Scand. J. Dent. Res. 1975: 83: 302-305 (Key words: bone; collagenase; gingiva)
Trypsin activation of latent collagenase from several mammalian sources HENNING BIRKEDAL-HANSEN, CHARLES M. COBB, ROBERT E. TAYLOR AND HAROLD M. FULLMER Institute of Dental Research, School of Dentistry, University of Alabama in Birmingham, Birmingham, Alabama, U. S. A. ABSTRACT - Latent collagenase, subject to activation by trypsin, was found in culture fluids of cells and tissues from several mammalian sources. The activation requires exposure to enzymatically active trypsin and cannot be achieved by inhibited or by heat-inactivated trypsin. (Received for publication 7 March, accepted 4 May 1975)
Collagenases capable of cleaving undenatured collagen under physiologic conditions have been demonstrated in extracts and culture fluids of several mammalian cells and tissues. It has recently been demonstrated that collagenase may appear in a latent form, which can be activated by various means. Unidentified activators of latent collagenase have been found in extracts of dental, bacterial plaque (ROBERTSON, CoBB, TAYLOR & FULLMER 1974), in rheumatoid synovial fluid (KRUZE & WojTECKA 1972) and in culture fluids of tadpole skin (HARPER & GROSS 1972). Better defined chemically, however, is activation of collagenase with trypsin. Enzyme preparations from bone and fibroblast cultures have been activated in this way (VAES 1972, HOOK, HOOK & BROWN 1973). Attempts to apply the method to other collagenases have been unsuccessful (HARPER, BLOCH & CROSS JEFFREY
1971, BAUER, EISEN
&
1972). This paper reports the
presence of latent collagenase, subject to activation by trypsin, in culture fluids from gingiva, bone, skin, synovial tissue and macrophages.
lUlateriai and methods
Collagenase preparations were obtained from maintenance cultures of gingiva, skin, bone, synovial tissue and alveolar macrophages as follows: chronically inflamed human gingiva from seven patients subjected to periodontal surgery, non-inflamed bovine gingiva from eight newly slaughtered 1- to 2-year-old cows, human rheumatoid synovial tissue obtained from surgery on two patients, and skin of five newborn Charles River rats. These were sliced, processed and cultured in Tyrode antibiotic solution (100 |Xg/ ml streptomycin, 100 units/ml penicillin, 3.0 \igl ml amphotericin B, 4.0 |ig/ml gentamicin) essentially as described by FULLMER, GIBSON, LAZARUS, BLADEN & WHEDON (1969). The
culture fluids from each source were harvested daily or every second day, pooled and stored frozen. Femora and tibiae of five newborn Charles River rats were dissected, opened and
LATENT COLLAGENASE thoroughly washed in Tyrode antibiotic solution after removal of the marrow. The bones were incubated in 95 % O2, 5 % CO2 at 37°C in Tyrode antibiotic solution containing 1.0 iu/ ml heparin (Panheprin®, Abbot Laboratories) and 0.1 iu/ml parathyroid hormone (Parathyroid TCA powder, Sigma). The culture fluids were harvested at the end of the 4 d culture period, pooled and stored frozen. Alveolar macrophages were obtained from two 4 kg white New Zealand rabbits by the method of MYRVIK, LEAKE & FARISS (1961) 12 d after intravenous administration of 1 ml Freund's complete adjuvant. The cells were washed thoroughly in Tyrode antibiotic solution and cultured in 10 % CO2 in air at 37°C for 4 d using Dulbecco's modified Eagle medium containing antibiotics. The culture fluids were harvested daily, pooled and stored frozen. Sterility of all cultures was monitored directly by phase contrast microscopy and by incubating aliquots of the culture fluids on blood agar plates. The pooled culture fluids from each source were finally concentrated and fractionated by ammonium sulfate precipitation as described by FULLMER et al. (1969). The precipitates formed between 20 and 60 % saturated ammonium sulfate were dissolved in from 5 to 50 mi trisNaCl-CaCl2 buffer (0.03 M tris-, NaCl, 0.2 M NaCl, 5 mM CaCl2; pH 7.4) depending on the level of activity. The preparations were finally dialyzed against the same buffer and stored frozen. Manifest and latent collagenase activity was determined in samples ranging from 10 to 200 |xl in volume. The sample size was dependent on the level of activity in the individual preparations. The samples were made up to a total volume of 500 |A1 with tris-NaCl-Ca-Cl2 buffer and either activated for 10 min at room temperature with 4 |ig/ml TPCK-trypsin (Worthington) before addition of excess (60 [.(.g/ml) soybean trypsin inhibitor (Sigma) - or incubated for the same period of time with trisNaCI-CaCl2 buffer. Resultant collagenase activity was then determined by the WC-glycine radiofibril assay (ROBERTSON, TAYLOR & FULLMER 1972) in which the enzyme activity is quantitated by the release of soluble peptides from labeled gels of reconstituted collagen fibrils. The substrate was resistant to non-specific proteolytic digestion, trypsin (0.01 %) solubilizing only 7-10 % of the collagen during a 20 h assay. The gel lysis measured in unactivated preparations represents manifest collagenase activity. The difference between activated and
am.
unactivated preparations represents latent, trypsin activatable collagenase activity. In control experiments trypsin and soybean inhibitor were mixed before addition to the enzyme preparation or heat-inactivated trypsin was substituted for active trypsin. The effect of heparin and cyclic-AMP, previously shown to enhance collagenase activity (SAKAMOTO, GOLD-
HABER & GLIMCHER 1973, HARPER & TooLE 1973), was tested by adding these substances directly to the reaction mixture. Also a sterile extract of dental, bacterial plaque was prepared and tested for its ability to activate latent collagenase as previously described by ROBERTSON et al. (1974).
Results and discussion
All of the tissue- and cell culture preparations examined contained latent collagenase which was activated by trypsin (Fig. 1). Heat-inactivated trypsin (60°C,
TRYPSIN ACTIVATED UNACTIVATED
SKIN RAT
SYN HUM
MAC RAB
GING HUM
i1 GING BOV
BONE RAT
Fig. 1. Manifest and latent collagenase activity in culture fluids from rat skin, human rheumatoid synovial tissue (SYN HUM), rabbit alveolar macrophages (MAC RAB), human gingiva (GING HUM), bovine gingiva (GING BOV) and rat bone. Duplicate samples of pooled, concentrated and ammonium sulfate fractionated enzyme preparations were assayed for collagenase activity either directly (unactivated) or after activation for 10 min at room temperature with 4 |xg/ml trypsin followed by addition of excess soybean trypsin inhibitor. Complete lysis of collagen substrate represents 3,250 counts/min. The difference in the heights of the white and black bars represents latent, trypsin activatable collagenase activity.
304
BIRKEDAL-HANSEN, COBB, TAYLOR AND FULLMER
30 min) failed to activate latent collagenase as did trypsin premixed with soybean inhibitor. It therefore appears that the activation is dependent on brief exposure to small amounts of uninhibited, enzymatically active trypsin. Sterile extracts of dental, bacterial plaque also caused substantial activation of the enzyme preparations, but heparin (1—150 iu/ml) and cyclic-AMP (10^-10-3M) had no effect on these preparations. Comparing the preparations represented in Fig. 1 with other similar preparations revealed that though latent collagenase activity occurred consistently, the relative amounts of latent and manifest collagenase activity varied considerably from tissue to tissue and even from culture to culture of the same tissue. It has been suggested that trypsin activation of latent collagenase is strong evidence for the existence of a collagenase precursor, a procollagenase, which presumably is activated as a result of limited proteolysis by trypsin (VAES 1972, HOOK et al. 1973). Trypsin has been shown, however, to prevent serum inhibition of collagenase (SAKAMOTO et al. 1972), an effect which can probably be ascribed to binding of trypsin to alpha-2-macroglobulin. This globulin is known to inhibit mammalian collagenase as well as a series of other proteases. It therefore remains possible that the effect of trypsin in activating latent collagenase is release of inhibition, caused by small remnants of serum frequently present during the first days of culture (EISEN, BAUER & JEFFREY 1971). Activation of collagenase, whether by conversion of proenzyine to active form or by release of inhibition, could serve as an important regulating mechanism of collagenase activity in vivo. Acknowledgments — This project was supported by USPHS Research Grant DE-02670 from the National Institute of Dental Research, Na-
tional Institutes of Health, Bethesda, Maryland, USA, and by the Danish Medical Research Council (No. 512-2896).
References BAUER, E. A., EISEN, A. Z. & JEFFREY, J.
J.:
Regulation of vertebrate collagenase activity in vivo and in vitro. J. Invest. Dermatol. 1972: 59: 50-55. EISEN, A. Z., BAUER, E. A. & JEFFREY, J.
J.:
Human skin collagenase. The role of serum alpha-globulins in the control of activity in vivo and in vitro. Proc. Natl. Acad. Sci. (USA) 1971: 68: 248-251. FULLMER, H . M . , GIBSON, W . A., LAZARUS, G. S., BLADEN, H . A. & WHEDON, K . A.: The
origin of collagenase in periodontal tissues of man. /. Dent. Res. 1969: 48: 646-651. HARPER, E., BLOCH, K. J. & GROSS, J.:
The
zymogen of tadpole collagenase. Biochemistry 1971: 10: 3035-3041. HARPER, E . & GROSS, J.: Collagenase, procollagenase and activator relationships in tadpole tissue culture. Biochem. Biophys. Res. Commun. 1972: 48: 1147-1152. HARPER, E . & TOOLE, B. P.: Gollagenase and hyaluronidase stimulation by dibutyryl adenosine cyclic 3':5'-monophosphate. /. Biol. Chem. 1973: 248: 2625-2626. HOOK, R . M . , HOOK, C. W . & BROWN, S. I.:
Fibroblast collagenase. Partial purification and characterization. Invest. Ophthalmol. 1973: 12: 771-776. KRUZE, D . & WOJTECKA, E.: Activation of leukocyte collagenase proenzyme by rheumatoid synovial fluid. Biochim. Biophys. Acta 1972: 285: 436-446. MYRVIK, Q . N . , LE.AKE, E. S. & FARISS, B.:
Studies on pulmonary alveolar macrophages from the normal rabbit: a technique to produce them in a high state of purity. /. Immunol. 1961: 86: 128-132. ROBERTSON, P. B., COBB, C. M . , T.\YLOR, R . E. & FULLMER, H . M . : Activation of latent
collagenase by microbial plaque. /. Periodontal Res. 1974: 9: 81-83. ROBERTSON, P. B., TAYLOR, R . E. & FULLMER,
H. M.: A reproducible quantitative collagenase radiofibril assay. Clin. Chim. Acta 1972: 42: 43-45. S.\KAMOTO, S., GOLDHABER, P. &
GLIMCHER,
M. J.: Maintenance of mouse bone collagenase activity in the presence of serum pro-
LATENT COLLAGENASE tein by addition of trypsin. Proc. Soc. Exp Biol. (N.Y.) 1972: 139: 1038-1041. SAKAMOTO, S.,
GOLDHABER, P.
&
GLIMCHER,
M. J.: Mouse bone collagenase: the effect of heparin on the amount of enzyme released in Address: Institute of Dental Research School of Dentistry University of Alabama in Birmingham Birmingham Alabama 35294 U.S.A.
305
tissue culture and on the activity of the enzyme. Calcif. Tissue Res. 1973: 12: 247-258. VAES, G.: Release of collagenase as an inactive proenzyme by bone explants in culture. Biochem. J. 1972: 126: 275-289.
Trypsin activation of latent collagenase from several mammalian sources HENNING BIRKEDAL-HANSEN, CHARLES M. COBB, ROBERT E. TAYLOR AND HAROLD M. FULLMER Institute of Dental Research, School of Dentistry, University of Alabama in Birmingham, Birmingham, Alabama, U. S. A. ABSTRACT - Latent collagenase, subject to activation by trypsin, was found in culture fluids of cells and tissues from several mammalian sources. The activation requires exposure to enzymatically active trypsin and cannot be achieved by inhibited or by heat-inactivated trypsin. (Received for publication 7 March, accepted 4 May 1975)
Collagenases capable of cleaving undenatured collagen under physiologic conditions have been demonstrated in extracts and culture fluids of several mammalian cells and tissues. It has recently been demonstrated that collagenase may appear in a latent form, which can be activated by various means. Unidentified activators of latent collagenase have been found in extracts of dental, bacterial plaque (ROBERTSON, CoBB, TAYLOR & FULLMER 1974), in rheumatoid synovial fluid (KRUZE & WojTECKA 1972) and in culture fluids of tadpole skin (HARPER & GROSS 1972). Better defined chemically, however, is activation of collagenase with trypsin. Enzyme preparations from bone and fibroblast cultures have been activated in this way (VAES 1972, HOOK, HOOK & BROWN 1973). Attempts to apply the method to other collagenases have been unsuccessful (HARPER, BLOCH & CROSS JEFFREY
1971, BAUER, EISEN
&
1972). This paper reports the
presence of latent collagenase, subject to activation by trypsin, in culture fluids from gingiva, bone, skin, synovial tissue and macrophages.
lUlateriai and methods
Collagenase preparations were obtained from maintenance cultures of gingiva, skin, bone, synovial tissue and alveolar macrophages as follows: chronically inflamed human gingiva from seven patients subjected to periodontal surgery, non-inflamed bovine gingiva from eight newly slaughtered 1- to 2-year-old cows, human rheumatoid synovial tissue obtained from surgery on two patients, and skin of five newborn Charles River rats. These were sliced, processed and cultured in Tyrode antibiotic solution (100 |Xg/ ml streptomycin, 100 units/ml penicillin, 3.0 \igl ml amphotericin B, 4.0 |ig/ml gentamicin) essentially as described by FULLMER, GIBSON, LAZARUS, BLADEN & WHEDON (1969). The
culture fluids from each source were harvested daily or every second day, pooled and stored frozen. Femora and tibiae of five newborn Charles River rats were dissected, opened and
LATENT COLLAGENASE thoroughly washed in Tyrode antibiotic solution after removal of the marrow. The bones were incubated in 95 % O2, 5 % CO2 at 37°C in Tyrode antibiotic solution containing 1.0 iu/ ml heparin (Panheprin®, Abbot Laboratories) and 0.1 iu/ml parathyroid hormone (Parathyroid TCA powder, Sigma). The culture fluids were harvested at the end of the 4 d culture period, pooled and stored frozen. Alveolar macrophages were obtained from two 4 kg white New Zealand rabbits by the method of MYRVIK, LEAKE & FARISS (1961) 12 d after intravenous administration of 1 ml Freund's complete adjuvant. The cells were washed thoroughly in Tyrode antibiotic solution and cultured in 10 % CO2 in air at 37°C for 4 d using Dulbecco's modified Eagle medium containing antibiotics. The culture fluids were harvested daily, pooled and stored frozen. Sterility of all cultures was monitored directly by phase contrast microscopy and by incubating aliquots of the culture fluids on blood agar plates. The pooled culture fluids from each source were finally concentrated and fractionated by ammonium sulfate precipitation as described by FULLMER et al. (1969). The precipitates formed between 20 and 60 % saturated ammonium sulfate were dissolved in from 5 to 50 mi trisNaCl-CaCl2 buffer (0.03 M tris-, NaCl, 0.2 M NaCl, 5 mM CaCl2; pH 7.4) depending on the level of activity. The preparations were finally dialyzed against the same buffer and stored frozen. Manifest and latent collagenase activity was determined in samples ranging from 10 to 200 |xl in volume. The sample size was dependent on the level of activity in the individual preparations. The samples were made up to a total volume of 500 |A1 with tris-NaCl-Ca-Cl2 buffer and either activated for 10 min at room temperature with 4 |ig/ml TPCK-trypsin (Worthington) before addition of excess (60 [.(.g/ml) soybean trypsin inhibitor (Sigma) - or incubated for the same period of time with trisNaCI-CaCl2 buffer. Resultant collagenase activity was then determined by the WC-glycine radiofibril assay (ROBERTSON, TAYLOR & FULLMER 1972) in which the enzyme activity is quantitated by the release of soluble peptides from labeled gels of reconstituted collagen fibrils. The substrate was resistant to non-specific proteolytic digestion, trypsin (0.01 %) solubilizing only 7-10 % of the collagen during a 20 h assay. The gel lysis measured in unactivated preparations represents manifest collagenase activity. The difference between activated and
am.
unactivated preparations represents latent, trypsin activatable collagenase activity. In control experiments trypsin and soybean inhibitor were mixed before addition to the enzyme preparation or heat-inactivated trypsin was substituted for active trypsin. The effect of heparin and cyclic-AMP, previously shown to enhance collagenase activity (SAKAMOTO, GOLD-
HABER & GLIMCHER 1973, HARPER & TooLE 1973), was tested by adding these substances directly to the reaction mixture. Also a sterile extract of dental, bacterial plaque was prepared and tested for its ability to activate latent collagenase as previously described by ROBERTSON et al. (1974).
Results and discussion
All of the tissue- and cell culture preparations examined contained latent collagenase which was activated by trypsin (Fig. 1). Heat-inactivated trypsin (60°C,
TRYPSIN ACTIVATED UNACTIVATED
SKIN RAT
SYN HUM
MAC RAB
GING HUM
i1 GING BOV
BONE RAT
Fig. 1. Manifest and latent collagenase activity in culture fluids from rat skin, human rheumatoid synovial tissue (SYN HUM), rabbit alveolar macrophages (MAC RAB), human gingiva (GING HUM), bovine gingiva (GING BOV) and rat bone. Duplicate samples of pooled, concentrated and ammonium sulfate fractionated enzyme preparations were assayed for collagenase activity either directly (unactivated) or after activation for 10 min at room temperature with 4 |xg/ml trypsin followed by addition of excess soybean trypsin inhibitor. Complete lysis of collagen substrate represents 3,250 counts/min. The difference in the heights of the white and black bars represents latent, trypsin activatable collagenase activity.
304
BIRKEDAL-HANSEN, COBB, TAYLOR AND FULLMER
30 min) failed to activate latent collagenase as did trypsin premixed with soybean inhibitor. It therefore appears that the activation is dependent on brief exposure to small amounts of uninhibited, enzymatically active trypsin. Sterile extracts of dental, bacterial plaque also caused substantial activation of the enzyme preparations, but heparin (1—150 iu/ml) and cyclic-AMP (10^-10-3M) had no effect on these preparations. Comparing the preparations represented in Fig. 1 with other similar preparations revealed that though latent collagenase activity occurred consistently, the relative amounts of latent and manifest collagenase activity varied considerably from tissue to tissue and even from culture to culture of the same tissue. It has been suggested that trypsin activation of latent collagenase is strong evidence for the existence of a collagenase precursor, a procollagenase, which presumably is activated as a result of limited proteolysis by trypsin (VAES 1972, HOOK et al. 1973). Trypsin has been shown, however, to prevent serum inhibition of collagenase (SAKAMOTO et al. 1972), an effect which can probably be ascribed to binding of trypsin to alpha-2-macroglobulin. This globulin is known to inhibit mammalian collagenase as well as a series of other proteases. It therefore remains possible that the effect of trypsin in activating latent collagenase is release of inhibition, caused by small remnants of serum frequently present during the first days of culture (EISEN, BAUER & JEFFREY 1971). Activation of collagenase, whether by conversion of proenzyine to active form or by release of inhibition, could serve as an important regulating mechanism of collagenase activity in vivo. Acknowledgments — This project was supported by USPHS Research Grant DE-02670 from the National Institute of Dental Research, Na-
tional Institutes of Health, Bethesda, Maryland, USA, and by the Danish Medical Research Council (No. 512-2896).
References BAUER, E. A., EISEN, A. Z. & JEFFREY, J.
J.:
Regulation of vertebrate collagenase activity in vivo and in vitro. J. Invest. Dermatol. 1972: 59: 50-55. EISEN, A. Z., BAUER, E. A. & JEFFREY, J.
J.:
Human skin collagenase. The role of serum alpha-globulins in the control of activity in vivo and in vitro. Proc. Natl. Acad. Sci. (USA) 1971: 68: 248-251. FULLMER, H . M . , GIBSON, W . A., LAZARUS, G. S., BLADEN, H . A. & WHEDON, K . A.: The
origin of collagenase in periodontal tissues of man. /. Dent. Res. 1969: 48: 646-651. HARPER, E., BLOCH, K. J. & GROSS, J.:
The
zymogen of tadpole collagenase. Biochemistry 1971: 10: 3035-3041. HARPER, E . & GROSS, J.: Collagenase, procollagenase and activator relationships in tadpole tissue culture. Biochem. Biophys. Res. Commun. 1972: 48: 1147-1152. HARPER, E . & TOOLE, B. P.: Gollagenase and hyaluronidase stimulation by dibutyryl adenosine cyclic 3':5'-monophosphate. /. Biol. Chem. 1973: 248: 2625-2626. HOOK, R . M . , HOOK, C. W . & BROWN, S. I.:
Fibroblast collagenase. Partial purification and characterization. Invest. Ophthalmol. 1973: 12: 771-776. KRUZE, D . & WOJTECKA, E.: Activation of leukocyte collagenase proenzyme by rheumatoid synovial fluid. Biochim. Biophys. Acta 1972: 285: 436-446. MYRVIK, Q . N . , LE.AKE, E. S. & FARISS, B.:
Studies on pulmonary alveolar macrophages from the normal rabbit: a technique to produce them in a high state of purity. /. Immunol. 1961: 86: 128-132. ROBERTSON, P. B., COBB, C. M . , T.\YLOR, R . E. & FULLMER, H . M . : Activation of latent
collagenase by microbial plaque. /. Periodontal Res. 1974: 9: 81-83. ROBERTSON, P. B., TAYLOR, R . E. & FULLMER,
H. M.: A reproducible quantitative collagenase radiofibril assay. Clin. Chim. Acta 1972: 42: 43-45. S.\KAMOTO, S., GOLDHABER, P. &
GLIMCHER,
M. J.: Maintenance of mouse bone collagenase activity in the presence of serum pro-
LATENT COLLAGENASE tein by addition of trypsin. Proc. Soc. Exp Biol. (N.Y.) 1972: 139: 1038-1041. SAKAMOTO, S.,
GOLDHABER, P.
&
GLIMCHER,
M. J.: Mouse bone collagenase: the effect of heparin on the amount of enzyme released in Address: Institute of Dental Research School of Dentistry University of Alabama in Birmingham Birmingham Alabama 35294 U.S.A.
305
tissue culture and on the activity of the enzyme. Calcif. Tissue Res. 1973: 12: 247-258. VAES, G.: Release of collagenase as an inactive proenzyme by bone explants in culture. Biochem. J. 1972: 126: 275-289.
