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J Biol Chem, Vol. 273, Issue 40, 25796-25803, October 2, 1998
-INDUCED HYPOPHOSPHORYLATION OF THE
RETINOBLASTOMA PROTEIN*
,From the Division of Cardiology and the Research Service, Veterans Affairs Medical Center, San Francisco, California 94121, and the Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, California 94143
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ABSTRACT |
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Although responsible for only approximately
one-third of the overall myocardial mass, the interstitial fibroblasts
of the heart serve a fundamental role in establishing the functional integrity of myocardium and are the major source of myocardial extracellular matrix production. Their importance in clinical medicine
is underscored by the observation that fibroblast numbers increase in
response to several pathologic circumstances that are associated with
an increase in extracellular matrix production, such as long standing
hypertension and myocardial injury/infarction. Up to the present time,
however, there has been little information available on either the
kinetics of the cardiac fibroblast cell cycle, or the fundamental
mechanisms that regulate its entry into and exit from the cell cycle.
Previous work from our laboratory examining the effects of interleukin
(IL)-1
on myocardial growth and gene expression in culture indicated
that cardiac fibroblasts have a diminished capacity to synthesize DNA
in response to mitogen in the presence of this cytokine. The mechanism
of IL-1
action was not clear, however, and could have resulted from
action at several different points in the cell cycle. The
investigations described in this report indicate that IL-1
exerts
its effect on the fibroblast cell cycle at multiple levels through
altering the expression of cardiac fibroblast cyclins,
cyclin-dependent kinases, and their inhibitors, which
ultimately affect the phosphorylation of the retinoblastoma gene
product.
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INTRODUCTION |
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Interleukin-1
(IL1-1
) is a pleiotropic
17 Kd hormone-like polypeptide produced by a number
of cell types including mononuclear inflammatory cells, epithelial
cells, endothelial cells, mesangial cells, smooth muscle cells, and
fibroblasts (1). In the heart, expression of IL-1
by myocardial
cells has been detected in several clinical circumstances that are
characterized by immunologic myocardial injury (e.g.
myocarditis, transplant rejection, ischemia, and congestive heart
failure) (2-7). IL-1
expression during inflammatory myocardial
injury is not unique, however, since myocardial cells respond to other
pathologic stresses (such as pressure load) with the production of a
number of both known and potentially novel growth substances (8-18)
(reviewed in Long (19)). The production of these factors by myocardial cells is noteworthy since several growth factors and cytokines have
been implicated in the initiation and regulation of myocardial growth
under both developmental and pathologic conditions. In this regard,
work from both our laboratory and others has identified the
cardiac fibroblast as an important intracardiac source of IL-1
in both the acute response to hypoxia/ischemia as well as in
circumstances associated with chronic cardiac dysfunction (20, 21) and
IL-1
has been found to alter the growth of myocardial cells in
culture (22-24). Specifically, IL-1
increases cardiac myocyte
protein content while inhibiting cardiac fibroblast DNA synthesis (22).
However, despite the previous findings on myocardial [3H]thymidine incorporation, little work has been done to
localize the effects of IL-1
on myocardial cell cycle control.
In contrast to the cardiac myocyte, whose replicative capacity is limited in the adult heart, the cardiac fibroblast retains the ability to proliferate, and does so in response to many pathologic circumstances. As such, the growth of the heart after birth (both developmental, or "physiologic," growth as well as abnormal, or "pathologic," growth) is characterized by hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. This disparity in growth potential is important since the cardiac fibroblast makes up nearly two-thirds of the total myocardial cell numbers, is the major source of extracellular matrix (ECM) production in the heart, and appears to be the major producer of many of the cytokines known to have potent effects on cardiomyocyte growth, fibroblast proliferation, and ECM homeostasis (19). Despite the obvious importance of the cardiac fibroblast in myocardial repair, however, there is little information available on either the kinetics of the fibroblast cell cycle, or the factors that regulate its initiation and progression. In order for the cardiac fibroblast to become a potential target for therapeutic manipulation, the fundamental mechanisms that regulate its entry into (and exit from) the cell cycle as well as the factors responsible for ECM deposition must be identified.
For virtually all mammalian cells with proliferative potential, a
general scheme has emerged for the control of cell
proliferation in response to mitogen stimulation. This scheme involves
the cell cycle-specific stimulation of a binary system of cell
regulators consisting of a family of regulatory subunits (the cyclins),
which bind to (and help to activate) the catalytic subunits, the
cyclin-dependent protein kinases (Cdks) (reviewed in Refs.
25 and 26). The cyclins are believed to determine the subcellular
localization, substrate specificity, interaction with upstream
regulatory proteins, and timing of Cdk activation. In general, whereas
Cdk expression has been felt to be more or less constitutive, cyclin
expression is "cyclic," and responsive to proliferative signals. As
such, there are cyclins specific to the G1, S, and
G2/M phases of the cell cycle. With regard to the
cyclin:Cdks required for S phase entry (i.e. the D and E
cyclin:Cdks), the substrates that have been identified as critical for
cell cycle progression are members of the so-called "pocket
protein" family. Made up of three members, p107, p130, and the
quintessential pocket protein, the retinoblastoma gene product (Rb), it
is the phosphorylation of Rb by the cyclin:Cdk dimer that results in
release of the E2F transcription family and S phase entry (27-31).
Finally, proteins that block the action of specific cyclin-Cdk
complexes, the cyclin-dependent kinase inhibitors (CKIs),
act as negative regulators of the G1-cyclins and Cdks cause
G1 arrest when overexpressed in transfected cells (32)
(reviewed in Massague and Polyak (33). As such, there are several
levels at which a given antimitogenic stimulus may exert its control.
For example, transforming growth factor
, which also appears in
myocardium in response to injury, plays an important role in the
regulation of the cell cycle machinery by its action on cyclins, Cdks,
and CKIs (34-36).
As noted previously, the expression and effects of cytokines such as
IL-1
in the postinjury and remodeling heart suggest that these
peptides are likely to be involved in both of these processes.
Consistent with this notion, work from several investigators has
suggested that IL-1
exerts a profound effect on myocardial growth
and function in culture (22, 23, 37-42). With respect to the
proliferative potential of the cardiac fibroblast, previous work from
our laboratory investigating the effects of IL-1
on myocardial
growth and gene expression indicated that cultured cardiac fibroblasts
have a diminished capacity to synthesize DNA in response to mitogen in
the presence of this cytokine. The mechanism of IL-1
action was not
clear, however, and could have resulted from action at several
different points in the cell cycle. The investigations described below
were performed in an effort to explain the manner in which IL-1
exerted its effect(s) on fibroblast proliferation. Specifically, we
examined the effect of IL-1
on the expression of cyclins, Cdks,
CKIs, and Rb phosphorylation using the neonatal rat cardiac fibroblast
culture system.
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EXPERIMENTAL PROCEDURES |
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Materials--
Minimum essential medium with Hanks' salts was
obtained from Cell Culture Facility, University of California, San
Francisco. Calf serum was obtained from Hyclone Labs (Logan, UT).
Bovine serum albumin was obtained from Intergen (Purchase, NY).
Propidium iodide (PI), N-hexanoyl-D-sphingosine
(C6-ceramide), and N-acetyl-D-sphingosine (C2-ceramide) were obtained from (Sigma). [3H]Thymidine
(20 Ci/mmol) and [
-32P]ATP (10 mCi/ml) were obtained
from NEN Life Science Products. Recombinant mouse IL-1
was obtained
from Genzyme Corp. (Cambridge, MA).
NG-Monomethyl-L-arginine,
monoacetate salt (L-NMMA) was obtained from Calbiochem. For
immunoblotting, anti-cyclin D2 (rabbit polyclonal), anti-cyclin D3 (mouse monoclonal), anti-cyclin E (rabbit
polyclonal), anti-cyclin A (rabbit polyclonal), anti-p21/p27 (rabbit
polyclonal), anti-Cdk2 (rabbit polyclonal),
anti-Cdk4 (rabbit polyclonal) antibodies, gluthathione
S-transferase retinoblastoma (GST-Rb) fusion protein, protein A-agarose, and normal rabbit IgG were purchased from Santa Cruz, Inc. (Santa Cruz, CA). Histone H1 and p13-agarose
conjugated beads were purchased from Upstate Biotechnology (Lake
Placid, NY). Anti-Rb (mouse monoclonal) was from Pharmingen (San Diego, CA). Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies and enhanced chemiluminescence reagent
(ECL) were obtained from Amersham Pharmacia Biotech. Nitrocellulose membrane was obtained from Schleicher & Schuell.
Cell Culture--
Primary cultures of neonatal rat cardiac
fibroblasts were prepared as described previously (43). For flow
analysis and Western blotting experiments, fibroblasts were plated in
60-mm dishes (2.5 × 105/dish), grown for 48 h to
~80% confluency in growth medium (minimum essential medium, 5% calf
serum). Cells were then synchronized by 48 h of serum starvation
(minimum essential medium, 0.1% bovine serum albumin) and subsequently
treated with calf serum in the presence or absence of IL-1
and the
cell cycle kinetics determined by incorporation of
[3H]thymidine (1-h pulse with 10 µCi/ml) (22) and flow
cytometry as described below.
Sample Preparation for Flow Cytometry-- After treatment, cells were trypsinized at the indicated time with 500 ml of trypsin/EDTA (2 mg/ml/0.02%) for 2-5 min, scraped, and pelleted by centrifugation at 500 × g for 5 min. Cell pellets were fixed in 25% ethanol, 15 mM MgCl2 on ice for 30 min. The fixed cells were pelleted and resuspended in 0.5 ml of calcium- and bicarbonate-free Hanks' solution with Hepes (CBFHH) supplemented with 10 mg/ml RNase A and incubated at 37 °C for 30 min. Cellular DNA was stained with 10 mg/ml propidium iodide, and samples were filtered through a 70-mm nylon mesh to remove cell clumps. Samples were used immediately or kept at 4 °C until analysis. No differences were seen between fresh and stored samples.
Flow cytometric analysis was done using a FACScan benchtop cytometer (Becton and Dickinson Immunocytometry System, San Jose, CA) with a standard 15 milliwatt, 488 nm, air cooled, argon-ion laser and standard filter sets. Single cell populations were gated using forward scatter, an indicator of cell size versus side scatter, an indicator of cell granularity. The FL2 detector measures fluorescent light from PI, which emits a red color at 650-nm wavelength of the FACScan laser, and PI intensity is proportional to the DNA content of the cell. The FL2-PI area versus width plots distinguished true cycling G2/M cells from doublets or aggregates of G0/G1 cells by comparison to standardized area versus width plots and were adjusted in all experiments. Using Cell Quest Software (Becton and Dickinson Immunocytometry System), at least 20,000 cells were collected per sample at low flow rate (12 µl/min), and DNA data were analyzed with ModFit software (Verity Software House, Popsham, ME).Immunoblotting--
For analysis of cell cycle-specific protein
expression, cells treated with serum ± IL-1
were harvested in
ice-cold homogenization buffer (150 mM NaCl, 10 mM Tris, pH 7.4, 1.0 mM EDTA, 1.0 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride,
50 mM NaF, 20 µg/ml leupeptin, 20 µg/ml aprotinin,
0.01% Tween 20, 0.5 mM NaVO4, 20 mM
-glycerol phosphate) followed by sonication (2-s
cycle time for 10 s). Total cell extracts were cleared by
centrifugation (13,000 rpm for 5 min), and aliquots were removed for
analysis of protein concentration (44) and either used immediately or
frozen at
70 °C until use. Proteins were separated by SDS-PAGE in
10% discontinuous gels (7.5% gels for Rb detection) and transferred
onto nitrocellulose membranes for 60 min at 100 V (30 min at 100 V for
Rb). Equal protein loading for all samples was assured by Ponceau S
staining of the membrane following transfer. Thereafter, membranes were probed with peptide-specific primary antibodies at 1 µg/ml.
Horseradish peroxidase-linked goat anti-rabbit at 1:5000 dilution was
used as a secondary antibody for rabbit polyclonal antibodies;
horseradish peroxidade-conjugated anti-mouse IgG at 1:5000 was used for
the mouse monoclonal antibodies. The ECL detection system was used to
detect the antigen-antibody complexes, with average exposure times of
5-60 s. Signal intensities were quantified by using Image densitometric analysis (National Institutes of Health, Division of
Computer Research and Technology, Bethesda, MD).
In Vitro Kinase Assays--
For the analysis of the cell cycle
kinase activity, cells were treated as described previously and
harvested in ice-cold homogenization buffer. Lysed cells were
precleared for 60 min at 4 °C with 20 µl of protein A-agarose and
5 µg of normal rabbit IgG. Precleared lysates were subsequently
incubated with 20 µl p13-agarose-conjugated beads for
2 h at 4 °C and collected by centrifugation (4,000 rpm for 5 min). Beads were washed twice with homogenization buffer and were used
for in vitro kinase assay using either GST-Rb fusion protein
or histone H1 as a substrate. Kinase reactions were carried out in
kinase buffer (20 mM Hepes, pH 7.2, 100 mM
NaCl, 10 mM MgCl2, 0.5 mM
dithiothreitol, 25 mM ATP, and 5 µCi of [
-32P]ATP per reaction at 30 °C for 15 min. The reaction
was stopped by adding 2× Laemmli buffer, and the proteins were
separated by SDS-PAGE, dried, and autoradiographed. The bands
corresponding to the phosphorylated GST-Rb and histone H1 were
quantified by densitometric analysis and normalized to the control.
Statistics-- Results are given as mean ± S.D. Mean values for two groups were compared using Student's t test, or analysis of variance for more than two groups with a p value of <0.05 representing statistical significance.
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RESULTS |
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IL-1
Inhibits Cardiac Fibroblast DNA Synthesis under Both Basal
and Mitogen-stimulated Conditions--
Previous work in our laboratory
suggested that IL-1
had potent dose-dependent effects on
cardiac fibroblast DNA synthesis in culture; however, the kinetics of
this inhibitory response was unknown (22). Since the determination of
the time course of inhibitory effects can provide important clues as to
the cell cycle-specific site of action, we fist evaluated the
time-dependence of the IL-1
effect on mitogen-stimulated cardiac
fibroblast proliferation in more detail. As noted in Fig.
1A, the inhibitory effect was seen if IL-1
was added at any point up to 12 h after the
addition of mitogen (5% calf serum). If IL-1
was added after this
point, however, there was a diminution in its ability to prevent
[3H]thymidine incorporation. Similarly, in investigations
into the time course of "release" from IL-1
-induced block, the
peak of [3H]thymidine incorporation was also delayed
approximately 12 h (Fig. 1B). These findings were quite
similar to that seen in the response of mink lung cells (Mv1Lu) to
transforming growth factor
and suggested that the IL-1
effect
may be due to a similar mechanism and site of action (36).
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IL-1
Prevents Cardiac Fibroblast Exit from
G0/G1--
Although the time course of IL-1
inhibition defined by [3H]thymidine incorporation
suggested a site of action at the G1/S restriction point,
it was critical to define this with certainty. Flow cytometric analysis
allows for the measurement of DNA content in isolated cardiac
fibroblasts on a per cell basis and is the technique of choice for the
determination of cell cycle kinetics. In preliminary experiments, the
normal cell cycle distribution of cardiac fibroblasts was determined at
various times after mitogen stimulation (range from 3 to 48 h). We
found that the highest percent of cells in S phase was at 20 h
(data not shown). Using flow cytometric analysis of these cells, we
have determined the site of inhibition by IL-1
on cardiac
fibroblasts cell cycle progression following their co-treatment with
IL-1
and calf serum. As indicated in Fig.
2, IL-1
prevents fibroblast entry into
S phase by arresting them at the G1/S interphase.
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IL-1
Effects Are Not Mediated through Prostaglandin, Nitric
Oxide, or Sphingomyelin/Ceramide Pathways--
As is true for most
peptide growth factors/cytokines, the biological action of IL-1
is
mediated through specific cell surface receptors. In this regard, the
stimulation of IL-1 receptors has been shown to initiate a variety of
signaling pathways, including cyclooxygenase/prostaglandins (45),
nitric oxide (46), and sphingomyelinase (47-50). In order to
understand which of these mechanisms might be involved in the arrest of
cardiac fibroblast proliferation seen in our culture system, we
performed a series of experiments in which inhibitors of these various
pathways were included during the IL-1
treatment. Neither the nitric
oxide synthase inhibitor L-NMMA, nor the cyclooxygenase
inhibitor indomethacin had any effects on basal levels of DNA synthesis
and were incapable of blocking the IL-1
effect. The percentage of
cells in S phase for these experiments was 33 ± 3, 19 ± 2, 15.6 ± 1.6, and 16 ± 1.8 for serum, serum/IL-1
,
serum/IL-1
/L-NMMA, and serum/IL-1
/indomethacin, respectively (n = 4, p = NS for all
inhibitors). In view of the previous suggestion that the production of
ceramide from sphingomyelin was associated with an inhibition of
proliferation in other cells types (51), additional experiments aimed
at understanding the role of the sphingomyelinase pathways were also
performed. Using the putative down-stream effectors of sphingomyelin
breakdown (C2- and C6-ceramide), we found that the cardiac fibroblasts
were relatively refractory to these agents when examined under
conditions of serum treatment similar to that used for IL-1
. The
percentage S phase in these experiments for serum- and
serum/C6-ceramide-treated cells was 30.31 ± 3.1, and 28.20 ± 2.0, respectively (n = 4, p = NS).
In light of our previous report suggesting that the growth effect of
IL-1
on cardiac myocytes was due to the action of a down-stream
tyrosine kinase pathway (22), we attempted to address the effects of
tyrosine kinase inhibition (both genestein and tyrphostin) on the
IL-1
effect. Unfortunately, we were unable to either confirm or
refute the effects of tyrosine kinase inhibition on the cardiac
fibroblasts due to the basal effects of this class of inhibitor on
cardiac fibroblasts proliferation. The percentage S phase in these
experiments for serum, serum/tyrophastin-, serum/IL-1
-, and
serum/IL-1
/tyrophastin-treated cells was 37.1 ± 2.5, 19.7 ± 1.4, 21.2 ± 2, and 18.3 ± 1.6, respectively
(n = 4).
IL-1
Decreases the Phosphorylation State of Rb in Cardiac
Fibroblasts--
Studies of cell cycle regulation have identified the
pocket protein Rb as a critical checkpoint control protein for the
G1-to-S phase transition (27, 28, 31, 52, 53).
To determine whether the phosphorylation status of Rb correlated with
the extent of growth inhibition as judged by accumulation of cells in
the G0/G1 phase of the cell cycle shown by
fluorescein-activated cell sorter analysis, we performed Western blot
analysis. As shown in Fig. 3A,
in untreated cardiac fibroblasts, Rb was in the hypophosphorylated form
(110 kDa), causing it to migrate faster than the hyperphosphorylated form in SDS-PAGE (pRb, 120 kDa). Investigations into the time course of
5% calf serum treatment indicate that the shift in phosphorylation state of Rb reaches a plateau by 24 h. Cells that remained
serum-starved over the 24-h treatment were similar to 0 h cells
with Rb remaining in the hypophosphorylated form. As shown in Fig.
3B, cells co-treated with IL-1
showed a
dose-dependent decrease in the expression of the higher
mobility form of Rb. Using scanning densitometry, the ratio of the fast
migrating form (Rb) to the relatively slow migrating forms (pRb) can be
determined. When applied to our studies, we found that in calf
serum-treated cells, 70 ± 8% of the Rb protein was in the
pRb form (hyperphosphorylated) form, whereas cells co-treated with
IL-1
contained only 22 ± 2.5% in the pRb form (Fig.
3C). Of note was the finding that cells treated with IL-1
had a decrease in the overall level of detectable Rb. Similar effects
were observed when Mv1Lu cells were treated with transforming growth
factor
1 (36) and is of unclear etiology, although it may reflect
the relatively short half-life for Rb (54).
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induced
G0/G1 growth arrest have shown that SV40 large
T-antigen can rescue the cell from the G0/G1
block by bypassing the need for the Rb protein in the G1/S
transition (36). To further address the role of Rb in the
IL-1
-induced cell cycle arrest, we examined the effect of IL-1
in
the SV40 large T-antigen-transformed cardiac fibroblast cell line
(TxNMCs) whose [3H]thymidine incorportion had been shown
previously to be unaffected by IL-1
(22). In our culture system,
IL-1
did not inhibit S phase entry of T-antigen-transformed cardiac
fibroblasts (percentage S phase in serum and serum/IL-1
-treated
cells 18.3 ± 2.0 and 20.4 ± 2.3, respectively,
n = 4, p = NS), confirming the
necessity of a functional Rb protein for the antiproliferative effect
of IL-1
to be manifest.
IL-1
Decreases in Vitro GST-Rb Phosphorylation--
Although
the changes in Rb mobility shown in Fig. 3 were likely the result of
decrease in the activity of the G1/S
cyclin-dependent kinases, it was critical to confirm that
this was indeed the case. To assess kinase activity, cardiac
fibroblasts were treated with calf serum ± IL-1
for 20 h
followed by in vitro kinase reaction using either
recombinant GST-Rb or histone H1. The p13-conjugated agarose beads were utilized since this protein associates specifically with several of the cyclin:Cdk complexes (55-58). As shown in Fig. 4B, in vitro GST-Rb
phosphorylation increases by 4.2 ± 0.7-fold in serum-treated
cells versus controls. In contrast, Rb phosphorylation increases only 1.7 ± 0.5-fold in serum/IL-1
-treated cells
versus controls. Overall, these results indicate that
IL-1
reduces cyclin-dependent kinase activity by 60%.
Similar results were obtained using histone H1 as a substrate (data not
shown).
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IL-1
Inhibits Mitogen-stimulated Fibroblast Cyclin and Cdk
Expression--
Once we determined that IL-1
exerted its inhibitory
effect at the G1/S restriction point with a decrease in
cyclin-dependent kinase activity and phosphorylation state
of Rb (Fig. 3, B and C), it was important to
identify the potential mechanism(s) of this effect. In an effort to
clarify this point, we examined the effects of IL-1
on the protein
expression of G1/S cyclins (D2, D3,
E, and A) and their catalytic subunits, Cdk2 and Cdk4. For these
experiments, cardiac fibroblasts were treated in an identical fashion
as those subjected to flow and Rb analyses. As shown in Fig.
5B, expression of cyclins
D2, D3, A, and Cdks 2 and 4 protein increase by
1.84 ± 0.1-, 1.49 ± 0.03-, 6.19 ± 0.4-, 4.31 ±
0.80-, 2.05 ± 0.17-, and 1.90 ± 0.21-fold versus
controls, respectively, in the serum-stimulated cells. In contrast,
levels of these proteins in cells co-treated with IL-1
were
0.66 ± 0.1-, 0.76 ± 0.01-, 3.32 ± 0.1-, 1.89 ±
0.16-, 0.48 ± 0.03-, and 1.03 ± 0.02-fold compared with the
control, respectively. The decrease in cyclin/Cdk protein was specific
for IL-1
treatment since reprobing of these membranes showed an
increase in inducible nitric oxide synthase protein compared with both
control and serum-treated cells as shown previously (data not shown)
(59).
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IL-1
Increases p27 and p21 Cyclin Kinase Inhibitor Levels in
Cardiac Fibroblasts--
The cyclin kinase inhibitors, p21 and p27,
have been shown to interact with cyclin D, E, and A subunit as a
mechanism of their actions (60-62). For this reason, we determined the
extent to which the Cdk inhibitors p21 and p27, contributed to the
IL-1
inhibitory activity in cardiac fibroblasts. Serum-starved
fibroblasts were treated with 5% calf serum in the presence of
increasing concentration of IL-1
. After 20 h, cells were
harvested and protein subjected to SDS-PAGE and Western blotting using
antibody that recognizes both p21 and p27 proteins. As indicated in
Fig. 6A, p27 protein levels
are strongly increased by IL-1
even in the presence of mitogen-rich
growth medium. In contrast, p21 protein levels increase in both
IL-1
- and serum-treated cells. Densitometric analyses of additional
investigations into the effect of IL-1
on these CKIs are shown in
Fig. 6B, indicating a preferential increase in p27 protein
levels in IL-1
treated cells. Confirming that the increase in p27
protein was specific for IL-1
-treated cells, reprobing of the
membranes indicate the expected decrease in G1 cyclin
expression in the same extracts (but an increase in serum treated
cells, data not shown).
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DISCUSSION |
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Although the interstitial cells of the heart comprise only approximately one-third of the overall myocardial mass they serve a fundamental role in establishing the functional integrity of the myocardium and are the source of myocardial ECM production. Furthermore, the cardiac fibroblast is also the source of a variety of important growth factors/cytokines that can act via paracrine and autocrine mechanisms to affect both myocyte and fibroblast growth/gene expression, respectively (19). Their importance in clinical medicine is underscored by the observation that fibroblast numbers increase in response to several pathologic circumstances that are associated with an increase in ECM production, such as long standing hypertension and myocardial injury/infarction. The importance of the cardiac interstitium in maintaining overall cardiac health has lead to the term "interstitial heart disease," indicating that certain disease states may predominantly involve the extracellular space (reviewed in Weber (63)). For example, in hypertensive heart disease, the interstitial compartment undergoes growth that may exceed that of the cardiac myocytes. Rather than cellular hypertrophy, however, interstitial growth takes the form of cellular hyperplasia and an increase in collagens I, III, IV, and fibronectin. This situation ultimately results in hypertrophied hearts, with an increase in both interstitial and myocyte mass (64-67). Similarly, following myocardial infarction, increases in non-myocyte numbers, collagen content, and myocardial stiffness have also been seen (68-71).
Unfortunately, the mechanism(s) underlying the interstitial cell fibroproliferative response to injury are not known with certainty. Recent work from a number of groups investigating the deposition of ECM has suggested that both locally produced and circulating vasoactive peptides (i.e. angiotensin II/aldosterone, norepinephrine, and endothelin-1) play important roles in myocardial remodeling under several pathologic circumstances. Specifically, studies using both the administration of these agonists (or their peptide-specific antagonists) as well as mechanical stretch have shown alterations in fibroproliferative response that support such a cause-effect role (70, 72-78). Additional in vitro work with some of the cytokines produced by the heart in response to injury have indicated that these substances are equally important in ECM homeostasis (76, 79-85).
Previous work in our laboratory with the cytokine IL-1
, one of the
factors expressed during myocardial injury, indicate that this peptide
has profound effects on both cardiac myocyte and cardiac fibroblast
growth and gene expression (22). Although the mechanism(s) of the
unique myocyte-specific effects of IL-1
have been explored in detail
elsewhere (24), our preliminary findings of an inhibitory effect of
IL-1
on cardiac fibroblast DNA synthesis in culture had not been
elucidated further. In view of the critical role played by the cardiac
fibroblast in the process(es) of myocardial remodeling post injury,
however, this remained an important area of research. It was the
purpose of the investigations described in the present report to gain
additional insight into the growth regulation of the cardiac fibroblast
and expand our previous observations in a way that would help to
understand the mechanism(s) of the antiproliferative effect of IL-1
.
The ultimate goal of research into the mechanisms of fibroblast growth
control is in the development of novel therapeutic approaches to
instances of myocardial injury, which could target the interstitium of
the heart as well as the contractile unit of the heart, the cardiac myocyte.
Entry into the cell cycle upon growth stimulation requires a number of coordination of events from the membrane to the nucleus. As expected, the cardiac fibroblast, like other cell types, enters the cell cycle in response to mitogen-induced signals. They do so by inducing G1/S phase genes, specifically the cyclins and their respective kinase partners, which are required for cell growth and differentiation (86). These protein complexes, which are activated in an ordered fashion, alter the ratio of phosphorylated to dephosphorylated Rb and subsequently the initiation of specific events such as DNA replication and subsequent cell division (reviewed in Morgan (87)).
The major finding of the studies reported here is that IL-1
appears
to exert its inhibitory effect on the cardiac fibroblast at the
G1/S interphase by preventing the phosphorylation of the retinoblastoma gene product, a key regulator of the G1/S
transition in most mammalian cells. More specifically, IL-1
appears
to prevent the post-translational modification of Rb in response to
mitogen by a dual action on the expression and activity of the cyclins and cyclin-dependent kinases necessary for cell cycle
progression as well as that of the G1 cyclin kinase
inhibitor p27.
As with most other growth factors, IL-1
effects are mediated through
the IL-1 receptor (reviewed in Refs. 88 and 89). However, little is
known about the intracellular secondary signals that occur after IL-1
treatment. In an attempt to understand the mechanism through which
IL-1
exerts its antiproliferative effects, we investigated the role
of a number of previously described IL-1
signaling pathways in our
cultured cardiac fibroblasts. Given the prior evidence for the role of
NO and prostaglandin pathways in the inhibitory effects of IL-1
in
other cell types (45, 90-92), as well as the known effect of both NO
and the cGMP pathway in cardiac fibroblasts (93), it was critical to
determine whether our observations were also due to these mediators.
Neither the nitric oxide synthase inhibitor L-NMMA nor the
cyclooxygenase inhibitor indomethacin was capable of blocking the
IL-1
antiproliferative effect. Furthermore, we found that the
IL-1
-induced cell cycle arrest was probably not due to the
sphingomyelinase pathway (as determined by lack of response to
C2- or C6-ceramide (49, 94, 95). Unfortunately,
we were unable to either confirm or refute the effects of tyrosine
kinase inhibition on the cardiac fibroblasts due to the basal effects
of this class of inhibitors on cardiac fibroblasts proliferation. A
role for a tyrosine kinase intermediate in the IL-1
-induced cell
cycle arrest is suggested, however, by the preliminary finding that the
induction of p27 by IL-1
is prohibited by both genestein and
tyrphostin.2
In this regard, it is noteworthy that one of the stress kinase members
of the mitogen-activated protein kinase family known to be associated
with cytokines in other cell types, namely p38/HOG-1, is a dual
specificity kinase (inducing both serine/threonine and tyrosine
phosphorylations), that has been implicated in the induction of a
G1/S arrest in NIH/3T3 cells through the activation of the Cdc42 pathway (96) and possibly involving expression of cyclin D1 (97). Future investigations using the overexpression of
dominant negative p38/HOG-1 may help in confirming the role for this
kinase family in the IL-1
effect. In addition, the importance of the CKIs in the IL-1
-mediated effect on cardiac fibroblast cell cycle kinetics could be further elucidated by taking advantage of genetically altered animals lacking expression of the p21 and/or p27 genes (98-101).
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ACKNOWLEDGEMENTS |
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We thank M. Paningbatan for technical assistance, and Dr. P. Simpson for continued advice and support.
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FOOTNOTES |
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* This work was supported in part by the National Institutes of Health Grants HL58974 and HL59428 (to C. S. L.) and the Department of Veterans Affairs Research Service (to C. S. L.) in conjunction with the Center for Biomedical Laboratory Science Department at San Francisco State University.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. Section 1734 solely to indicate this fact.
Recipient of a Sarnoff Endowment Research Fellowship during the
tenure of this work.
§ Present address: Dept. of Medicine, University of California, San Diego, La Jolla, CA 92093.
¶ To whom correspondence should be addressed: Cardiology Section, Box 0960, Denver Health Medical Center, 777 Bannock St., Denver, CO 80204. Tel.: 303-436-5499, Fax: 303-436-7739; E-mail: clong{at}dhha.org.
The abbreviations used are: IL, interleukin; ECM, extracellular matrix; Cdk, cyclin-dependent protein kinase; CKI, cyclin-dependent kinase inhibitors; Rb, retinoblastoma; PI, propidium iodide; L-NMMA, NG-Monomethyl-L-arginine, monoacetate saltGST, glutathione S-transferasePAGE, polyacrylamide gel electrophoresis.
2 F. Koudssi and C. S. Long, unpublished observations.
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REFERENCES |
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