Vol.
174,
February
No.
3, 1991
14,
1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
CALMODULIN-DEPENDENT ACTIVITIES
COLIAGENASE
IN
M.RICHARDl*,
AND PROTEOGLYCANASE
CHONDROCYrES
OSTEOARTHRITIC PBROQUETl,
1204-1207
FROM
HUMAN
CARTILAGE
E.VIGNON2, M.J.PESCHARDl, and P.LOUISOTl
J.P.CARRET3
’ Department of General and Medical Biochemistry INSERM-CNRS
U189
Lyon-Sud Medical School BP12 69921 - OULLINS Cedex, France 2 Rheumatology Division, Edouard Herriot Hospital 69437 - Lyon Cedex , France 3 Orthopedic Surgery, Jules Courmont Hospital 69310 - Pierre-Benite, Received
December
19,
France
1990
Chondrocyte metalloproteinases appear to play a major role in the development of osteoarthritis. The intracellular post-traductional mechanisms regulating collagenase and proteoglycanase are not known. Calmodulin antagonists including phenothiazine and sulfonamide derivatives significantly increased proteoglycanase activity and decreased collagenase activity. H-7, a specific inhibitor of protein kinase C, had no effect on the two metalloproteinase activities, and calmodulin was ineffective in in vitro assays upon metalloproteinase activities. We postulate that collagenase and proteoglycanase activities are controlled by calmodulin-dependent regulation. 0 1991Academic Press,Inc.
The cartilage matrix macromolecules consist mainly of proteoglycans, collagens and minor amounts of non collagenous proteins. Chronic progressive destruction of articular joint structure is a characteristic pathological feature
of
osteoarthritis,
Neutral
metalloproteinases
(collagenase
and
proteoglycanase) are involved in this articular degeneration process (1,5). Their activities may be regulated by control mechanisms which would operate at different levels including traductional and post-traductional post-traductional
effects, phosphorylation
events. Among
and glycosylation are the most
common for enzymes. However, to date there is very little information on the *To whom all correspondence should be addressed. Abbreviations : H-7, 1-(5isoquinolinylsulfonyl)-2-methylpiperazine; W-5, N-(6aminohexyl)-1 -naphtalene-sulfonamide; W-7, N-(6-aminohexyl)-5-chloro-
1-naphtalene-sulfonamide; 0006-291X/91 Copyright All rights
TFP, trifluoperazine.
$1.50
0 1991 by Academic Press. Inc. of reproduction it1 arzy form reserved.
1204
Vol.
‘174,
No.
3, 1991
intracellular
BIOCHEMICAL
post-traductional
metalloproteinases. inhibition
AND
metalloproteinase
it has been shown
kinase
collagenase production Ca++/calmodulin-dependent
activation
(6).
protein
kinase
C
known
we
have
MATERIALS
that tyrosine
phosphatase
with
(7)
tested
) (H-7), and
the
(TFP), N-(6-aminohexyl)-1
N-(6-aminohexyl)-5-chloro-1
chondrocyte the
To assess if protein enzymes are involved
regulation
COMMUNICATIONS
regulating
correlated
isoquinolinylsulfonyl-2-methylpiperazine trifluoperazine
RESEARCH
mechanisms
Recently
or tyrosine
BIOPHYSICAL
inhibition
of
kinase C and in chondrocyte
the
effect
of
1-(5-
the most specific
inhibitor
of calmodulin
antagonists
effect
-naphtalene
sulfonamide
naphtalene-sulfonamide
(W-5)
of and
(W-7).
and METHODS
TFP, W-5, W-7, H-7 and calmodulin were purchased from Sigma. Stock solutions of each antagonist was made up in 95% (v/v) ethanol. Controls were performed to verify that the solvent alone was without effect when added to the assays. Human osteoarthritic cartilage was obtained from femoral heads or knee joints at the time of total hip or knee replacement (average age 64 range 56-72). Articular chondrocytes were isolated by sequential enzymatic digestion (trypsin and collagenase) of cartilage slices as described (8, 9). The cells were washed twice with DMEM medium without foetal calf serum to eliminate collagenase (No collagenolytic activity was found in the last supernatant). The chondrocytes (8xlO6/in ml DMEM medium) were seeded into 24-well microtiter plates and treated for 60 min. at 37°C (with the different drugs) in an incubator 95% air, 5% C02. The chondrocytes were collected, centrifuged (1 ,lOOg, 5 in 20 mmol/l Tris-HCI buffer pH 7.40 min. , washed twice and resuspended (10 6’ cells/ml buffer). Cells were homogenized with a Potter-Elvehjem and broken up by ultrasonic treatment in ice with a probe (Sonimass type 25 T, 120 volts, 4x30 sec.). The homogenate was used for enzymatic determinations. In in vitro experiment assays, compounds were directly added to the chondrocyte homogenate during enzymatic kinetics. Collagenolytic activity (spontaneously active form) was determined using [3H]-acetylated soluble type II collagen as a substrate (10) Collagen was [3H]-acetylated with [3H]-acetic anhydride as in 11. Using polyacrylamide gel electrophoresis the labelled collagen degradated by chondrocyte homogenate and by a pure collagenase were found to be similar while pronase digestion gave different products. Proteoglycanase (spontaneously active form) was measured by the released of soluble radioactive fragments from labelled proteoglycans extracted from normal human articular cartilage (10) and [3H]-acetylated (11). No radioactivity was released by a specific chemical desacetylation of glycosaminoglycans from labelled proteoglycans. The measured radioactivity was thus specific to the proteoglycan core protein degradation. RESULTS
and DISCUSSION As shown
marked
decrease
control).
Same
(respectively less
on collagenase products
antagonists,
activity
strongly
on metalloproteinase
effect on both collagenase
W-7
(respectively
increased
five and eight times). A weak calmodulin
effective
significant
in table 1, calmodulin
activities.
25 and 15 percent
proteoglycanase inhibitor, W-5,
On the opposite
and proteoglycanase 1205
and TFP, induced
activities.
of
activity was much
H-7
had no
Vol.
174, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 EFFECTS OF CALMODULIN ANTAGONISTS and H-7 ON CHONDROCYTE METALLOPROTEINASE ACTIVITIES
COLIAGENASE
PROTEOGLYCANASE
H-7
w-5
w-7
TFP
97213 NS
60212
25?15
15rlO
p
174,
February
No.
3, 1991
14,
1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
CALMODULIN-DEPENDENT ACTIVITIES
COLIAGENASE
IN
M.RICHARDl*,
AND PROTEOGLYCANASE
CHONDROCYrES
OSTEOARTHRITIC PBROQUETl,
1204-1207
FROM
HUMAN
CARTILAGE
E.VIGNON2, M.J.PESCHARDl, and P.LOUISOTl
J.P.CARRET3
’ Department of General and Medical Biochemistry INSERM-CNRS
U189
Lyon-Sud Medical School BP12 69921 - OULLINS Cedex, France 2 Rheumatology Division, Edouard Herriot Hospital 69437 - Lyon Cedex , France 3 Orthopedic Surgery, Jules Courmont Hospital 69310 - Pierre-Benite, Received
December
19,
France
1990
Chondrocyte metalloproteinases appear to play a major role in the development of osteoarthritis. The intracellular post-traductional mechanisms regulating collagenase and proteoglycanase are not known. Calmodulin antagonists including phenothiazine and sulfonamide derivatives significantly increased proteoglycanase activity and decreased collagenase activity. H-7, a specific inhibitor of protein kinase C, had no effect on the two metalloproteinase activities, and calmodulin was ineffective in in vitro assays upon metalloproteinase activities. We postulate that collagenase and proteoglycanase activities are controlled by calmodulin-dependent regulation. 0 1991Academic Press,Inc.
The cartilage matrix macromolecules consist mainly of proteoglycans, collagens and minor amounts of non collagenous proteins. Chronic progressive destruction of articular joint structure is a characteristic pathological feature
of
osteoarthritis,
Neutral
metalloproteinases
(collagenase
and
proteoglycanase) are involved in this articular degeneration process (1,5). Their activities may be regulated by control mechanisms which would operate at different levels including traductional and post-traductional post-traductional
effects, phosphorylation
events. Among
and glycosylation are the most
common for enzymes. However, to date there is very little information on the *To whom all correspondence should be addressed. Abbreviations : H-7, 1-(5isoquinolinylsulfonyl)-2-methylpiperazine; W-5, N-(6aminohexyl)-1 -naphtalene-sulfonamide; W-7, N-(6-aminohexyl)-5-chloro-
1-naphtalene-sulfonamide; 0006-291X/91 Copyright All rights
TFP, trifluoperazine.
$1.50
0 1991 by Academic Press. Inc. of reproduction it1 arzy form reserved.
1204
Vol.
‘174,
No.
3, 1991
intracellular
BIOCHEMICAL
post-traductional
metalloproteinases. inhibition
AND
metalloproteinase
it has been shown
kinase
collagenase production Ca++/calmodulin-dependent
activation
(6).
protein
kinase
C
known
we
have
MATERIALS
that tyrosine
phosphatase
with
(7)
tested
) (H-7), and
the
(TFP), N-(6-aminohexyl)-1
N-(6-aminohexyl)-5-chloro-1
chondrocyte the
To assess if protein enzymes are involved
regulation
COMMUNICATIONS
regulating
correlated
isoquinolinylsulfonyl-2-methylpiperazine trifluoperazine
RESEARCH
mechanisms
Recently
or tyrosine
BIOPHYSICAL
inhibition
of
kinase C and in chondrocyte
the
effect
of
1-(5-
the most specific
inhibitor
of calmodulin
antagonists
effect
-naphtalene
sulfonamide
naphtalene-sulfonamide
(W-5)
of and
(W-7).
and METHODS
TFP, W-5, W-7, H-7 and calmodulin were purchased from Sigma. Stock solutions of each antagonist was made up in 95% (v/v) ethanol. Controls were performed to verify that the solvent alone was without effect when added to the assays. Human osteoarthritic cartilage was obtained from femoral heads or knee joints at the time of total hip or knee replacement (average age 64 range 56-72). Articular chondrocytes were isolated by sequential enzymatic digestion (trypsin and collagenase) of cartilage slices as described (8, 9). The cells were washed twice with DMEM medium without foetal calf serum to eliminate collagenase (No collagenolytic activity was found in the last supernatant). The chondrocytes (8xlO6/in ml DMEM medium) were seeded into 24-well microtiter plates and treated for 60 min. at 37°C (with the different drugs) in an incubator 95% air, 5% C02. The chondrocytes were collected, centrifuged (1 ,lOOg, 5 in 20 mmol/l Tris-HCI buffer pH 7.40 min. , washed twice and resuspended (10 6’ cells/ml buffer). Cells were homogenized with a Potter-Elvehjem and broken up by ultrasonic treatment in ice with a probe (Sonimass type 25 T, 120 volts, 4x30 sec.). The homogenate was used for enzymatic determinations. In in vitro experiment assays, compounds were directly added to the chondrocyte homogenate during enzymatic kinetics. Collagenolytic activity (spontaneously active form) was determined using [3H]-acetylated soluble type II collagen as a substrate (10) Collagen was [3H]-acetylated with [3H]-acetic anhydride as in 11. Using polyacrylamide gel electrophoresis the labelled collagen degradated by chondrocyte homogenate and by a pure collagenase were found to be similar while pronase digestion gave different products. Proteoglycanase (spontaneously active form) was measured by the released of soluble radioactive fragments from labelled proteoglycans extracted from normal human articular cartilage (10) and [3H]-acetylated (11). No radioactivity was released by a specific chemical desacetylation of glycosaminoglycans from labelled proteoglycans. The measured radioactivity was thus specific to the proteoglycan core protein degradation. RESULTS
and DISCUSSION As shown
marked
decrease
control).
Same
(respectively less
on collagenase products
antagonists,
activity
strongly
on metalloproteinase
effect on both collagenase
W-7
(respectively
increased
five and eight times). A weak calmodulin
effective
significant
in table 1, calmodulin
activities.
25 and 15 percent
proteoglycanase inhibitor, W-5,
On the opposite
and proteoglycanase 1205
and TFP, induced
activities.
of
activity was much
H-7
had no
Vol.
174, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 EFFECTS OF CALMODULIN ANTAGONISTS and H-7 ON CHONDROCYTE METALLOPROTEINASE ACTIVITIES
COLIAGENASE
PROTEOGLYCANASE
H-7
w-5
w-7
TFP
97213 NS
60212
25?15
15rlO
p
