(Received for publication, June 26, 1995; and in revised form, September 13, 1995)
From the
In heart muscle, the cytokine-inducible isoform of nitric oxide
synthase (NOS2) is expressed in both cardiac myocytes and microvascular
endothelial cells (CMEC). mRNA levels for both NOS2 and for
osteopontin, a multifunctional extracellular matrix phosphoprotein
containing an RGD integrin binding domain, are increased in cardiac
muscle following intraperitoneal injection of adult rats with
lipopolysaccharide. In vitro, interleukin-1 and
interferon-
increased osteopontin mRNA levels in CMEC as well as
NOS2 expression in both CMEC and cardiac myocytes. However, osteopontin
mRNA levels in heart muscle in vivo, and in cardiac myocytes
and CMEC in vitro, also are increased 10-30-fold by the
synthetic glucocorticoid dexamethasone, an agent that suppresses
cytokine induction of NOS2 in both cell types. The hexapeptide GRGDSP,
which interrupts binding of RGD-containing proteins to cell surface
integrins, increased NOS2 mRNA, while a synthetic osteopontin peptide
analogue decreased NOS2 mRNA and protein levels in both
cytokine-pretreated cardiac myocytes and CMEC cultures. Also,
transfection with a full-length antisense-osteopontin cDNA in
cytokine-pretreated CMEC decreased endogenous osteopontin mRNA and
increased NOS2 mRNA levels. These results suggest that osteopontin
could regulate the location and extent of NOS2 induction in the heart.
Increased expression of osteopontin also may be one mechanism by which
glucocorticoids suppress NOS2 activity in cardiac myocytes and
microvascular endothelial cells.
Among the cellular constituents of heart muscle, both
microvascular endothelial cells (CMEC) ()and cardiac
myocytes exhibit a marked induction of the cytokine-inducible nitric
oxide synthase (iNOS or NOS2) in response to soluble inflammatory
mediators in vitro and in vivo in experimental animal
models that mimic systemic sepsis or regional or global myocardial
inflammation(1, 2, 3, 4, 5, 6, 7, 8) .
Within the heart, for example, high levels of NO produced by cardiac
myocytes or by CMEC following induction of NOS2 causes impaired myocyte
contractile function that may contribute to the heart failure
characteristic of the systemic inflammatory response syndrome or
advanced cardiac allograft
rejection(8, 9, 10) . Unless the expression
and activity of NOS2 are spatially and temporally regulated, NO and
other highly reactive nitrogen oxide radicals can induce nonspecific
cellular toxicity that may contribute to the death of the
organism(11, 12, 13, 14) .
The
regulation of NOS2 activity in most tissues is primarily at the
transcriptional level, although post-transcriptional and
post-translational regulatory mechanisms have been
described(12, 14) . In addition to interrupting
selected components of the immune response that trigger NOS2 induction,
glucocorticoids have been shown to suppress NOS2 activity. In
ventricular myocytes and CMEC exposed to interleukin-1 (IL-1
)
and interferon-
(IFN-
), for example, pretreatment with
dexamethasone decreases NOS2 mRNA and protein
abundance(4, 5) . The mechanism(s) by which
glucocorticoids suppress NOS2 induction in the presence of cytokines is
unclear.
Other agents have also been shown to regulate the extent of
induction of NOS2 by inflammatory mediators. Among these is
osteopontin, a relatively ubiquitous extracellular matrix
phosphoprotein that contains an RGD integrin-binding motif. Its
function appears to be determined by the specific tissue and cell type
from which it is secreted(15, 16) . Hwang et al.(17) have recently shown that exogenous recombinant human
osteopontin decreased both NOS2 mRNA abundance and enzyme activity in
primary cultures of renal proximal tubular epithelial cells that had
been exposed to lipopolysaccharide (LPS) endotoxin and IFN-. It
was unclear, however, what regulated endogenous production and
secretion of osteopontin in these cells.
Within the heart, it is not known which cell types express osteopontin nor what role, if any, osteopontin could have in the regulation of NOS2 expression in cardiac cells. In this report, we demonstrate that CMEC and ventricular myocytes constitutively express osteopontin mRNA in vivo and in primary culture, and that dexamethasone markedly increases osteopontin mRNA and secretion by both cell types. The data suggest that the suppression of NOS2 by glucocorticoids in both ventricular myocytes and in CMEC could be mediated in part by this multifunctional extracellular matrix phosphoprotein.
CMEC from adult rat hearts were isolated as
described by Nishida et al.(21) . Briefly, after
removing the atrial and valvular tissue, and right ventricle, the left
ventricle was immersed in 70% ethanol for 10 s to devitalize epicardial
mesothelial and endocardial endothelial cells. After peeling off the
outer to of the ventricular wall, the remaining tissue was minced
finely and treated with collagenase and trypsin in
Ca-free Hanks' balanced salt solution (Life
Technologies, Inc.). Dissociated cells were washed and resuspended in
DMEM containing 20% fetal calf serum (Life Technologies, Inc.) and
plated on laminin (10 µg/ml)-coated dishes at a density of 2,500
cells/cm
. After 1 h of plating, the cells were washed twice
with DMEM to remove loosely adherent cells. These primary isolates have
been documented to contain >90% endothelial cells, with a phenotype
at low passage number consistent with their microvascular origin, as
described previously(21) .
In some studies, cardiac myocytes and non-myocyte cell fractions were isolated from 300-g male Sprague-Dawley rats that had been injected intraperitoneally with LPS (from Salmonella typhimurium, 4 mg/kg; Sigma) and/or dexamethasone (1.2 mg/kg), while control animals received only phosphate-buffered saline injections. When animals received both reagents, the LPS was injected 1 h after dexamethasone. Animals were sacrificed 8 or 16 h following injection(s), and ventricular myocytes and the non-myocyte fractions were obtained after density gradient sedimentation as described above.
Figure 1:
Constitutive expression of osteopontin
in normal adult rat heart. A, Poly(A) mRNA
was isolated from the ventricular muscle of two normal adult rat
hearts. Five µg of the RNA were electrophoresed and analyzed by
Northern blot using
P-labeled osteopontin cDNA as a probe.
Each band represents the hybridization signal from one heart. B, CMEC were isolated from the ventricular muscle of adult rat
hearts according to the methods described under ``Experimental
Procedures.'' Total cellular RNA (15 µg) isolated from
confluent serum-starved CMEC (lane 1) or from 24-h plated
primary isolates of myocytes (lane 2) were electrophoresed on
formaldehyde agarose gels and analyzed by Northern blot using
P-labeled rat osteopontin cDNA
probe.
The regulation of
osteopontin expression is tissue-specific and complex in those cell
types in which it has been examined(15, 16) . In
osteoblasts, agents that induce bone resorption including inflammatory
mediators such as TNF-, IL-1
and LPS, also induce osteopontin
expression and secretion(26) . To determine whether osteopontin
expression could be enhanced in cardiac myocytes and CMEC,
respectively, cells were exposed to a combination of cytokines
(rhIL-1
and rmIFN-
) for 16 h. As shown in Fig. 2A, incubation with this combination of cytokines
approximately doubled CMEC osteopontin mRNA abundance compared to
control CMEC cultures, whereas osteopontin mRNA remained low and
unchanged as analyzed by Northern blot in cytokine-pretreated adult
ventricular myocyte primary isolates at 16 h (Fig. 2B).
Figure 2:
Regulation of osteopontin mRNA levels by
cytokines and dexamethasone in cardiac cells. Confluent 3-h
serum-starved CMEC (A) or primary isolates of ventricular
myocytes (B) were exposed to vehicle alone (lane 1)
or to a combination of cytokines (rhIL-1, 4 ng/ml +
rmIFN-
, 500 U/ml, lane 2) or dexamethasone alone (3
µM) (lane 3) for 16 h. The cells in lane 4 were pretreated with dexamethasone for 1 h, and then with the
combination of cytokines for 16 h. Total RNA was extracted, and 15
µg of RNA were analyzed by Northern blot using
P-labeled rat osteopontin cDNA as a probe. The filters
were then hybridized to an 18 S probe to normalize for loading
differences. The data are expressed as normalized osteopontin mRNA
levels as a percent of control levels in the absence of dexamethasone
or cytokines.
Since glucocorticoids are known to suppress the induction of
inflammatory cytokine-induced gene expression in many cell types,
including cardiac myocytes(5) , cytokine-treated CMEC and
ventricular myocytes were also pretreated with dexamethasone, which we
presumed would suppress any cytokine-induced expression of osteopontin
mRNA as has been shown, for example, in rat osteoblast
cells(27) . Unexpectedly, as shown in Fig. 2,
dexamethasone markedly enhanced osteopontin mRNA abundance in both CMEC
and adult ventricular myocytes, regardless of whether IL-1 and
IFN-
were present in the incubation medium. Cells treated with a
combination of dexamethasone with cytokines tended to have lower levels
of osteopontin mRNA than cells treated with dexamethasone alone,
although cultures treated with both classes of reagents still expressed
osteopontin mRNA at levels that were 10-15-fold higher than base
line. To determine whether similar changes in osteopontin mRNA
abundance could be detected in vivo, adult rats were injected
intraperitoneally with either LPS or dexamethasone (or both). Myocyte
and non-myocyte fractions were obtained, and total RNA was isolated and
analyzed by Northern blot. A faint band of the appropriate size for
osteopontin mRNA was detected in both myocyte and non-myocyte fractions
from normal hearts. This signal could be increased severalfold at 16 h
following injection of dexamethasone or of LPS, or with a combination
of these agents (Fig. 3). A detectable increase above base-line
osteopontin mRNA levels could be detected within 8 h of injection of
LPS, with or without dexamethasone (data not shown).
Figure 3:
Regulation of osteopontin mRNA levels in
myocyte and non-myocyte fractions of hearts from adult rats injected in vivo with dexamethasone and/or LPS. Animals were injected
intraperitoneally with either dexamethasone (1.2 mg/kg; 16 h before
sacrifice) or with LPS (4 mg/kg; 8 h before sacrifice), or both
reagents. Animals were sacrificed to isolate myocyte and non-myocyte
fractions, and total RNA was prepared. Northern analyses were performed
as described in Fig. 2using a P-labeled rat
osteopontin cDNA. The 18 S hybridization signal was used to determine
the relative amount of RNA loaded per lane. Data are shown for one
animal in each treatment group. The experiment was performed twice with
similar results.
To verify that
these changes in mRNA abundance were paralleled by similar directional
changes in osteopontin synthesis and secretion, the media conditioned
by control or dexamethasone-treated cells were concentrated and
analyzed by Western blot using a monoclonal anti-osteopontin antibody.
As shown in Fig. 4A, a band corresponding to
osteopontin (approximately 62 kDa) was detected in medium conditioned
by CMEC. The intensity of this band was increased significantly in
conditioned medium from dexamethasone-treated CMEC. In
myocyte-conditioned medium, a slightly higher molecular mass band
(approximately 69 kDa) was detected by the same antibody by Western
blot. This band is within the range of sizes that has been reported for
this glycosylated phosphoprotein (i.e. 44-75
kDa)(16) . As in CMEC, dexamethasone increased by approximately
3-fold the intensity of this 69-kDa band in proteins from ventricular
myocytes (Fig. 4B). Similar data were obtained by
immunoprecipitation of conditioned media from both cell types following
metabolic labeling with [S]methionine, using a
polyclonal anti-peptide osteopontin antiserum prepared as described
previously (23) , followed by SDS-PAGE (data not shown).
Figure 4:
Western blot analysis of osteopontin in
media conditioned by CMEC and ventricular myocytes. Confluent
serum-starved CMEC (A) or primary isolates of ventricular
myocytes (B) were treated with vehicle alone
(MeSO, lane ``C'') or with dexamethasone
(3 µM, DEX) for 16 h in serum-free medium. The
conditioned media were collected and concentrated, and 12 µg of
protein from the concentrate were separated by SDS-PAGE and transferred
to nitrocellulose membranes. Equivalent loading and transfer of protein
was checked by Ponceau S staining of the membrane. Osteopontin was
detected by immunoblotting using a monoclonal anti-osteopontin
antibody. The arrow indicates the position of
osteopontin.
Figure 5:
Concentration-effect relationship of
dexamethasone on osteopontin mRNA abundance in cardiac cells.
Confluent, serum-starved CMEC (A) or ventricular myocyte
primary isolates (B) were treated over a range of
dexamethasone concentrations (30 nM to 15 µM).
Total RNA was isolated after 16 h of treatment and analyzed by Northern
blot with P-labeled rat osteopontin and 18 S probes. The
density of each osteopontin signal was divided by that of corresponding
18 S rRNA and is expressed as a percentage of the normalized
osteopontin hybridization signal in the absence of dexamethasone for
both cell types.
Figure 6:
Time course of increase in osteopontin
mRNA levels by dexamethasone. Confluent serum-starved CMEC (A)
or ventricular myocyte primary isolates (B) were treated with
dexamethasone (3 µM) for the time periods indicated. Total
RNA was extracted and 15 µg of the RNA was analyzed by Northern
blot using P-labeled osteopontin and 18 S probes. The
signal density of osteopontin mRNA was divided by that of 18 S rRNA,
and the data are expressed as a percentage of the normalized
osteopontin hybridization signal at 0 h.
Figure 7:
Dexamethasone and osteopontin mRNA
stability. Confluent serum-starved CMEC were exposed to either vehicle
or 3 µM dexamethasone for 16 h before adding actinomycin D
(10 µg/ml). Total RNA was isolated at the indicated time points.
Northern blot analyses were performed using 15 µg of total RNA per
lane and P-labeled rat osteopontin and 18 S probes. The
signal density of osteopontin mRNA was divided by that of 18 S rRNA.
The data, expressed as natural logarithm of osteopontin mRNA at
successive time points (R
) normalized to
the maximal level at time 0 (R
), from four
independent experiments are plotted as a function of time (mean
± S.E.).
Figure 8:
Regulation of NOS II mRNA abundance by
GRGDSP peptide in cardiac cells. Confluent serum-starved CMEC cells (A) or primary isolates of myocytes (B) were
pretreated for 1 h with the indicated concentrations of GRGDSP peptide (RGD) and then exposed to a combination of cytokines
(rhIL-1, 4 ng/ml + rmIFN-
, 500 units/ml) for 16 h. Total
RNA (15 µg) was used for Northern analysis using
P-labeled NOS2 and 18 S probes. The normalized NOS2 mRNA
hybridization signals are presented as a percentage of NOS2 mRNA
abundance with IL-1
and IFN-
alone. No NOS2 mRNA was
detectable by Northern blot in the absence of cytokines regardless of
whether or not the GRGDSP peptide was present (mean ± S.E.; data
shown are the averages of three
experiments).
These data suggested that some matrix protein attachments, possibly involving endogenous osteopontin, could suppress the extent of NOS2 expression in both cell types. To determine whether osteopontin did affect the extent of induction of this NO synthase isoform, microvascular endothelial cells and ventricular myocytes were treated with a 20-mer synthetic peptide analogue (OPP) based on the rat osteopontin sequence that spans the RGD integrin-binding motif. OPP has been shown to be functional for osteopontin signaling and mimics recombinant human osteopontin in renal tubular epithelial cells(17) . As shown in Fig. 9, 20 nM OPP decreased NOS2 transcript levels by approximately 50% in cytokine-pretreated microvascular endothelial cells. In cytokine-treated primary isolates of adult ventricular myocytes, there was about a 40% decline in NOS2 mRNA abundance at 20 nM and 50 nM OPP. This decline in NOS2 transcript with OPP also was accompanied by a reduction in NOS2 protein by approximately 20 and 50% in CMEC and adult ventricular myocytes, respectively, as detected by immunoblot analysis using an anti-NOS2 monoclonal antibody (data not shown).
Figure 9:
Regulation of NOS II gene expression by a
synthetic OPP. Confluent serum-starved CMEC (A) or primary
isolates of ventricular myocytes (B) were pretreated for 1 h
with the indicated concentrations of a 20-amino acid synthetic peptide
(OPP) and then exposed to a combination of cytokines (rhIL-1, 4
ng/ml + rmIFN-
, 500 units/ml) for an additional 16 h in the
continuous presence of OPP. Total RNA was extracted and analyzed by
Northern blot using
P-labeled NOS2 and 18 S probes. The
hybridization signal density of NOS2 was divided by that of 18 S, and
normalized NOS2 mRNA levels are expressed as a percent of maximal NOS2
mRNA levels following exposure to cytokines
alone.
Figure 10:
Transfection of CMEC with an antisense
rat osteopontin cDNA construct increases NOS2 mRNA levels. CMEC primary
cultures that were approximately 50% confluent were transfected with an
antisense osteopontin cDNA construct (AS OP cDNA) using
Lipofectin for 6 h. As a control, cells were mock-transfected with
Lipofectin alone. The media were supplemented with 10 ml of DMEM
containing 20% serum for 48 h. The cells were then serum-starved for 3
h before exposure to rhIL-1 (4 ng/ml) and rmIFN-
(500
units/ml) for a subsequent 16 h. Total RNA was isolated and analyzed by
Northern blot using
P-labeled osteopontin, NOS2, and 18 S
probes. The normalized NOS2 and osteopontin hybridization signals are
expressed relative to the NOS2 and osteopontin mRNA levels in CMEC
treated with cytokines alone, the mean of which was set to 100% (*p < 0.01 compared to control NOS2 signal;**p < 0.02
compared to control osteopontin signal; mean ± S.E. of three
experiments).
Osteopontin, originally identified in its role of facilitating resorption of bone hydroxylapatite by osteoclasts, is now known to be synthesized in many different cell types including luminal epithelial cells in many organs and by smooth muscle in a number of tissues including the vasculature(27, 30, 31, 32, 33) . Osteopontin mRNA has been inconsistently detected in normal rat heart(18, 27, 34) . Murry et al.(35) have reported that osteopontin expression was markedly increased in a subset of infiltrating macrophages around and within zones of myocardial injury induced by a transdiaphragmatic freeze-thaw technique. Similarly, Williams et al.(36) have recently reported that osteopontin mRNA is increased markedly in hearts of hamsters with a heritable cardiomyopathy, which they attributed to infiltrating tissue macrophages.
Osteopontin may play an important role in several cardiovascular disease processes, including atherosclerosis, aortic valve calcification, as well as repair of myocardial injury as reviewed by Giachelli et al.(37) . An increase in osteopontin levels was observed in rat carotid arteries following vascular injury induced by experimental balloon angioplasty(18) . Indeed, this extracellular matrix phosphoprotein may play a more general role in the immune response than mediating chemotaxis of phagocytic cells; for example, the early T-cell activation gene 1 (Eta1) that is expressed following nonspecific activation of several lymphocyte subclasses has been identified as being osteopontin(38) . Osteopontin also can stimulate lymphocyte immunoglobulin production, suggesting that it may function as a cytokine in some circumstances(16) . Murry et al.(35) also demonstrated that osteopontin expression could be detected in other tissues and cell types following injury, including regenerating skeletal muscle cells. Within the heart, osteopontin mRNA and protein has been detected by in situ hybridization and immunohistochemistry only within macrophages in injured muscle, but not in the extracellular matrix or other cell types (35, 36) . Since the only known functions of osteopontin are related to its role as an extracellular matrix phosphoprotein, Murry et al.(35) speculated that extracellular osteopontin protein levels and presumably levels in otherwise normal heart muscle were below the level of detection by the immunohistochemical techniques they employed.
Denhardt and
Guo(16) , in a recent review, have emphasized the cell type
specificity of the regulation of osteopontin gene expression.
Osteopontin is expressed constitutively in arterial vascular smooth
muscle cells and osteoblasts and its expression is increased by peptide
growth factors such as basic fibroblast growth factor and transforming
growth factor- and by phorbol esters (16) and decreased by
glucocorticoids, at least in the osteoblast cell line ROS
17/2.8(27) . In contrast, we find that glucocorticoids increase
osteopontin expression and protein secretion in ventricular myocytes
and CMEC in primary culture. This differential responsiveness to
glucocorticoids may be due to the fact that the osteopontin promoter is
known to contain two glucocorticoid response elements, as well as two
AP-1 sites (16, 39, 40) . Glucocorticoids, by
binding to steroid hormone receptors, can directly repress
AP-1-mediated transcriptional activation(41) .
In addition
to increasing osteopontin mRNA levels and protein content in
ventricular myocytes and CMEC, dexamethasone also decreases NOS2 mRNA
abundance and activity in both cell types(4, 5) . The
mechanisms by which glucocorticoids regulate cytokine-induced gene
expression are complex and differ among specific cell
types(42) . However, the temporal association between
osteopontin expression and decreased NOS2 mRNA levels suggests that
endogenous osteopontin in the extracellular matrix could regulate NOS2
activity in these cells. This hypothesis is supported by the
observation that the synthetic 20-mer osteopontin peptide analogue
(OPP) decreased NOS2 mRNA and protein levels in both cell types and
that transfection of CMEC with an antisense osteopontin cDNA decreased
endogenous osteopontin mRNA levels while increasing NOS2 mRNA abundance
in response to IL-1 and IFN-
. The specific intracellular
signaling pathways initiated by osteopontin binding to
v
3 (or
other) integrins that result in decreased NOS2 mRNA abundance are not
known. Integrin recruited and autophosphorylated focal adhesion kinase,
which can subsequently activate either ras or protein kinase
C-dependent pathways, has been shown to synergistically enhance some
cellular responses to cytokines(43) . It is possible that
osteopontin acts to interrupt downstream signaling by other
extracellular matrix proteins (such as fibronectin, with which
osteopontin is known to
interact)(25, 44, 45) . Regardless of the
specific mechanisms, it is likely that the increased expression,
synthesis, and secretion of osteopontin induced by specific cytokines
or glucocorticoids contributes to the spatial and temporal regulation
of nitric oxide production by NOS2 in cardiac muscle.