking drugs, by interfering with this mechanism [49], could prevent ,B-agonist mediated sup pression of collagen production and thus contribute to an ov erproduction of collagen which could result in fibrosis. 2. Certain inflammatory states, characterized by the devel opment of fibrosis, may also result from interference with the normal ,B-agonist mediated mechanisms that suppress collagen production by increasing intracellular degradation. In these conditions, however, the interference in the regulation of col lagen production is likely mediated by the proteolytic enzymes released by inflammatory cells resulting in proteolytic attack of fibroblast cell surface ,B-agonist receptors. As a result, in the
July 1982
INTRACELLULAR DEGRADATION OF NEWLY
presence of inflammation, some cells may overproduce type I collagen since these cells could not respond to suppressive /3agonist regulation of collagen production. In support of this hypothesis, the in vitro treatment of lung fibroblasts with either trypsin or leukocyte elastase results in the loss of isoproterenol mediated suppression of type I collagen [50]. 3. Bleomycin, a chemotherapeutic drug used in the treat ment of certain cancers, can cause pulmonary fibrosis. Experi mental studies with this drug have demonstrated that many changes are induced in affected lung tissue, including inflam mation, increased collagen synthesis, and a 3-fold increase in intracellular degradation of newly synthesized collagen [51]. Although it is currently unknown which effects are primary and which are secondary, (or even which cell types are being af fected in this disease process), it is likely that the changes in intracellular degradation of newly synthesized collagen contrib ute to the altered connective tissue formation caused by bleo mycin exposure. Diabetes mellitus is associated with a variety of abnormalities of connective tissue including the atrophy of skin. Schneir et al, have demonstrated, in vivo, that the skin of diabetic rats degrades a much larger portion of newly synthesized collagen than does normal skin, and that this increase in degradation can account for much of the atrophy seen in the skins of these animals [23,24]. Although the mechanism which leads to the increased degradation of newly synthesized collagen in the diabetic skin and the relationship of this process to other atrophic conditions are unknown, it is possible that such pro cesses could be important in the pathogenesis of these clinically significant conditions. Scurvy is the clinical syndrome which follows the prolonged deficiency of ascorbic acid. Without this vitamin, collagen can not be adequately hydroxylated and, as a result, abnormal, nonhelical collagen chains are formed [52]. Since a large portion of these molecules are degraded within the cell, collagen pro duction is dramatically reduced in scurvy [36]. Moreover, as noted above, the pathway of intracellular degradation can be overwhelmed, resulting in the secretion of some abnormal chains which, conceivably, could be incorporated into extracel lular matrix and result in abnormal connective tissue. It is therefore likely that in scurvy the decreased production of normal molecules and the secretion of abnormal molecules both contribute to the connective tissue abnormalities present.
REFERENCES 1. P rockop DJ, Kivirikko KI, Tuderman L, Guzman NA: The biosyn thesis of collagen and its disorders. New Engl J Med 301:13-23, 77-85, 1979
2. Rucker RB, Tinker S: Structure and metabolism of arterial elastin. Int Rev Exp Pathol 7:1-47, 1977
3. Reid KBM, Porter RR: Subunit structure of subcomponent CIq of the first component of human complement. Biochem J 155:19-23, 1976 4. Rosenberry T, Richardson J: Structure of 18S and 1 4S acetylcho linesterase. Biochemistry 16:3550-3558, 1977 5. Bradley KH, McConnell SD, Crystal RG: Lung collagen composi tion and synthesis: Characterization and changes with age . •1 BioI Chern 249:2674-2683, 1974 6. Daughattay WH, Mariz IK: The formation of free hydroxyproline by rat cartilage in vitro. J BioI Chern 237:2831-2835,1962 7. Stern B, Glimcher MJ, Goldhaber P: The effect of various oxygen tensions on the synthesis and degradation of bone collagen in tissue culture. Proc Soc Exp BioI Med 121:869-872, 1965 8. Grant ME, Kefalides NA, Prockop DJ: The biosynthesis of base ment membrane collagen in em bryonic chick lens. J BioI Chern 247:3545-3551, 1972 9. Hurych J, Chvapil M, The role of free hydroxyproline in the biosynthesis of collagen. Biochim Biophys Acta 107:91-96, 1965 10. Kibrick AC, Singh DK: Hydroxyproline excreted in the urine: Its source from collagen of tissues after [14] C proline in rats with and without administration of prednisone . J Clin End Metab 38:594-601, 1974 11. Laitinen 0: The metabolism of collagen and its hormonal control in the rat. Acta Endocrinologica suppl 120:1-84, 1967m 12. Kivirikko KI: Urinary excretion of hydroxyproline in health and disease. Int Rev Connective Tissue Res 5:93-163, 1970
SYNTHESIZED
COLLAGEN
81s
13. Bienkowski RS, Baum BJ, Crystal RG: Fibroblasts degrade newly synthesized collagen within the cell before secretion. Nature 276:413-416, 1978 14. Bienkowski RS, Cowan MJ, McDonald JA, Crystal RG: Degrada tion of newly synthesized collagen. J BioI Chern 253:4356-4363, 1978 15. Prockop DJ, Peterkovsky B, Udenfriend S: Studies on the intra cellular localization of collagen synthesis in the intact chi c k embryo. J BioI Chern 237:1581-1584, 1962 16. Green NM, Lowther DA: Formation of Collagen Hydroxyproline in vitro. Biochem J 71:55-66, 1959 17. Roszkowski M, Sauk JJ: The role of intracellular lysosomal en zymes in the autocellular-surveillance of unhydroxylated colla gens in dermal and gingival fibroblasts. J Dent Res 60:1045-1052, 1981 18. Diegelmann RF, Cohen IK, Guzelian PS: Rapid degradation of newly synthesized collagen by primary cultures of adult hepato cytes. Biochem Biophys Res Commun 97:819-826,1980 19. Steinmann BU, Martin GR, Baum BI, Crystal RG: Synthesis and degradation of collagen by skin fibroblasts from patients with osteogenesis imperfecta . FEBS lett 101:269-272, 1979 20. Krieg 1', Horlein D, Wiestner M, Muller PK: Aminoterminal exten sion peptides from type I pro collagen normalize excessive colla gen synthesis of scleroderma fibroblasta. Arch Dermatol Res 263:171-180,1978 21. Krane SM, Munoz AJ, Harris ED Jr: Collagen-like fragments: Excretion in urine of patients with Paget's disease of bone . Science 157:713-716, 1967 22. Avioli LV, Prockop DJ : Collagen degradation and the response to parathyroid extract in the intact rhesus monkey. J Clin Invest 46:217-224, 1967 23. Schneir M, Bowersox J, Ramamurthy N, Yavelow J, Murray J, Edlin-Folz E, and Golub L: Response of rat con nective tissues to streptozotocin-diabetes. Tissue specific effects on collagen me tabolism. Biochim Biophys Acta 583:95-102, 1979 24. Schneir M, Golub L: The effect of streptozotocin-induced diabetes on collagen catabolism, Streptozotocin: Fundamentals and Ther apy. Edited by Agarwal. Elsevier-/North-Holland, Biomedical Press, 1981, pp 160-182 25. Barnes MJ, Constable BJ, Morton LF, Kodicek K Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid deficient guinea pigs. Biochem J 119:575-585, 1970 26. Harris ED Jr, Cartwright EC: Mammalian collagenases in Pro teases, Mammalian Cells and Tissues. Edited by AJ Barrett. North Holland, Amsterdam , 1977, pp 249-284 27. Halban PA, Wollheim CB: Intracellular degradation of insulin stores by rat pancreatic islets in vitro. An alternative pathway for homeostasis of pancreatic insulin content. J BioI Chern 255:6003-6006, 1980 28. Halban PA, Wollheim CB, Blondel B, Niesor E, Renold AK Per turbation of hormone storage and release induced by cyprohep tadine in rat pancreatic islets in vitro. Endocrinology 104:1096-1106, 1979 29. Morrissey JJ, Cohn DV: Secretion and degradation of parathor mone as a function of intracellular maturation of horm one pools . J Cell BioI 83:521-528, 1979 30. Habener JF, Kemper B, Potts JT Jr.: Calcium dependent intracel lular degradation of parathyroid hormone: A possible mechanism for the regulation of hormone stores. Endocrinology 97:431-441, 1975 31. M orissey JJ, Hamilton JW, Cohn DV: The secretion of parathor mone and glycosylated proteins by parathyroid cells in culture. Biochem Biophys Res Comm 82:1279-1286,1978 32. Shenai R, Wallis M: Biosynthesis and degradation of prolactin in the rat anterior pituitary gland Time course of incorporation of label in vitro and evidence for rapid degradation . Biochem .1 182:735-743,1979 33. Fietzek PP, Kuhn K: The prim ary structure of collagen. lnt J Connect Tissue Res 7:1-60, 1976 34. Inouye K, Sakakibara S, Prockop DJ: Effects of the stereo config uration of the hydroxyl group in 4 -hydroxyproline on the triple helical structures formed by homogeneous peptides resembling collagen. Biochem Biophys Acta 420:133-141, 1976 35. Berg RA, Schwartz ML, Crystal RG: Regulation of the production of secretory proteins: Intracellular degradation of newly synthe sized "defective" collagen. Proc Nat! Acad Sci 77:4746-4750,1980 36 . Berg RA, Steinmann B, Rennard SI, Crystal RG: Ascorbate defi ciency results in decreased collagen production: underhydroxy lation leads to increased intracellular degradation and decreased production of triple helical molecules, submitted for publication 37. Vistica DT, Ahrens FA, Ellison WR: The effects of lead upon collagen synthesis and proline hydroxylation in the swiss mouse
31'6 fibroblast. Arch Biochem Biophys 179:15-23, 1977 38. Ramaley PB, Rosenbloom J: Inhibition of proline and lysine hy droxylation prevents normal extrusion of collagen by 31'6 fibro blasts in culture. FEBS lett 15:59-64, 1971 39. Tolstoschev P, Berg RA, Rennard SI, Bradley KH, Trapnell BC, Crystal RG: Procollagen production and procollagen messenger
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40.
41.
42. 43.
44.
45.
46.
47.
48.
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RNA levels and activity in human lung fibroblasts during perIods of rapid and stationaly growth. J BioI Chern 256:3135-3140, 1981 Berg RA, Schwartz ML, Rome LH, Crystal RG: Lysosomal function in the degradation of defective collagen in cultured lung fibro blasts, submitted for pu blication Karim A, Cournil I, Leblond CP: Immunohistological localization of procollagens II Electron microscopic distribution of procolla gen I antigenicity in the odontoblasts and predentin of rat incisor teeth by a direct method using peroxi clase linked antibodies . J Histochem Cytochem 27:1070-1083,1979 Locke L, Sykes AK: The role of the Golgi complex in the isolation and digestion of organelles. Tissue and Cell 7:143-158, 1975 Baum BJ , Moss ,1, Breul SD, Berg RA, Crystal RG: Effect of cyclic AMP on the intracellular degradation of newly synthesized col lagen. J BioI Chern 255:2843-2847, 1980 Rennard S, Saltzman L, Moss J, Fells G, Gadek J, Hom B, Hun ninghake G, Crystal H: Modulation of fibroblast production of collagen types I and III: Effects of PGEI and isoproterenol. Fed Proc 40:1813, 1981 Lapiere CM, Nusgens B, P ierard GE: Interaction between collagen type I and type III in conditioning bundles organization. Connect Tissue Res 5:21, 1977 Sakamoto M, Sakamoto S, Brickley-Parsons D, Glimcher M.l: Collagen synthesis and degradation in embryonic chick-bone explants. J Bone ,It Surg 61-A:1042-1052, 1979 Kream BE, Rowe DW, Gworek SC, Raisz LG: Parathyroid hormone alters collagen cynthesis and proc ollagen mRNA levels in fetal rat calvaria. Proc Natl Acad Sci 77:5654-5658, 1980 Sauk ,JJ Jr.: Collagen synthesis and turnover following particle
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49.
50.
51. 52. 53. 54.
55. 56. 57.
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phagocytosis in dermal fi b roblasts. Biochim Biophys Acta 607:161-170, 1980 Berg RA, Moss J, Baum BJ, Crystal RG: Regulation of collagen production by the B-adrenergic system. J Clin Invest 67:1457-1462, 1981 Rennard S, Berg R, Moss J, Saltzman L, Hom B, Stier L, Gadek, Fells G, Crystal R: Protease mediated alteration of collage n production by lung fibroblasts. Am Rev Resp Dis 119:224, 1981 Clark JG, Overton JE, Marino BA, Uitto J, Starcher BC: Collagen biosynthesis in bleomycin-induced fibrosis in hamsters. J Lab Clin Med 96:943-953, 1980 Barnes M.l: Function of ascorbic acid in collagen metabolism. Ann NY Acacl Sci 258:264-277, 1975 Steinberg ,/: The turnover of collagen in fibroblast cultures. J Cell Sci 12:217-234, 1973 Jimin ez SA, Dehm P, Olsen BR, Prockop DJ: Intracellular collagen and proto collagen from embryonic tendon cells. ,J BioI Chern 248:720-729, 1973 Steinberg J: Collagen turnover and the growth state in 3T6 fibro blast cultures. Lab Invest 39:491-496, 1978 Prockop D J: Isotopic studies on collaged degradation and the urine excretion of hydroxy proline. ,1 Clin Invest 43:453-4()o. 1964 Lindstedt S, Proc kop DJ: Isotopic studies on urinary hydroxypro line as evidence for rapidly catabolized forms of collagen in the young rat. J BioI Chern 236: l:l99-1403, 1961 Phelps P, Avioli LV, Pro ckop DJ: Isotopic studies on collagen degradation in man: Effect of triiodothyronine on hydroxypro line- [14]C excretion. Clin Res 15:467, 1967
Supplement 1 Printed in U.S.A.
THE .JOURNAL OF INVESTIGATIVE DERMATOLOGY, 79:778-828, 1982
Copyright ©
1982
Vol. 79,
by The Williams & Wilkins Co.
Intracellular Degradation of Newly Synthesized Collagen STEPHEN 1. RENNARD, M.D., LARUE E. STIER, M.S., AND RONALD G. CRYSTAL, M.D. Pulmonary Branch, National Heart, Lung, and Blood Institute Bethesda, Maryland,
The intracellular degradation of newly synthesized collagen is a cellular pathway that accounts for the destruction of 10-60% of collagen synthesized by a vari ety of cell types prior to secretion. This pathway can serve in a regulatory role to limit the secretion of defec tive molecules, and, in response to some extracellular mediators, regulates the amount and type of collagens secreted. In addition, this pathway may contribute to the pathogenesis of a variety of conditions affecting the extracellular matrix including fibrosis, diabetes mellitus, and scurvy.
Collagen is the most abundant extracellular structural protein of higher organisms. Although the process by which cells pro duce collagen has been described in considerable detail, the mechanisms which regulate the production of this macromole cule are not fully understood. Recent evidence indicates that in addition to tra �scriptional and translational controls, collagen producing cells can modulate collagen production by degrading a portion of newly synthesized collagen prior to secretion. In this context, this review will describe: (1) the pathway of intracellular degradation of collagen including the experimental evidence that demonstrates this pathway in various cells and tissues; (2) the role of intracellular degradation as an important mechanism by which cells regulate both the quality and the quantity of collagen produced; and (3) the relationship between the intracellular degradation of newly synthesized collagen and some disease states. THE PATHWAY OF INTRACELLULAR DEGRADATION OF COLLAGEN: TECHNICAL CONSIDERATIONS Certain aspects of collagen biosynthesis provide important means for quantifying the intracellular degradation of newly synthesized collagen (see reference 1 for review). Specifically, the modification of prolyl and lysyl residues in the nascent polypeptide of the newly synthesized procollagen chain serve as specific markers for collagen (Figure). In this regard, the hydroxylation of proline and lysine are considered to be rela tively specific for collagen in most tissues. Thus, although it is known that hydroxylation of prolyl residues does occur in elastin [2] and in the collagenous regions of the C1q component of complement [3] and acetylcholinesterase [4], quantitatively, the vast amount of hydroxyproline is found in collagen. Indeed, the hydroxyproline content of tissues is generally utilized as a measure of collagen content, and the error due to hydroxypro line from other sources has been estimated to be less than 5% [5]. Importantly, since hydroxyproline and hydroxylysine are fonned only after the synthesis of the polypeptide chain of the collagen molecule [1], the presence of small peptide fragments containing these modified amino acids can be utilized as a marker of collagen degradation. The analysis of such low mo lecular weight hydroxylated derivatives forms the basis for studies regarding intracellular degradation of collagen.
Reprint requests to: Stephen l. Rennard, Pulmonary Branch, Na tional Heart, Lung. and Blood Institute, Bethesda, MD 20205.
U.S.A.
Historical Aspects
A large number of early studies demonstrated that, following the addition of labeled proline to cells or tissues in vitro or to animals in vi vo, there was rapid formation of low molecular weight peptides containing hydroxyproline (Table). Although at the time the significance of these low molecular weight hydroxyproline containing molecules was unclear, it is now recognized that they represented rapid degradation of newly synthesized collagenous chains. Moreover, although the major ity of these studies were not designed to address the question of the site of the rapid degradation of newly synthesized colla gen, as will be described below, considerable experimental evidence exists in these reports in support of an intracellular site for this process. Experimental Proof of an Intracellular Site for the Degradation of Newly Synthesized Collagen
The evidence indicating that rapid degradation of newly synthesized collagen occurs at an intracellular location is now clearly established. Although early investigators considered the possibility that the rapid formation of low molecular weight hydroxyproline containing species indicated intracellular deg radation of newly synthesized collagen [6-12], it was the studies of Bienkowski and colleagues that definitively demonstrated the intracellular location for the degradation of newly synthe sized collagen [13,14]. Utilizing cultured lung fibroblasts [1:3], these studies demonstrated that, following the addition of ra diolabeled proline to culture medium, low molecular weight hydroxyproline was found within 8 minutes in the cells but not in the culture medium. Since collagen synthesis, processing, and secretion requires about :30 minutes, the early presence of hydroxyproline in low molecular weight form dearly indicated collagen degradation prior to secretion. In addition, these workers performed several important con trol experiments. The possibility that the low molecular weight hydroxyproline containing species represented partially com pleted collagen chains which had undergone hydroxylation while still bound to the ribosome was exluded by two experi ments: (1) The majority of hydroxyproline was found to be in a relatively homogeneous population of very small fragments (partially completed nascent chains would likely be present as a range of variably sized fragments); and (2) timed experiments showed that the small hydroxyproline containing species were not converted to complete collagen chains. The possibility that phagocytosis of extracellular collagen contributed to the for mation of low molecular weight hydroxyproline was excluded by studies demonstrating that exogenous collagen added to cultures could be recovered intact. The possibility that some unusual metabolic pathway might be producing labeled hy droxyproline de novo was excluded since the amount of degra dation observed in these studies (10-:30%) was about the same whether hydroxyproline or hydroxylysine was utilized as a marker. Lastly, extracellular degradation was demonstrated to be minimal since (1) the test cells produced no active collagen ase, (2) the addition of extracellular protease inhibitors had no effect on the collagen degradation observed, and (:3) exogenous collagen added to these cultures was recovered intact. Taken together, these observations indicated that a portion of collagen molecules are degraded rapidly after synthesis and 778
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RENNARD ET AL
788
fetal mouse calvaria [7], an d guinea pig granulomata [16]. The rapid formation of low molecular weight hydroxyproline has also been demonstrated in cells derived from tendon (Berg RA, personal communication), lens [8], gingiva [17], liver [18], and skin [9,17,19,20], as well as from lung [13]. The Widespread Nature of Intracellular Degradation:
vivo •
Translation (Synthesis)
r
•
Intracellular
Degradation
Fragments
•
Modification of Amino Acids
•
Addition of Carbohydrate
Assembly of
Procollagen Molecules • Se cre t io n •
Cleavage of � P rocollage n "
�__ " �
Extensions
Co :2 ., u
�
)( UJ
Schematic outline of the pathway of collagen production; major intracellular and extracellular steps are indicated. The posttranslational modifications to proline and lysine residues occur prior to intracellular
in
Studies
A variety of in vivo studies also offer evidence in support of the widespread nature of intracellular degradation of newly synthesized collagen (Table). The experimental design of most of these studies involves the injection of radiolabeled proline into live animals and evaluating the specific activity and the time course of hydroxyproline excreted in the urine. These kinetic studies suggest that significant amounts of urinary hy droxyproline originate from a pool of collagen with a half life of no more than several hours in man [21] or in rat [10]. In addition, labeled hydroxyproline is detectab le in the urine soon after the labeled proline is administered in monkey [22] and guinea pig [to], in agreement with the concept that a pool of rapidly degraded collagen exists in vivo. Moreover, the specific activity of the excreted hydroxyproline in these studies is very high suggesting that little degradation of mature, unlabeled collagen is taking place and that this rapid degradation is selective for newly synthesized collagen chains. Further in vivo evidence in support of intracellular degrada tion in specific tissues has been presented by Schneir and colleagues who have detected low molecular weight hydroxy proline in skin, aorta, and intestine after the injection of labeled proline into rats [23,24]. Two lines of evidence strongly suggest that his in vivo collagen degradation was due to an intracellular process. First, the high specific activity of the low molecular
degradation; low molecular weight fragments containing these hydrox ylated aminoacids can be utilized as indicators of intracellular degra
TABLE 1. Rapid degradation of newly synthesized collagen
dation.
excluded the possibility that the low molecular weight hydrox yproline represents the degradation of secreted collagen either extracellularly or foll owing phagocytosis. Widespread Nature
of Intracellular Degradation: in
Experimental Design
In vitro
Skin (emCalvaria (embryo)
Studies
Mandible
been
,
,
Percentage of newly synthesized collagen degraded
Reference
16
Chick
bryo)
vitro
The rapid degradation of newly synthesized collagen has demonstrated in a number of in vitro studies (Table) . Although the early studies which demonstrated the rapid deg radation of newly synthesized collagen and the rapid formation of hydroxyproline following the addition of radioactive proline to various experimental systems were not specific ally designed to demonstrate the intracellular degradation of newly synthe sized collagen they do present evidence which supports the concept that rapid, likely intracellular, degradation of collagen occurs in many tissues. For example, Daughaday and Mariz reported the presence of low molecular weight hydroxyproline within 2 hr after the addition of labeled proline to rat cartilage in culture [6]. They showed that this material accumulated at a linear rate for the duration of the labeling period, and, in addition, demonstrated that an extracellular pool of collagen could not be the source of the low molecular weight hydroxy proline. Taken together, these data are similar to that of Bien kowski, Baum, and Crystal for lung fibroblast cultures [13] and suggest that in rat cartilage there is also rapid intracellular degradation of a portion of newly synthesized collagen. Similar evidence in support of the intracellular degradation of newly synthesized collagen also exists for other tissues and cells (Table). Low molecular weight hydroxyproline has been observed within 15 min following the addition of labeled proline to cultures of fetal chick skin [9] and to intact chick em bryos [15]. Furthermore, low molecular weight hydroxyproline accu lumates at a constant rate in c u l tures of fetal chick skin [9]
Species
Tissue
Rat
25-30
Mouse
20-30
7
40
46
15-25
6
Chick
Cartilage
Rat
Lens
Chick
Granuloma
Guinea pig
Lung
Human Rabbit Hamster
Embryo
Chick
26
4,47
8
16 20-40
14
6-24
51
10
15
(whole) Cells
In vitro
Fibroblast skin
In vivo
30-32
Human
17,48
30
9, 19
Calf
30
Mouse
Chick
Lens
Chick
Lung
Human
Gingiva
Mouse
30
17
3T6
Mouse
20,60
38
Hepatocyte
Rat
Whole ani-
Rat
mal
34
8
10,30
13, 35,39
37
55
45 90
37 18
10,11,56
Guinea pig
57
Monkey
22
Man Skin
Rat Guinea pig
Intestine
Rat
Aorta
Rat
h 8 h 10 9h
58,21 23, 24 25
2:3 23
not available. Values probably represent an underestimate; see text for discussion.
" Rapid degradation detected but quantitative data h
20 17,53
Tendon
July 1982
INTRACELLULAR
DEGRADATION
OF NEWL Y
SYNTHESIZED
COLLAGEN
798
weight hydroxyproline peptides indicated a maximal amount of degradation soon after synthesis. Second, although an increase in the percentage of rapid degradation was present in skin of diabetic rats, in mixing experiments in vitro, diabetic skin did not cause the degradation of newly synthesized collagen pro duced by normal skin, thus excluding an extracellular mecha nism for collagen degradation.
suprising. However, the available evidence indicates that the intracellular degradation of collagen is a widespread process which can occur simultaneously with extracellular collagen degradation. It is likely that these 2 pathways for the destruc tion of collagen molecules play very different roles in the metabolism of collagen.
Quantitive Studies of Intracellular Degradation
Collagen
In general, all quantitative studies of intracellular degrada tion in normal cells for which data are available are quite similar, demonstrating 10-40% of newly synthesized collagen being degraded (Table). The hepatocyte appears to be an exception to this rule; hepatocyes in culture degrade 90% of newly synthesized collagen [18]. It is not known whether this high level of intracellular degradation found in hepatocyte cultures reflects a special property of these cells, a general property of epithelial cells, or if this result depends on the specific conditions utilized in this study. Interestingly, in spite of technical limitations of making such measurements in vivo, the available quantitative data for in vivo estimates of intracellular collagen degradation agree well with the in vitro estimates. For example, Schneir et al [23,24] have estimated intracellular degradation to be 8% in skin and 9% in intestine based on a 4 hr labeling period, in good agree ment with Barnes et al [25] who observed 9% of hydroxyproline to be present in low molecular weight form in guinea pig skin in vivo. While these values are somewhat lower than those obtained by in vitro methods, both of these investigators pres ent evidence that the low molecular weight species are cleared rapidly from tissues. This is consistent with the urinary excre tion data and suggests that the estimates of 8-9% represent a lower limit for the rapid degradation of newly synthesized collagen.
Recent studies indicate that collagen is not the only secreted protein subject to intracellular degradation. Insulin [27,28], parathyroid hormone [29-31], and prolactin [32] all appear to be subject to intracellular degradation within the cell prior to secretion. Interestingly, in the case of these hormones, the degradation appears to be selective for recently synthesized molecules that are resident within an intracellular storage pool and appears to regulate the amount available for secretion in response to a variety of physiologic stimuli.
Intracellular Degradation Compared to Extracellular Degradation
It is quite clear that the intracellular degradation of newly synthesized collagen and the extracellular degradation of col lagen by specific neutral proteases are distinct processes. How ever, in some tissues, intracellular and extracellular degradation of collagen appear to take place simultaneously. For example, in gingival fibroblasts [17], in intact chick lens [8], and in cultured lens cells [8], labeled proline is rapidly converted to low molecular weight hydroxyproline suggesting collagen deg radation. However, the addition of extracellular protease inhib itors results in a decrease in the amount degraded, indicating extracellular collagen degradation. Importantly, however, some collagen degradation occurs even in the presence of these extra cellular protease inhibitors. Thus, although Grant, Kefalides, and Prockop found that the addition of serum completely blocked the degradation of labeled extracellular collagen added to cultured lens cells, the degradation of newly synthesized collagen still constituted greater than 20% of collagen synthesis, and therefore must represent an intracellular process [8]. Sim ilarly, Roszkowski and Sauk observed that extracellular pro tease inhibitors could block much, but not all, of the degrada tion of newly synthesized collagen in gingival cell cultures [17]. Studies of the specific activity of the hydroxyproline found in low molecular weight fragments under these conditions in dicated that the most recently synthesized collagen was being degraded while older, extracellular collagen, if degraded at all, was degraded at a much lower rate. Thus, extracellular protease inhibitors could decrease extracellular collagen degradation while the simultaneous intracellular degradation of newly syn thesized collagen was unaffected. That many cell types can produce proteases capable of de grading collagen is well known [26], and that some extracellular degradation of collagen might occur in some systems in which intracellular degradation is simultaneously occurring is not
Intracellular Degradation of Secreted Proteins Other Than
ROLE OF INTRACELLULAR DEGRADATION OF NEWLY SYNTHESIZED COLLAGEN IN THE CELLULAR CONTROL OF COLLAGEN PRODUCTION Current concepts suggest that the intracellular degradation of newly synthesized collagen serves two distinct roles in mod ulating collagen production. First, intracellular collagen degra dation provides a mechanism for the destruction of defective molecules before their secretion, thus preventing the incorpo ration of defective molecules into the extracellular matrix. Second, intracellular degradation provides one means to regu late the amount of collagen and the relative amounts of collagen types produced by cells in response to certain extracellular mediators. Degradation of Defective Molecules
The formation of the triple helical structure of the collagen molecule requires severe constraints on the amino acid com position of the component polypeptide chains [1,33]. Since collagen is a large molecule with nearly 1,000 amino acid residues per chain, even a very small percentage of errors would result in the production of significant amounts of abnormal molecules. For example, every third amino acid must be a glycine to allow the chains to properly fold into a triple helix [1,33]; substitution of a glycine residue with another amino acid results in a molecule that can not assume a normal conforma tion. In addition, the hydroxyprolyl residues in the collagen chain are also required for a stable triple helical structure [1,33]. Thus, it is possible that intracellular degradation of newly synthesized collagen could function as a cellular "quality control" mechanism to rapidly destroy any abnormal molecules that might be synthesized. Support for the hypothesis that intracellular degradation selectively disposes of abnormal collagen comes from 3 lines of evidence. First, incubation of cell" with the analogues of proline such as cis-hydroxyproline or azetidine result in the incorporation of these analogues into collagen molecules that can not assume a stable triple helix [34]. In both situations, there is an increase in the amount of collagen degraded within the cell [13,35]. Second, when hydroxylation of proline is blocked, cells pro duce abnormal, underhydroxylated collagen; these circum stances are associated with an increase in the proportion of newly synthesized collagen which is rapidly degraded [36-38]. For example, in the absence of ascorbate, lung fibroblasts degrade up to 60% of newly synthesized collagen [36], a 4- to 6fold increase in baseline intracellular collagen degradation. In terestingly, some of the abnormal collagen chains produced by these cells are still secreted, suggesting that the mechanism of intracellular degradation may have a limited capacity, i.e., in the absence of ascorbate, abnormal molecules can still "overflow" into the extracellular space. If such "overflow" oc-
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ET AL
curs in cell types which also secrete proteases capable of extra cellular collagen degradation, this may account for the increase in extracellular degradation of collagen sometimes observed when cells produce abnormal collagen. Third, lung fibroblasts underhydroxylate collagen during the log phase of growth; this condition is associated with a nearly 3-fold increase in the proportion of newly synthesized collagen degraded intracellularly over that observed when cells achieve confluency [39]. Taken together, these experiments clearly support the con cept that abnormal collagen molecules can be detected within the cell and shunted into a degradative pathway. Intracellular Site of Degradation of Defective Collagen Molecules
Studies with lung fibroblasts have demonstrated that the increase in intracellular degradation that occurs in association with the formation of abnormal collagen chains is due to a lysosomal process [35,40]. For example, inhibitors of lysosomal proteolytic activity block the increased intracellular degrada tion that occurs when cells make defective collagen. Moreover, the decrease in production of low molecular weight hydroxy proline containing fragments in the presence of inhibitors of lysosomal function is accompanied by an accumulation of hy droxyproline containing material within lysosomes [35]. Al though one study of ascorbate deficient rat skin fibroblasts showed no decrease in the percentage of intracellular degrada tion with lysosomal inhibitors [17], in this case, only the me dium and an acetic acid extract of the cell layers was analyzed, and therefore, intralysosomal material was not fully accounted for, i.e., the effect of lysosomal inhibitors may have been un derestimated. Further support of the localization of intracellular degrada tion to a lysosomal site derives from studies of Karim, Cournil, and Leblond [41] which demonstrate material within lysosom9s that was antigenically cross reactive with procollagen. This material appears to reach lysosomes by way of the endoplasmic reticulum and the Goigi apparatus where collagen containing microvesicular bodies are formed. Interestingly, such microves icular bodies have also been implicated in mediating the lyso somal destruction of other intracellular components [42]. Although the available evidence supports the concept that a lysosomal process is involved in the increased intracellular degradation of newly synthesized collagen that occurs with the production of defective molecules, lysosomal inhibitors do not inhibit all intracellular degradation [17,35,40]. In the )Jresence of these agents approximately 10% of newly synthesized colla gen molecules are still degraded. Thus, in some circumstances, intracellular extralysosomal processes may be important for the intracellular degradation of collagen. Intracellular Degradation of Newly Synthesized Collagen as a Mechanism for Control of Collagen Production
Intracellular degradation of newly synthesized collagen is one means by which cells regulate collagen production in response to exogenous mediators. For example, intracellular degradation is important in the response of lung and skin fibroblasts to stimuli which affect the cAMP system [43]. Agents which elevate cAMP in these cells also cause an increase in the intracellular degradation of newly synthesized collagen with a corresponding decrease in the amount of collagen :-;ecreted. Moreover, the effect of elevated levels of cAMP appears to be selective in increasing the degradation of type I collagen, i.e., the production of type III collagen by these cells appears to be unaffected by cAMP levels [44]. In this context, since certain properties of the extracellular matrix may critically depend on the ratio of collagen types present, intracellular degradation may play an important role in regulating matrix composition [45]. Although intracellular degradation of collagen has been noted
in bone [7,46] (Kream BE and Raisz LG, personal communi cation) and cartilage [6], tissues where collagen production is sensitive to hormonal control, the relative role of intracellular degradation in the control of collagen production in these sites is just beginning to be understood. In cultured bone, for exam ple, intracellular degradation appears to be less important than other mechanisms for the control of collagen production [47]. Thus, the quantitative importance of intracellular degradation in the control of collagen synthesis is likely to be different in various tissues. Control of Intracellular Degradation
Several lines of evidence suggest that widely differing cellular events can lead to the intracellular degradation of newly syn thesized collagen. First, agents which increase cAMP within cells do not affect the degree of prolyl hydroxylation [43]; i.e., the effects of cAMP on collagen degradation are not mediated through changes in the conformation of the collagen molecule. Second, under circumstances where collagen is underhydroxy lated, production of both types I and III collagen is reduced [36] (Rennard SI, Berg RA, and Crystal RG, unpublished observations); in contrast, the cAMP mediated effect is specific in decreasing the amount of type I [44]. Thus the cell can increase intracellular degradation of newly synthesized collagen in a different fashion in response to different stimuli. Third, intracellular degradation has also been observed to be increased in skin fibroblasts following a phagocytic stimulus [48] although it is unknown whether this event affects either intracellular cAMP or the degree of hydroxylation of collagen. Together, these results strongly suggest that more than one cellular pathway leads to the intracellular degradation of newly synthe sized collagen. INTRACELLULAR DEGRADATION OF NEWLY SYNTHESIZED COLLAGEN AND DISEASE STATES Very few disease states have been studied with regard to a pathogenic role for intracellular degradation of newly synthe sized collagen. Although it is possible that abnormal intracel lular degradation of collagen on a congenital basis may contrib ute to one of the primary heritable collagen disorders, this phenomenon has only been studied in skin fibroblasts from patients with osteogenesis imperfecta, a group of inherited disorders characterized by the underproduction of type I col lagen. In these cells, intracellular degradation of collagen is normal and does not appear to contribute to the disease process [19]. In acquired diseases, however, current evidence suggests a role for intracellular degradation in 3 distinct disease states: fibrosis, diabetes mellitus, and scurvy. Fibrosis is a state characterized by an abnormal accumulation of connective tissue and can result from a wide variety of causes. Several separate lines of evidence suggest a role for intracellular degradation in certain types of fibrosis. 1. Fibrosis has been associated with ,B-blocking drugs such as propanolol or practolol, and may be due to a pharmacologic interruption in the control of intracellular degradation of col lagen [49]. As detailed above, ,B-agonists, by increasing intra cellular cAMP levels, induce an increase in the intracellular degradation of collagen and reduce collagen production by certain fibroblasts [43,44]. ,B-blocking drugs, by interfering with this mechanism [49], could prevent ,B-agonist mediated sup pression of collagen production and thus contribute to an ov erproduction of collagen which could result in fibrosis. 2. Certain inflammatory states, characterized by the devel opment of fibrosis, may also result from interference with the normal ,B-agonist mediated mechanisms that suppress collagen production by increasing intracellular degradation. In these conditions, however, the interference in the regulation of col lagen production is likely mediated by the proteolytic enzymes released by inflammatory cells resulting in proteolytic attack of fibroblast cell surface ,B-agonist receptors. As a result, in the
July 1982
INTRACELLULAR DEGRADATION OF NEWLY
presence of inflammation, some cells may overproduce type I collagen since these cells could not respond to suppressive /3agonist regulation of collagen production. In support of this hypothesis, the in vitro treatment of lung fibroblasts with either trypsin or leukocyte elastase results in the loss of isoproterenol mediated suppression of type I collagen [50]. 3. Bleomycin, a chemotherapeutic drug used in the treat ment of certain cancers, can cause pulmonary fibrosis. Experi mental studies with this drug have demonstrated that many changes are induced in affected lung tissue, including inflam mation, increased collagen synthesis, and a 3-fold increase in intracellular degradation of newly synthesized collagen [51]. Although it is currently unknown which effects are primary and which are secondary, (or even which cell types are being af fected in this disease process), it is likely that the changes in intracellular degradation of newly synthesized collagen contrib ute to the altered connective tissue formation caused by bleo mycin exposure. Diabetes mellitus is associated with a variety of abnormalities of connective tissue including the atrophy of skin. Schneir et al, have demonstrated, in vivo, that the skin of diabetic rats degrades a much larger portion of newly synthesized collagen than does normal skin, and that this increase in degradation can account for much of the atrophy seen in the skins of these animals [23,24]. Although the mechanism which leads to the increased degradation of newly synthesized collagen in the diabetic skin and the relationship of this process to other atrophic conditions are unknown, it is possible that such pro cesses could be important in the pathogenesis of these clinically significant conditions. Scurvy is the clinical syndrome which follows the prolonged deficiency of ascorbic acid. Without this vitamin, collagen can not be adequately hydroxylated and, as a result, abnormal, nonhelical collagen chains are formed [52]. Since a large portion of these molecules are degraded within the cell, collagen pro duction is dramatically reduced in scurvy [36]. Moreover, as noted above, the pathway of intracellular degradation can be overwhelmed, resulting in the secretion of some abnormal chains which, conceivably, could be incorporated into extracel lular matrix and result in abnormal connective tissue. It is therefore likely that in scurvy the decreased production of normal molecules and the secretion of abnormal molecules both contribute to the connective tissue abnormalities present.
REFERENCES 1. P rockop DJ, Kivirikko KI, Tuderman L, Guzman NA: The biosyn thesis of collagen and its disorders. New Engl J Med 301:13-23, 77-85, 1979
2. Rucker RB, Tinker S: Structure and metabolism of arterial elastin. Int Rev Exp Pathol 7:1-47, 1977
3. Reid KBM, Porter RR: Subunit structure of subcomponent CIq of the first component of human complement. Biochem J 155:19-23, 1976 4. Rosenberry T, Richardson J: Structure of 18S and 1 4S acetylcho linesterase. Biochemistry 16:3550-3558, 1977 5. Bradley KH, McConnell SD, Crystal RG: Lung collagen composi tion and synthesis: Characterization and changes with age . •1 BioI Chern 249:2674-2683, 1974 6. Daughattay WH, Mariz IK: The formation of free hydroxyproline by rat cartilage in vitro. J BioI Chern 237:2831-2835,1962 7. Stern B, Glimcher MJ, Goldhaber P: The effect of various oxygen tensions on the synthesis and degradation of bone collagen in tissue culture. Proc Soc Exp BioI Med 121:869-872, 1965 8. Grant ME, Kefalides NA, Prockop DJ: The biosynthesis of base ment membrane collagen in em bryonic chick lens. J BioI Chern 247:3545-3551, 1972 9. Hurych J, Chvapil M, The role of free hydroxyproline in the biosynthesis of collagen. Biochim Biophys Acta 107:91-96, 1965 10. Kibrick AC, Singh DK: Hydroxyproline excreted in the urine: Its source from collagen of tissues after [14] C proline in rats with and without administration of prednisone . J Clin End Metab 38:594-601, 1974 11. Laitinen 0: The metabolism of collagen and its hormonal control in the rat. Acta Endocrinologica suppl 120:1-84, 1967m 12. Kivirikko KI: Urinary excretion of hydroxyproline in health and disease. Int Rev Connective Tissue Res 5:93-163, 1970
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13. Bienkowski RS, Baum BJ, Crystal RG: Fibroblasts degrade newly synthesized collagen within the cell before secretion. Nature 276:413-416, 1978 14. Bienkowski RS, Cowan MJ, McDonald JA, Crystal RG: Degrada tion of newly synthesized collagen. J BioI Chern 253:4356-4363, 1978 15. Prockop DJ, Peterkovsky B, Udenfriend S: Studies on the intra cellular localization of collagen synthesis in the intact chi c k embryo. J BioI Chern 237:1581-1584, 1962 16. Green NM, Lowther DA: Formation of Collagen Hydroxyproline in vitro. Biochem J 71:55-66, 1959 17. Roszkowski M, Sauk JJ: The role of intracellular lysosomal en zymes in the autocellular-surveillance of unhydroxylated colla gens in dermal and gingival fibroblasts. J Dent Res 60:1045-1052, 1981 18. Diegelmann RF, Cohen IK, Guzelian PS: Rapid degradation of newly synthesized collagen by primary cultures of adult hepato cytes. Biochem Biophys Res Commun 97:819-826,1980 19. Steinmann BU, Martin GR, Baum BI, Crystal RG: Synthesis and degradation of collagen by skin fibroblasts from patients with osteogenesis imperfecta . FEBS lett 101:269-272, 1979 20. Krieg 1', Horlein D, Wiestner M, Muller PK: Aminoterminal exten sion peptides from type I pro collagen normalize excessive colla gen synthesis of scleroderma fibroblasta. Arch Dermatol Res 263:171-180,1978 21. Krane SM, Munoz AJ, Harris ED Jr: Collagen-like fragments: Excretion in urine of patients with Paget's disease of bone . Science 157:713-716, 1967 22. Avioli LV, Prockop DJ : Collagen degradation and the response to parathyroid extract in the intact rhesus monkey. J Clin Invest 46:217-224, 1967 23. Schneir M, Bowersox J, Ramamurthy N, Yavelow J, Murray J, Edlin-Folz E, and Golub L: Response of rat con nective tissues to streptozotocin-diabetes. Tissue specific effects on collagen me tabolism. Biochim Biophys Acta 583:95-102, 1979 24. Schneir M, Golub L: The effect of streptozotocin-induced diabetes on collagen catabolism, Streptozotocin: Fundamentals and Ther apy. Edited by Agarwal. Elsevier-/North-Holland, Biomedical Press, 1981, pp 160-182 25. Barnes MJ, Constable BJ, Morton LF, Kodicek K Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid deficient guinea pigs. Biochem J 119:575-585, 1970 26. Harris ED Jr, Cartwright EC: Mammalian collagenases in Pro teases, Mammalian Cells and Tissues. Edited by AJ Barrett. North Holland, Amsterdam , 1977, pp 249-284 27. Halban PA, Wollheim CB: Intracellular degradation of insulin stores by rat pancreatic islets in vitro. An alternative pathway for homeostasis of pancreatic insulin content. J BioI Chern 255:6003-6006, 1980 28. Halban PA, Wollheim CB, Blondel B, Niesor E, Renold AK Per turbation of hormone storage and release induced by cyprohep tadine in rat pancreatic islets in vitro. Endocrinology 104:1096-1106, 1979 29. Morrissey JJ, Cohn DV: Secretion and degradation of parathor mone as a function of intracellular maturation of horm one pools . J Cell BioI 83:521-528, 1979 30. Habener JF, Kemper B, Potts JT Jr.: Calcium dependent intracel lular degradation of parathyroid hormone: A possible mechanism for the regulation of hormone stores. Endocrinology 97:431-441, 1975 31. M orissey JJ, Hamilton JW, Cohn DV: The secretion of parathor mone and glycosylated proteins by parathyroid cells in culture. Biochem Biophys Res Comm 82:1279-1286,1978 32. Shenai R, Wallis M: Biosynthesis and degradation of prolactin in the rat anterior pituitary gland Time course of incorporation of label in vitro and evidence for rapid degradation . Biochem .1 182:735-743,1979 33. Fietzek PP, Kuhn K: The prim ary structure of collagen. lnt J Connect Tissue Res 7:1-60, 1976 34. Inouye K, Sakakibara S, Prockop DJ: Effects of the stereo config uration of the hydroxyl group in 4 -hydroxyproline on the triple helical structures formed by homogeneous peptides resembling collagen. Biochem Biophys Acta 420:133-141, 1976 35. Berg RA, Schwartz ML, Crystal RG: Regulation of the production of secretory proteins: Intracellular degradation of newly synthe sized "defective" collagen. Proc Nat! Acad Sci 77:4746-4750,1980 36 . Berg RA, Steinmann B, Rennard SI, Crystal RG: Ascorbate defi ciency results in decreased collagen production: underhydroxy lation leads to increased intracellular degradation and decreased production of triple helical molecules, submitted for publication 37. Vistica DT, Ahrens FA, Ellison WR: The effects of lead upon collagen synthesis and proline hydroxylation in the swiss mouse
31'6 fibroblast. Arch Biochem Biophys 179:15-23, 1977 38. Ramaley PB, Rosenbloom J: Inhibition of proline and lysine hy droxylation prevents normal extrusion of collagen by 31'6 fibro blasts in culture. FEBS lett 15:59-64, 1971 39. Tolstoschev P, Berg RA, Rennard SI, Bradley KH, Trapnell BC, Crystal RG: Procollagen production and procollagen messenger
828
40.
41.
42. 43.
44.
45.
46.
47.
48.
RENNARD ET AL
RNA levels and activity in human lung fibroblasts during perIods of rapid and stationaly growth. J BioI Chern 256:3135-3140, 1981 Berg RA, Schwartz ML, Rome LH, Crystal RG: Lysosomal function in the degradation of defective collagen in cultured lung fibro blasts, submitted for pu blication Karim A, Cournil I, Leblond CP: Immunohistological localization of procollagens II Electron microscopic distribution of procolla gen I antigenicity in the odontoblasts and predentin of rat incisor teeth by a direct method using peroxi clase linked antibodies . J Histochem Cytochem 27:1070-1083,1979 Locke L, Sykes AK: The role of the Golgi complex in the isolation and digestion of organelles. Tissue and Cell 7:143-158, 1975 Baum BJ , Moss ,1, Breul SD, Berg RA, Crystal RG: Effect of cyclic AMP on the intracellular degradation of newly synthesized col lagen. J BioI Chern 255:2843-2847, 1980 Rennard S, Saltzman L, Moss J, Fells G, Gadek J, Hom B, Hun ninghake G, Crystal H: Modulation of fibroblast production of collagen types I and III: Effects of PGEI and isoproterenol. Fed Proc 40:1813, 1981 Lapiere CM, Nusgens B, P ierard GE: Interaction between collagen type I and type III in conditioning bundles organization. Connect Tissue Res 5:21, 1977 Sakamoto M, Sakamoto S, Brickley-Parsons D, Glimcher M.l: Collagen synthesis and degradation in embryonic chick-bone explants. J Bone ,It Surg 61-A:1042-1052, 1979 Kream BE, Rowe DW, Gworek SC, Raisz LG: Parathyroid hormone alters collagen cynthesis and proc ollagen mRNA levels in fetal rat calvaria. Proc Natl Acad Sci 77:5654-5658, 1980 Sauk ,JJ Jr.: Collagen synthesis and turnover following particle
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50.
51. 52. 53. 54.
55. 56. 57.
58.
phagocytosis in dermal fi b roblasts. Biochim Biophys Acta 607:161-170, 1980 Berg RA, Moss J, Baum BJ, Crystal RG: Regulation of collagen production by the B-adrenergic system. J Clin Invest 67:1457-1462, 1981 Rennard S, Berg R, Moss J, Saltzman L, Hom B, Stier L, Gadek, Fells G, Crystal R: Protease mediated alteration of collage n production by lung fibroblasts. Am Rev Resp Dis 119:224, 1981 Clark JG, Overton JE, Marino BA, Uitto J, Starcher BC: Collagen biosynthesis in bleomycin-induced fibrosis in hamsters. J Lab Clin Med 96:943-953, 1980 Barnes M.l: Function of ascorbic acid in collagen metabolism. Ann NY Acacl Sci 258:264-277, 1975 Steinberg ,/: The turnover of collagen in fibroblast cultures. J Cell Sci 12:217-234, 1973 Jimin ez SA, Dehm P, Olsen BR, Prockop DJ: Intracellular collagen and proto collagen from embryonic tendon cells. ,J BioI Chern 248:720-729, 1973 Steinberg J: Collagen turnover and the growth state in 3T6 fibro blast cultures. Lab Invest 39:491-496, 1978 Prockop D J: Isotopic studies on collaged degradation and the urine excretion of hydroxy proline. ,1 Clin Invest 43:453-4()o. 1964 Lindstedt S, Proc kop DJ: Isotopic studies on urinary hydroxypro line as evidence for rapidly catabolized forms of collagen in the young rat. J BioI Chern 236: l:l99-1403, 1961 Phelps P, Avioli LV, Pro ckop DJ: Isotopic studies on collagen degradation in man: Effect of triiodothyronine on hydroxypro line- [14]C excretion. Clin Res 15:467, 1967