(Received for publication, November 1, 1994; and in revised form, January 3, 1995)
From the
Experiments were designed to clarify the role of several
proteins, junB, retinoblastoma protein (RB), and the
transforming growth factor- (TGF-
) receptors that are
potential intermediates in TGF-
activation of the
2(I)
collagen promoter. Treatment of NIH-3T3 cells with TGF-
increased
the activity of a transiently transfected murine
2(I) collagen
promoter (nucleotides -350 to +54) fused to a luciferase
reporter gene 9-fold. Co-transfection of a junB stimulated the
basal activity of the
2(I) collagen promoter 93-fold,
respectively. Expression of antisense junB RNA attenuated the
effect of TGF-
. Simian virus 40 large T antigen, an inhibitor RB
function, did not prevent TGF-
effects on the
2(I) collagen
promoter. A chimeric receptor containing the extracellular domain of
the colony-stimulating factor-1 receptor and the intracellular domain
of the type I TGF-
receptor enhanced
2(I) collagen promoter
activity 4.8-fold, whereas a similar chimera containing the type II
receptor intracellular domain had much weaker effects. Similar results
were obtained with a plasminogen activator inhibitor-1 promoter,
previously shown to be activated by TGF-
through AP-1 elements. We
conclude that TGF-
activates the
2(I) collagen and
plasminogen activator inhibitor-1 promoters in NIH-3T3 cells through junB and the type I TGF-
receptor kinase domain.
Excessive deposition of extracellular matrix (ECM) ()in progressive renal disease, liver cirrhosis, and lung
fibrosis results in permanent organ dysfunction (1, 2, 3, 4) . One of the major
regulators of ECM deposition in fibrotic disease is transforming growth
factor-
(TGF-
)(1, 2, 3, 4) , a 25-kDa
homodimeric protein that belongs to a superfamily of multifunctional
cytokines(5) . Among the effects of TGF-
that enhance ECM
deposition are increased transcription of the type I collagen genes (6, 7, 8) and a protease inhibitor that
reduces ECM degradation, plasminogen activator inhibitor-1
(PAI-1)(9) . However, the mechanism whereby TGF-
affects
the above genes is incompletely understood. The goal of the present
study was to determine the role of potential intermediates, junB, retinoblastoma protein (RB), and the type I and type II
TGF-
-receptor kinase domains, in TGF-
action on the
2(I)
collagen and PAI-1 promoters.
Binding sites for the transcription
factor AP-1 were classically described as being protein kinase C
response elements(10) . More recently, TGF- has also been
shown to activate AP-1 elements in the PAI-1(11) ,
TGF-
1(12) , TIMP-1(13) , and artificial
promoters(14, 15) . TGF-
induces the expression
of c-fos, junB, and c-jun which bind to AP-1
sites(14, 16, 17, 18, 19, 20, 21) .
Interestingly, junB expression was increased to a greater
extent than c-jun expression by TGF-
in several
reports(14, 16, 17, 18, 19, 20) ,
including two out of three studies in NIH-3T3
cells(14, 20) . junB, which is about 50%
homologous to c-jun(22) , could mediate both the
positive and negative effects of TGF-
on gene
expression(23, 24) . However, junB has not
been previously reported to transactivate the
2(I) collagen
promoter. Rather, the effects of TGF-
on type I collagen promoters
have been attributed to nuclear factor-1(25) , SP1 (26, 27) , and unidentified transcription
factors(26, 28) . Thus, it is of interest to determine
whether junB might be involved in mediating the effects of
TGF-
on the
2(I) promoter.
TGF--mediated growth
arrest of epithelial cells in the G
phase of the cell cycle
is manifested by inhibition of retinoblastoma protein (RB)
phosphorylation(29) . Altered phosphorylation of RB causes it
to release the proteins bound to it, including the transcription factor
E2F-1 (30) and an inhibitor of SP1 (31) . Thus, changes
in RB phosphorylation, in response to TGF-
, could influence the
expression of ECM genes. Experiments utilizing simian virus 40 (SV40)
transformed cell lines, in which RB function is blocked(32) ,
or in cells lacking RB (33) show that TGF-
is still able
to stimulate PAI-1 gene expression. Nevertheless, it is important to
study the role of RB in TGF-
activation of type I collagen
promoters, since TGF-
modulates the activity of these promoters
partly through SP1(26, 27) .
Three TGF-
receptors designated types I-III were originally detected by
I-TGF-
cross-linking and subsequently cloned. The
type III TGF-
receptor is a proteoglycan, betaglycan, that is
involved in ligand presentation(34) . The type I and II
TGF-
receptors, found on most cell types, possess intrinsic
serine-threonine kinase
activity(35, 36, 37) . TGF-
-resistant,
mutant mink lung cells developed by Massague and
colleagues(15) , demonstrate that both the type I and type II
TGF-
receptors are required for TGF-
-mediated changes in
PAI-1 gene expression and growth inhibition. Furthermore, the type II
TGF-
receptor has recently been shown to phosphorylate the type I
TGF-
receptor in response to TGF-
(38) . However,
these experiments do not indicate whether the type I or type II
receptor or both are able to phosphorylate the downstream substrates
that participate in the activation of type I collagen genes by
TGF-
.
We report here that junB is necessary for
TGF- to increase transcription from the a2(I) collagen and PAI-1
promoters and that inhibition of RB function by SV40 large T antigen
did not prevent TGF-
from acting on either promoter. Construction
of chimeric receptors showed that the type I TGF-
-receptor kinase
domain was able to activate both promoters.
Figure 1:
Effect of TGF- on deletion mutants
of the
2(I) collagen promoter. NIH-3T3 cells were transfected with
14 µg of plasmid DNA containing the indicated segments of the
2(I) collagen promoter fused to a luciferase reporter gene along
with a
-galactosidase expression vector (TK
) and then
stimulated with TGF-
(4 ng/ml) or vehicle. Luciferase was assayed
after 21 h in serum-free media and the results expressed relative to
that in cells treated with vehicle for each construct. Each value
(average ± S.E.) represents the results from three independent
experiments. The asterisk indicates luciferase activity from
TGF-
-stimulated cells is statistically greater than the luciferase
activity measured in vehicle-treated cells transfected with the same
construct (Student's t test, p <
0.01).
Figure 2:
Effect of junB on deletion mutants of the
2(I) collagen promoter. NIH-3T3 cells were co-transfected with 14
µg of plasmid DNA containing the indicated segments of the
2(I) collagen promoter fused to a luciferase reporter gene, either
pRcCMV or the junB expression vector pJUNB, and a
-galactosidase expression vector, TK
. Luciferase was assayed
after 21 h in serum-free media and the results expressed relative to
each construct transfected with the pRcCMV vector. Each value (average
± S.E.) represents the results from three independent
experiments. The asterisk indicates luciferase activity from
pJUNB transfected cells is statistically greater than the luciferase
activity measured in pRcCMV-transfected cells that were also
co-transfected with the same
2(I) collagen reporter gene construct
(Student's t test, p <
0.05).
Figure 3:
Lack
of inhibition of TGF- effects on the
2(I) collagen and PAI-1
promoters by SV40 large T antigen. A, NIH-3T3 cells were
co-transfected with 14 µg of pH 6 (nt -350 to +54 of the
2(I) collagen promoter fused to luciferase), either pSG5,
expression vectors for SV40 T antigen, pTAG, or a non-RB binding SV40 T
antigen mutant (pMUTTAG), and a
-galactosidase expression vector
(TK
or CMV
). The cells were stimulated with TGF-
(4
ng/ml) for 21 h in serum-free media and luciferase measured. The
results are expressed relative to the luciferase activity in
vehicle-treated cells transfected with pSG5. Each value (average
± S.E.) represents the results from three independent
experiments. The asterisk indicates luciferase activity from
TGF-
-stimulated cells is statistically greater than the luciferase
activity measured in vehicle-treated cells transfected with the same
constructs (Student's t test, p < 0.01). B, similar experiments were performed with pPAI-1 (nt
-699 to +54 of the PAI-1 promoter fused to
luciferase).
Figure 4:
Comparison of the function of the type I
and type II TGF- receptor intracellular domains. NIH-3T3 cells
were co-transfected with 6 µg of pH 6 (nt -350 to +54 of
the
2(I) collagen promoter fused to a luciferase reporter gene) or
pPAI-1 (nt -699 to +54 of the PAI-1 promoter fused to a
luciferase reporter gene), 7 or 14 µg of either pCHIMONE, a chimera
containing the extracellular and transmembrane domains of the CSF-1
receptor, and the type I TGF-
-receptor kinase domain, pCHIMTWO a
chimera containing the type II TGF-
-receptor kinase domain or pKN,
a chimera containing a kinase-negative mutant of the type I TGF-
receptor intracellular domain, and 5 µg of the
-galactosidase
expression vector TK
. Luciferase was measured after 21 h in
serum-free media, and the results are expressed relative to pH 6 or the
PAI-1 promoter co-transfected with a similar amount of pKN. Each value
(average ± S.E.) represents the results from three independent
experiments. The asterisk indicates luciferase activity from
pCHIMONE- or pCHIMTWO-transfected cells is statistically greater than
the luciferase activity measured in pKN-transfected cells that were
also co-transfected with the same reporter gene construct
(Student's t test, p <
0.05)
Expression vectors were prepared by ligating the relevant cDNA into a blunt-ended HindIII site of pRcCMV (Invitrogen, San Diego, CA). The cDNAs for pJUN, pJUNB, pFOS, and pTAG were obtained as follows: a 2.6-kilobase EcoRI c-jun cDNA from pJAC.1 (44) (ATCC), a 1570-base pair HindIII-EcoRI junB cDNA fragment from ATCC clone 465.20(22) , a 2.1-kilobase EcoRI rat c-fos cDNA from pc-fos (45) , a kind gift of Dr. Peter Maxwell and Dr. Tom Curran, and a 2.9-kilobase BamHI SV40 T antigen from pBRSV (ATCC). pREVJUN, pREVJUNB, and pREVFOS express c-jun, jun B, and c-fos cDNA in the reverse orientation, respectively. For pREVFOS c-fos cDNA was amplified by PCR from a murine fibroblast cDNA library (Stratagene, La Jolla, CA).
Site-directed mutagenesis was performed as
described(39) . Mutant sequences were confirmed by dideoxy
sequencing with a Sequenase kit (United States Biochemical, Cleveland,
OH).The following mutations were produced: pNFN, in which 5`-TGGCA-3`,
bases -305 to [minus[301 from the murine 2(I)
collagen promoter, was changed to 5`-TTTAA-3`; pMUTTAG, in which lysine
107 from SV40 large T antigen was changed to glutamine(46) ;
pKN, in which lysine 232 from the ALK5 (35) type I
TGF-
-receptor kinase domain incorporated in the chimeric receptor
described below was changed to arginine; and pMUTPLA2, in which
serine-505 of murine cPLA
(47) (a generous gift of
J. Knopf, The Genetics Institute, Cambridge, MA) was changed to
alanine.
pCSF was created by ligating cDNA encoding the
extracellular and transmembrane domains of the murine colony
stimulating factor-1 (CSF-1) receptor (48) amplified by PCR
from a murine fibroblast cDNA library (Stratagene) into the EcoRI and BglII sites of the expression vector, pSG5
(Stratagene). A BamHI site was included in the 3` end of the
CSF-1 cDNA. To create pCHIMONE and pCHIMTWO, cDNA encoding the
intracellular domain of the type I TGF- receptor, ALK-5 (also
called R4)(35, 37) , and the type II receptor (36) , respectively, were amplified by PCR from a human
placenta cDNA library (Stratagene) and cloned into the BamHI
site of pCSF.
Exposure of NIH-3T3 cells to TGF- enhanced
luciferase activity driven by the -350 to +54 segment of the
2(I) collagen promoter and two smaller segments (-224 to
+54 and -108 to +54) 9-, 5-, and 5-fold, respectively (Fig. 1), consistent with previous reports on type I promoters (25, 26, 27, 28) and nuclear run-off
studies of the type I collagen
genes(6, 7, 8) . The specificity of the
response is shown by the minimal effect of TGF-
on the -41
to +54 segment of the
2(I) collagen promoter (Fig. 1).
Furthermore, TGF-
did not increase the activity of the thymidine
kinase promoter linked to
-galactosidase that was used to control
for transfection efficiency. Thus, TGF-
affected two regions of
the
2(I) collagen promoter, -350 to -224 and
-108 to -41.
To determine the role of junB,
c-jun, and c-fos in TGF- signaling, we
co-transfected the
2(I) collagen promoterreporter gene noted above
with vectors directing the expression of these transcription factors in
the sense or the antisense orientation. As depicted in Table 1,
co-expression of c-jun or junB with the
2(I)
collagen promoter resulted in 4- and 93-fold increases in luciferase
activity, respectively. TGF-
did not increase the activity of the
2(I) collagen promoter co-transfected with the junB expression vector further. Expression of c-fos did not
affect the basal activity of the
2(I) collagen promoter, but
augmented the effect of TGF-
from 3.8- to 6.2-fold.
Antisense
RNA effectively reduces the expression of endogenous genes (50) . The results of experiments aimed at inhibiting junB expression are summarized in Table 1. Co-transfection of an
expression vector for antisense junB RNA diminished the action
of TGF- on the
2(I) collagen promoter from 3.8- to 1.2-fold (Table 1). Expression of antisense c-jun or c-fos RNA had no effect. Luciferase activity driven by the
2(I)
collagen promoter was elevated 11-fold by expression of antisense junB, but only 1.8-fold by expression of antisense c-jun RNA. This effect is discussed below. To evaluate for nonspecific
effects, an inactive mutant of cPLA
, in which serine 505
was mutated to alanine(48) , was co-transfected. It had no
effect on basal or TGF-
-stimulated expression of the reporter gene (Table 1). Similar results were obtained with the human PAI-1
promoter (nucleotides (nt): -699 to +64), except that junB had a slightly smaller effect (53-fold) and c-jun had a greater effect (22-fold) (Table 1).
The effect of junB on several deletion mutants of the 2(I) collagen
promoter was tested. As shown in Fig. 2, co-transfection of junB increased activity of the -350 to +54,
-224 to +54, -108 to +54, and -41 to
+54 segments of the
2(I) collagen promoter 50-, 62-, 22-, and
2.7-fold, respectively. Thus, the effects of junB are specific
and involve one of the segments, (-108 to -41), of the
2(I) collagen promoter that is affected by TGF-
.
It was
previously reported that a 3-base pair mutation in an nuclear
factor-1-binding site in the murine 2(I) collagen promoter
abolished TGF-
effects on this promoter(25) . We tested
the same mutation under the present experimental conditions. In
accordance with previous results, the 3-base pair mutation reduced
basal promoter activity to 26%, but the mutation did not alter the
effect of TGF-
on the
2(I) collagen promoter (not shown).
We have shown that junB is a mediator of TGF-
action on the murine
2(I) collagen promoter. First, junB or c-jun transactivated the
2(I) collagen reporter
gene. Consistent with a role for junB in TGF-
action,
expression of c-fos augmented the effect of TGF-
from
3.8- to 6.2-fold, presumably by dimerizing with junB (Table 1). Importantly, expression of antisense junB RNA, but not antisense c-jun or c-fos RNA,
diminished the effect of TGF-
(Table 1). The relevance of
the effects of junB on the
2(I) collagen promoter are
further reinforced by the similar results obtained when a promoter
previously shown to be regulated by TGF-
through AP-1 sites that
can bind junB, the PAI-1 promoter, was tested (Table 1).
The results of the present study do not distinguish between a
requirement for new junB transcription versus dephosphorylation and activation of pre-existing junB.
Cycloheximide, a protein synthesis inhibitor, abolishes the effect of
TGF- on type I collagen mRNA levels in some
studies(6, 51) , but not
others(52, 53) . These observations suggest that
depending on the experimental conditions, TGF-
is able to exert
its effects by increasing the synthesis of new transcription factors,
such as junB, or by modifying pre-existing transcription
factors. By analogy with other agonists, it is possible that TGF-
may activate junB by dephosphorylating it near its DNA-binding
domain (54) .
Other factors, in addition to junB,
may be involved in TGF- activation of the
2(I) collagen
promoter. The effects of c-jun and junB on the
2(I) collagen promoter may be indirect, since the murine
2(I)
collagen promoter fragment tested has no consensus AP-1 sites,
unlike the PAI-1 promoter. Furthermore, a role for other transcription
factors, in addition to junB, in mediating the effects of
TGF-
is not excluded by the present experiments.
Phorbol esters
(activators of protein kinase C) and platelet-derived growth factor
(PDGF) are known to induce the expression of both c-jun and junB(55) but not the type I collagen genes. Addition
of phorbol esters or epidermal growth factor, which also activates a
receptor tyrosine kinase like PDGF, to fibroblasts inhibits type I
collagen gene expression(56, 57) . Therefore, the
ability of PDGF or phorbol esters to elevate c-jun and junB may be opposed by other effects that inhibit 2(I)
collagen gene expression. Alternatively, TGF-
may provide a second
signal to the
2(I) collagen gene, in addition to altered junB activity, which is not available to cells exposed to PDGF or
phorbol esters.
Expression of antisense junB RNA increased
2(I) collagen or PAI-1 promoter activity. This paradoxical effect
may be explained by the release of c-jun from inactive
c-jun-junB heterodimers, when the level of junB is reduced. The cells used in the present experiments probably
contained significant levels of c-jun and junB, since
they were serum starved for only 3 h prior to the addition of
TGF-
. However, the effect of antisense junB was larger than that
of expressing c-jun indicating there is an additional unexplained
effect of expressing junB cDNA.
The TGF-1 gene
contains a functional AP-1 site in its promoter(12) . However,
the effects of c-jun and junB are not likely to be
due to increased release of active TGF-
from NIH-3T3 fibroblasts.
Most cells release TGF-
in a latent form that requires activation
by proteases(40) . In particular, NIH-3T3 fibroblasts
transformed by p21
were found to secrete only latent
TGF-
(58) . Furthermore, mannose 6-phosphate, which has
previously been shown to inhibit TGF-
processing, was included in
the cell culture media during the transfections(40) .
The
present results differ from some previous reports on the 2(I)
collagen promoter. A mutation in the
2(I) collagen promoter
nuclear factor-1 site around -300, previously described to
eliminate the effect of TGF-
(25) , did not prevent
TGF-
activation of the
2(I) collagen promoter under the
present experiments. We observed a 2-fold decrease in the effect of
TGF-
when the
2(I) collagen promoter was deleted from
-350 to -224 to +54 consistent with a recent report on
the human
2(I) collagen promoter(26) . However, we also
detected an additional effect of TGF-
in the -108 to
-39 region, which appeared to be mediated through junB.
The discrepancies between the present data and previous results with
the
2(I) collagen promoter are probably due to differences in the
experimental conditions and the cell types studied.
The results in Fig. 3demonstrate that inhibition of RB function does not block
TGF- action on the
2(I) collagen or the PAI-1 promoters.
Indeed, the enhancement of
2(I) collagen and PAI-1 promoter
activity by SV40 T antigen, but not a non-RB-binding SV40 large T
antigen mutant, raises the possibility that RB may tonically inhibit
these promoters, perhaps by modifying SP1 activity(31) . Since
SV40 large T antigen has many potential effects, further experiments
will be required to settle this point.
This is the first report
concerning an active chimera between the extracellular domain of a
tyrosine kinase receptor and the intracellular domain of a
serine-threonine kinase receptor. The results indicated that the type I
TGF--receptor kinase domain is sufficient to convey the TGF-
signal to the
2(I) and PAI-1 promoters (Fig. 4). A similar
chimera containing the kinase domain from the type II TGF-
receptor had a lesser effect and was active only when large amounts
were transfected. The recently reported phosphorylation of the type I
TGF-
receptor in response to TGF-
(38) and the
apparent determination of the specificity of responses to TGF-
family members by the type I rather than the type II TGF-
receptor (38) are consistent with the present experiments. However, the
data presented here do not contradict the observed requirement for
intact type I and type II TGF-
receptors in vivo as
demonstrated by TGF-
signaling in mutant cell lines(15) .
Under normal circumstances, the type II TGF-
receptor is needed
for ligand binding to the type I receptor and to phosphoylate the type
I receptor(38) . In fact, the increased activation of the a2(I)
collagen or PAI-1 promoter noted when both chimeric receptors were
transfected together could be due to phosphorylation of the type I
receptor by the type II receptor(38) ,
In summary, the
results we obtained suggest that TGF- action on the
2(I)
collagen and PAI-1 promoters involves the type I TGF-
-receptor
kinase domain and junB, but not changes in RB function.