Vol. 188, No. 2, 1992 October 30, 1992
TETRACYCLINES
BIOCHEMICAL
INHIBIT
AND BIOPHYSICAL
INTRACELLULAR
MUSCLE PROTEOLYSIS
Bruce S. Schneider’, Jonathan Maimon’, N. S. Ramamurth~,
RESEARCH COMMUNICATIONS Pages 767-772
IN VITRO
Lome M. Golub2,
and Robert A. Greenwald’
‘Department
of Medicine, Long Island Jewish Medical Center, New Hyde Park,NY 11042 (The Long Island Campus for the Albert Einstein College of Medicine) 2Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York, Stony Brook,NY 11794
Received September 8, 1992
Tetracycline antibiotics (TETs) have a recently discovered novel action: inhibition of extracellular metalloproteinase activity, especially that of collagenase and gelatinase. This property, now confirmed in 8 different laboratories using >40 tissue sources, includes natural and semi-synthetic TETs as well as a chemically modified TET (CMT) devoid of antimicrobial activity. We have used 14C-Tyr biosynthetically labelled intracellular proteins in L6 myoblast culture as a test system to assess intracellular proteolysis. Starvation accelerates proteolysis, which can be suppressed by agents such as insulin or serum. Minocycline, doxycycline, and CMT all retarded the rate of intracellular protein degradation in a dose dependent manner. These agents also demonstrated marked synergism with insulin. A CMT derivative (pyrazole) stripped of one of its metal chelation sites and lacking anti-collagenase activity, also lost its antiproteolytic effect. CMT at physiologic concentrations (I 5 pg/ml) had no effect on protein synthesis, but at 15 pg/rnl (pharmacologic), a suppressive effect was noted. These findings demonstrate that TETs can inhibit protein degradation as well as synthesis in a mammalian muscle-derived cell line. 0 1992Academic PIf2SS.1°C.
Tetracyclines (TETs) are widely known and used as antibiotics in both humans and animals. In addition to the bacteriostatic/antimicrobial
properties of these drugs, an extensive
literature has developed over the past 9 years indicating that TETs are also potent inhibitors of connective tissue degrading enzymes, specifically the neutral, metal-dependent
enzymes
collagenase (MMP-1 and/or MMP-8) and gelatinase (MMP-2 and/or MMP-9). The initial observation of this phenomenon
was reported by Golub et. al. in 1983 [l] and was based on
study of collagenase levels in the gingival tissues and skin of diabetic rats treated with TETs. In the ensuing years, the observation has been confirmed in at least 8 different laboratories and 0006-291X/92 $4.00 767
Copyright 0 1992 by Academic Press, Inc. AN rights of reproduction in any form reserved.
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
has been extended to matrix metalloproteinases
from over 40 different tissues as well as to
unique TET derivatives devoid of antimicrobial
activity [2,3,4]. Since tetracyclines are taken up
by muscle in vivo [5], we determined whether TETs were capable of inhibiting intracellular protein degradation in a mammalian muscle cell line.
Materials
and Methods
Doxycycline (Dox) and minocycline (Min) were obtained from Sigma Chemical, St. Louis, MO. The non-antimicrobial tetracycline referred to as CMT (4-dedimethylaminotetracycline), a potent inhibitor of MMPs, was prepared and used as previously described [4]. The system used to determine rates of degradation and synthesis of intracellular proteins has been previously described [6]. In brief, L6 myoblasts were plated at a density of 0.5 x 16 cells/well in 96-well plates in 1 ml and grown to confluence over 3 d in Eagle’s MEh? fortified with 15% fetal calf serum and 2 mM glutamine. To determine the rate of protein synthesis in cells at confluence, the wells were rinsed repeatedly with PBS with calcium and th$ cells incubated at 37°C in a humidified atmosphere of 5% CO in air in Ham’s F12 containing, 1% BSA, 0.02 I.tCi L’4-C-tyrosine (53.8 mCi/mmole, NEN), plus the desired concentrations of tetracyclines/ and or human recombinant insulin (Lilly). All conditions were assessed in quadruplicate wells. Following incubation for 24 hr, the medium was removed, the cells were I washed with ice cold PBS-calcium, and 0.8 ml 1% Triton X-100 in distilled water was added 20 min at 37”. The solubilized cellular material and retained media were counted separately 14C in 2 ml Aquasol 2 in a Packard Tri-carb model 4530 liquid scintillation counter with automatic efficiency program for dpm. As previously described, in this system, >99% of the radioactivity in the medium after incubation is soluble in ice cold 20% TCA and >98% of the radioactivity in the cell pellet is TCA precipitable; this holds true for both synthesis and degradation experiments. To determine thetf;ate of protein degradation, myoblasts were grown to confluence in the presence of 0.1 PCi of -C-tyrosine per well. At confluence, the medium was removed, the cells washed repeatedly, and reincubated in serum-free Ham’s F12 medium plus 1% BSA containing 2 mM non-labelled Tyr which prevents reutilization of the released 14-C-Tyr [6]. At time points from 4-30 hr, media and cells were harvested and counted in quadruplicate as described above. Protein degradation was calculated as per cent supematant dpm/total (supernatant + cells) dpm. Total protein in the cells was measured by the method of Smith et. al. [7] with BCA (Pierce Chemical.)
Results When G6 myoblasts are incubated in Ham’s media containing 1% bovine serum albumin but without added fetal calf serum, the basal rate of protein degradation in 24 hr generally ranges from 3545%. Representative data for various additives are shown in Table I. Addition
of insulin reduced the rate of protein degradation in a dose dependent manner. Both
Min and Dox, 30 pg/ml, had an effect essentially equivalent to that of 0.1 pg/mI insulin (3437%); CMT’, 64 bg/ml, had an even greater effect (50% reduction of the degradation
rate,
down to 23%). Across several experiments, the lowest, consistently effective dose of any TET
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table I. Effects of ins& and three different tetracyclines on basal intraceUular protein degradation in L-6 myobWs after 24 hr lncubatlon. All concentrations in #g/rnf. Results are expressed as per cent refease of labelled tyrodne f S.E.M. P vafues for insulin are comparisons to buffer control: p values for tetracydine runs are comparisons to equivalent sample with insdin alone at the same concentration. Ins = insulin, CMT = 4 dedimethylaminotetracycline, Min = minocydii, Dox = doxycydine. % %-Tyr
Additive None
release
Q
TETRACYCLINES
BIOCHEMICAL
INHIBIT
AND BIOPHYSICAL
INTRACELLULAR
MUSCLE PROTEOLYSIS
Bruce S. Schneider’, Jonathan Maimon’, N. S. Ramamurth~,
RESEARCH COMMUNICATIONS Pages 767-772
IN VITRO
Lome M. Golub2,
and Robert A. Greenwald’
‘Department
of Medicine, Long Island Jewish Medical Center, New Hyde Park,NY 11042 (The Long Island Campus for the Albert Einstein College of Medicine) 2Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York, Stony Brook,NY 11794
Received September 8, 1992
Tetracycline antibiotics (TETs) have a recently discovered novel action: inhibition of extracellular metalloproteinase activity, especially that of collagenase and gelatinase. This property, now confirmed in 8 different laboratories using >40 tissue sources, includes natural and semi-synthetic TETs as well as a chemically modified TET (CMT) devoid of antimicrobial activity. We have used 14C-Tyr biosynthetically labelled intracellular proteins in L6 myoblast culture as a test system to assess intracellular proteolysis. Starvation accelerates proteolysis, which can be suppressed by agents such as insulin or serum. Minocycline, doxycycline, and CMT all retarded the rate of intracellular protein degradation in a dose dependent manner. These agents also demonstrated marked synergism with insulin. A CMT derivative (pyrazole) stripped of one of its metal chelation sites and lacking anti-collagenase activity, also lost its antiproteolytic effect. CMT at physiologic concentrations (I 5 pg/ml) had no effect on protein synthesis, but at 15 pg/rnl (pharmacologic), a suppressive effect was noted. These findings demonstrate that TETs can inhibit protein degradation as well as synthesis in a mammalian muscle-derived cell line. 0 1992Academic PIf2SS.1°C.
Tetracyclines (TETs) are widely known and used as antibiotics in both humans and animals. In addition to the bacteriostatic/antimicrobial
properties of these drugs, an extensive
literature has developed over the past 9 years indicating that TETs are also potent inhibitors of connective tissue degrading enzymes, specifically the neutral, metal-dependent
enzymes
collagenase (MMP-1 and/or MMP-8) and gelatinase (MMP-2 and/or MMP-9). The initial observation of this phenomenon
was reported by Golub et. al. in 1983 [l] and was based on
study of collagenase levels in the gingival tissues and skin of diabetic rats treated with TETs. In the ensuing years, the observation has been confirmed in at least 8 different laboratories and 0006-291X/92 $4.00 767
Copyright 0 1992 by Academic Press, Inc. AN rights of reproduction in any form reserved.
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
has been extended to matrix metalloproteinases
from over 40 different tissues as well as to
unique TET derivatives devoid of antimicrobial
activity [2,3,4]. Since tetracyclines are taken up
by muscle in vivo [5], we determined whether TETs were capable of inhibiting intracellular protein degradation in a mammalian muscle cell line.
Materials
and Methods
Doxycycline (Dox) and minocycline (Min) were obtained from Sigma Chemical, St. Louis, MO. The non-antimicrobial tetracycline referred to as CMT (4-dedimethylaminotetracycline), a potent inhibitor of MMPs, was prepared and used as previously described [4]. The system used to determine rates of degradation and synthesis of intracellular proteins has been previously described [6]. In brief, L6 myoblasts were plated at a density of 0.5 x 16 cells/well in 96-well plates in 1 ml and grown to confluence over 3 d in Eagle’s MEh? fortified with 15% fetal calf serum and 2 mM glutamine. To determine the rate of protein synthesis in cells at confluence, the wells were rinsed repeatedly with PBS with calcium and th$ cells incubated at 37°C in a humidified atmosphere of 5% CO in air in Ham’s F12 containing, 1% BSA, 0.02 I.tCi L’4-C-tyrosine (53.8 mCi/mmole, NEN), plus the desired concentrations of tetracyclines/ and or human recombinant insulin (Lilly). All conditions were assessed in quadruplicate wells. Following incubation for 24 hr, the medium was removed, the cells were I washed with ice cold PBS-calcium, and 0.8 ml 1% Triton X-100 in distilled water was added 20 min at 37”. The solubilized cellular material and retained media were counted separately 14C in 2 ml Aquasol 2 in a Packard Tri-carb model 4530 liquid scintillation counter with automatic efficiency program for dpm. As previously described, in this system, >99% of the radioactivity in the medium after incubation is soluble in ice cold 20% TCA and >98% of the radioactivity in the cell pellet is TCA precipitable; this holds true for both synthesis and degradation experiments. To determine thetf;ate of protein degradation, myoblasts were grown to confluence in the presence of 0.1 PCi of -C-tyrosine per well. At confluence, the medium was removed, the cells washed repeatedly, and reincubated in serum-free Ham’s F12 medium plus 1% BSA containing 2 mM non-labelled Tyr which prevents reutilization of the released 14-C-Tyr [6]. At time points from 4-30 hr, media and cells were harvested and counted in quadruplicate as described above. Protein degradation was calculated as per cent supematant dpm/total (supernatant + cells) dpm. Total protein in the cells was measured by the method of Smith et. al. [7] with BCA (Pierce Chemical.)
Results When G6 myoblasts are incubated in Ham’s media containing 1% bovine serum albumin but without added fetal calf serum, the basal rate of protein degradation in 24 hr generally ranges from 3545%. Representative data for various additives are shown in Table I. Addition
of insulin reduced the rate of protein degradation in a dose dependent manner. Both
Min and Dox, 30 pg/ml, had an effect essentially equivalent to that of 0.1 pg/mI insulin (3437%); CMT’, 64 bg/ml, had an even greater effect (50% reduction of the degradation
rate,
down to 23%). Across several experiments, the lowest, consistently effective dose of any TET
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table I. Effects of ins& and three different tetracyclines on basal intraceUular protein degradation in L-6 myobWs after 24 hr lncubatlon. All concentrations in #g/rnf. Results are expressed as per cent refease of labelled tyrodne f S.E.M. P vafues for insulin are comparisons to buffer control: p values for tetracydine runs are comparisons to equivalent sample with insdin alone at the same concentration. Ins = insulin, CMT = 4 dedimethylaminotetracycline, Min = minocydii, Dox = doxycydine. % %-Tyr
Additive None
release
Q
