Collagen Degradation in Rat Skin but Not in Intestine During Rapid Growth

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Proc. Natl. Acad. Sci. USA Vol. 74, No. 4, pp. 1436-1439, April 1977 Biochemistry Collagen degradation in rat skin but not in intestine during rapid growth: Effect on collagen types I and III from skin (connective tissue/metabolism/isotopes/hydroxyproline/proline) LEROY KLEIN AND JESUDIAN CHANDRARAJAN Departments of Surgery and Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 Communicated by Oscar D. Ratnoff, January 14, 1977

ABSTRACT Metabolic degradation of prelabeled collagen Isolation and Preparation of Skin and Intestine. From each in whole body skin and whole intestine was compared to that litter, five pups were killed at 1 day after birth, three at 14 days, of types I and III collagens from skin in young, rapidly growing rats. Pregnant rats were given [3Hlproline during the last week and two at 42 days. The entire skin from each rat was removed of gestation; and after birth, littermates were compared. Be- as previously described (11), finely minced, and thoroughly tween the second and sixth weeks of age, there was a 43% loss mixed. The minced skin from each rat was weighed and pooled of radioactivity from dermal collagen but no significant loss of with skin from its littermates for the three age groups and stored radioactivity from intestinal collagen. Pepsin treatment solu- at -70°. The entire intestine, from the gastroduodenal sphincter bilized 90% of the dermal collagen but only 12% of intestinal to the rectum, was removed from each rat, the surrounding collagen. Skin from 2- and 6-week-old rats yielded the same proportions of type I and type III collagens (type I, 82%; type loose connective tissue was dissected off, and the intestinal III, 18%). The relative losses of total radioactivity from types contents were flushed out with running water. The cleaned I and III were similar to each other (50 and 44%, respectively) intestine was finely minced, thoroughly mixed, weighed, pooled and to the loss from whole skin. Because types I and III colla- with intestine from littermates for the three age groups, and gens are known to be present in both skin and intestine, the stored at -70°. Weighed aliquots of skin and intestine were marked degradation of both collagen types in skin but not in the taken (150, 400, and 600 mg for the age groups of 1, 14, and 42 intestine may be related to the amount and kind of intermo- days, respectively) for the initial chemical and isotopic analysis lecular crosslinks present. of hydroxyproline (12). From an additional litter, six pups were Connective tissues or organs contain either single or multiple killed at 1 day after birth and six at 14 days of age. The entire types of collagen. Bone and tendon contain only type I collagen skin from each rat was removed for chemical and isotopic (1), cartilage only type II collagen (2), and renal glomeruli only analysis of hydroxyproline without pooling the skins. type IV collagen (3). In contrast, skin, intestine, liver, spleen, Pepsin Solubilization of Collagen. Finely minced skin or kidney, lung, aorta, uterus, umbilical cord, placental mem- intestine (1000 mg) was suspended in 100 ml of 0.5 M acetic branes, myocardium, and scar tissue contain two types of col- acid. Pepsin (Worthington) was added at a concentration of 1 lagen, I and III (1). The functional significance of single and mg/ml (13) and the samples were stirred for 24 hr at 8°. The multiple types of collagen in tissues is unknown. incubation mixture was centrifuged at 30,000 X g for 1 hr at During embryonic growth of skin, there is a change from 40. The incubation was repeated overnight with additional predominantly type III collagen to predominantly type 1 (4). pepsin if a large residue remained after digestion and centrif- This change has been attributed to a more rapid destruction of ugation. The supernatant was dialyzed against 4 liters of 0.02 type III collagen (4), a slower rate of degradation of type III M Na2HPO4, and the resultant collagen precipitate was sepa- collagen (5), or independent degradation of different types of rated by centrifugation at 30,000 X g for 1 hr. The residue after collagen (6). Recently, we have shown (7) a large turnover of pepsin digestion was analyzed chemically and isotopically collagen of calvarial fibrous tissue (type I) in young, rapidly (12). growing rats. With growth remodeling of long bones, degra- Fractionation of Type I and Type III Collagens. The col- dation of collagen was equivalent in bone (type I) and cartilage lagen precipitate was suspended in 10 ml of 2 M guanidine-HCI (type II) but less than that in calvaria (7). The differences in containing 0.05 M Tris-HCl, pH 7.5, and was denatured by degradation rates suggest that other factors besides type of warming to 450 for 30 min. The resultant solution was centri- collagen play a role in its destruction. fuged at 30,000 X g for 40 min at 250. A 5-ml aliquot of the In this paper, collagen degradation is quantified for two supernatant was applied to a calibrated column (1.6 X 115 cm) tissues, skin and intestine, because both contain the same two of agarose beads (Bio-Gel A-Sm, 100-200 mesh). The column types of collagen, I and III (1), although rat skin contains a large was equilibrated and eluted with 2 M guanidine-HCl/0.05 M amount of soluble collagens (8, 9) whereas human and rat in- Tris-HCI, pH 7.5, at 220 with a flow rate of 10 ml/hr. Fractions testine have negligible amounts (8, 10). The results demonstrate (3 ml) were collected and the absorbance was measured at 230 that, during rapid growth, there is a large degradation of dermal nm in a Gilford spectrophotometer (model 2400). The residue collagen and its constituent types I and III compared to little, after guanidine and heat denaturation was analyzed chemically if any, turnover of intestinal collagen. and isotopically (12). Quantification of Type I and Type III Collagens. The MATERIALS AND METHODS relative quantities of type I and type III collagens solubilized by pepsin were determined by tracing the appropriate Bio-Gel Labeling of Animals. During the last 7 days of gestation (7), A-5m elution patterns on paper, cutting out the traced outlines, five pregnant Fischer rats were injected daily with 100 or 500 and weighing the individual pieces of paper. Aliquots (1 ml) ACi of L-[2,3-3H]proline (specific activity, 500 mCi/mmol; of the column fractions from each of the two peaks were pooled New England Nuclear), 50 or 250 ,utCi in the morning and 50 and analyzed for hydroxyproline (12).and proline (14). The or 250 ,Ci in the evening, subcutaneously into the dorsum. amount of collagen was calculated based on types I and III 1436 Downloaded by guest on September 28, 2021 Biochemistry: Klein and ChandraRajan Proc. Natl. Acad. Sci. USA 74 (1977) 1437

Table 1. Weight of and collagen mass in skin and intestine Table 2. Hydroxyproline and its radioactivity in collagen of growing rats of whole skin from rat littermates at birth Collagen per Specific Total Weight per rat* (g) rat* (mg) radioactivity radioactivity Age Total in in (wk) n Skin Intestine Skin Intestine hydroxy- hydroxy- hydroxyproline proline proline 0 20 0.595 0.126 0.830 0.044 Rat (pg/rat) (dpm/,p g) (dpm/rat) + 0.029 ± 0.021 ± 0.032 ± 0.003 2 12 4.91 0.866 11.73 0.546 Male 1310 474 621,000 + 0.22 ± 0.031 ± 0.42 ± 0.030 Male 1340 486 651,000 6 8 12.38 4.49 85.70 5.88 Female 1360 471 640,000 + 0.72 ± 0.42 ± 2.15 ± 0.32 Female 1310 494 647,000 Female 1375 456 627,000 Data are derived from four litters and are expressed as mean i SEM. For each litter, whole skin and intestine were from five rats at Female 1320 471 622,000 birth, from three rats at 2 weeks of age, and from two rats at 6 weeks Mean 1340 475 635,000 of age. n = number of samples analyzed. SEM ±11 ±5.4 ±5,400 * Expressed as wet weight and hydroxyproline per rat, respective- CV(%) ±2.0 ±2.8 ±2.1 ly. A pregnant Fischer rat was injected with 100 yCi of L-[2,3-3H]- collagens containing 13.7% and 17.9% hydroxyproline (15), proline daily during the last 7 days ofgestation. SEM = standard error respectively. The molar ratio of proline to hydroxyproline was of the mean; CV = coefficient of variation. calculated for each type of collagen as an index of its identity and relative purity. Pepsin solubilization of skin from newborn rats gave poor yields. Isotopic Analyses of Tissues and Collagen Types. Weighed Similar treatment of intestine solubilized 15% and 9% of the aliquots of pooled skin and pooled intestine were hydrolyzed intestinal collagen, and 7% and 6% of the total radioactivity, for hydroxyproline analysis or were used for pepsin digestion. as peptides. Two components were separated by chromatog- Specific radioactivity of collagen from pooled skin and intestine raphy on a column of Bio-Gel A-5m (Fig. 1). Chromatographic and from types I and III collagens was obtained from the two peak I had an approximate molecular weight of 100,000, chromatographic fractions with the highest concentration of suggesting that this collagen was type I. The earlier peak (peak hydroxyproline (16). Aliquots (1 ml) of the fractions were mixed III) had an approximate molecular weight of 300,000; after with 10 ml of Scintisol (Isolab Inc.) for counting of radioactivity reduction and alkylation, it was a single component with a in a liquid scintillation counter (model 3003, Packard Instru- molecular weight similar to that of a chains, suggesting that this ments). Total radioactivity of the weighed aliquot of tissue was collagen was type III. Similar proportions of the total collagen calculated by multiplying the specific radioactivity in hy- were found to be type I for 2- and 6-week-old skin (83% and droxyproline by the amount of hydroxyproline determined 82%, respectively) (Table 4), compared to 30% type I and 70% chemically in all of the chromatographic fractions containing type III collagen in skin at birth. The proportions calculated by hydroxyproline. Total radioactivity in hydroxyproline for whole using absorbance at 230 nm agreed with those obtained by skin and intestine and for types I and III collagens from skin was hydroxyproline analysis. calculated by converting the aliquot's radioactivity to the total Because types I and III collagens contained different amounts amount per rat. Hydroxyproline was used as an index of col- of proline and hydroxyproline, the molar ratio of these two lagen in the intestine because histological (17) and chemical (18) amino acids was used to confirm the presence of the two types analysis shows that elastin is a minor component compared to of collagens. As shown in Table 5, the molar ratios obtained collagen. from the two chromatographic peaks were different from each other and corresponded to the reported molar ratios of purified RESULTS types I and III collagens obtained from rat skin (15). Collagen Accumulation and Degradation During Growth. Isotopic Kinetics of Types I and III Collagens. Both whole Growth of rats from birth to 2 weeks of age resulted in 14- and skin and intestine showed similar decreases in specific radio- 12-fold increases in dermal and intestinal collagen, respectively, activity of hydroxyproline from birth to 6 weeks of age (Table and growth from 2 to 6 weeks of age resulted in 7- and 11-fold 6). Types I and III collagens from skin had a somewhat smaller increases (Table 1). The increases in collagen mass due to specific radioactivity than that observed in the corresponding growth for both skin and intestine were twice as large as the whole skin, but this was not statistically significant except for increment for wet weight. The total amount of hydroxyproline and its radioactivity, in whole skin at birth, varied among the Table 3. Collagen turnover in rat skin and intestine six littermates by +2% (Table 2). The total radioactivity in hydroxyproline isolated from whole skin and the entire intes- Age Whole skin Whole intestine tinal tract decreased 43% and 4%, respectively, from 2 to 6 (wk) (dpm x 10-3)* (dpm x 10-3)* weeks of age (Table 3). The relative loss in skin collagen radioactivity was similar to that observed (7) during 4 weeks of 2 3450±115 138±9 growth remodeling of whole long bones (48%) and calvarial 6 1971 ± 95 133 ± 11 bones (52%). A -43% -4% I Relative Amount of Types and III Collagens from Rat Four pregnant Fischer rats were injected with 500,Ci of L-[2,3- Skin. Pepsin treatment of skin from 2- and 6-week-old rats 3H]proline daily during the last 7 days of gestation. solubilized 87% and 92% of the dermal collagen, respectively, * Total radioactivity in hydroxyproline per rat expressed as mean ± and 83% and 86% of the total radioactivity in hydroxyproline. SEM; n = 8 for the four litters. Downloaded by guest on September 28, 2021 1438 Biochemistry: Klein and ChandraRajan Proc. Natl. Acad. Sci. USA 74 (1977)

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I-- I I I I -- r - 20 30 40 50 6 20 30 40 50 60 FRAcliON FRAcfON FIG. 1. Chromatographic fractionation of type I and type III collagens on a Bio-Gel A-5m column (1.6 X 115 cm). The sample was eluted with 2 M guanidine.HCl/0.05 M Tris.HCl, pH 7.5; 3-ml fractions each were collected and the absorbance at 230 nm was monitored. The horizontal bar indicates fractions pooled for specific radioactivity values in hydroxyproline and proline. (A) Skin from 2-week-old rat. (B) Skin from 6-week-old rat. type I collagen at 6 weeks of age. The specific radioactivity of collagen (10) by neutral salt and acetic acid media, as well as type I collagen was lower than that of type III collagen, but this by the relative resistance of intestinal collagen to pepsin solu- difference was not statistically significant. bilization reported in the present study. An alternate possibility The total radioactivity of hydroxyproline in types I and III is that the primary structures of types I and III collagens in the collagens from skin showed a similar decrease (50% and 44%, intestine differ from their primary structures in skin. respectively) (Table 7) from 2 to 6 weeks of age. This decrease The use of pepsin to solubilize collagen types by the removal was similar in degree to that observed in whole skin; thus, the of terminal aldehydic crosslinks has made possible studies on degradation rate of types I and III collagens from skin was the relative effectiveness of collagenase (19, 20) or trypsin (21) similar to that of whole skin or bone (7). In contrast there was to destroy collagen types at the molecular level, but these studies no degradation of intestinal collagen and, thus, of its constituent may not give an insight into the destruction of collagen at the types of collagen. tissue (supramolecular) level which contains crosslinks. Unlike the preponderance of type III collagen found in DISCUSSION newborn rat skin (Table 4), the similarity in the ratios of type The present study showed that collagen from rat skin was de- I to type III collagen at 2 and 6 weeks of age suggests that both graded during rapid growth, as previously found (7) for sin- types are synthesized at a similar rate after birth. This constancy gle-type collagens from rat calvarial and long bones (7). Both in the ratio of type I to III soon after birth agrees with previous types I and III collagens that were extracted from rat skin were findings in human skin (6), in which the ratio is constant from degraded at a similar rapid rate. In contrast, we were unable 3 months to 78 years of age. to observe any degradation in intestinal collagen. Although rat In dermal and intestinal collagens, differences in growth rates skin and intestine (1) contain the same two types of collagens, and reutilization of radioactive proline need to be considered the marked difference in their catabolism can be more easily as a possible explanation of the apparent metabolic stability of explained by a difference in the number or kind of intermo- intestinal collagen. Table 1 demonstrates a similar growth rate lecular crosslinks present in their collagens. This difference in for dermal and intestinal collagen and thus a similar rate of the degree of crosslinking is supported by the high extractibility accumulating radioactive proline. Since both the skin and the of skin collagen (8, 9) and the low extractibility of intestinal intestine depend on local and systemic sources of radioactive proline, it is highly unlikely that the intestine was reusing more Table 4. Relative amounts of types I and III collagen radioactivity, because the major sources of radioactive proline in rat skin, by two methods reside peripherally in muscle, skin, and bone. Determination Absorbance at Hydroxyproline Table 5. Molar ratio of 230 nm* analysist proline to hydroxyproline in collagen Age Type I Type III Type I Type III Age (wk) n (% of total) (% of total) (wk) n Type I Type III 0 2 30.0± 3.0 70.0 0 3 1.16± 0.04 0.89± 0.01 2 3 83.3 ± 0.7 16.7 80 20 2 3 1.23 ± 0.01 0.90 ± 0.02 6 4 82.0± 1.0 18.0 84 16 6 4 1.27 ± 0.02 0.89 ± 0.01 Theoretical* 1.19 0.75 * Mean + SEM. t Based on types I and III collagens containing 13.7% and 17.9% hy- Data are expressed as mean ± SEM. droxyproline, respectively (15). * Obtained from ref. 15. Downloaded by guest on September 28, 2021 Biochemistry: Klein and ChandraRajan Proc. Natl. Acad. Sci. USA 74 (1977) 1439 Table 6. Specific radioactivity* wound strength and collagen mass observed during the early in intestinal and dermal collagens in growing rats healing phase of the large intestine (10). The marked turnover of dermal and skeletal (7) collagens Skint with growth is in contrast to collagen's metabolic inertness in Age Intestine the slowly growing or nongrowing rat (25), thus emphasizing (wk) n intactt Intact Type I Type III the need to study collagen turnover in an active, growing rat. 0 3 2710 2270 2160 2130 It appears that the peripheral musculoskeletal collagens (bone, ± 60 ± 65 ±107 ±104 tendon, skin) are metabolically active, while at the same time 2 3 254 296 270 282 the visceral collagens (intestine, artery, and kidney) (L. Klein, ± 12 ± 11 ± 6.1 ±14 unpublished data) are inert during growth. Like intestinal 6 4 23.9 24.0 15.7 22.5 wounds, arterial wounds heal rapidly (26). In the nongrowing ± 1.5 ± 1.4 ± 1.6 ±2.5 state in humans, continuous collagen degradation could be the basis for atrophy of peripheral collagens and metabolic inertness See Table 3 for details of labeling. for hypertrophy (fibrosis) of visceral collagens. * Hydroxyproline in dpm/,gg. Data are expressed as mean + SEM. t Specific radioactivity varied (2470-2850) and (2110-2570) for in- We thank Mrs. Alice Yessayan, Mrs. Corinne Pulphus, and Mrs. testine and skin, respectively, among the four litters of rats. Anita Pettigrew for their technical assistance. This work was supported by U.S. Public Health Service Grants AG-00258 and AG-00361. of specific radioactivity in newly synthesized salt-soluble col- 1. Epstein, E. H., Jr. & Munderloh, N. H. (1975)J. Biol. Chem. 250, lagens from rat skins, from birth to 8 weeks of age, showed that 9304-9312. the reutilization of radioactive proline was high during the first 2. Miller, E. J. (1971) Biochemistry 10, 1652-1658. week of life but reached a low level by 2 weeks of age (L. Klein, 3. Kefalides, N. A. (1971) Biochem. Biophys. Res. Commun. 45, unpublished data). The lack of significant reutilization of ra- 226-234. dioactive proline can also be seen in the decreases of total ra- 4. Bailey, A. J. & Robins, S. P. (1972) FEBS Lett. 21,330-334. dioactivity observed in the skeletal collagens (7) of rats that were 5. Shuttleworth, C. A. & Forrest, L. (1975) Eur. J. Biochem. 55, prelabeled in utero, as in the present study. 391-395. In order for the quantities of radioactivity to be compared 6. Epstein, E. H., Jr. (1974) J. Biol. Chem. 249,3225-3231. in a meaningful way from one age to another, two requirements 7. Klein, L. & Zika, J. M. (1976) Calcif. Tissue Res. 20,217-227. must be met: (i) littermates must contain an equivalent amount 8. Orekhovitch, V. N. & Shpikiter, V. 0. (1958) Science 127, of radioactivity per mass of and a unit 1371-1376. tissue, (ii) reproducible 9. Cadavi1, N. G., Denduchis, B. & Mancini, R. E. (1963) Lab. In- of mass must be defined anatomically and proved chemically. vest. 12, 598-605. Equivalent levels of hydroxyproline and its total radioactivity 10. Irvin, T. T. & Hunt, T. K. (1974) Surg. Gynecol. Obstet. 138, can be seen among littermates for total skin (Table 2) as well 741-746. as for calvarial and long bones (7). Total skin and intestine as 11. Zika, J. M. & Klein, L. (1971) Biochim. Biophys. Acta 229, reproducible units of mass are defined within the statistical 509-515. limits given in Table 1 for the three age groups. 12. Klein, L. (1970) Stand. Methods Clin. Chem. 6,41-56. The insignificant loss of radioactivity from the intestine 13. Chung, E. & Miller, E. J. (1974) Science 183, 1200-1201. during rapid growth can be interpreted in one of two ways: (i) 14. Summer, G. K. & Roszel, N. 0. (1965) Clin. Chem. 11, 455- intestinal collagen is metabolically inert, or (ii) if 458. remodeling 15. Gallop, P. M. & Paz, M. A. (1975) Physiol. Rev. 55,418-587. of intestine occurs with growth, then intestinal collagen is 16. Klein, L. & Weiss, P. H. (1966) Proc. Natl. Acad. Sci. USA 56, conserved (16). Little evidence is available as to whether in- 277-284. testinal connective tissues remodel with growth. Unlike the slow 17. Hass, G. M. (1939) Arch. Pathol. 27,334-365. healing of skin wounds which are metabolically active (22), the 18. Neuman, R. E. & Logan, M. A. (1950) J. Biol. Chem. 186, exceedingly rapid healing observed in intestinal wounds (10, 549-556. 23,24) suggests that intestinal collagen has little, if any, turnover 19. Robertson, P. B. & Miller, E. J. (1972) Biochim. Biophys. Acta and thus appears to be metabolically inert under these condi- 289,247-250. tions. Consistent with this interpretation is the maintenance of 20. Miller, E. J., Harris, E. D., Jr., Chung, E., Finch, J. E., McCros- kery, P. A. & Butler, W. T. (1976) Biochemistry 15,787-792. 21. Miller, E. J., Finch, J. E., Jr., Chung, E., Butler, W. T. & Rob- Table 7. Turnover in collagen types from whole rat skin ertson, P. B. (1976) Arch. Biochem. Biophys. 173,631-637. 22. Madden, J. W. & Peacock, E. E. (1971) Ann. Surg. 174, 511- Age Type I Type III Total (I + III) 518. (wk) n (hydroxyproline, dpm x 10-3)* 23. Hermann, J. B., Woodward, S. C. & Pulaski, E. J. (1964) Surg. Gynecol. Obstet. 119,269-275. 2 3 2528 ± 76 701 ± 45 3228 ± 56 24. Scheinin, T. M. & Viljanto, J. (1966) Ann. Med. Exp. Fenn. 44, 6 4 1268 ± 142 394 ± 41 1660 ± 180 49-51. 'A -50% -44% -49% 25. Thompson, R. C. & Ballou, J. E. (1956) J. Biol. Chem. 223, 795-809. See Table 3 for details of labeling. 26. Lowenberg, R. I. & Shumacker, H. B. (1949) Arch. Surg. 59, * Total radioactivity data are presented as mean ± SEM. 74-83. Downloaded by guest on September 28, 2021