(Received for publication, December 27, 1994; and in revised form, February 1, 1995)
From the
The POU transcription factor Pit-1 activates the prolactin gene
in pituitary lactotrophs and may integrate responses of the gene to
external signals. To study the role of Pit-1 in dopaminergic inhibition
of the prolactin gene, we transiently transfected Pit-1 and dopamine D2
receptor vectors into a series of heterologous cell lines and examined
dopamine regulation of the prolactin gene promoter. Regulation was
Pit-1-dependent in all cell lines tested. Moreover, dopamine
responsiveness was cell type-specific: stimulatory in fibroblasts
(COS-7) and muscle-type cells (P19/MeSO-induced) and
inhibitory in pancreatic endocrine (RIN, InR1-G9) and neural-like
(P19/retinoic acid-induced) cells. Because dopaminergic responses in
Pit-1-transfected RIN cells paralleled those in pituitary GH4 cells,
the islet cell line was used to test for sequences in Pit-1 that
mediate negative hormone signals. Dopamine responsiveness of the Pit-1
transactivation domain (residues 8-80) was examined using a
chimeric LexA construct. LxPit-1, LxSp1, and Lx-glucocorticoid receptor
fusions all activated basal transcription, but only LxPit-1 was
regulated by dopamine. Regulatory responses of LxPit-1 and full-length
Pit-1 were quantitatively similar. In addition, gain-of-function
G
mutants that inhibit Pit-1-dependent promoters in GH4
cells also suppressed selectively Pit-1- or LxPit-1-dependent promoters
in RIN cells. This demonstrates that Pit-1 can function as a specific
target for distinct inhibitory G protein signals. Interestingly, Pit-1
sequences N-terminal to the DNA-binding POU domain appear to be
sufficient in mediating regulation by these pathways.
Pit-1 (GHF-1) is a member of the POU class of
homeodomain-containing transcription factors and has a critical role in
the developmental appearance of specific pituitary cell types (reviewed
in (1) and (2) ). The predominant Pit-1 isoform in
mammals is 291 amino acids long and consists of a 142-residue
C-terminal POU domain that mediates DNA binding as well as homo- and
heterodimerization, and an N-terminal sequence important for
transactivation(3, 4, 5) . Pit-1 activates
transcription of the prolactin (PRL) ()gene by binding to
specific sites in PRL 5`-sequences(6, 7, 8) .
PRL promoter activity in pituitary cells is disrupted by mutation of
these sites(9) . Moreover, in vitro analyses of Pit-1
mutants from Snell mice (10) and from hypoprolactinemic
patients with combined pituitary hormone deficiency (11, 12) have confirmed the importance of Pit-1
integrity for activation of the PRL gene. Because of its pivotal role
in PRL gene transcription, regulation of Pit-1 function in pituitary
lactotroph cells could provide an effective mechanism for positive or
negative control of PRL production.
The tuberoinfundibular neurons
of the hypothalamus project to the pituitary stalk and regulate the
release of PRL(13) . Regulation is predominantly inhibitory and
is mediated by dopamine (DA) D2 receptors present on lactotroph cells.
Activation of D2 receptors leads also to rapid decreases in the
incorporation of [H]Leu into PRL protein (14) and suppression of PRL gene transcription(15) . We
have shown that inhibition of the PRL gene by DA is conferred by
proximal promoter sequences and that binding sites for Pit-1 are
important in this response(16) . The ability of tandemly linked
Pit-1 sites to confer dopaminergic responses in the absence of other
PRL promoter elements suggests possible direct effects of DA on Pit-1
function.
To establish the importance of Pit-1 in D2 receptor
regulation of the PRL promoter, we have transfected promoter/luciferase
constructs into cell lines lacking endogenous Pit-1 and examined DA
responsiveness following introduction of recombinant Pit-1. We noted
previously (17) that responses of the PRL promoter to D2
receptor activation were not only Pit-1-dependent, but also cell
type-specific. Whereas in pituitary GH4 cells high affinity
Pit-1-binding sites conferred inhibitory regulation by D2 receptors, in
Ltk fibroblasts transfected with Pit-1, regulation at
these sites was stimulatory. In this study, we identify cell types in
which Pit-1 reconstitutes the negative response of the PRL
promoter to activated D2 receptors. Furthermore, by use of chimeric
transcription factors, we show that N-terminal sequences of Pit-1 can
mimic the negative response of a full-length Pit-1 to DA.
Figure 1:
Cell type-specific and Pit-1-dependent
regulation of the PRL promoter by DA in heterologous cell lines. A
schematic diagram of the constructs used for transfection is shown.
Cell lines were transiently transfected with the PRL promoter construct
-422P and an expression vector for the D2 receptor. Each
transfection included either the Pit-1 expression vectors or an
identical vector lacking the Pit-1 cDNA. Values represent the means
± S.E. for three or more independent experiments. For P19 cells
that were differentiated using MeSO or retinoic acid, each
experiment indicates a separate differentiation event. Bars illustrate the response to DA; basal promoter activity (with or
without Pit-1) has been assigned a value of 1. Activation of basal
promoter activity by the addition of Pit-1 was as follows: COS-7
= 13.5 ± 1.4-fold, undifferentiated P19 = 7.1
± 1.0-fold, Me
SO (DMSO)-treated P19
= 5.9 ± 0.8-fold, retinoic acid (RA)-treated P19
= 6.3 ± 1.5-fold, RIN = 2.7 ± 0.4-fold, and
InR1-G9 = 4.6 ± 1.2-fold. Significant differences in DA
regulation between paired values (with or without Pit-1) are indicated
by asterisks (Student's t test):***, p < 0.0001;**, p < 0.004; *, p < 0.007.
On the yaxis, 2 indicates a 100% increase,
and -2 indicates 50% inhibition. CMV,
cytomegalovirus.
In the absence of Pit-1, DA
failed to regulate the activity of the -422P PRL promoter in each
of the transfected cell lines (Fig. 1). Cotransfection of a
Pit-1 vector elevated basal promoter activity (see legend to Fig. 1) and reconstituted DA responsiveness in a cell
type-specific manner. In COS-7 cells, PRL promoter function was
stimulated about 2-fold by DA, a response similar to that observed in
Ltk cells(17) .
Undifferentiated P19 cells
provided an interesting system in which D2 receptor activation failed
to regulate the PRL promoter in either the absence or presence of Pit-1 (Fig. 1). These cells are induced by MeSO or
retinoic acid to differentiate into muscle- and neural-like phenotypes,
respectively(18) . Following conversion of P19 cells into
muscle-type cells, D2 receptor activation stimulated PRL promoter
activity 40-50% in the presence of Pit-1. Because high affinity
[
H]spiperone-binding sites were detectable in
undifferentiated P19 cells transiently transfected with D2 receptor
expression vector, (
)these functional differences cannot be
explained solely on the basis of D2 receptor expression, processing, or
transport to cell membranes. The data could suggest that a signaling
step distal to the D2 receptor and dependent on
Me
SO-induced differentiation is required for the
stimulatory transcriptional response to DA. In contrast to these cells,
P19 cells exposed to retinoic acid exhibited a consistent inhibitory
response to DA that was dependent on Pit-1 (Fig. 1). Together
with our earlier data(17) , these results demonstrate that
unique patterns of differentiation can lead to opposite transcriptional
responses of a specific target gene to D2 receptor activation.
To determine whether Pit-1 would mediate an inhibitory response to DA in cells functionally similar to GH4 cells, we tested two endocrine cell lines of pancreatic origin: RIN and InR1-G9. In the absence of Pit-1, the PRL promoter was weakly active in both pancreatic cell lines, but unresponsive to DA (Fig. 1). Cotransfection of the Pit-1 vector elevated basal PRL promoter activity, but in marked contrast to fibroblasts, mediated a 25% (InR1-G9) to 40% (RIN 1056A) inhibitory response to DA. Expression of Pit-1 in these endocrine cells therefore reconstitutes a pituitary-like signaling pathway that specifically mediates dopaminergic suppression of PRL promoter activity.
The
basis for cell type-specific differences in regulation of the PRL
promoter by D2 receptors is not yet clear. As activation of transfected
D2 receptors leads to a reduction in cAMP in most cell lines studied (16, 23, 24, 25, 26, 27, 28) ,
regulation of cAMP may not adequately explain the cell type-specific
differences in D2 receptor-regulated transcription. By contrast,
DA-induced changes in [Ca]
and/or phospholipid turnover do parallel more closely the
cell-specific pattern of regulation observed for the PRL promoter. In
fibroblasts, D2 receptor activation stimulates accumulation of inositol
phosphates (24) , elevates
[Ca
]
(17, 24) ,
and activates PRL promoter activity(17) . It is noteworthy that
these responses are characteristic also of GH4 cells treated with
stimulatory hormones such as TRH(29) . Furthermore,
multimerized Pit-1-binding sites confer stimulation in both DA-treated
fibroblasts and TRH-treated GH4 cells (17, 30, 31) , suggesting a similar
regulatory mechanism at the transcriptional level. In contrast to
fibroblasts, pituitary lactotrophs and GH4ZR7 cells treated with DA
exhibit reduced [Ca
]
due to
inhibition of voltage-dependent L-type Ca
channels(17, 24, 32) . L-type
Ca
channels are present in other excitable cell
types, including pancreatic endocrine cells, and may contribute to the
differential pattern of DA-regulated transcription in fibroblast and
islet cell lines. Interestingly, in P19 cells, both Me
SO
and retinoic acid induce excitable cell phenotypes(18) ;
however, only in the neurally differentiated cells was an inhibitory
Pit-1-dependent response observed after DA treatment.
Extracellular signals, including DA, could affect
Pit-1-dependent transcription by one or more mechanisms. First, Pit-1
levels could be altered by regulation of the pit-1 gene
promoter (33, 34, 35) or of Pit-1 protein
stability(36) . In vivo, this mechanism may be most
important in a developmental context, contributing to the establishment
of specific anterior pituitary cell types. Second, Pit-1 interactions
at coregulatory DNA sites could be affected. This is best illustrated
by the hormone-dependent synergism between Pit-1 and members of the
nuclear hormone receptor family, including estrogen (37, 38) and retinoic acid (34) receptors.
Pit-1 interactions with factors of other classes can also coordinate
stimulatory hormone signals(39, 40) . Third, Pit-1 can
be phosphorylated by specific protein kinases, resulting in selective
changes in DNA binding activity. The predominant acceptor sites are
Ser-115 and Thr-220, with the latter (near the N terminus of the
homeodomain) being critical for phosphorylation-dependent DNA
interaction(41) . In the case of the thyrotropin -subunit
gene, phosphorylation-enhanced binding of Pit-1 to the thyrotropin
promoter provides an interesting model for TRH-stimulated
transcription(42) .
Of these three mechanisms, we cannot exclude the involvement of a coregulatory interaction in the DA response as co-occupancy can occur on Pit-1-binding sites as short as 26 base pairs (e.g. see (5) ). However, as determined by mobility shift assays and as discussed previously(17) , DA does not detectably alter Pit-1 levels or binding to high affinity sites within the 5-h time period required for maximal transcriptional inhibition. This could suggest that DA suppresses the transactivating function of Pit-1, rather than regulating its DNA binding activity. As the major transactivation domain of Pit-1 has been localized to the N-terminal region(3, 4) , a chimeric transcription factor containing Pit-1 residues 8-80 fused to the Escherichia coli LexA DNA-binding domain (LxPit-1) was tested for DA responsiveness. Regulation was monitored using a reporter plasmid that contained two LexA operator sequences. To ensure that regulation was specific for Pit-1 N-terminal sequences, and not the LexA domain, a number of chimeric LexA transcription factors (Fig. 2A) were compared to LxPit-1. In addition, chimeras were tested in multiple cell lines to determine whether Pit-1 N-terminal sequences might contribute to the characteristic cell-specific pattern of regulation observed with full-length Pit-1.
Figure 2:
Dopaminergic regulation of LexA
operator-dependent transactivation. A, transcription units of
the luciferase reporter and chimeric LexA expression vectors are
illustrated. Plasmids are described under ``Experimental
Procedures'' and the references cited. Lex-DBD represents
the LexA DNA-binding domain. Each cell line was transiently transfected
with 10 µg of 2xLexA/luciferase, 5 µg of D2 receptor expression
vector, and 5 µg of either the LexA fusion construct or an empty
vector(-) as indicated in B-D. CMV,
cytomegalovirus; LTR, long terminal repeat; GR,
glucocorticoid receptor; RSV, Rous sarcoma virus. B, responsiveness of LexA fusion constructs to DA in COS-7
cells. Cells transfected with the LxGR fusion expression vectors were
first treated with 0.1 µM dexamethasone (Dex) or
vehicle for 10 min, followed by DA (1 µM final
concentration) for 5 h. Each value represents the means ± S.E.
of three or more independent experiments. ***, p < 0.0001 versus empty vector control (Student's t test).
Activation of constitutive 2xLexA/luciferase activity by the LexA
fusion constructs was as follows: LxPit-1 = 6.1 ±
0.9-fold, LxGR = 1.6 ± 0.4-fold, LxGR +
dexamethasone = 7.3 ± 1.0-fold, LxRel = 18.6
± 3.1-fold, and LxSp1 = 13.1 ± 1.1-fold. C, responsiveness of LexA fusion constructs to DA in
undifferentiated (leftpanel) and
MeSO-treated (right panel) P19 cells. Values
represents the means ± S.E. of three or more independent
experiments. Left panel, activation of constitutive
2xLexA/luciferase activity in undifferentiated P19 cells was as
follows: LxPit-1 = 1.8 ± 0.3-fold, LxGR = 1.6
± 0.3-fold, LxGR + dexamethasone = 9.4 ±
1.7-fold, and LxSp1 = 14.1 ± 0.9-fold. Right
panel, each experiment with Me
SO-treated P19 cells
represents an independent differentiation event.***, p <
0.0001 versus empty vector control (Student's t test). Activation of basal activity by LexA chimeras was as
follows: LxPit-1 = 2.1 ± 0.3-fold, LxGR = 2.3
± 0.3-fold, LxGR + dexamethasone = 7.6 ±
1.1-fold, and LxSp1 = 10.5 ± 0.6-fold. D,
responsiveness of LexA fusion constructs to DA in pancreatic RIN and
InR1-G9 cells. Values represent the means ± S.E. of three or
more independent experiments. Left panel, Mann-Whitney
test.**, p < 0.02; *, p < 0.03 versus empty vector control. Activation of basal activity by LexA fusions
was as follows: LxPit-1 = 2.1 ± 0.3-fold, LxGR =
2.3 ± 0.6-fold, LxGR + dexamethasone = 8.9 ±
1.1-fold, LxRel = 22.2 ± 1.3-fold, and LxSp1 =
15.7 ± 0.8-fold. Right panel, Student's t test.**, p < 0.008 versus empty vector
control. Activation of basal activity by LexA fusions was as follows:
LxPit-1 = 3.6 ± 1.0-fold, LxRel = 15.9 ±
2.7-fold, and LxSp1 = 12.3 ±
2.2-fold.
A DA response was not detected in control COS-7 cells transfected with only the D2 receptor expression vector and the 2xLex reporter (Fig. 2B). However, in cells cotransfected with the LxPit-1 expression vector, basal luciferase activity was enhanced 6-fold, and the addition of DA caused a 1.7-fold stimulation. This stimulatory effect was quantitatively similar to that observed with full-length Pit-1 and the PRL promoter in COS-7 cells (Fig. 1). In contrast to LxPit-1, each of the other chimeric factors (i.e. LxGR, LxRel, and LxSp1) elevated basal transcription of the reporter construct, but none was able to mediate a DA response. Because the LxGR construct contains the glucocorticoid receptor ligand-binding domain, full transcriptional function is dependent on the presence of glucocorticoids(19) . Accordingly, dexamethasone activated LxGR-dependent transcription 7-8-fold (see legend to Fig. 2B); however, no further changes in LxGR function were seen upon treatment with DA (Fig. 2B). In the same experiments, LxPit-1 activity was unaffected by the addition of dexamethasone (data not shown).
In P19 cells, full-length Pit-1 can
mediate a transcriptional response to DA only once cell differentiation
has occurred. As shown in Fig. 2C, the chimeric factor
LxPit-1 exhibited a similar pattern of regulation, in which no DA
response was detected in undifferentiated P19 cells (leftpanel), but a 50% increase was seen following
MeSO induction (rightpanel). LxSp1 and
LxGR (with or without dexamethasone) were unable to mediate a response,
as noted in COS-7 cells. Moreover, basal luciferase levels were
activated equally by LxPit-1 in undifferentiated and
Me
SO-induced P19 cultures (see legend to Fig. 2C), indicating that DA regulation is not strictly
proportional to the transactivating potential of LxPit-1. These data
demonstrate the specificity of Pit-1 N-terminal sequences in mediating
regulation by DA. Interestingly, in two preliminary experiments (data
not shown), retinoic acid-induced P19 cells exhibited negative
LxPit-1-dependent regulation by DA (approximately 20% inhibition),
further demonstrating that the cell type-specific pattern of regulation
is determined by Pit-1 N-terminal sequences.
Consistent with its function in COS-7 and P19 cells, the LxPit-1 chimera mimicked the regulatory pattern of Pit-1 in DA-treated RIN and InR1-G9 cells. As shown in Fig. 2D (leftpanel), LxPit-1 mediated a 40-50% decrease in Lex sitedependent reporter activity in RIN cells, which is quantitatively comparable to the DA effect in cells cotransfected with -422P/luciferase and Pit-1 (Fig. 1). A negative response similar to that of RIN cells was seen in InR1-G9 cells expressing LxPit-1 (Fig. 2D, rightpanel). In both cell lines, transcriptional activation by LxGR or LxSp1 was unaffected by DA. A modest response to DA was observed with LxRel specifically in RIN cells.
Activated mutants of G subtypes that interact with lactotrophs (32) and GH4
cells (44) D2 receptors can mimic the inhibitory actions of DA
on the PRL promoter(17) . Transfection of the same mutants (i.e. G
and G
) into RIN
cells suppressed the activity of a Pit-1-activated PRL promoter by
25-30% (Fig. 3). To determine if N-terminal sequences of
Pit-1 were sufficient to mediate regulation, G
expression vectors were cotransfected with the LxPit-1 construct
and the 2xLex reporter. G
and G
mutants inhibited LxPit-1-activated luciferase expression to the
same extent as with a full-length Pit-1 (Fig. 3). Control
transfections with the LxSp1 construct demonstrated that regulation was
dependent on Pit-1 residues 8-80.
Figure 3:
LxPit-1-dependent inhibition by activated
G mutants in RIN cells. Cells were electroporated with
the D2 receptor expression vector (5 µg), either the
-422P/luciferase or 2xLexA/luciferase reporter (10 µg), and
expression vectors for Pit-1 (5 µg), LxPit-1 (5 µg), LxSp1 (5
µg), G
(2.5 µg), or G
(2.5
µg) or the appropriate empty vector controls as shown. Values
represent the means ± S.D. of three independent experiments.***, p < 0.008 (Mann-Whitney test). Activation of basal activity
by the addition of Pit-1 (2.1 ± 0.7-fold), LxPit-1 (1.8 ±
0.8-fold), and LxSp1 (13.9 ± 3.6-fold) was as
indicated.
In summary, our study demonstrates that Pit-1 can function as a terminal second messenger for inhibitory G protein signals and that the transactivation domain is important in this response. The selective ability of Pit-1 N-terminal sequences to transfer DA regulation to LexA operator sites shows that neither the Pit-1 POU domain nor the Pit-1 DNA-binding site is entirely necessary for DA responsiveness. Protein/protein interactions involving the Pit-1 N-terminal domain are likely to be essential for DA regulation. It remains to be determined whether inhibition of transactivation requires direct modification of Pit-1 residues within the 72-amino acid sequence or an indirect mechanism, such as recruitment or dissociation of a DA-regulated accessory protein. Finally, the ability of LxPit-1 to respond to hormone signals in a number of different cell types indicates that factors associated with the transcriptional regulatory mechanism are not restricted to the pituitary.
Note Added in Proof-Recent analysis of Pit-1 mutants has shown that phosphorylation at Ser-115 and Thr-220 is not a requirement for Pit-1-mediated signaling in hormone-stimulated cells (Okimura, Y., Howard, P. W., and Maurer, R. A.(1994) Mol. Endrocrinol.8, 1559-1565; Fischberg, D. J., Chen, X.-h., and Bancroft, C.(1994) Mol. Endocrinol.8, 1566-1573). Interestingly, Okimura et al., and Howard and Maurer (Howard, P. W., and Maurer, R. A.(1994) J. Biol. Chem.269, 28662-28669) reported that a chimeric factor containing full-length Pit-1 fused to a GAL4 DNA-binding domain was unable to mediate responses to cAMP or TRH, respectively. Whether differences in hormone responses of GAL4-Pit-1 and LexA-Pit-1-(8-80) reflect differences in signaling pathways, heterologous DNA-binding domains, or DNA recognition sites or are related to the length of the chimeric Pit-1 sequence needs to be explored.