From the Department of Medicine, Division of Nephrology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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We recently demonstrated that transforming growth
factor- (TGF-
) stimulates phosphorylation of the type I inositol
1,4,5-trisphosphate receptor (Sharma, K., Wang, L., Zhu, Y., Bokkala,
S., and Joseph, S. (1997) J. Biol. Chem. 272, 14617-14623), possibly via protein kinase A (PKA) activation in murine
mesangial cells. In the present study, we evaluated whether TGF-
stimulates PKA activation. Utilizing a specific PKA kinase assay, we
found that TGF-
increases PKA activity by 3-fold within 15 min of
TGF-
1 treatment, and the enhanced kinase activity was completely
reversed by the inhibitory peptide for PKA (PKI; 1 µM).
In mesangial cells transfected with a PKI expression vector, enhanced
PKA activity could not be demonstrated with TGF-
1 treatment.
TGF-
1 was also found to stimulate translocation of the
-catalytic
subunit of PKA to the nucleus by Western analysis of nuclear protein as
well as by confocal microscopy. TGF-
1-mediated phosphorylation of
cAMP response element-binding protein was completely reversed by H-89
(3 µM), a specific inhibitor of PKA. Stimulation of
fibronectin mRNA by TGF-
1 was also attenuated in cells
overexpressing PKI. We thus conclude that TGF-
stimulates the PKA
signaling pathway in mesangial cells and that PKA activation
contributes to TGF-
stimulation of cAMP response element-binding
protein phosphorylation and fibronectin expression.
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INTRODUCTION |
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Transforming growth factor-
(TGF-
)1 has been
established as an important cytokine intimately involved in tissue
fibrosis. In a variety of kidney diseases characterized by excess
glomerular matrix deposition, TGF-
has been found to be up-regulated
(1). Diabetic kidney disease is characterized by accumulation of a variety of matrix molecules, including fibronectin (2). We have
demonstrated that TGF-
1 is stimulated in the diabetic kidney and
that neutralization with anti-TGF-
antibodies significantly inhibits
diabetes-induced fibronectin gene expression (3). Although stimulation
of fibronectin by TGF-
is well recognized (4, 5), the signaling
pathways by which TGF-
regulates fibronectin gene expression are
poorly understood.
Recent studies have demonstrated that TGF- initially binds to its
type II receptor and then forms a heteromeric complex with the type I
receptor (reviewed in Ref. 6). Cross-phosphorylation of the type I
receptor by the type II receptor is critical for subsequent
phosphorylation of various members of the recently described Smad
family of proteins. Phosphorylation of Smad2 and Smad3 has been
demonstrated to play an important role in mediating the effects of
TGF-
on cell proliferation (7, 8); however, their role in
stimulation of matrix molecules such as fibronectin is unclear. The
protein kinase A (PKA) pathway induces many effects on cells similar to
TGF-
, and in particular, activation of both TGF-
and PKA
stimulates fibronectin production, at least in part, by stimulating
gene transcription (4). A critical step involved in the transcriptional
gene regulation by the PKA pathway is the phosphorylation of the cAMP
response element-binding protein (CREB). Interestingly, CREB
phosphorylation by TGF-
has been demonstrated in several cell types
(9-11), and the consensus cAMP response element (CRE, TGAGTCA) has
been implicated in mediating the effects of TGF-
on the fibronectin
promoter in rat mesangial cells (12) and on the cyclin A promoter in
Chinese hamster lung fibroblasts (13). Although these findings would
suggest that the PKA pathway is involved in TGF-
signaling, this
possibility was considered unlikely primarily because cAMP levels were
not elevated following TGF-
treatment (9, 10).
PKA is composed of two regulatory subunits and two catalytic subunits
in its inactive state (14). Elevation of cAMP levels leads to
dissociation of the heteromeric complex, thus allowing the free
catalytic subunit to be active as a serine/threonine kinase in the
cytoplasm and nucleus. Until recently, activation of PKA without a rise
in cAMP levels was not thought to occur. In an elegant study by Zhong
et al. (15), the -catalytic subunit of PKA was found to
be bound to I
B in the cytoplasm, rather than by one of its
regulatory subunits; degradation of I
B led to activation of the
-catalytic subunit. Therefore, it is apparent that other mechanisms
are operant to allow for PKA activation independently of cAMP. In
support of a role for the PKA pathway being involved in TGF-
signaling, we recently demonstrated that TGF-
-induced phosphorylation of the type I inositol 1,4,5-trisphosphate receptor in
mesangial cells appeared to be mediated by PKA (16). In the present
study, we demonstrate that TGF-
stimulates PKA activity without
elevating intracellular cAMP levels in mesangial cells and that
inhibition of PKA attenuates TGF-
-induced stimulation of CREB
phosphorylation and fibronectin gene expression.
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EXPERIMENTAL PROCEDURES |
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Materials--
[-32P]ATP was purchased from NEN
Life Science Products. An enhanced chemiluminescence system was
purchased from Amersham Pharmacia Biotech. TGF-
1 was purchased from
R&D Systems (Minneapolis, MN). All other reagents were from Sigma
unless otherwise noted.
Cell Culture-- An SV40-transformed murine glomerular mesangial cell line, which has been previously described (17), was primarily used in these studies. These cells retain many of the differentiated characteristics of mesangial cells in primary culture. We also performed a limited series of experiments in rat glomerular mesangial cells that were conducted between passages 4 and 6 and in the mink lung epithelial cell line (CCL-64, obtained from American Type Culture Collection, Rockville, MD).
In Vitro Kinase Assay for PKA Activity--
Murine mesangial
cells (MMCs) were grown to confluence in Dulbecco's modified Eagle's
medium (DMEM) with 10% fetal calf serum and then growth-arrested for
24 h in serum-free DMEM. Cells were treated with agonists for
various periods of time, washed with PBS, and harvested with cold
extraction buffer containing 25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM
-mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotonin, 0.5 mM phenylmethylsulfonyl fluoride. Protein concentrations of
the crude lysates were quantitated, and equal amounts of protein were
added to a reaction mixture containing 40 mM Tris-HCl, pH
7.4, 20 mM MgCl2, 0.1 mg/ml bovine serum
albumin, 100 µM biotinylated PKA peptide substrate
(Kemptide, LRRASLG; Promega, Madison, WI), 3000 Ci/mmol
[
-32P]ATP, and 0.5 mM ATP per reaction.
Experiments were performed in parallel with the addition of a PKA
inhibitor peptide (TTYADFIASGRTGRRNAIHD, 1 µM; Promega).
The reaction was allowed to proceed for 5 min at 30 °C and then
terminated by the addition of 2.5 M guanidine hydrochloride. Five µl of sample were spotted onto
streptavidin-coated discs, washed repeatedly, dried in an oven, and
placed in scintillation vials for radioactive counting. To visually
demonstrate that the Kemptide peptide was indeed phosphorylated, the
in vitro kinase reaction was also performed with
fluorescently tagged Kemptide (Promega). Phosphorylation of this
peptide alters the peptide's net charge from +1 to
1, allowing the
phosphorylated peptide to be separated from the unphosphorylated
peptide on an agarose gel at neutral pH.
Transfection of MMCs with Protein Kinase A Inhibitor (PKI) Expression Vector-- The expression vectors for PKI and mutant PKI driven by the Rous sarcoma virus promoter were obtained from Dr. Richard Maurer (Oregon Health Sciences University, Portland, OR) (18). The mutant PKI sequence codes for glycine rather than arginine at positions 20 and 21. The arginines at these sites are essential for inhibition of catalytic subunit activity. Subconfluent MMCs were transfected with 10 µg of plasmid and Superfect transfection reagent (QIAGEN Inc., Chatsworth, CA) for 3 h. Cells were washed and rested in serum-free DMEM for an additional 16 h prior to the addition of agonists.
Western Blot Analysis--
MMCs were treated with TGF- and/or
the PKA inhibitor H-89 (3 µM, 30 min) prior to the
addition of TGF-
in serum-free DMEM. Cell extracts were prepared as
described previously (16). Nuclear extracts were prepared
according to the method of Schreiber et al. (19).
Total cellular or nuclear protein was quantitated using the Bio-Rad DC
protein assay, and 20 µg of protein were resolved on a 10%
SDS-polyacrylamide gel and transferred to nitrocellulose membrane.
Immunoblotting was performed with antibodies against the
-catalytic
subunit of PKA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
anti-phospho-CREB antibody (Upstate Biotechnology, Inc., Lake Placid,
NY), anti-CREB antibody, anti-I
B-
antibody, or anti-NF-
B p65
antibody (Santa Cruz Biotechnology, Inc.). Immunoreactive bands were
detected using the enhanced chemiluminescence system.
Confocal Analysis--
MMCs were grown in 6-well tissue culture
dishes on No. 1 coverslips pretreated with poly-D-lysine
(0.1 mg/ml for 5 min) with DMEM containing 10% fetal calf serum. After
cells were adherent, they were rested in serum-free DMEM overnight and
then exposed to TGF-1 (10 ng/ml) or forskolin (10 µM)
for 15 min. After exposure to agonists, cells were washed with PBS
three times and fixed with 3.7% formaldehyde for 10 min at room
temperature. After repeated washing, cells were permeabilized with
0.05% Triton X-100 in PBS (PBS/TX) for 10 min at room temperature,
washed with PBS/TX three times, blocked with 4% normal goat serum in
PBS/TX for 10 min, and then incubated with anti-
-catalytic antibody
(1:200) for 30 min at 37 °C. Cells were washed with PBS/TX and
blocked again with 4% normal goat serum, and secondary antibody
(fluorescein-conjugated goat anti-rabbit antibody, 1:500 dilution;
Rockland Inc., Gilbertsville, PA) was applied for 30 min at 37 °C.
After repeated washing, cells were post-fixed with 3.7% formaldehyde
for 10 min at room temperature and washed, and coverslips were placed
on glass slides and mounted with SlowFade (Molecular Probes, Inc.,
Eugene, OR). Slides were visualized with a confocal microscope
(courtesy of Dr. James Keen, Thomas Jefferson University), and
representative regions were photographed. Control cells, stained only
with secondary antibody, showed minimal fluorescence.
Measurement of cAMP--
Cyclic AMP levels were measured in MMCs
after treatment with TGF-1 (10 ng/ml; 5, 15, and 30 min) or
forskolin (10 µM, 15 min) using a Biotrak cAMP enzyme
immunoassay kit (Amersham Pharmacia Biotech). MMCs were trypsinized and
resuspended in PBS with 65% (v/v) ethanol. The cell precipitates were
centrifuged, the supernatants were drawn off, and the extracts were
dried in a vacuum oven. Extracts were resuspended in assay buffer,
acetylated, and assayed for cAMP following the instructions supplied by
the manufacturer. MMCs were assayed in the absence and presence of
isobutylmethylxanthine (100 µM) 10 min prior to the
addition of TGF-
1 or forskolin.
Northern Analysis--
To assess whether TGF-1-induced
fibronectin mRNA expression was affected by inhibition of PKA, MMCs
transiently transfected with PKI expression vector or mutant PKI were
treated with TGF-
1 (10 ng/ml) for 24 h and washed with ice-cold
PBS, and total RNA was isolated using acid guanidinium
thiocyanate/phenol/chloroform (20). Twenty µg of total RNA were
loaded onto a 1.2% agarose gel containing 2.2 M
formaldehyde, electrophoresed, and transferred onto nylon membrane. The
probe for murine fibronectin has been described previously (3).
Hybridization and washing conditions were performed as described
previously (21). To standardize for loading, membranes were stripped
and reprobed with a
-actin cDNA probe (kindly provided by Dr.
Pamela A. Norton). Densitometric analysis was performed as described
previously (16), and mRNA levels were calculated relative to those
of
-actin.
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RESULTS |
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TGF- Stimulates PKA Activity--
MMCs were treated with
TGF-
1 for various time points, and an in vitro kinase
assay was performed utilizing a biotinylated substrate for PKA (Fig.
1A). The reaction mixture was
spotted onto avidin-coated discs to bind only the biotinylated peptide, thus increasing the specificity of the assay. With 15 min of
administration of TGF-
1, PKA activity was increased by 3-fold
relative to control values and remained elevated at 30 min. The
addition of a specific peptide inhibitor of the catalytic subunit of
PKA (PKI) completely blocked the increased kinase activity in
TGF-
-treated samples, demonstrating that the increased kinase
activity was specific for PKA (Fig. 1A). A dose-response
relationship was noted, with maximal stimulation of PKA noted at a
concentration of 10 ng/ml TGF-
1 (Fig. 1B). To further
demonstrate that PKA activity was being stimulated by TGF-
1 or
forskolin, cells were transiently transfected with an expression vector
for the PKI peptide to inhibit PKA activity or a PKI peptide that was
mutated in its PKA catalytic recognition site. TGF-
or forskolin
stimulated PKA activity in cells expressing the mutant PKI peptide, but
not in MMCs expressing wild-type PKI (Fig. 1C). To visually
demonstrate that the peptide substrate for PKA was indeed
phosphorylated by TGF-
1, a fluorescently tagged peptide substrate
was employed in the in vitro kinase assay. Phosphorylation
of the peptide promotes migration to the positive electrode on an
agarose gel. TGF-
1 treatment for 5 and 15 min (lanes 3 and 4) stimulated migration of this peptide to the
positive electrode (Fig. 1D).
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TGF- Stimulates Translocation of
-Catalytic Subunit of
PKA--
Activation of PKA leads to dissociation of the catalytic
subunit from its regulatory subunit and translocation to the nucleus (14). To demonstrate that PKA is activated and that the catalytic subunit is dissociated from its inhibitory regulatory subunit, nuclear
proteins were isolated from control and TGF-
-treated MMCs, and
immunoblot analysis was performed with an antibody against the
-catalytic subunit of PKA. Fig. 3
demonstrates accumulation of the catalytic subunit in the nucleus with
TGF-
1 treatment. Confocal microscopy (Fig.
4) also demonstrated a redistribution of
the
-catalytic subunit from a primarily perinuclear distribution in
untreated MMCs (Fig. 4A) to a nuclear localization in
TGF-
1-treated (Fig. 4B) and forskolin-treated (Fig.
4C) MMCs. As compared with control cells, TGF-
1- and
forskolin-treated cells demonstrated a similar redistribution of the
-catalytic subunit diffusely into the cytoplasm.
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TGF- Does Not Increase Intracellular cAMP Levels or Affect
NF-
B/I
B--
Increased intracellular cAMP levels enhance binding
of cAMP to the regulatory subunits of PKA and promote dissociation from the catalytic subunit. Therefore, cAMP levels were measured following TGF-
1 treatment after various time intervals. MMCs stimulated with
TGF-
1 for 5, 15, or 30 min did not raise intracellular cAMP levels
(Fig. 5). Forskolin treatment did cause a
marked increase in cAMP levels in MMCs, as would be expected. In the
presence of the phosphodiesterase inhibitor isobutylmethylxanthine,
TGF-
also did not increase cAMP levels (data not shown).
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TGF--induced CREB Phosphorylation Is Blocked by Inhibition of
PKA--
To determine the functional significance of TGF-
-induced
PKA activation, we evaluated if CREB was phosphorylated in response to
TGF-
1 in MMCs by performing immunoblot analysis of nuclear protein
with anti-phospho-CREB antibody. Fig. 7
demonstrates enhanced CREB phosphorylation (43-kDa band) in nuclear
protein isolated from TGF-
1-treated cells as compared with control
cells. Pretreatment of MMCs with H-89, a relatively specific inhibitor
of PKA, was sufficient to completely inhibit TGF-
-induced CREB
phosphorylation. Immunoblot analysis of the same membrane with an
antibody that recognizes total CREB revealed that TGF-
1 did not lead
to accumulation of CREB in the nucleus (data not shown).
Anti-phospho-CREB antibody is raised against the phosphoserine in a
portion of the CREB protein that shares 100% homology with the
phosphoserine site of the transcription factor ATF-1 (22); therefore,
this protein may also be demonstrated on immunoblotting. The band at 38 kDa corresponds to phospho-ATF-1 and was also found to be markedly
stimulated in nuclear extracts from TGF-
1-treated cells (Fig. 7).
Inhibition of PKA by H-89 also completely prevented TGF-
-induced
phosphorylation of ATF-1. Treatment of MMCs transiently transfected
with the PKI expression vector failed to demonstrate increased CREB or
ATF-1 phosphorylation in response to TGF-
1 treatment (data not
shown). Thus, CREB phosphorylation by TGF-
1 appears to be largely
dependent on activation of PKA.
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TGF--induced Fibronectin mRNA Stimulation Is Attenuated by
Inhibition of PKA--
Fibronectin gene stimulation is considered to
be controlled, at least in part, by CREs located in the fibronectin
promoter, and both TGF-
and PKA stimulation may stimulate the
fibronectin promoter via CRE sites (4, 12, 23). To assess if PKA may be
involved in stimulation of steady-state fibronectin mRNA levels by
TGF-
, we performed Northern analysis of MMCs transiently transfected with an expression vector for either wild-type PKI or mutant PKI (Fig.
8). MMCs overexpressing mutant PKI
treated with TGF-
1 for 48 h demonstrated a 2.5-fold stimulation
of steady-state fibronectin mRNA levels (lane 4 as
compared with lane 3), whereas MMCs overexpressing wild-type
PKI had a blunted effect (lane 2 as compared with lane 1) with TGF-
1 treatment (Fig. 8).
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DISCUSSION |
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Our major conclusions are that TGF-1 stimulates PKA in
mesangial cells and that the PKA pathway plays an important role in TGF-
1-induced CREB phosphorylation and TGF-
stimulation of
fibronectin gene expression. Surprisingly, we found PKA activation by
TGF-
in the absence of a detectable rise in cAMP levels.
Our results demonstrating TGF--induced activation of PKA are in
contrast to other published reports in different cell types. Prior
studies excluded the PKA pathway in TGF-
1 signaling in mink lung
epithelial cells and rat mesangial cells primarily because TGF-
did
not elevate intracellular cAMP levels (9, 10). Separate studies in
Chinese hamster ovary cells (13) and murine embryonic palatal cells
(11), using overexpression of the regulatory subunit to block PKA
activity (13) and a competitive analogue of cAMP
((Rp)-cAMP) (11), respectively, concluded that
TGF-
-induced CREB phosphorylation occurred independently of the PKA
pathway. However, the recent demonstration by Zhong et al.
(15) that PKA may be activated without cAMP elevation sets an important precedent that may provide an explanation to justify the different results. This group reported that the
-catalytic subunit of PKA is
constitutively associated with I
B, likely via an ankyrin repeat region of I
B, and degradation of I
B leads to stimulation of
-catalytic subunit kinase activity. These authors further suggested that other proteins containing ankyrin repeat sites may also function to bind the
-catalytic subunit of PKA and to regulate its activity independently of intracellular cAMP. Therefore, failure to observe an
increase in cAMP-mediated kinase activity is not adequate evidence to
exclude the involvement of the PKA pathway in a given cellular process.
It should be noted that in two of the above-mentioned studies (11, 13),
PKA activity was measured by in vitro kinase assays after
10-15 min of exposure to TGF- and was found not to be increased. The in vitro kinase assays utilized either Kemptide
substrate peptide (13) or a nonspecific peptide (11) as a phosphate acceptor from [32P]ATP with subsequent washing on
phosphocellulose filters. The net phosphorylation detected would
reflect the amount of phosphorylated target peptide substrate that
binds to the phosphocellulose filter as well as other proteins that are
present in the cell lysate that have basic residues (24). In addition,
overall phosphatase activity in the cell lysate may also be reflected
in this assay. As TGF-
has been shown to activate serine/threonine
kinases (6, 25) as well as various phosphatases (26), this assay method may not be conclusive. Our studies used two separate in
vitro kinase assays wherein the Kemptide substrate is biotinylated
or fluorescently labeled, thus assuring that phosphorylation of only the peptide substrate of interest would be measured. To enhance specificity, only the PKI-inhibitable fraction of kinase activity was
taken to reflect PKA activity. In addition, we demonstrate translocation of the
-catalytic subunit to the nucleus with TGF-
treatment by immunoblot analysis as well as confocal microscopy, providing an independent means of demonstrating PKA activation.
The mechanism(s) by which TGF- stimulates PKA activation remains
unclear. Our studies failed to demonstrate that I
B protein underwent
degradation in whole cell lysate or that NF-
B was translocated to
the nucleus with TGF-
treatment. This would suggest that TGF-
may
affect degradation of another protein that associates with and inhibits
the
-catalytic subunit of PKA, possibly via ankyrin repeat sites. In
a study in which platelet-derived growth factor-BB was found to
stimulate PKA without raising intracellular cAMP levels (27), it was
suggested that phosphorylation of regulatory subunits may affect
affinity of cAMP. Additionally, it is conceivable that localized
intracellular compartments of cAMP may increase in response to TGF-
,
without a concomitant increase in total cellular cAMP. These
possibilities are presently being explored in our laboratory.
TGF--induced PKA activation likely contributes to CREB and ATF-1
phosphorylation. CREB phosphorylation by TGF-
has been demonstrated
by several independent studies (9-11). In a study employing murine
embryonic palatal cells (11), TGF-
-induced phosphorylation of CREB
was demonstrated with an anti-phospho-CREB antibody specific for serine
133 (22). Serine 133 is not only the site of CREB phosphorylation by
PKA, but also the site of CREB phosphorylation by mitogen-activated
protein kinase-stimulated CREB kinase and calcium-calmodulin kinase.
Evidence against the role of the latter two pathways in mediating
TGF-
-induced CREB phosphorylation was provided in the above study
(11). The present study demonstrates that TGF-
-induced CREB
phosphorylation, employing the same anti-phospho-CREB antibody, is
completely attenuated in mesangial cells when pretreated with the
relatively specific inhibitor of PKA, H-89. Of note, H-89 inhibits the
catalytic activity of PKA and is therefore not dependent on regulatory
subunit binding of the catalytic subunit. Similar results were also
found with the PKI peptide, which also binds to the free catalytic
subunit of PKA. In addition, the phospho-CREB antibody also recognizes phospho-ATF-1 (22), and this nuclear transcription factor was also
found to be phosphorylated by TGF-
1 treatment and reversed with PKA
inhibition. The role of CREB phosphorylation in mediating gene
regulation by TGF-
is unclear; however, a recent study in Drosophila demonstrated that a CREB-binding site was
important in mediating decapentaplegic (the
Drosophila homologue of TGF-
) stimulation of the homeotic
gene Ultrabiothorax (28).
TGF-1 stimulation of the fibronectin promoter appears to require a
consensus CRE (TGACGTCA) site (4, 23), and both CREB and ATF-1 derived
from mesangial cells bind to this site (10). CREB phosphorylation
enhances CREB binding to CREB-binding protein, thus activating gene
transcription via the CRE (29). Inhibiting CREB phosphorylation would
thus inhibit CREB binding to CREB-binding protein and may inhibit
TGF-
-induced stimulation of fibronectin gene transcription. Our
finding that inhibition of PKA in mesangial cells (by overexpressing
PKI) prior to TGF-
treatment attenuates stimulation of fibronectin
mRNA levels supports this hypothesis. The role of other
transcription factors that are regulated by PKA and that bind to the
CRE of the fibronectin promoter, such as ATF-1 and ATF-2, may also be
relevant to TGF-
stimulation of fibronectin gene transcription. In
addition, it is likely that cross-talk among the PKA pathway, the
mitogen-activated protein kinase pathway, and the Smad pathway
participates in mediating specific responses to TGF-
in various cell
types.
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ACKNOWLEDGEMENT |
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We thank Gyorgy Hajnoczky for critical review of this manuscript.
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FOOTNOTES |
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* This work was supported by Grant KO8 DK02308 (to K. S.) from the National Institutes of Health.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.
To whom correspondence should be addressed: Dept. of Medicine,
Div. of Nephrology, Thomas Jefferson University, Suite 353, JAH 1020 Locust St., Philadelphia, PA 19107. Tel.: 215-503-6950; Fax:
215-923-7212; E-mail: sharma1{at}jeflin.tju.edu.
1
The abbreviations used are: TGF-,
transforming growth factor-
; PKA, protein kinase A; CREB, cAMP
response element-binding protein; CRE, cAMP response element; MMCs,
murine mesangial cells; DMEM, Dulbecco's modified Eagle's medium;
PBS, phosphate-buffered saline; PKI, protein kinase A inhibitor.
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REFERENCES |
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