(Received for publication, August 30, 1995)
From the
In response to changes in vascular homeostasis, endothelial
cells secrete endothelin-1 (ET-1), which in turn regulates gene
expression and phenotype in underlying vascular cells. We characterized
a nuclear signaling cascade in which Src protein-tyrosine kinases link
the ET-1 receptor to induction of c-fos transcription. A
dominant negative SrcK- kinase mutant blocked ET-1-stimulated
c-fos transcription. Expression of the COOH-terminal Src
kinase (Csk), which represses Src kinases, also blocked induction of
c-fos transcription by ET-1. Activation of the c-fos promoter by ET-1 required both the CArG DNA sequence of the
c-fos serum response element and the Ca/cAMP
response element. In contrast, Src-induced c-fos transcription
required only the CArG cis-element, demonstrating a divergence
in signals regulating c-fos transcription. Thus, Src kinases
contribute to a nuclear signaling cascade linking an ET-1 receptor to
the CArG element of the c-fos serum response element. A
Src-based pathway might play a more general role to propagate ET-1
nuclear signals that regulate cell growth and development. In addition,
these results point to a widening role for nonreceptor protein-tyrosine
kinases in propagating signals from G protein-coupled receptors.
The function and phenotype of vascular cells is controlled in
part by diffusible, endothelial-derived mediators such as endothelin-1
(ET-1)()(1, 2, 3) . ET-1 is a
21-amino acid vasoconstrictor peptide that also regulates gene
expression and growth of vascular and nonvascular cells in culture (4, 5, 6, 7, 8) .
Accumulating evidence suggests that ET-1 contributes to growth and
compensatory remodeling of the vasculature in vivo(9, 10, 11, 12) , and gene
targeting studies demonstrate a critical role for ET-1 nuclear
signaling in cell differentiation and development (13, 14, 15) . ET-1 binds to heterotrimeric G
protein-coupled receptors and evokes a nuclear signaling cascade that
activates immediate early gene transcription(16) .
Transcription of immediate early genes is thought to convert
ET-1-dependent nuclear signals into phenotypic changes in target cells.
However, the molecular mechanisms linking ET-1 receptors to immediate
early gene transcription remain unclear.
An ET-1 nuclear signaling cascade might involve nonreceptor protein-tyrosine kinases. ET-1 rapidly stimulates pp60 c-Src and pp125 focal adhesion kinase activity, resulting in tyrosine phosphorylation of specific cellular proteins(17, 18, 19, 20, 21) . A role for c-Src in ET-1 signaling is also supported by the observation that ET-1-stimulated phosphoinositide turnover is greatly amplified in v-src-transformed fibroblasts(22) . Protein tyrosine phosphorylation is required for induction by ET-1 of the c-fos immediate early gene(19) , which is one of the earliest genomic responses to ET-1(19, 23, 24) . These observations, and the finding that v-Src elevates c-fos transcription (i.e. see (25) ), suggest that the pathways that propagate ET-1 signals to the nucleus might involve c-Src.
In this study, we investigated whether c-Src or other Src
family kinases participate in a nuclear signaling cascade linking G
protein-coupled ET-1 receptors to the c-fos promoter in
mesangial cells. We report here that ET-1-stimulated c-fos transcription is blocked by expression of a dominant negative
c-Src mutant and by the COOH-terminal Src kinase (Csk), which both
repress c-Src kinase activity. Moreover, full activation of the
c-fos promoter by ET-1 requires c-Src-dependent pathways
regulating the CArG box of the c-fos SRE and c-Src-independent
pathways regulating the Ca/cAMP response element.
Figure 1:
Expression of the dominant negative
SrcK- mutant blocks ET-1-stimulated c-fos transcription. A, schematic representation of the -356wt/fos luciferase (LUC) reporter and expression plasmids transfected into
mesangial cells. Transcription of v-src and c-src inserts is driven by the M-murine leukemia virus long terminal
repeat (LTR), whereas expression of the SrcK- insert is
driven by the SV40 enhancer/promoter. B, left,
mesangial cells transfected with the -356wt/fosLUC reporter (1
µg) with and without the -SrcK- expression plasmid (2 µg)
were rendered quiescent in DMEM, 0.5% FBS. In all experiments promoter
strength was held constant by inclusion of the appropriate amount of
expression plasmid lacking a cDNA insert. As a negative control, cells
were also transfected with 1 µg of the pGL3LUC parent vector
lacking the c-fos promoter fragment. Cells were then left
untreated (- - - ) or were treated with ET-1 (100
nM) or FBS (10%) for 16 h before cell lysis and measurement of
luciferase activity. Data are normalized for -galactosidase
activity and are expressed relative to untreated cells transfected with
-356wt/fosLUC (- - - -). Mesangial cells
were also transfected with a vector expressing
Raf-1 (31) plus and minus the SrcK- plasmid. Right,
mesangial cells transfected with -356wt/fosLUC (1 µg) and
vectors expressing SrcK- (2 µg) or v-Src (2 µg) were
treated as above with ET-1 (100 nM, 16 h). Typical relative
light unit values ranged from 500 to 2000 in 20 µl of lysates from
untreated cells transfected with
-356wt/fosLUC.
For transient transfection
with the wild-type and point mutant -356fosCAT reporters, cells
in 100-mm Petri dishes (1.0 10
cells/plate) were
transiently transfected as described above, but with 10 µg of the
-356fosCAT reporters, 5 µg of pRSV
Gal, and
pUC19 carrier DNA to a total of 30 µg of DNA/plate. Where
indicated, cells were co-transfected with 10 µg of plasmids
expressing either v-Src or c-Src (30) (see Fig. 1A) or treated with ET-1 or FBS as described
above. CAT activity was quantified by liquid scintillation counting of
thin layer chromatography
C spots of the mono- and
diacetylated forms of chloramphenicol and normalized for
-galactosidase activity as described previously(37) .
We used cultured rat mesangial cells in this study to
investigate the potential role of c-Src in activation of the c-fos promoter by ET-1. Mesangial cells derive from the glomerular
microvasculature of the kidney, and ET-1 apparently contributes to the
compensatory growth of mesangial cells and remodeling of glomerular
capillaries following renal injury(8, 9) . Previous
experiments using cultured mesangial cells showed that receptors of the
ET subtype stimulate c-Src activity and tyrosine
phosphorylation of cellular proteins(19, 38) .
Moreover, induction of c-fos mRNA by ET-1 is blocked by
protein-tyrosine kinase inhibitors(19) . These results suggest
that the signaling mechanisms that transduce the ET-1 signal from
receptor to the nucleus are intact in mesangial cells and might involve
c-Src protein-tyrosine kinases (17, 19, 20) .
To determine whether c-Src contributes to c-fos promoter activation by ET-1, we subcloned a genomic DNA fragment containing nucleotides -356 to +109 of the murine c-fos gene into a plasmid to drive transcription of a luciferase gene (Fig. 1A). When this reporter construct (-356wt/fos LUC) was transiently transfected into mesangial cells, ET-1 stimulated a 3.6-fold increase in luciferase activity (Fig. 1B). These results demonstrate that the -356wt/fos LUC reporter contains the necessary cis-elements to confer responsiveness to ET-1. Moreover, transfection with a luciferase vector without the c-fos promoter sequence (Fig. 1B, pGL3LUC) confirmed that the increase in transcription observed with ET-1 was not due to activation of spurious regulatory elements in the luciferase vector.
We next employed a dominant negative mutant strategy (39) to
determine whether c-Src contributes to activation of c-fos transcription by ET-1. A kinase-inactivating mutation in c-Src
(Lys
Met, SrcK-) forms a dominant negative
c-Src protein that blocks signaling by platelet-derived growth factor
and epidermal growth factor in NIH 3T3
fibroblasts(27, 28) . Mesangial cells were
co-transfected with the -356wt/fos LUC reporter and a vector
expressing SrcK- under control of the SV40 promoter/enhancer (Fig. 1A) followed by stimulation with ET-1. The
dominant negative SrcK- mutant prevented the increase in
c-fos transcription in cells stimulated by ET-1 (Fig. 1B, 3.6-fold versus 1.2-fold with
SrcK-). The wild-type SrcK+ construct (2 µg) (27) did not inhibit ET-1-stimulated c-fos transcription (data not shown). SrcK- failed to decrease
transcription in cells transfected with the pGL3LUC control reporter (Fig. 1B), and SrcK- only partially inhibited
c-fos transcription in cells stimulated by fetal bovine serum (Fig. 1B, 6.2-fold versus 4.4-fold with
SrcK-). The ability of SrcK- to inhibit ET-1-stimulated
c-fos transcription suggests a role for c-Src in ET-1 nuclear
signaling.
Two additional experiments confirmed that SrcK- did
not simply produce its blockade by a general inhibition of
transcription. First, mesangial cells were co-transfected with the
-356wt/fos LUC reporter and a vector expressing a constitutively
activated c-Raf-1 mutant (Raf-1) that lacks the 303
NH
-terminal amino acids(31) . c-Raf-1 acts
downstream of c-Src in a signaling pathway that increases c-fos transcription (see (40, 41, 42) for
review), and therefore SrcK- would not be predicted to inhibit
c-Raf-1-induced transcription. Consistent with this prediction,
Raf-1 stimulated a 3.0-fold increase in c-fos transcription that was not inhibited by SrcK- (Fig. 1B). Second, the actions of dominant negative
mutants should be reversible(39) , and we demonstrated that
expression of v-Src ( (30) and Fig. 1A)
reversed inhibition of ET-1-stimulated c-fos transcription by
SrcK- (Fig. 1B). Taken together, these results
strongly suggest that the dominant negative actions of SrcK- are
specific and that c-Src contributes to activation of c-fos transcription by ET-1.
To obtain independent evidence that
c-Src propagates an ET-1 signal to the nucleus, we transfected cells
with a vector expressing COOH-terminal Src kinase (Csk)(29) .
Csk is a protein-tyrosine kinase containing SH2 and SH3 domains that
phosphorylates the COOH-terminal tyrosine (i.e. Tyr) of c-Src and other Src family tyrosine
kinases(43) . Phosphorylation of the COOH-terminal tyrosine
suppresses Src kinase activity, and gene targeting and other studies
demonstrate that Csk negatively regulates Src family tyrosine kinase
activity in
vivo(29, 44, 45, 46) . Csk
expression blocked the increase in c-fos transcription in
cells treated with ET-1 (Fig. 2, 4.1-fold versus 1.2-fold with Csk). Csk only partially inhibited FBS-stimulated
c-fos transcription and had no effect on
-Raf-1-stimulated transcription. These results support the
hypothesis that c-Src or other Src family kinases contribute to an ET-1
signaling cascade that increases c-fos transcription.
Figure 2:
Expression of the Src-inactivating
COOH-terminal Src kinase (Csk) blocks ET-1-stimulated
c-fos transcription. Mesangial cells were transfected with
-356wt/fosLUC as described above plus or minus a plasmid
expressing Csk (1 µg) under transcriptional control of the
cytomegalovirus promoter/enhancer(29) . Cells were then treated
with ET-1 (100 nM, 16 h) or FBS (10%, 16 h) before assay of
luciferase activity. In some experiments cells were transfected with
the vector expressing Raf-1 plus or minus Csk. Data are corrected
for
-galactosidase and normalized to control values with
-356wt/fosLUC alone (- - -
-).
We next asked which cis-elements of the c-fos promoter are activated by the ET-1/Src-based signaling cascade. Plasmids expressing either c-Src or v-Src (Fig. 1A) were co-transfected with a plasmid in which a genomic c-fos promoter sequence (bp -356 to +109) was subcloned upstream of a chloramphenicol acetyltransferase reporter gene (-356wt/fos CAT)(33) . Both c-Src and v-Src increased c-fos transcription (Fig. 3A, 2.4-fold versus 5.2-fold, respectively), demonstrating that this -356 to +109 c-fos promoter fragment contains Src-responsive cis-elements. We therefore transfected mesangial cells with a series of point mutant -356wt/fos CAT reporters(33, 34, 35, 36) . These extensively characterized point mutations prevent binding of cognate trans-acting factors to their respective cis-elements, thereby enabling analysis of the function of specific cis-elements in activation of the c-fos promoter by ET-1 and Src (see Table 1).
Figure 3:
Src-based pathways propagate ET-1 signals
to the CArG element of the c-fos SRE but not to the Ca/CRE. A, mesangial cells in 100-mm Petri dishes were transfected
with the -356wt/fosCAT reporter plasmid and with plasmids
expressing v-Src (10 µg, lane 2) or c-Src (10 µg, lane 3). After transfection, cells were held in DMEM, 0.5% FBS
for 24 h and lysed for measurement of CAT activity (i.e. 48 h
after DNA addition). Radioactive bands corresponding to acetylated and
nonacetylated [H]chloramphenicol were excised
from the thin layer plate and counted to calculate CAT activity.
Activity was corrected for
-galactosidase expression and expressed
relative to values for null transfection (lane 1). Similar
results were observed in three independent experiments. B,
mesangial cells were transfected with point mutant c-fos promoter constructs linked to CAT (10 µg each, Table 1).
As indicated in the legend, cells were also co-transfected with a
plasmid expressing v-Src (10 µg, Fig. 1A) or
treated with ET-1 (100 nM, 16 h) or FBS (10%, 16 h) as
described in the legend to Fig. 1B. These point
mutations are discussed in the text and have been extensively
characterized and the sequences are
published(33, 34, 35, 36) . Mutant cis-elements of the c-fos promoter are indicated
schematically at the left, where a strike-through indicates
that cognate trans-acting factors cannot bind to the point
mutant cis-element. Transfection efficiencies were not always
equivalent between sets transfected with different plasmids, thus the
results were normalized to control values with the identical plasmid.
Standard error bars were omitted for clarity but were never greater
than 15% of the mean value. Abbreviations: SIE, sis-inducible
element; Ets, E26 transformation-specific; CArG,
CA-rich G; SRE, serum response element; FAP,
AP-1/CRE-like element; Ca/CRE, Ca
/cAMP
response element.
Mutation of the
CArG sequence of the c-fos SRE, which binds dimers of serum
response factor, prevented transcriptional activation by ET-1 and v-Src (Fig. 3B, pm12). That FBS-stimulated transcription of
the pm12fos CAT reporter plasmid demonstrated that the mutant construct
was transcriptionally active. A promoter containing point mutations in
the sis-inducible element, AP-1/CRE-like element, and
Ca/CRE (Ca/CRE), but not in the SRE, were not
responsive to ET-1 but were responsive to v-Src (Fig. 3B,
pm3.6.9). Although promoters containing five tandem repeats of the
SRE responded to v-Src, ET-1 failed to stimulate transcription of these
constructs (Fig. 3B, 5
SRE). Point mutation of
the Ets DNA sequence of the c-fos SRE, which recognizes p62
ternary complex factors (i.e. Elk-1 or Sap-1), did not inhibit
c-fos transcription stimulated by ET-1 or v-Src (Fig. 3B, pm18). In mesangial cells, the ability of
v-Src to stimulate c-fos transcription in the absence of
Ets-binding proteins is consistent with our previous observation that
ET-1-stimulated c-fos transcription did not require Ets
DNA-protein interactions (37) and substantiate the apparently
cell type-specific requirement for p62 ternary complex factors in
c-fos promoter
regulation(25, 47, 48, 49) . Taken
together, these results argue that c-fos transcription
stimulated by ET-1 and v-Src requires the CArG DNA sequence of the SRE.
Unlike v-Src, however, DNA sequences in addition to the CArG box are
necessary for ET-1-stimulated c-fos transcription.
Point
mutations in the c-fos Ca/CRE, which binds homo- or
heterodimers of CREB and ATF, block c-fos transcription
stimulated by cAMP and
[Ca]
(36, 50, 51) .
ET-1 does not increase intracellular cAMP in mesangial
cells(52) , but elevation of
[Ca
]
is an important effector
in ET-1 signaling(16) . We therefore tested whether the Ca/CRE
was required for ET-1 and Src nuclear signaling to the c-fos promoter. A c-fos promoter with Ca/CRE point mutations
was not responsive to ET-1 but was stimulated by v-Src (Fig. 3B, pm3). Thus, ET-1 signaling to the c-fos promoter requires the Ca/CRE. Moreover, Src or effectors upstream
of Src represent a point of divergence for nuclear signals stimulating
c-fos transcription in cells treated with ET-1.
We previously demonstrated that a dominant negative Ras mutant (Asn-17-c-Ha-Ras(32) ) blocks activation of the c-fos SRE by ET-1(37) . Because Ras has been reported to be downstream of c-Src in NIH 3T3 cells(53, 54) , we asked whether Asn-17-c-Ha-Ras would block Src-stimulated transcription from the -356 to +109 c-fos promoter fragment in mesangial cells. As expected(37) , Asn-17-c-Ha-Ras inhibited c-fos transcription in cells treated with ET-1 (Fig. 4). Asn-17-c-Ha-Ras also inhibited c-fos transcription stimulated by c-Src (Fig. 4), thereby confirming that Ras lies downstream of Src in this pathway.
Figure 4: A dominant negative Ras mutant (Asn-17-c-Ha-Ras) blocks c-fos transcription stimulated by ET-1 and c-Src. Mesangial cells were transiently transfected with the -356wt/fosLUC reporter plus or minus a plasmid expressing a dominant negative Ras mutant under transcriptional control of the Moloney-murine leukemia virus terminal repeat. As indicated, cells were treated with ET-1 or co-transfected with a vector expressing c-Src as described in the legend to Fig. 1B. Promoter strength was held constant in all experiments by addition of expression plasmids without cDNA inserts.
ET-1 evokes nuclear signaling cascades leading to differential expression of genes that control cell growth and/or differentiation. Previous studies have shown that ET-1 receptors activate nonreceptor protein-tyrosine kinases such as c-Src and focal adhesion kinase, but it is not clear whether nonreceptor protein-tyrosine kinases contribute to nuclear signaling by ET-1. The goal of these studies was to determine whether c-Src or Src family kinases contribute to transcriptional activation of the c-fos immediate early gene by ET-1. Our results, based on inhibition with dominant negative SrcK- mutants and Csk, strongly suggest that c-Src contributes to a nuclear signaling pathway linking ET-1 receptors to c-fos transcription. c-Src appears to participate in a pathway specifically linking ET-1 receptors to the CArG cis-element of the c-fos SRE. Although stimulation of c-fos transcription by ET-1 signaling also requires the Ca/CRE, c-Src does not appear to participate in this limb of the pathway (Fig. 5).
Figure 5:
Schematic representation of the nuclear
signaling pathway linking ET-1 receptors to c-fos transcription in mesangial cells of the glomerular
microvasculature. ET-1 binds to a G protein-coupled ET receptor subtype (19, 38) and a bifurcating
pathway activates the CArG element of the SRE and the Ca/CRE.
ET-1-activated c-Src (or a closely related Src family kinase)
propagates the signal leading from the ET
receptor to the
CArG sequence, which binds SRF, but does not appear to regulate events
occurring at the Ca/CRE. Ras apparently acts downstream of the ET
receptor and c-Src in the pathway leading to CArG activation (see
also (37) ).
As it is possible that
both SrcK- and Csk inhibit other closely related members of the
Src family of protein-tyrosine kinases such as Fyn and
Lyk(27, 28, 44, 46) , we cannot
formally rule out possible involvement of other Src-related kinases in
ET-1 nuclear signaling. However, we have been unable to demonstrate
stimulation of other Src-related kinases by ET-1 in mesangial cells.
For example, mesangial cells contain abundant levels of p62 c-Yes, but
ET-1 does not stimulate c-Yes protein-tyrosine kinase activity. ()We therefore think that it is likely that c-Src
contributes to transduction of the ET-1 signal to the c-fos promoter in these cells.
We demonstrated that full activation of the c-fos promoter by ET-1 also required the Ca/CRE, which binds homo- or heterodimers of CREB and ATF. Point mutations in the Ca/CRE that prevent binding of CREB/ATF (36) blocked ET-1-stimulated c-fos transcription. The dual requirement of the c-fos SRE and Ca/CRE for c-fos promoter activation by ET-1 is similar to nerve growth factor-induced c-fos transcription, which also requires the c-fos SRE and Ca/CRE(58) . Our data are also consistent with recent results by Robertson et al.(59) in transgenic mice, where regulation of c-fos transcription by external stimuli required multiple, interdependent cis-elements in the c-fos promoter.
Our experiments also provide additional support for the importance of ET-1-evoked signals in initiating immediate early gene transcription in vascular and nonvascular cells. The results do not exclude, however, the importance of other second messenger systems to regulate immediate early gene transcription and cell growth following exposure to ET-1. Indeed, we and others (19, 60) have found that protein kinase C is required for mitogenic signaling by ET-1 and for enhancement by ET-1 of EGF-induced transformation. Nonetheless, c-Src activation is an early step following ET-1 receptor activation and is temporally situated to transduce signals leading to long term changes in gene expression and vascular cell phenotype. Src kinases might also contribute to ET-1 nuclear signals that regulate determination of cell fate and development(13, 14, 15) . Finally, these results also point to a widening role for nonreceptor protein-tyrosine kinases in propagating signals from G protein-coupled receptors.