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HEMATOPOIESIS
From the Departament de Fisiologia (Biologia del
Macròfag), Facultat de Biologia and Fundació August Pi i
Sunyer, Campus de Bellvitge, Universitat de Barcelona, Barcelona,
Spain.
Decorin is a small proteoglycan that is ubiquitous in the
extracellular matrix of mammalian tissues. It has been extensively demonstrated that decorin inhibits tumor cell growth; however, no data
have been reported on the effects of decorin in normal cells. Using
nontransformed macrophages from bone marrow, results of this study
showed that decorin inhibits macrophage colony-stimulating factor
(M-CSF)-dependent proliferation by inducing blockage at the
G1 phase of the cell cycle without affecting cell
viability. In addition, decorin rescues macrophages from the induction
of apoptosis after growth factor withdrawal. Decorin induces the expression of the cdk inhibitors p21Waf1 and
p27Kip1. Using macrophages from mice where these genes have
been disrupted, inhibition of proliferation mediated by decorin is
related to p27Kip1 expression, whereas p21Waf1
expression is necessary to protect macrophages from apoptosis. Decorin
also inhibits M-CSF-dependent expression of MKP-1 and extends the
kinetics of ERK activity, which is characteristic when macrophages
become activated instead of proliferating. The effect of decorin on
macrophages is not due to its interaction with epidermal growth factor
or interferon- Stimulated monocytes and macrophages secrete a
diverse set of mediators that influence cellular immune functions and
inflammation. These mediators include proinflammatory and
anti-inflammatory cytokines, prostaglandins, leukotrienes, and reactive
oxygen metabolites.1 At the inflammatory sites,
proteoglycans are both secreted by activated mononuclear leukocytes and
released as a result of extracellular matrix (ECM) degradation. Thus,
proteoglycans, which are major constituents of the ECM, are another
class of molecules produced by monocytes and
macrophages2,3 that are potential modulators of the
immune response.
Decorin belongs to a family of small leucine-rich
proteoglycans4,5 and is found in the ECM of several of
tissues such as skin,6,7 cartilage,8,9 and
bone.10 The biologic importance of these molecules is
unclear. In vitro binding studies have shown that some of them interact
with several types of collagen11,12 and act as important
regulators of collagen fibrillogenesis. In support of this hypothesis,
a decorin-deficient mouse was found to have fragile skin with an
abnormal organization of collagen fibers.13 Decorin may
also affect the production of other ECM components by regulating the
activity of transforming growth factor- Different observations have revealed that decorin is involved in the
control of cell proliferation. The forced expression of decorin in
Chinese hamster ovary (CHO) cells leads to a decreased growth rate,
lower saturation density, and altered morphology.19 It has
been suggested that decorin causes these effects by sequestering TGF- Besides the antioncogenic role of decorin, a protective role of decorin
in fibrotic diseases has been observed.26,27 However, there are no data regarding the effects of decorin on normal cells. We
have analyzed the role of decorin in the control of macrophage proliferation. We have used primary bone marrow-derived macrophage (BMDM) cultures, which provide a homogeneous population that responds to physiologic proliferative or activating stimuli.28
Decorin inhibits macrophage colony-stimulating factor
(M-CSF)-dependent proliferation of macrophages and induces the
expression of p21Waf1 and, in contrast with other cellular
models, also p27Kip1. Moreover, decorin increases both the
adhesion of these cells and their resistance to die after withdrawal of
growth factor. The effects of decorin in macrophages are not mediated
through interaction with EGF or interferon- Reagents
Cell culture
Antibodies and constructs The analysis of p21Waf1 and p27Kip1 expression by Western blotting was performed with monoclonal antimouse p21Waf1 and p27Kip1 antibodies (BD Pharmingen). Antibodies to cdk-4 and cyclin D1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated antimouse IgG (Cappel, Turnhout, Belgium) was used as secondary antibody. A primary antibody against mouse -actin was used as loading control
and purchased from Sigma. The antibody against cdk-2 (M-2) used for the
analysis of cdk-2 activity and the polyclonal antimouse
1-integrin antibody were obtained from Santa Cruz Biotechnology.
The pMH117 plasmid corresponds to the mouse p21Waf1 full-length complementary DNA (cDNA) cloned in pEx-lox and was kindly provided by Dr J. Massague (Sloan Kettering Institute, HHMI, New York, NY). The MKP-1 probe was obtained from Dr R. Bravo (Bristol-Myers Squibb, Princeton, NJ). The probe for the 18S ribosomal RNA (rRNA) was obtained as described.30 Proliferation assay Cell proliferation was measured as previously described31 with minor modifications. The cells were deprived of M-CSF for 18 hours and then 105 macrophages were incubated for 24 hours in 24-well plates (3424 MARK II; Costar, Cambridge, MA) in 1 mL complete medium in the presence or absence of the indicated reagents. In some cases, the plates were precoated with the indicated ECM proteins. After this period, the media was removed and replaced with 0.5 mL media containing [3H]-thymidine (0.037 MBq). After 6 additional hours of incubation at 37°C, the media was removed and the cells were fixed in ice-cold 70% methanol. After 3 washes in ice-cold 10% trichloroacetic acid, the cells were solubilized in 1% sodium dodecyl sulfate (SDS), 0.3 N NaOH. Radioactivity was counted by liquid scintillation using a 1500 Tri-Carb Packard scintillation counter. Each point was performed in triplicate and the results were expressed as the mean ± SD.In parallel experiments, 1 × 106 cells were plated in 35-mm cell culture dishes and after 24 to 48 hours of culture in the indicated conditions, the viable cells were collected and counted by trypan blue exclusion using a hemocytometer. Again, each experiment was performed 3 times and the results were expressed as the mean ± SD. Apoptosis assay Low molecular apoptotic DNA created by internucleosomal cleavage was measured as described,32 using an enzyme-linked immunosorbent assay (ELISA) technique based on the detection of histone-associated DNA fragments (Cell Death Detection ELISA Plus, Roche Diagnostics, Mannheim, Germany). Each point was performed in triplicate, and the result was expressed as the mean ± SD.Analysis of DNA content with DAPI Cells (106) were previously subjected to a specific treatment and then the DNA content was analyzed as described previously.33 Twelve thousand cells were counted for each histogram, and cell cycle distributions were analyzed with the Multicycle program (Phoenix Flow Systems, San Diego, CA)Adhesion analysis Cell adhesion to the substrate was analyzed by crystal violet staining. Flat-bottomed ELISA plates were precoated in 50 µL/well phosphate-buffered saline (PBS) containing the indicated amount of each matrix protein, or bovine serum albumin (BSA) as a control, overnight at 4°C or for 2 hours at 37°C. After coating, the wells were blocked with PBS and 1.5% BSA for 1 hour at 37°C. Then, 10 000 cells/well were cultured for only 30 to 60 minutes due to the high capacity of macrophages to attach themselves at any surface. The cells were washed twice in PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. After 3 washes by immersion with twice-distilled water, the cells were stained in a solution of 0.1% crystal violet in twice-distilled water for 20 minutes. After 3 new washes, the plates were dried at 37°C, developed by adding 0.1 M HCl for 5 minutes, and quantified using an ELISA reader at 630 nm. Each sample was analyzed in triplicate and the results were represented as the mean ± SD.Protein extraction and Western blot analysis Western blot analysis was performed as previously described.32 Cell lysates (100 µg/lane) were loaded. The analysis of p21Waf1 and p27Kip1 expression was performed with monoclonal antimouse p21Waf1 and p27Kip1 antibodies (BD Pharmingen). Antibodies to cdk-4 and cyclin D1 were obtained from Santa Cruz Biotechnology. A primary antibody against mouse-actin was used as loading control
and purchased from Sigma. Peroxidase-conjugated antimouse IgG was used
as secondary antibody. All antibody incubations were performed for 1 hour at room temperature.
Determination of ERK activity by in-gel kinase assay Total protein (50 µg) was separated by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 0.1 mg/mL myelin basic protein (MBP) (Sigma) copolymerized in the gel. After electrophoresis, SDS was removed by washing the gel with 2 changes of 20% 2-propanol in 50 mM Tris-HCl (pH 8.0) for 1 hour at room temperature. The gel was then incubated with 50 mM Tris-HCl (pH 8.0) containing 5 mM-mercaptoethanol (buffer A) for 1 hour at room
temperature. The proteins were denatured by incubating the gel with 2 changes of 6 M guanidine-HCl for 1 hour at room temperature and then
renatured by incubating with 5 changes of buffer A containing 0.04%
Tween-20 for 16 hours at 4°C. To perform the phosphorylation assay,
the gel was first equilibrated in 40 mM Hepes-NaOH (pH 7.4) containing
2 µM DTT, 0.1 mM EGTA, 15 mM MgCl2, 300 µM sodium orthovanadate for 30 minutes at 25°C and then incubated for 1 hour in
the same solution containing 50 µM adenosine triphosphate (ATP) and
3.7 MBq 32P- -ATP (ICN Pharmaceuticals, Costa
Mesa, CA). The reaction was stopped by washing the gel with 5%
trichloroacetic acid containing 10 mM sodium pyrophosphate to inhibit
phosphatase activity. The gel was dried, exposed to x-ray films (Kodak)
and quantified using a Bio-Rad Molecular Analyst System (Bio-Rad Labs,
Richmond, CA).
Northern blot analysis Total cellular RNA (20 µg), extracted with the acid guanidinium thiocyanate-phenol-chloroform method was separated in 1% agarose with 5 mM MOPS (3-[N-morpholino]propanesulfonic acid), pH 7.0/1 M formaldehyde buffer, transferred overnight to a GeneScreen membrane (Life Science Products, Boston, MA) and fixed by UV irradiation (150 millijoule). All the probes were labeled with 32P- -dCTP (ICN Pharmaceuticals) using the oligolabeling
kit method (Amersham Pharmacia). After incubating the membranes for 18 hours at 65°C in hybridization solution (20% formamide, 5 ×
Denhart, 5 × standard sodium citrate [SSC], 10 mM EDTA, 1% SDS, 25 mM Na2HPO4, 25 mM
NaH2PO4, 0.2 mg/mL salmon sperm DNA, and
106 cpm/mL 32P-labeled probe), they were
exposed to Kodak films.
Cdk-2 activity Quiescent macrophages were cultured in 60-mm plates precoated with 10 µg/mL BSA or decorin and stimulated with 1000 U/mL M-CSF for the indicated times. The analysis of cdk-2 activity was performed as described elsewhere, without modifications.33
In the present work, we used macrophages obtained from bone marrow
cultures, because they represent a homogeneous population of
macrophages that require M-CSF to proliferate and survive. Under the
effect of M-CSF, macrophages proliferate in a dose-dependent manner.
When decorin (10 µg/mL) was precoated to the plates, macrophage proliferation was inhibited (Figure 1A).
This effect was dose-dependent, and macrophage proliferation was
completely inhibited at a concentration of 100 µg/mL decorin (Figure
1B). It is important to note that the indicated concentrations of
decorin correspond to the concentration of the precoating solution and
that we are not able to quantify the amount of decorin adsorbed to the
plate after precoating, but other proteins under the same conditions
bound less than 10% to 20%. Decorin also inhibits macrophage
proliferation in the presence of either recombinant M-CSF or
granulocyte-macrophage colony-stimulating factor (GM-CSF) proteins
(Figure 1C).
The inhibitory effect of decorin was confirmed by flow cytometry (Figure 1D) and by cell counting (Figure 1E). The distribution of the DNA content of cells stained with DAPI showed that macrophages treated with decorin were blocked mainly at the G1 phase of the cell cycle (73%), whereas macrophages growing in normal conditions showed a distribution corresponding to an active proliferating population (51% cells at G1 phase; Figure 1C). Moreover, the inhibition of proliferation was not due to a lower cell viability because we did not detect any subdiploid peak corresponding to apoptotic cells (Figure 1C) or a decrease in the cell number after 48 hours of culture in the presence of 100 µg/mL decorin (Figure 1E). It has been reported that decorin inhibits proliferation through the
expression of p21Waf1 in certain tumor cellular
models.25,34 Therefore, we analyzed the expression of this
cdk inhibitor in macrophages treated with decorin. Western blot
analysis showed that decorin induced the expression of
p21Waf1 in a time- and dose-dependent manner (Figure
2A). However, and in contrast with other
cellular models, decorin also induced in macrophages the expression of
another cdk inhibitor, p27kip1 (Figure 2A). No differences
were observed in the expression of cyclin D1, E, cdk-2, and
cdk-4 protein analyzed by Western blot (data not shown). Moreover, the
analysis of cdk-2 activity measured as in vitro phosphorylation of
histone H1 showed that treatment of macrophages with decorin inhibits
cdk-2 activity (Figure 2B). To characterize the involvement of each of
these 2 molecules in the inhibitory effect of decorin, we used
macrophages obtained from mice where these genes have been disrupted by
homologous recombination. Decorin inhibited proliferation in
macrophages from p21Waf1 knock-out mice but no effect was
observed in macrophages from p27Kip1 knock-out mice (Figure
2C). Thus, although decorin in macrophages induced the expression of
both p21Waf1 and p27Kip1, only
p27Kip1 was involved in the inhibition of proliferation by
decorin, in contrast with what has been reported in tumor
cells.34
Due to these differences between primary macrophages and tumor cells,
and because it has been described that decorin inhibits tumor cell
growth through its interaction with the EGF receptor,22,25 the activation of the ERK pathway and the expression of
p21Waf1, we analyzed the involvement of this pathway in our
model. Both IFN-
The differences observed between the treatment with decorin and EGF
suggest that the inhibition of macrophage proliferation induced by
decorin could not be mediated through its interaction with the EGF
receptor, because this growth factor does not seem to modulate
macrophage proliferation in BMDMs. However, IFN- Macrophages growing in vitro adhere to the surface of the plates, and it has been described in several cellular models that this adhesion is important for the proliferation and viability of the cells.36,37 Because decorin is a component of the ECM, we decided to test how this proteoglycan affects macrophage adhesion and if this was important in modulating proliferation and viability. We observed that decorin enhanced the adhesion of macrophages to the
surface of plastic dishes (Figure 4A).
Moreover, macrophages growing in dishes precoated with decorin showed a
higher degree of spreading than nontreated macrophages, and their
ameboid morphology changed to a more complex morphology with pseudopoda
ramification (Figure 4B).
Macrophages bind with different affinities to several proteins present
in the ECM. We found that BMDMs showed a higher level of binding to
plates precoated with fibronectin and vitronectin, whereas laminin
reduced their adhesion (Figure 5A). We
then tested how macrophage adhesion affected their proliferation. Using
an inhibitor of cellular adhesion, P-OH-M, that blocked macrophage adherence to the dishes (Figure 5A), we showed that these cells must
adhere to proliferate (Figure 5B). Surprisingly, a strong adhesion
could also decrease macrophage proliferation. Macrophages growing on a
fibronectin surface, to which they attach strongly, proliferate less
than macrophages growing on a control BSA-precoated surface (Figure
5B). Macrophages cultured on a surface to which they attach slightly
(ie, laminin-coated surface) showed a higher level of proliferation
than control cells. Similar results were obtained using vitronectin or
collagen I (data not shown). We could not dismiss the fact that the
different effects on proliferation were due to signaling through
different integrin receptors. However, our results suggest that,
although macrophage anchorage is necessary to proliferate, the
proliferation of macrophages could be modulated by their degree of
adhesion.
So far, our results showed that both decorin and fibronectin enhance macrophage adhesion and inhibit M-CSF-dependent proliferation. To determine if the effect of decorin on macrophage proliferation is mediated by its effect on adhesion, we checked the mechanism by which adhesion to fibronectin modulates macrophage proliferation. When the expression of several components of the machinery that
regulate progression through the G1 phase of the cell cycle was analyzed, we found that both decorin and fibronectin did not modify
the expression of either cyclin D1, cdk4 (Figure
6A), cdk-2, or cyclin E protein
expression (data not shown). However, whereas decorin induced the
expression of both cdk inhibitors p21Waf1 and
p27Kip1, fibronectin only induced p27Kip1
expression (Figure 6A). Similar to decorin, fibronectin also inhibited
cdk-2 activity (data not shown). The adhesion of macrophages to a
fibronectin-coated surface also extended ERK activity in response to
M-CSF (Figure 6B) by inhibiting MKP-1 expression (Figure 6C). Also, the
inhibitory effect of fibronectin on macrophage proliferation was
abolished in macrophages from p27Kip1 knock-out mice but
not in those from p21Waf1 knock-out mice (Figure 6D). This
suggested that an increased macrophage adhesion probably inhibited
macrophage proliferation, and this correlated with an extended ERK
activity and the induction of p27Kip1.
Because fibronectin inhibited proliferation in a way similar to decorin, and macrophages could produce fibronectin, we checked whether the effects of decorin on macrophage adhesion and proliferation were only mediated by the release of fibronectin induced by decorin. Decorin modulates the interactions of matrix molecules, such as
fibronectin, with the cells.16,17 Therefore, we analyzed the effect of decorin on macrophage adhesion to fibronectin. The treatment of macrophages with decorin enhanced their adhesion to a
fibronectin-coated surface but did not modify their adhesion to
laminin-coated surfaces (Figure 7).
Because adhesion analysis is performed for only 30 to 60 minutes, it is
unlikely that the effect of decorin will be mediated through an
increase of fibronectin secretion induced by decorin. Moreover,
Northern blot analysis of fibronectin expression in macrophages
demonstrated that decorin did not induce or increase fibronectin
expression induced by M-CSF in macrophages (data not shown). This
suggests that decorin binds to fibronectin through a different domain
than the one recognized by macrophages and also that decorin and
fibronectin are probably recognized by different receptors on
macrophages.
To definitively discard that the effects of decorin could be mediated
by the secretion of fibronectin or through fibronectin receptors, we
blocked the fibronectin signal pathway using
anti-
Also, attachment to the ECM may modulate cell viability.35
Previously, we had reported that the expression of p21Waf1
together with a blockage of the cell cycle protected macrophages from
apoptosis.33 Because decorin increased macrophage adhesion to the ECM, induced p21Waf1 expression, and inhibited
proliferation, we decided to further explore the role of decorin in the
control of macrophage survival. From the experiments described in
Figure 1, we concluded that decorin did not induce apoptosis in BMDMs.
Instead, decorin protected macrophages from apoptosis induced by growth
factor withdrawal (Figure 9). In contrast
with its effect on macrophage proliferation, the increase in adhesion
induced by decorin was not the mediator of this event, because the
culture of macrophages on a fibronectin-coated surface did not protect
macrophages from apoptosis induced by M-CSF starvation (Figure 9).
Similar to our previous observations with IFN-
Macrophages are derived from undifferentiated stem cells in the
bone marrow and through the blood they reach the different tissues
where, in most cases, they undergo apoptosis. In the presence of
specific growth factors or cytokines, macrophages proliferate, become
activated, or differentiate. To carry out their functional activities,
macrophages must become activated. After interacting with IFN- At the inflammatory sites, different proteoglycans (such as decorin) are secreted by macrophages.39 Therefore, we wanted to know its effect on BMDMs, a homogeneous population of nontransformed cells.28 Decorin inhibits M-CSF-dependent proliferation of macrophages and inhibits apoptosis induced by growth factor withdrawal. After the treatment with decorin, we detected the induction of both cdk inhibitors (p21Waf1 and p27Kip1). Using mice with these genes disrupted by homologous recombination, we have found that p27Kip1 is responsible for the antiproliferative effect of decorin, whereas p21Waf1 is required to induce protection against apoptosis. The inhibition of M-CSF-dependent proliferation in macrophages by decorin confirms previous observations in several tumor cells.34 However, the mechanism of inhibition is different from what has been described because in tumor cells the growth-suppressive properties of decorin require a functional cdk inhibitor p21Waf1.34 Our observation is in accordance with the fact that fibrillar collagen, a molecule that interacts closely with decorin both in vitro40 and in vivo,13 inhibits smooth muscle cell proliferation by inducing the expression of p27Kip1.41 In addition, we have observed that binding of macrophages to fibronectin, another protein of the ECM that binds to decorin, also inhibits macrophage proliferation through the induction of p27Kip1. We have found other differences compared to tumor cells. It has also
been reported that decorin causes a rapid phosphorylation of the EGF
receptor and a concurrent activation of the ERK signal pathway, which
leads to a protracted induction of endogenous p21Waf1 and,
ultimately, cell cycle arrest.22,25 Although we did not detect a direct activation of the ERK pathway by decorin, the latter
extended the ERK activity induced by M-CSF. In previous works we have
described that this elongation is important for the regulation of
macrophage proliferation and activation.42,43 Besides, we
have observed that decorin does not interact with EGF or IFN- An increasing number of observations indicate that proteoglycans can
regulate cell proliferation through interaction with various growth
factors.44 The expression of decorin in CHO cells has a
dramatic effect on their morphology and growth
properties.19 This effect is partly caused by the ability
of decorin to bind to TGF- The growth of adherent cells such as fibroblasts or macrophages
requires signals not only from growth factor receptors but also from
integrins.36,45,46 That is also true for macrophages, because the total inhibition of their adhesion blocks macrophage proliferation. However, we thereby show for the first time that the
level of this adhesion has a strong effect on the modulation of the
level of macrophage proliferation. A strong attachment induced by
decorin or fibronectin is enough to reduce macrophage proliferation,
whereas a slight adhesion such as that induced by laminin increases
proliferation. The use of anti- A few examples have been described for the G1 phase blockage mediated by cellular adhesion. This effect is caused by the increase of the expression of p27Kip1, which inhibits cyclin E-cdk2 kinase activity.41,47,48 Cell-to-cell contact induces the cells to stop proliferating during normal organ development. More recently it has become clear that such contact-mediated growth arrest is caused by the up-regulation of p27Kip1. This is shown in the p27Kip1 knock-out mice, which are generally bigger and have a significantly expanded hematopoietic progenitor pool.49,50 We have observed that the inhibitory effect on macrophage proliferation induced by adhesion to some ECM components (ie, decorin or fibronectin) is also mediated through the expression of p27Kip1. In addition, the signals mediated by the ECM are important for cell
survival. A laminin-rich ECM is a potential survival factor for
differentiated mammary alveolar epithelial cells both in vivo and in
vitro.51 It has been suggested that the laminin-rich ECM
acts through Our results could have physiologic relevance. Although the
concentration of decorin precoating solution used in our studies (10-100 µg/mL) could seem slightly higher than that estimated to
occur in collagenous matrices (5-12.5 µg/mL) found in
vivo,22 the amount of decorin adsorbed to the plate should
be significantly lower. Macrophages play a critical role during
inflammation. From blood, macrophages reach the inflammatory foci and
remain there until inflammation disappears.54 In the
tissues, macrophages need to survive in the absence of growth factors.
Whereas stimulated Th1 cells remain at the inflammatory loci and
produce IFN-
We specially thank Dr E. Ruoslahti and Dr Richard Maki from The
Burnham Institute, La Jolla, CA, for the kind gift of all the purified
decorin used in this work as well as for all their support. We also
thank Dr J. Roberts from HHMI, Seattle, WA, and Dr Modolell from the
Max Plank Institute, Freiburg, Germany, for the
p21Waf1/p27Kip1 and IFN-
Submitted January 29, 2001; accepted June 6, 2001.
Supported by grants from Comisión Interministerial de Ciencia y Tecnologia (CICYT) (SAF98-102 and PM 98/0200) to A.C. J.X. and A.F.V. are recipients of fellowships from the Comissió Interdepartamental de Recerca i Innovació Tecnològica. M. Comalada is a recipient of a fellowship from the Fundació August Pi i Sunyer.
J.X., M.C., and M.C. are equal contributors to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. |
Top
Abstract
Introduction
,
2 ng/mL), GM-CSF (
, 10 ng/mL), or control (
). (D) Decorin blocks
the cell cycle at G1 phase. The 106 macrophages
were cultured in the presence of 1000 U/mL M-CSF in 35-mm Petri dishes
precoated with BSA or with 100 µg/mL decorin for 24 hours. DNA
content was measured by DAPI staining and flow cytometry. Cell cycle
distribution was analyzed using the Multicycle program (Phoenix Flow
Systems). (E) Counting of viable cells cultured in 100 µg/mL
decorin-precoated plates for 24 to 48 hours. The cells were
counted by trypan blue exclusion using a hemocytometer. Each point was
performed in triplicate, and the results were represented as the
mean ± SD.
