From the
Transforming growth factor-
TGF-
The human lung carcinoma cell line, A549, was obtained from
the American Type Culture Collection and grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal calf
serum and at 37 °C and 5% CO
For Northern blotting and analysis of steady-state mRNA
levels for PAI-1 and fibronectin (FN), confluent cultures of A549 cells
were treated with the various test agents for either 3 h (PAI-1) or 5 h
(FN). Total cellular RNA was extracted using the Trizol reagent (Life
Technologies, Inc.) following the manufacturer's protocols.
Fifteen micrograms of total cellular RNA were fractionated on 1%
agarose gels containing formaldehyde followed by transfer to Boehringer
Mannheim nylon membranes. The membranes were hybridized to
A549 human lung carcinoma cells are growth-inhibited by
TGF-
Following transfection, A549 cells were placed into serum-free DMEM
and treated with 100 pM TGF-
Simultaneous treatment of
transfected cells with increasing concentrations of the PKC inhibitor,
staurosporin (Fig. 2A), and 100 pM TGF-
The observation that specific
antagonists can block the expression of a transgene in response to
TGF-
TGF-
We thank Dr. Joan Massagué for generously
providing the p3TP-Lux plasmid. We thank Cindy Richard for assistance
in preparation of this manuscript and Dr. Gary Stein for critical
reading and discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TGF-
) is a
multifunctional peptide that elicits a wide variety of responses in
cells. TGF-
binds to cell surface receptors that contain
cytoplasmic serine/threonine kinase domains. Here we provide evidence
that both phospholipase C and protein kinase C (PKC) are involved in
the TGF-
activation of transcription and luciferase expression
from the p3TP-Lux plasmid. Down-regulation of PKC prevents TGF-
1
induction of luciferase expression. Staurosporin and Calphostin C,
inhibitors of PKC, block the ability of TGF-
1 to initiate
transcription of the luciferase gene. Further, D609, an inhibitor of
phosphatidylcholine-phospholipase C (PC-PLC), and secondarily PKC also
blocks TGF-
1-induced transcription of the transgene in A549 cells
while the phosphatidylinositol-PLC pathway inhibitor U73122 is without
effect. TGF-
elevates steady-state mRNA levels for the endogenous
PAI-1 and fibronectin genes. Treatment of cells with calphostin C or
D609 prevents the TGF-
-induced increase in these mRNAs. Together,
these results suggest that PC-PLC and PKC are in a TGF-
signaling
pathway that results in elevated gene expression.
(
)
has been implicated in
activities ranging from embryogenesis and differentiation to promoting
wound healing in vivo. In vitro, TGF-
enhances
extracellular matrix production as well as cell adhesion, inhibits the
proliferation of epithelial and hematopoietic progenitor cells, acts as
an immunosuppressive agent, and modulates the differentiation of a
variety of cell types including adipocytes, keratinocytes, myoblasts,
and hematopoietic progenitors
(1, 2) . Growth suppression
has been correlated to down-regulation of c-myc expression
(3) , blockade of the phosphorylation of pRb
(retinoblastoma gene product) in late G
(4) , and
inhibition of cyclin-dependent
kinases
(5, 6, 7) . However, the steps between
receptor binding and these nuclear events remain obscure. TGF-
initiates responses through binding to cell surface receptor proteins
(type I and type II receptors) that contain serine/threonine kinase
motifs in their cytoplasmic domains
(8) . This implies initiation
of a phosphorylation cascade as early steps in receptor signaling.
Several lines of evidence indicate that type I and type II receptors
form heterodimeric signaling
complexes
(9, 10, 11) . Recently, the
Massagué laboratory
(12) reported that TGF-
binds to
the type II receptor, which then recruits and phosphorylates the type I
receptor on its cytoplasmic domain. The type II receptor appears to be
constitutively phosphorylated. In addition, homodimers of type II
receptors have been observed in the absence and presence of
TGF-
(13) . The nature and identity of additional
intermediates in the early postreceptor signal transduction pathways
utilized by TGF-
remain to be elucidated. Here we provide evidence
that both phosphatidylcholine-specific phospholipase C (PC-PLC) and
protein kinase C (PKC) may be early intermediates in TGF-
signaling pathways that lead to activation of gene transcription.
. Porcine TGF-
1 was
obtained from R& Industries, Minneapolis, MN. Pathway inhibitors
and toxins were obtained from Calbiochem with the exception of U73122,
which was from Biomol Inc., Plymouth Meeting, PA. Luciferase assay kit
was purchased from Promega, Madison, WI. Luciferase activity was
measured using an Analytical Luminescence Laboratory Monolight 2010
luminometer. Emitted light was measured over a 30-s interval.
Chloramphenicol acetyltransferase (CAT) activity was assessed by thin
layer chromatography and scanning of plates on a Betagen Betascope 603
blot analyzer. For transfection, A549 cells were plated at 1.5
10
cells/100-mm plate and allowed to grow overnight. The
next morning cells were co-transfected with 15 µg/plate of the
p3TP-Lux plasmid and 2 µg of pCAT-control plasmid (Promega) via the
DEAE-dextran method
(14) . Briefly, cells were washed 3 times
with phosphate-buffered saline followed by incubation with plasmid DNA,
100 µg/ml DEAE-dextran, and 4 µM chloroquine in
serum-free DMEM. After a 3-h incubation, cultures were treated with 10%
glycerol in serum-free DMEM for 90 s followed by washing with
phosphate-buffered saline. Transfected cells were allowed to recover
for 24 h prior to induction. All subsequent treatments were done in
serum-free DMEM supplemented with 100 pM TGF-
1 and
various inhibitors. Cells were extracted in reporter lysis buffer
(Promega) to permit analysis of both luciferase activity and CAT
activity.
P-labeled probes for human PAI-1 or rat fibronectin. The
PAI-1 probe (PAI-1 oligonucleotide probe from Oncogene Sciences) was
end-labeled with
P using T4 polynucleotide kinase. The FN
cDNA probe was labeled by the random prime method using a kit from
DuPont NEN.
(15). We have used this cell line to begin to address
TGF-
signal transduction. A TGF-
-responsive promoter linked
to the luciferase gene, the p3TP-Lux plasmid, obtained from Dr. Joan
Massagué
(10) was utilized as a marker for activation of
gene expression. The p3TP-Lux plasmid consists of the TGF-
response element from the PAI-1 gene and three tandemly arranged
phorbol ester response elements (TREs) placed 5` to the luciferase
gene. This plasmid is responsive to both TGF-
and phorbol esters.
1. A kinetic analysis
(Fig. 1A) of the response of the transgene to TGF-
1
indicates a rapid initiation in transcription with an approximately
2-fold elevation in luciferase activity within 1 h. Luciferase activity
continued to rise to 8-10-fold at 6 h and approximately
20-40-fold at 18-20 h (not shown).
Figure 1:
A, kinetic analysis of
TGF- response. Transfected A549 cells were transferred to
serum-free DMEM supplemented with 150 pM TGF-
1. At the
indicated times cells were harvested and processed for luciferase
analysis and CAT activity using the Promega kit according to the
manufacturer's protocols. The results shown are the average of
triplicate cultures and normalized for CAT activity and protein
content. TGF-
induces a rapid (within 2 h) elevation in luciferase
expression that continues to rise to approximately 10-fold by 6 h and
over 20-fold by 18 h (not shown). B, down-regulation of PKC
blocks TGF-
activation. Transfected A549 cells were treated with
200 ng/ml PMA (in DMEM with 10% fetal bovine serum) for 40 h to
down-regulate PKC. The medium was replaced with serum-free DMEM
supplemented with 100 pM TGF-
1 or 100 ng/ml PMA. After a
3-h incubation the cells were harvested, extracts were prepared, and
luciferase activity was determined. The results shown represent the
average of triplicate samples and are normalized for protein content.
Both TGF-
1 and PMA induce a 3-5-fold induction of luciferase
activity in parallel control cultures, but following PKC
down-regulation, neither TGF-
1 nor PMA could induce expression of
the transgene.
To address the role
of PKC in TGF- signaling, transfected A549 cells were treated with
200 ng/ml phorbol myristate acetate (PMA) for 40 h to down-regulate
PKC
(16) . For induction, medium was replaced with serum-free
DMEM alone or supplemented with 100 pM TGF-
1 or 100 ng/ml
PMA. Following a 3-h incubation, cell extracts were prepared and
assayed for luciferase activity. Fig. 1B shows that in
control cultures in which PKC was not down-regulated both TGF-
1
and PMA induce elevated luciferase activity. In contrast, following
down-regulation of PKC, neither TGF-
1 nor PMA was capable of
inducing expression of the transgene.
1
result in a nearly complete inhibition of luciferase activity by the
highest concentration of staurosporin tested. Half-maximal inhibition
was observed at approximately 50 nM staurosporin. A second
inhibitor of PKC, calphostin C, also inhibited the TGF-
response,
although a higher concentration was required to obtain maximal
inhibition. Calphostin C binds to the regulatory subunit of PKC while
staurosporin appears to interact with the ATP-binding site (17, 18).
This difference in mode of action or differences in drug stability and
half-lives may account for varied effectiveness of the drugs. The
ability of PKC inhibitors to block TGF-
1-induced gene expression
together with the lack of response following down-regulation of PKC
suggests that protein kinase C is a likely early downstream mediator of
TGF-
responses, which result in altered gene expression.
Figure 2:
Antagonists of protein kinase C inhibit
TGF- response. p3TP-Lux-transfected cells were treated
simultaneously with 100 pM TGF-
1 and the indicated
concentrations of either staurosporin (A) or calphostin C
(B). After 3 h, cells were harvested and processed as
described in the legend to Fig. 1. The results shown are the average of
determinations made from triplicate cultures. The dashedlines indicate the level of luciferase activity in
uninduced cultures.
Diacylglycerol (DAG), the endogenous activator of PKC, is generated
by the hydrolysis of phospholipids. We examined the ability of a newly
described inhibitor of phospholipase C, D609, to block
TGF-'s ability to activate transcription of the luciferase
gene. D609 blocks PC-PLC
(19) and secondarily inhibits the
activation of PKC by preventing the generation of DAG.
p3TP-Lux-transfected A549 cells were preincubated in serum-free medium
with the indicated concentrations of D609 for 1 h. 100 pM
TGF-
1 was added, and the incubation was continued for an
additional 4 h after which time cells were harvested, processed, and
assayed for luciferase activity. The results shown in Fig. 3A indicate that D609 treatment resulted in half-maximal inhibition
at 6-7 µg/ml and complete inhibition of TGF-
-activated
transcription of the transgene at 25-50 µg/ml.
U73122
(20) , an inhibitor of phosphatidylinositol-PLC-mediated
responses, was incapable of blocking TGF-
-activated luciferase
expression (Fig. 3B). We have consistently observed that
U73122 treatment can augment the TGF-
induction of luciferase.
Thus, direct inhibition of protein kinase C via either down-regulation
or staurosporin or calphostin C treatment as well as indirect
inhibition of protein kinase C via D609 block TGF-
1-induced
transcription from the p3TP-Lux plasmid. These results imply that both
PC-PLC and PKC are important intermediates in the generation of a
signal by TGF-
.
Figure 3:
D609, a phosphatidylcholine-specific
phospholipase C inhibitor blocks TGF- responsiveness. A,
transfected A549 cells were pretreated for 1 h in serum-free DMEM with
the indicated concentrations of D609. 100 pM TGF-
1 was
added and the incubation continued for an additional 4 h. The cells
were harvested, extracted, and assayed for protein content and
luciferase activity as described under ``Materials and
Methods.'' The results shown are the average of triplicate
cultures and indicate that D609 completely inhibits the TGF-
effect. The dashedline indicates the level of
luciferase activity in uninduced cultures. B, transfected A549
cells were treated with the indicated concentrations of U73122 and 100
pM TGF-
1. Following a 4-h incubation, the cultures were
harvested and assayed for luciferase and CAT activity. The results are
the averages of duplicate cultures.
The above results are in contrast to the
results obtained using inhibitors of G proteins in this assay. Neither
cholera toxin nor pertussin toxin was capable of inhibiting TGF-1
activation of transcription of the p3TP-Lux plasmid (not shown).
Cholera toxin inhibits G
while pertussis toxin inhibits
G
, both regulators of adenylate cyclase activity. We
conclude that PC-PLC and protein kinase C are important intermediates
in TGF-
signaling pathways, but toxin-inhibitable G proteins
(G
and G
) are not essential for TGF-
activation of transgene expression in A549 cells. Others have reported
that these G proteins may be involved in TGF-
stimulation of
proliferation of AKR-2B fibroblasts
(21, 22, 23) suggesting that different cell types and responses may
utilize different signaling pathways.
can be extended to endogenous genes encoding PAI-1 and
fibronectin. Expression of both genes as measured by steady-state mRNA
levels is elevated by TGF-
1 treatment. Co-treatment with
TGF-
1 and either calphostin C or D609 blocks the elevation in
PAI-1 and fibronectin mRNA levels (Fig. 4). Thus, one pathway
utilized by TGF-
to enhance expression of extracellular matrix
genes includes PC-PLC and PKC.
Figure 4:
Endogenous PAI-1 and fibronectin gene
expression also inhibited. Examination of steady-state mRNA levels by
Northern blotting reveals that both calphostin C and D609 inhibit the
ability of TGF-1 to elevate mRNA levels for PAI-1 and fibronectin.
A, A549 cells were incubated for 3 h in serum-free DMEM
containing test agents. Total cellular RNA was isolated and
fractionated on 1% agarose gels containing formaldehyde and processed
from Northern blotting and detection of PAI-1 mRNA. Lanes:
1, untreated; 2, 100 pM TGF-
1;
3, 100 pM TGF-
1 + 200 nM
calphostin C; 4, 100 pM TGF-
1 + 20
µg/ml D609. Following development of the autoradiograph, the blot
was stripped in boiling dH
O and reprobed for
glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA to assess
uniformity of RNA between samples. B, A549 cells were treated
as above but for 5 h. Following electrophoresis, the agarose gel was
incubated in 50 mM NaOH prior to transfer to facilitate
efficient transfer of the 7-8-kilobase FN mRNA. The NaOH
treatment likely accounts for the diffuse appearance of the signals for
FN mRNA and glyceraldehyde-phosphate dehydrogenase
mRNA.
Receptor tyrosine kinases also
interact with PLC isoforms. These receptors activate
phosphatidylinositol-PLC such as PLC-
(24) . Hydrolysis of
phosphatidylinositol generates DAG, which activates PKC but also
produces IP
(inositol 3-phosphate), which stimulates
release of Ca
by binding to IP
receptors.
Receptor tyrosine kinases through PLC-
are capable of activating
PKC and affecting Ca
-sensitive enzymes and pathways.
PC-PLC hydrolyzes PC to yield DAG and phosphocholine
(25) ; thus,
while PKC is activated, the absence of IP
would not result
in release of Ca
. PC-PLC has recently been shown to
be important in signaling by tumor necrosis factor
receptors and
activation of the transcription factor
NF-
B
(19, 26) . PC-PLC may also be important in the
mitogenic response to platelet-derived growth factor and in
ras-transformed cells
(27, 28) . Diaz-Meco
et al.(29) have suggested that TGF-
may prevent
the coupling of ras p21 to PC hydrolysis, thus contributing to
the antiproliferative response of TGF-
1.
utilizes
multiple signaling pathways to generate the diversity of responses
observed. For example, activation of transcription may involve a
different pathway than growth inhibition. Others have reported that
TGF-
rapidly activates the ras protooncogene
(30) and MAP kinase
(31) . ras activity may be
required for PC-PLC activation
(32) , and recently it was
reported that MAP kinase may be in a pathway downstream of
PKC
(33) . Selective activation of pathways by TGF-
may be
cell type-dependent. Last, overlap exists between signaling pathways
utilized by TGF-
receptors and receptor tyrosine kinases.
, transforming growth factor-
; DMEM, Dulbecco's
modified Eagle's medium; PC-PLC, phosphatidylcholine-specific
phospholipase C; PKC, protein kinase C; CAT, chloramphenicol
acetyltransferase; PAI-1, plasminogen activator inhibitor-1; FN,
fibronectin; PMA, phorbol myristate acetate; DAG, diacylglycerol;
IP
, inositol 3-phosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.