(Received for publication, April 13, 1995; and in revised form, July 5, 1995)
From the
In previous work we suggested that a kidney-specific
transcription factor LFB3 cooperates with cAMP-response element
(CRE)-binding proteins within a cAMP regulatory unit comprised of three
protein-binding domains and located 3.4 kilobase pairs upstream of the
urokinase-type plasminogen activator (uPA) gene in LLC-PK cells (Menoud, P.-A., Matthies, R., Hofsteenge, J., and Nagamine,
Y.(1993) Nucleic Acids Res. 21, 1845-1852). The two
domains contain a CRE-like sequence, and the third domain is recognized
by LFB3. The absolute requirement of LFB3 as well as the cooperation
among the three domains for cAMP regulation were confirmed by transient
transfection assays in F9 teratocarcinoma cells, in which the level of
LFB3 was negligible. Suspecting a possible feedback regulation of LFB3
mRNA expression during cAMP-dependent uPA gene induction in
LLC-PK
cells, we measured LFB3 mRNA levels after cAMP
treatment and found a strong reduction. This reduction was not due to a
change in template activity of the LFB3 gene because run-on
transcription showed no significant change in LFB3 gene transcription.
RNA synthesis inhibitor-chase experiments indicated that the
down-regulation was post-transcriptional. Interestingly, when the
inhibitor was added at the same time as cAMP, the cAMP-induced decrease
in LFB3 mRNA levels was abrogated, suggesting that on-going RNA
synthesis is required for the decrease. Similar effects on LFB3 mRNA
metabolism were observed with all agents that induce uPA mRNA in
LLC-PK
cells, including
12-O-tetradecanoylphorbol-13-acetate, okadaic acid,
colchicine, and cytochalasin. We discuss the significance of this
regulation in uPA gene expression.
Signal transduction, a process of successive activation of various molecules, is subject to various levels of regulation. In many cases, for the sake of homeostasis, activated molecules are sequestered from the pathway by desensitization of membrane-bound receptors(1, 2) , degradation of activated molecules(3, 4) , inactivation of activated molecules by dephosphorylation(5, 6) , or by a feedback mechanism(7) . Cross-talk between different signaling pathways is also an important mechanism for bestowing flexible and versatile regulation on a given pathway. This can be either positive or negative and occurs at various steps in the pathway in a cell-specific manner (for reviews, see (8, 9, 10) ). Therefore, in addition to the identification of successively activated components of a signaling pathway and the elucidation of the mechanism of activation of each component, it is also very important to know how the activity of each component is modulated by molecules not immediately upstream in the pathway. In this way, the nature of a signaling pathway may be understood in a more physiologically relevant context.
We have been
studying urokinase-type plasminogen activator (uPA) ()gene
regulation in LLC-PK
cells, a cell line derived from pig
kidney epithelia(11) . In these cells, the uPA gene is induced
through independent signaling pathways by various signals such as
cAMP(12) , 12-O-tetradecanoylphorbol-13-acetate
(TPA)(13) , the protein phosphatase 1/2A inhibitor okadaic
acid(14, 15) , and cytoskeletal
reorganization(13, 16) . The pig uPA gene has a
cAMP-inducible enhancer located 3.4 kb upstream of the transcription
start site(12) . This enhancer is comprised of three
protein-binding domains, A, B, and C. Domains A and B contain a core
sequence of the cAMP response element (CRE) but require the adjoining C
domain to confer full cAMP responsiveness on a heterologous
promoter(12, 17) . The C domain has no CRE and cannot
mediate cAMP responsiveness when used in isolation. We have purified
the protein binding to the C domain (17) and found it to be the
pig equivalent of mouse LFB3(18) . It is also known as
HNF1
(19) or vHNF1(20) . LFB3 is a
tissue-specific transcription factor highly expressed in kidney cells (18) with a structure closely related to the liver-specific
transcription factor HNF1
. Both HNF1
and LFB3 recognize the
same DNA sequence, at least in
vitro(17, 21) , although the domain C sequence is
quite different from the consensus HNF1
recognition sequence. It
is still not known which genes besides the uPA gene are the targets of
LFB3 in kidney cells, or how the expression of LFB3 is regulated. As
LFB3 is apparently involved in cAMP-dependent uPA gene regulation in
LLC-PK
cells, we were interested to know whether
cAMP-evoked signaling affected the expression of LFB3 in these cells.
Indeed, we have shown that cAMP treatment strongly reduces LFB3 mRNA
levels, suggesting a feedback mechanism via LFB3 in cAMP-dependent uPA
gene regulation in LLC-PK1 cells(17) . In the present study, we
verify the involvement of LFB3 in cAMP-induction of the uPA gene and
show that not only cAMP but also other agents that induce uPA gene
expression strongly reduce the amount of LFB3 mRNA. These agents are
12-O-tetradecanoylphorbol-13-acetate, okadaic acid,
colchicine, and cytochalasin B. Our results suggest the involvement of
LFB3 in uPA gene regulation by cAMP at different levels.
Figure 1:
Cooperative
role of domain C with neighboring domains A and B in uPA gene induction
by cAMP signaling. a, luciferase gene constructs containing
different parts of a cAMP-inducible enhancer of the uPA gene which is
composed of domains A, B, and C. The positions of apparent protein
contacts as determined by methylation interference experiments are
indicated by stars. Mutated domains and sequences are
indicated by lowercase letters. b, Transient
transfection assays in LLC-PK cells. Luciferase constructs
(1 µg) were induced either by 1 mM 8-Br-cAMP or by
transfecting together with 0.5 µg of pCEV (CEV), a vector
expressing a catalytic subunit of the cAMP-dependent protein kinase. c, the role of LFB3 was tested by transient cotransfection
assays in F9 cells using luciferase constructs (pTATA or pABC-TATA; 1 µg) and LFB3 expression vector (1 µg)
with or without pCEV (CEV) (0.5 µg). Assays were done in
duplicate and mean values are shown with error
bars.
We previously cloned the domain C-binding protein and found it to be the pig equivalent of mouse LFB3(17) . We therefore examined the effect of LFB3 on the above templates by transient coexpression assays in F9 cells, which have a negligible level of endogenous LFB3. We used only the catalytic subunit to activate the signaling because endogenous cAMP-dependent protein kinase is not responsive to cAMP in F9 cells by an unknown mechanism(29) . Fig. 1c shows that in F9 cells pABC-TATA was strongly induced by the catalytic subunit only when LFB3 was coexpressed. The control pTATA was not affected. These results unambiguously indicate the cooperation among three protein-binding domains and the involvement of LFB3 in cAMP regulation through the ABC site.
Figure 2: LFB3 mRNA levels. Total RNA was prepared from cells pretreated with 1 mM Br-cAMP or 100 ng/ml TPA for 2 h or 0.5 µM colchicine, 10 µM cytochalasin B, or 125 nM okadaic acid for 4 h. Samples (5 µg each) were analyzed for the levels of LFB3 and uPA mRNAs by Northern blot hybridization.
With the exception of Br-cAMP, all the other agents induce uPA gene via the activation of AP1, acting on the PEA3/AP1 site located 2 kb upstream of the transcription initiation site(13, 15) . Therefore, in the following experiments we compared in particular Br-cAMP and TPA.
Figure 3:
Domain C binding activity. Domain C
binding activity in the nucleus was tested by electromobility shift
assays using crude nuclear extracts from LLC-PK cells
pretreated for 7 h with 1 mM Br-cAMP, 100 ng/ml TPA, or both.
As controls, the same extracts were tested for domains A and B,
mPEA3/AP1, and SP1 binding activities.
Figure 4:
Nuclear run-on transcription. Nuclei were
isolated from LLC-PK cells untreated or pretreated with
various agents for 90 min. Nuclear transcription was performed in the
presence of a radioactive precursor and specific transcripts were
analyzed by filter hybridization.
Figure 5:
Stability of LFB3 mRNA. Effects of Br-cAMP (a and b) and TPA (c and d) on the
stability of LFB3 mRNA were examined by RNA synthesis inhibitor chase
experiments. In a and c, Br-cAMP and TPA,
respectively, were added at the same time of DRB. In b and d, Br-cAMP and TPA, respectively, were added 1 h before DRB.
, DRB;
, TPA or Br-cAMP;
, DRB plus TPA or
Br-cAMP.
Figure 6:
Effect of TPA pretreatment on cAMP
induction of pABC-TATA. LLC-PK cells were transfected with
pABC-TATA. At 20 h after transfection cells were treated with or
without TPA for 7 h, and then induced with or without 0.1 mM Br-cAMP for 4 h. Assays were done in duplicate and mean values are
shown with error bars.
LFB3 is an enhancer-binding protein augmenting basal
expression of a gene that contains its cognate cis-element. We
found in the induction of the uPA gene by cAMP in LLC-PK cells that LFB3 is a positive regulator cooperating with
CRE-binding proteins within a composite cAMP-responsive enhancer (17) (this work). Our results also suggest that LFB3 is
involved in a down-regulating phase of cAMP-induced uPA gene
expression. We have previously shown that uPA gene induction by cAMP is
transient; the rate of uPA gene transcription reaches optimal after
2-4 h of cAMP treatment but declines thereafter (32) . It
may be that in uPA gene regulation LFB3 acts as a negative feedback
regulator by decreasing its own concentration in response to cAMP. This
throws new light on LFB3, which has been implicated as a factor
coupling hormonal regulation and tissue-specific regulation of uPA gene
expression in kidney epithelial cells(17) .
The decrease in domain C binding activity seems to be due to a decrease in LFB3 protein levels. The decrease was also observed with TPA, and it may also be the case for colchicine, cytochalasin B, and okadaic acid, which all decreased LFB3 mRNA levels (see below). These agents induce uPA gene expression in LLC-PK1 cells via activation of the transcription factor AP1, although the mechanism of AP1 activation by each agent is different(13, 14, 15) . Thus, in addition to the features mentioned above, LFB3 may mediate negative cross-talk between cAMP-dependent signaling and AP1-activating signaling pathways in uPA gene regulation. Indeed, pretreatment with TPA significantly reduced cAMP induction of the luciferase gene driven by an enhancer consisting of domains A, B, and C. The decrease in DNA binding by LFB3 in the cells seems to be due to the reduction in the protein levels. We cannot formally exclude the possibility that the decrease is due to a post-translational modification of the protein; however, this is in any case not the main cause because we also detected a strong reduction in LFB3 mRNA levels. The possible role of LFB3 in cAMP-dependent uPA gene regulation through the ABC site revealed by this work is summarized in Fig. 7.
Figure 7: Working model for the role of LFB3 in cAMP-dependent uPA gene regulation through the ABC site. LFB3 is a kidney-enriched transcription factor and plays a role as both positive and negative regulator of cAMP induction of the uPA gene through the ABC site. It allows cAMP induction by cooperating with CRE-binding proteins (CRE-BP) on the ABC site of the uPA gene promoter. But later on, it also mediates a negative feedback regulation by cAMP and TPA by decreasing its protein levels, which is due to enhanced degradation of LFB3 mRNA.
The decrease in DNA-binding activity evoked by treatment with the uPA inducers in these cells was specific to the domain C binding protein, LFB3, and not a general effect, because DNA binding of the proteins recognizing domains A and B and of the ubiquitous transcription factor SP1 remained constant. Furthermore, the DNA-binding activity to the mutated PEA3/AP1-oligonucleotide, which contains an active AP1 site mediating the action of TPA, colchicine, cytochalasin and okadaic acid, was increased by Br-cAMP as well as by TPA. We have not elaborated the mechanism of the increase in PEA3/AP1-binding activity, i.e. whether it is transcriptional or post-transcriptional. It is worthwhile to mention that the peptide hormone calcitonin, which raises intracellular cAMP concentrations, strongly enhances de novo synthesis of c-Fos and c-Jun(13) , raising the interesting possibility of a cross-regulation of the TPA-dependent signaling pathway by the cAMP-dependent signaling pathway at the transcription step. The cAMP signal by itself does not utilize the PEA3/AP1 site to increase uPA gene expression(13) . We do not know yet whether the enhancement of c-Fos together with c-Jun levels exerts positive effects on PEA3/AP1 site-mediated uPA gene expression, because the overexpression of c-Fos had no effect on uPA gene induction in NIH3T3 cells(33) .
The decrease in LFB3 mRNA levels is mainly attributable to induced mRNA instability. We did not detect changes in the LFB3 gene transcription rate, but we did observe that LFB3 mRNA degradation increased in the presence of TPA or Br-cAMP. Interestingly, however, enhanced instability was observed only when DRB was added 1 h after TPA or Br-cAMP treatment, suggesting that some RNA transcripts or their translation products are involved in LFB3 mRNA metabolism. It may be that TPA or Br-cAMP induces a factor, RNA or protein essential for LFB3 mRNA degradation, or that an RNA or a protein of short half-life is involved in LFB3 mRNA degradation, at least at an early stage. A requirement for on-going RNA synthesis in mRNA degradation has been reported for several mRNAs, such as those for c-fos,(34) , c-myc(35) , collagenase(36) , and the transferrin receptor(37) . We have shown that an RNA instability-regulating site in the 3`-UTR of uPA mRNA requires on-going RNA synthesis for its activity (31) and that the importance of this site in overall uPA mRNA degradation may depend on cell type (38) . In none of these cases is it known how on-going RNA synthesis contributes to mRNA degradation.
Several
instability-determining sequences have been identified in many mRNAs.
These include sequences located in the 3`-untranslated region, such as
the iron-responsive element in the transferrin receptor
mRNA(37, 39) , sequences in the unstable yeast MFA2
mRNA (38) and AU-rich sequences in various oncogene and
lymphokine mRNAs (40, 41, 42, 43, 44) . But
instability-determining elements have also been identified in coding
regions, e.g. c-myc(35) and c-fos(45) mRNAs. We tested the 3`-UTR and protein-coding
regions of LFB3 mRNA in a system developed for the study of uPA mRNA
degradation by inserting these sequences in an otherwise stable globin
mRNA(31) ; however, the stability of recombinant globin mRNAs
was not affected by TPA or Br-cAMP. ()It may be that
regulatory sequences reside in 5`-UTR or the 3` extreme which we have
not tested or that the globin mRNA context interfered with TPA- and
Br-cAMP-induced mRNA degradation.
Whether the cAMP and TPA signals utilize the same mechanism to induce LFB3 mRNA destabilization is not yet established, although it is plausible considering that induced LFB3 mRNA instability by either agent requires ongoing RNA synthesis and that signal transductions induced by the two agents are related. In the cell, cAMP and TPA activate distinct signaling pathways but are otherwise quite related. Both agents trigger signaling by activating serine/threonine kinases, and the transcription factors that are eventually activated by these signals are also related; the cAMP and TPA signals activate CREB/ATF and AP1, respectively, which are highly related transcription factors containing basic/leucine zipper domains, recognize highly similar sequences, and can cross-dimerize (for reviews, see (8) and (46) ). A protein responsible for induced LFB3 mRNA degradation could be phosphorylated and regulated by cAMP-dependent protein kinase as well as by protein kinase C. Alternatively, the two different but related transcription factors may exert their effects at a post-transcriptional step by interacting with the same RNA sequence or RNA-binding protein. It should be remembered that colchicine, cytochalasin B, and okadaic acid also reduce LFB3 mRNA (Fig. 2) and that these agents do not require protein kinase C to activate AP1 and induce the uPA gene(13, 15) . Identification of regulatory sequences in LFB3 mRNA and the corresponding binding proteins should help answer these questions.
We have shown that uPA inducers reduce LFB3 mRNA levels. Is there
any physiological significance in this apparent linkage, or is this
reverse regulation fortuitous, using very common signaling pathways?
uPA is a secreted protease which plays an important role in various
extracellular proteolytic processes (for reviews, see (47, 48, 49, 50) ), but its
unchecked expression may have deleterious effects on producing organs
or nearby organs(37) . As LFB3 is an abundant transcription
factor in kidney (18) ()and is involved in uPA gene
regulation, it may have evolved so that the kidney cells use LFB3 as
one means to control the level of uPA expression.