Leukemia inhibitory factor and oncostatin M stimulate
collagenase-3 expression in osteoblasts
Samuel
Varghese,
Kyung
Yu, and
Ernesto
Canalis
Departments of Research and Medicine, Saint Francis Hospital and
Medical Center, Hartford 06105; and The University of Connecticut
School of Medicine, Farmington, Connecticut 06030
 |
ABSTRACT |
Leukemia inhibitory
factor (LIF) and oncostatin M (OSM) have multiple effects on skeletal
remodeling. Although these cytokines modestly regulate collagen
synthesis in osteoblasts, their effects on collagenase expression and
collagen degradation are not known. We tested whether LIF and OSM
regulate the expression of matrix metalloproteinases (MMPs) and tissue
inhibitors of metalloproteinases (TIMPs) in osteoblast-enriched cells
isolated from fetal rat calvariae. LIF and OSM increased collagenase-3
(MMP-13) mRNA and immunoreactive protein levels in a time- and
dose-dependent manner. LIF and OSM enhanced the rate of transcription
of the collagenase gene and stabilized collagenase mRNA in
transcriptionally arrested cells. LIF and OSM failed to regulate the
expression of gelatinase A (MMP-2) and B (MMP-9). LIF and OSM modestly
stimulated the expression of TIMP-1 but did not alter the expression of
TIMP-2 and -3. In conclusion, LIF and OSM stimulate collagenase-3 and
TIMP-1 expression in osteoblasts, and these effects may be involved in
mediating the bone remodeling actions of these cytokines.
cytokines; collagen degradation; matrix metalloproteinases; tissue
inhibitors of metalloproteinases; gelatinases
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INTRODUCTION |
CYTOKINES PRESENT IN the bone microenvironment
influence various aspects of bone remodeling (21). Because most bone
cells reside in the vicinity of bone marrow, the cytokines produced by
bone marrow cells act as paracrine factors for bone. Some cytokines are
also produced by stimulated bone cells, thus causing additional autocrine effects in the skeletal tissue. Physiological levels of
cytokines may be relevant for the normal bone remodeling process; however, increased production of cytokines such as interleukin (IL)-1
and IL-6 has been implicated as a causative factor for bone loss in
diseases such as osteoporosis (21).
Leukemia inhibitory factor (LIF) and oncostatin M (OSM) are members of
the IL-6 family of cytokines that share a common signal transducing
receptor component, glycoprotein 130 (gp130). LIF binds LIF receptors
(LIFR), whereas OSM can bind a specific OSM receptor (OSMR) as well as
LIFR (34). The cellular actions of LIF and OSM are elicited by the
heterodimerization of LIFR or OSMR with gp130 to generate intracellular
signals for the JAK/STAT signal transduction pathway. LIF and OSM are
primarily produced by cells of hematopoietic lineage, such as activated
monocytes and T lymphocytes. LIF is also synthesized by parathyroid
hormone (PTH)-stimulated osteoblasts (8). LIF and OSM are modest
mitogens for osteoblastic cells, and they regulate the expression of
important osteoblastic markers such as type I collagen and alkaline
phosphatase (10, 29, 36). The actions of LIF and OSM on bone resorption are somewhat controversial, and depending on the culture model stimulatory and inhibitory effects have been reported (10, 19, 29, 36).
Based on the actions of LIF and OSM in bone cultures, these cytokines
are regarded as potent bone remodeling agents with pleiotropic effects
on bone formation and resorption.
Collagenases are matrix metalloproteinases (MMPs) that degrade
components of extracellular matrix (25). Three mammalian collagenases,
collagenase-1, -2 and -3 (MMP-1, -8, and -13), are known, and these
proteinases can degrade fibrillar collagen at neutral pH. Collagenase-3
was originally identified in human breast carcinoma and was found to be
expressed in nonmalignant human cells, including chondrocytes and
osteoblasts (33). Collagenase-3 expression is stimulated in rodent
osteoblasts by several bone remodeling agents, including PTH, IL-1, and
IL-6 (7, 13, 26). Collagenase is synthesized and secreted as a
proenzyme and is activated by other proteases in the extracellular
matrix by proteolytic cleavage. Bone cells also synthesize gelatinases A (72-kDa gelatinase or MMP-2) and B (92-kDa gelatinase or MMP-9), which can complement collagenase activity by the activation of procollagenase and by further degradation of collagen fragments that
are generated by the initial cleavage of intact collagen fibrils by
collagenase (18). The biological activity of collagenase is suppressed
by tissue inhibitors of metalloproteinases (TIMPs), which are also
expressed by osteoblastic cells (7).
Because OSM and LIF have important effects on bone remodeling, we
postulated that they may regulate the synthesis of proteases that
regulate collagen turnover. In this study, we examined the regulation
of collagenase-3, gelatinases A and B, and TIMP-1, -2, and -3 by LIF
and OSM in primary cultures of osteoblast-enriched (Ob) cells isolated
from fetal rat calvariae.
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MATERIALS AND METHODS |
Cell culture. Fetuses were removed
from 22-day-pregnant Sprague-Dawley rats (Charles River Breeding
Laboratories, Wilmington, MA) and were killed by blunt trauma to the
nuchal area, following a protocol approved by the Animal Care and Use
Committee of Saint Francis Hospital and Medical Center. Ob cells from
parietal bones of fetal rat calvariae were isolated as described
previously (24). Briefly, bones were digested with bacterial
collagenase (Worthington, Freehold, NJ), and the cells from the third
to fifth digestions were pooled and plated at 6,000 to 12,000 cells/cm2 in Dulbecco's modified
Eagle's medium (DMEM) supplemented with nonessential amino acids, 100 µg/ml L-ascorbic acid, 20 mM HEPES (all from Life
Technologies, Grand Island, NY), and 10% fetal bovine serum (Summit
Biotechnology, Fort Collins, CO) and cultured at 37°C in a
humidified C02 incubator. At
confluence, the medium was replaced with serum-free DMEM for 16-24
h, and cells were exposed to control medium with or without test
agents. Recombinant mouse LIF (Genzyme, Cambridge, MA) was dissolved in
serum-free DMEM containing 0.1% bovine serum albumin (BSA; Sigma
Chemical, St. Louis, MO), and recombinant human OSM (R&D Systems,
Minneapolis, MN) was added directly to serum-free DMEM. For
determination of mRNA stability, cells were incubated with control
medium with or without LIF and OSM for 16 h before the addition of
5,6-dichlorobenzimidazole riboside (DRB; Sigma), which was dissolved in
ethanol and diluted 1:200; an equal amount of ethanol was present in
control and test cultures. For nuclear run-off assays, cells were grown
to subconfluence, trypsinized, replated, grown to confluence,
serum-deprived for 16 h, and exposed to control and test solutions. At
the end of the culture, the cell layer was extracted for isolation of
total RNA for Northern blot analysis, and nuclei were isolated to
determine rates of transcription. Medium was collected and stored at
20°C after the addition of polyoxyethylene sorbitan
monolaurate (Pierce Chemical, Rockford, IL) to a final concentration of
0.1% for procollagenase determination.
Northern blot analysis. Total RNA was
isolated from Ob cells by the method of Chomczynski and Sacchi (4) or
by using an RNeasy kit (Qiagen, Chatsworth, CA) and was fractionated on
a 1% agarose (Sigma)-formaldehyde (Life Technologies) gel containing 100 µg/ml ethidium bromide (Sigma) as described (4, 31). After
electrophoresis, RNA was transferred onto a Biotrans nylon membrane
(ICN Biomedicals, Aurora, OH) by capillary action. The integrity and
equal gel loading of RNA and efficiency of transfer were assessed by
visualizing 28S and 18S ribosomal RNA bands under ultraviolet (UV)
light. RNA was cross-linked to nylon membranes using a CL-1000 UV
cross-linker (UVP, San Gabriel, CA). cDNA fragments were isolated by
restriction endonuclease (New England Biolabs, Beverly, MA) digestion
of plasmid clones containing a 2.6-kilobase (kb) rat collagenase-3 cDNA
(kindly provided by Dr. Cheryl Quinn, St. Louis University School of
Medicine, St. Louis, MO), a 0.8-kb murine TIMP-1 cDNA, a 0.7-kb murine
TIMP-2 cDNA, a 0.75-kb murine TIMP-3 cDNA (all kindly provided by Dr.
Dylan Edwards, University of Calgary Health Sciences Center, Calgary,
AB, Canada), a 1.1-kb human gelatinase A cDNA (American Type Culture
Collection, Rockville, MD), a 0.78-kb murine gelatinase B cDNA (kindly
provided by Dr. Ghislain Opdenakker, University of Leuven, Brussels,
Belgium), and a 0.8-kb rat glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA (kindly provided by Dr. Ray Wu, Cornell
University, Ithaca, NY; see Refs. 14-16, 22, 26, 35). cDNAs were
radiolabeled by the random-priming method using
[
-32P]dATP and
[
-32P]dCTP (3,000 Ci/mmol; DuPont, Wilmington, DE) and DNA polymerase large fragment (New
England Biolabs), and the hybridization of the RNA blots was performed
for 16-24 h at 42°C with radiolabeled cDNAs in the presence of
50% formamide (Sigma) as described (31). The final wash was performed
at 55°C in 0.15 M sodium chloride-0.015 M sodium citrate, pH 7 (1× SSC)-0.1% sodium dodecyl sulfate (SDS; all from
Sigma) for the detection of collagenase-3, TIMP-1, -2, and -3, and
GAPDH mRNAs. The final wash was performed at 62°C in 0.2×
SSC-0.1% SDS for the detection of gelatinase A and B mRNAs. mRNA bands
were visualized by autoradiography using Kodak X-AR film (Eastman
Kodak, Rochester, NY) in the presence of a DuPont Cronex Lightning Plus
intensifying screen. The intensity of RNA bands was quantified by
densitometric tracing of the autoradiographs. Changes in collagenase-3,
TIMP-1, -2, and -3, and gelatinases A and B were normalized for changes
in GAPDH. For each test condition, Northern blot analysis was performed
using samples from two or more independent cultures.
Nuclear run-off assay. Nuclei were
isolated from Ob cells after Dounce homogenization in Tris buffer
containing 0.5% nonionic detergent IGEPAC CA630 (Sigma; see Ref. 1).
Nascent transcripts were radiolabeled by incubation of nuclei at room
temperature for 30 min in a reaction buffer containing 250 µCi (800 Ci/mmol) of
[
-32P]UTP (DuPont),
500 µM ATP, CTP, and GTP (all from Life Technologies), and 150 units
RNasin (Promega, Madison, WI).
[32P]RNA was isolated
by treatment with DNase I (Life Technologies) and proteinase K
(Boehringer Mannheim, Indianapolis, IN), followed by phenol-chloroform
extraction and ethanol precipitation using ammonium acetate. Linearized
plasmid DNA containing 1 µg of cDNA for rat collagenase-3 and mouse
ribosomal 18S RNA (American Type Culture Collection) or 1 µg of pUC18
(Life Technologies) plasmid DNA was immobilized on a Biotrans nylon
membrane using a slot-blot apparatus (Life Technologies). Equal counts
of [32P]RNA from each
sample were hybridized to immobilized cDNAs at conditions identical to
Northern hybridization and were washed in 1× SSC-0.1% SDS at
55°C. Hybridization of nascent transcripts was visualized by
autoradiography and quantitated by densitometry. Nuclear run-off assay
was performed two times for 1 h treatment with LIF and OSM and one time
for 4 and 16 h treatment with LIF.
Western immunoblot analysis. Aliquots
of equal volume from control and test culture medium were fractionated
on a 10% polyacrylamide (Boehringer Mannheim) gel by electrophoresis
using denaturing conditions and were transferred onto an Immobilon-P
membrane (Millipore, Bedford, MA; see Ref. 1). After blocking with 2%
BSA, the membrane was exposed to a 1:1,000 dilution of rabbit antiserum
raised against rat collagenase (11; kindly provided by Dr. John J. Jeffrey, Albany Medical College, Albany, NY), followed by the addition
of goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma). The blots were washed and developed with a horseradish peroxidase chemiluminescence detection reagent (DuPont). The chemiluminescent bands were visualized after exposure to DuPont Reflection film employing a reflection intensifying screen. Western blot analysis was
performed using samples from two independent cultures.
Statistical methods. Data on
collagenase mRNA decay were analyzed by linear regression, and the
slopes of the regression lines obtained for control and LIF- or
OSM-treated cells were compared for significant differences using the
method of Sokal and Rohlf (32).
 |
RESULTS |
The effects of LIF and OSM on collagenase-3 transcripts were examined
by Northern blot analysis. After treatment of Ob cells with 50 ng/ml of
LIF for 2-24 h, collagenase mRNA levels were increased by 6- to
22-fold (Fig.
1A
and Table 1). To determine the dose-dependent changes in
collagenase-3 expression, Ob cells were exposed to LIF at doses of
0.1-100 ng/ml for 16 h (Fig. 1B and Table 2). LIF increased collagenase
mRNA levels at doses 1 ng/ml and higher. Exposure of Ob cells to OSM at
50 ng/ml for 2-24 h increased collagenase mRNA levels by 5- to
19-fold (Fig. 2A and
Table 1). After treatment with OSM at doses of 0.1-100 ng/ml for
16 h, collagenase mRNA levels were increased at doses of 10 ng/ml and
higher (Fig. 2B and Table 2).
Immunoreactive procollagenase secretion in the culture medium was
determined by Western blot analysis using a specific rabbit anti-rat
procollagenase antibody (Fig. 3). LIF and
OSM at 50 ng/ml for 16-24 h increased procollagenase levels 7- to
12-fold.

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Fig. 1.
Effect of leukemia inhibitory factor (LIF) on collagenase-3 mRNA levels
in cultures of osteoblast-enriched (Ob) cells.
A: Ob cell cultures were treated with
LIF at 50 ng/ml for 2, 4, 16, and 24 h. Total RNA from control
( ) or LIF (+)-treated cultures was analyzed by Northern
hybridization using 32P-labeled
collagenase-3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
cDNAs. B: Ob cell cultures were
exposed to LIF at different doses for 16 h. Total RNA from control
cultures (0) and from cultures treated with LIF at doses of 0.1, 1, 10, 50, and 100 ng/ml was analyzed by Northern hybridization with
32P-labeled collagenase-3 and
GAPDH cDNAs. Levels of collagenase-3 [matrix metalloproteinase
(MMP)-13] and GAPDH mRNAs from 1 of 3 independent cultures are
shown.
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Fig. 2.
Effect of oncostatin M (OSM) on collagenase-3 mRNA levels in cultures
of Ob cells. A: Ob cell cultures were
treated with OSM at 50 ng/ml for 2, 4, 16, and 24 h. Total RNA from
control ( ) or OSM (+)-treated cultures was analyzed by Northern
hybridization using 32P-labeled
collagenase-3 and GAPDH cDNAs. B: Ob
cell cultures were exposed to OSM at different doses for 16 h. Total
RNA from control cultures (0) and from cultures treated with OSM at
doses of 0.1, 1, 10, 50, and 100 ng/ml was analyzed by Northern
hybridization with 32P-labeled
collagenase-3 and GAPDH cDNAs. Levels of collagenase-3 (MMP-13) and
GAPDH mRNAs from 1 of 3 independent cultures are shown.
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Fig. 3.
Effects of LIF (A) and OSM
(B) on procollagenase secretion in
Ob cell cultures. Ob cell cultures were exposed to LIF or OSM at 50 ng/ml for 16 or 24 h. Western blot analysis was performed using equal
amounts of culture medium from control ( ) and test (+) cultures.
Procollagenase (arrow) was detected using rabbit anti-rat collagenase
antibody and horseradish peroxidase chemiluminescence detection system.
Procollagenase levels from 1 of 2 independent cultures are shown.
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To investigate whether the collagenase-3 gene was regulated by LIF and
OSM at the transcriptional level, we examined the changes in the
collagenase gene transcription rate by nuclear run-off assays (Fig.
4). Exposure of Ob cells to LIF at 10 ng/ml
for 1, 4, and 16 h increased the synthesis of nascent collagenase
transcripts by two-, three-, and sixfold, respectively. OSM at 50 ng/ml
caused a fourfold increase in the synthesis of nascent collagenase
transcripts after 1 h. Because changes in collagenase mRNA levels may
also be secondary to mRNA stabilization, we tested collagenase mRNA decay after transcriptional arrest by adding the RNA polymerase II
inhibitor DRB at 75 µM to Ob cells pretreated with or without LIF or
OSM at 50 ng/ml for 16 h. The levels of collagenase mRNA from control
and LIF-treated cultures were measured before and 3-12 h after
transcriptional arrest to determine the rate of collagenase mRNA decay
(Fig. 5). The half-life of collagenase mRNA
was 4 h in control cultures, and it was estimated to be 24 h in
LIF-treated cultures, by extrapolation of values obtained in the first
12 h. The levels of collagenase mRNA from control and OSM-treated cultures were measured in a similar manner up to 24 h after
transcriptional arrest (Fig. 6). The
half-life of collagenase mRNA was ~5 h in control cultures, and it
was estimated to be 30 h in OSM-treated cultures, by extrapolation of
values observed in the first 24 h.

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Fig. 4.
Effects of LIF (A) and OSM
(B) on collagenase-3 gene
transcription rates. Ob cells were treated with LIF at 10 ng/ml for 1, 4, and 16 h or OSM at 50 ng/ml for 1 h. Nascent transcripts were
labeled in vitro with
[ -32P]UTP, and
labeled RNA was hybridized to immobilized cDNAs for collagenase-3
(MMP-13) and 18S rRNA and vector DNA pUC18.
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Fig. 5.
Effect of LIF on collagenase-3 mRNA decay in Ob cell cultures. Ob cells
were exposed to control medium or 50 ng/ml LIF- containing medium for
16 h before the addition of 5,6-dichlorobenzimidazole riboside (DRB) at
75 µM. Total RNA, obtained 0-12 h after DRB addition, was
subjected to Northern blot analysis and hybridized with
32P-labeled collagenase cDNA. Data
from control ( ) and LIF treatment ( ), expressed as means ± SE
for 3-5 independent cultures as percentage of mRNA levels before
the addition of DRB, are shown. Inset:
representative experiment showing changes in collagenase mRNA after
addition of DRB from cultures treated with and without LIF.
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Fig. 6.
Effect of OSM on collagenase-3 mRNA decay in Ob cell cultures. Ob cells
were exposed to control medium or 50 ng/ml OSM-containing medium for 16 h before addition of DRB at 75 µM. Total RNA, obtained 0-24 h
after DRB addition, was subjected to Northern blot analysis and
hybridized with 32P-labeled
collagenase cDNA. Data from control ( ) and OSM treatment ( ),
expressed as means ± SE for 3-4 independent cultures as
percentage of mRNA levels before addition of DRB, are shown.
Inset: representative experiment
showing changes in collagenase mRNA after addition of DRB from cultures
treated with and without OSM.
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LIF and OSM at 100 ng/ml for 2-24 h did not alter the expression
of gelatinase A or B mRNA levels (data not shown). The regulation of
TIMP-1, -2, and -3 transcripts was evaluated after exposure to LIF and
OSM at 50 ng/ml for 2-24 h. OSM increased the levels of TIMP-1
mRNA by twofold and LIF by two- to fourfold (Fig.
7 and Table 3).
Neither LIF nor OSM changed the levels of TIMP-2 and -3 mRNA (data not
shown).

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Fig. 7.
Effects of LIF and OSM on tissue inhibitor of metalloproteinase
(TIMP)-1 mRNA levels in cultures of Ob cells. Ob cell cultures were
treated with LIF (A) or OSM
(B) at 50 ng/ml for 2, 4, 16, and 24 h. Total RNA from control ( ) or test (+) cultures was analyzed
by Northern hybridization using
32P-labeled TIMP-1 and GAPDH
cDNAs. Levels of TIMP-1 and GAPDH mRNAs from 1 of 2 (A) or 3 (B) independent cultures are
shown.
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 |
DISCUSSION |
The present study shows that OSM and LIF stimulate the expression of
collagenase-3 in osteoblasts in a time- and dose-dependent manner. The
induction of collagenase mRNA by OSM and LIF is similar to that
observed for IL-6 in the presence of exogenously added soluble receptor
(7, 13). Recently, OSM has been shown to stimulate collagenase-3 in
chondrosarcoma cells (5). Unlike IL-6, the receptors for LIF and OSM
are expressed in rodent osteoblastic cells, indicating that these cells
are direct targets of these cytokines (2). LIF and OSM regulate
collagenase expression at the transcriptional and posttranscriptional
level. LIF and OSM are potent mediators of the JAK/STAT signal
transduction pathway in osteoblasts (17, 20). Putative DNA elements
that can interact with the STAT family of nuclear factors are present
within the collagenase gene promoter, and it is possible that LIF and
OSM mediate transcriptional regulation of the collagenase gene through these regulatory sequences (27). The activator protein-1 and polyoma
viral enhancer PEA3 sites that are present in several MMP promoters,
including the collagenase-3, have been shown to be important in the
regulation of MMPs by growth factors and cytokines (23, 27). It remains
to be established if these regulatory sites within the collagenase-3
promoter influence the transcriptional regulation by LIF and OSM in
osteoblasts. Previous studies showed that collagenase-3 expression can
be regulated at the posttranscriptional level by altering mRNA
stability (37). LIF and OSM increase the stability of collagenase mRNA
in osteoblastic cells. Unlike LIF and OSM, IL-6 does not alter the
stability of collagenase mRNA (7). The degradation of several mRNA
species is found to be mediated by the presence of AU-rich elements
"AUUUA" (AREs; see Ref. 3). Collagenase mRNA contains three AREs
in the 3'-untranslated region and one ARE in the protein-coding
region; however, it is unclear if these RNA elements play a role in
regulating mRNA stability. It is conceivable that the cytokines, such
as LIF and OSM, and other stimulators of collagenase mRNA stability may
function by suppressing the normal regulatory function of AREs.
LIF and OSM stimulated the secretion of procollagenase. MMPs and
proteinases that are present in the bone matrix may be responsible for
the proteolytic activation of procollagenase in bone. Neither LIF nor
OSM altered the expression of gelatinase A and B, possible activators
of procollagenase-3 in osteoblastic cells. In contrast, earlier studies
indicated that IL-6 can stimulate gelatinase A in osteoblasts (7). The
activators of collagenase may also be generated by osteoclastic cells
that are recruited to the bone remodeling site, since osteoclasts
synthesize a number of proteolytic enzymes, including MMPs (38). As
observed for most stimulators of collagenase-3, LIF and OSM also
increase the expression of TIMP-1. Stimulation of TIMP-1 in osteoblasts
by LIF and OSM is consistent with the observation that these cytokines
increase the expression of TIMP-1 in fibroblasts (30). Expression of TIMP-2 and -3 in osteoblasts was unaffected by these cytokines. TIMPs
inhibit MMPs by forming a bimolecular complex with MMPs, thus
eliminating their endopeptidase activity. TIMP-1 binds active collagenase, but it does not appear to interact with latent
procollagenase. Therefore, the increase in TIMP-1 by LIF and OSM may
not affect procollagenase activation but may restrict collagenolytic
activity, providing an additional regulatory control for collagen breakdown.
The precise role of collagenase stimulation in bone remodeling mediated
by LIF and OSM is unclear. A recent report by Holliday et al. (9)
showed that the collagen fragments generated by collagenase can enhance
osteoclastic activity. Therefore, it is possible that the increase in
collagenase expression may be partly responsible for the stimulation of
bone resorption by LIF and possibly by OSM in rodent calvaria. Because
collagenase-3 is also present in human skeletal cells, stimulation of
collagenase by various cytokines may contribute to the pathogenesis of
diseases leading to matrix degradation and osteopenia. Although the
physiological levels of cytokines of the IL-6 family may be important
in the normal bone remodeling, the increased levels of these cytokines in bone and joints of patients with postmenopausal osteoporosis and
rheumatoid arthritis may promote connective tissue degradation and
resorption (12, 28). In addition to degrading collagen fibrils,
collagenase-3 can process other components of bone matrix. A recent
study has demonstrated that collagenase-3 cleaves insulin-like growth
factor (IGF) binding protein-5 by which it can generate peptides that
may regulate the actions of IGF-I and -II, important local stimulators
of bone formation (6). Thus collagenase may coordinate different
aspects of bone resorption and formation during the bone remodeling
cycle by proteolytic cleavage of different components of bone matrix.
In conclusion, OSM and LIF stimulate expression of collagenase-3 and
TIMP-1 in osteoblastic cells. These cytokines do not regulate the
expression of gelatinases A and B and TIMP-2 and -3. The stimulation of
collagenase-3 and TIMP-1 by LIF and OSM may regulate turnover of
collagen and other components of bone matrix that may affect both bone
formation and resorption.
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ACKNOWLEDGEMENTS |
We thank Dr. Cheryl Quinn for rat collagenase-3 cDNA, Dr. Dylan
Edwards for murine TIMP-1, 2 and 3 cDNAs, Dr. Ghislain Opdenakker for
murine 92-kDa gelatinase cDNA, Dr. Ray Wu for rat GAPDH cDNA, and Dr.
John Jeffrey for rat procollagenase antibody. We also thank Kristine
Sasala, Deena Durant, and Susan O'Lone for expert technical assistance.
 |
FOOTNOTES |
This work was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-21707 and by a grant from
the Charles H. Hood Foundation.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: S. Varghese, Dept. of
Research, Saint Francis Hospital and Medical Center, 114 Woodland St.,
Hartford, CT 06105.
Received 17 September 1998; accepted in final form 3 November
1998.
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