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Transcription of the human prolactin gene in mammary cells.
A. Baudhuin, I. Manfroid, C. Van de Weerdt , J. A. Martial, M. Muller*
Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Institut de
Chimie B6, B-4000 Sart-Tilman, Belgium.
Keywords: prolactin, mammary cells, regulation, Pit-1
*Address for correspondence:
Tel +32 4 3664437
Fax +32 4 3662968
E-Mail: [email protected]
INTRODUCTION
Human prolactin (hPRL) is a polypeptide hormone mostly produced by the anterior
pituitary gland, where its expression is modulated by numerous extracellular signals such as
TRH, EGF, thyroid hormones, glucocorticoids, estrogens and by second messengers Ca2+ and
cAMP (1). In mammals, PRL mainly promotes growth and development of the mammary
gland, as well as milk production and secretion through binding to its specific transmembrane
receptor (PRLR). It is also involved in mammary tumor development (2) and acts as a
mitogen on several human breast cancer lines (3,4). However, anticancer therapies designed
to block expression and secretion of pituitary lactogenic hormones (PRL and GH) had little
effect on the course of the disease (5), suggesting that extra-pituitary PRL expression might
be involved (6). Synthesis of PRL mRNA has been observed in T lymphocytes, decidualized
endometrium (7) and, interestingly, also in both normal and neoplastic human breast tissue (8)
and in human mammary carcinoma lines (4,6,8). The observation that hPRL antagonists
blocked mammary tumor cell growth (3,4) further supported the existence of an
autocrine/paracrine loop, where hPRL produced in mammary cells directly stimulates
mammary cell proliferation.
Nothing is known about the transcriptional regulation of the hPRL gene in mammary
cells. Two distinct transcription start sites are described for the hPRL gene (see Fig.1A), one
used in the pituitary starting with exon 1b, the other initiating a slightly larger hPRL mRNA
with an additional non-coding exon (exon 1a) in lymphocytes and endometrial cells (7). In
pituitary, transcription is mainly controlled by the pituitary-specific transcription factor Pit-1
and modulated by the estrogen receptor. Recently, activation of the MAPK pathway was
shown to induce binding of AP-1 to the proximal pituitary promoter (1,9). Lymphoid and
endometrial hPRL expression, independent of Pit-1, was shown to be controlled by cAMP or
cAMP and progesterone, respectively (1,7,10). Here we present the first extensive analysis of
the transcriptional regulation of the human prolactin gene in human mammary tumor cells.
RESULTS
Few studies are available concerning the promoter used in mammary cells and they give
conflicting results (8,11). We performed RT-PCR experiments to determine which type of
mRNA codes for the endogenous hPRL in several mammary cell lines (Fig.1B). We detected
a lymphocyte-type messenger in three of the cell lines, other data from the literature are also
included (* in Fig.1B). In addition, we studied the functionality of the two hPRL promoters in
several mammary cell lines by performing transient expression experiments using the
luciferase reporter gene controlled by each of the two hPRL gene promoters (see Fig.1A). In
most cell lines tested, the distal promoter displayed a higher activity in these transient assays
(Fig.1B). Taken together, these results argue for a preferential use of the lymphoid/decidual
promoter in different mammary cell lines. However, the precise determination of the
transcriptional start site used in each of these cell lines will depend on the more precise RNA
analysis experiments that are presently being performed.
As tumor cell lines often present substantial DNA rearrangements, we verified the
integrity of the hPRL gene locus by using a PCR approach on genomic DNA extracted from
the different cell lines. No major rearrangement was detected in the cell lines tested.
A more extensive analysis of the hPRL transcriptional regulation was performed in the
luminal epithelial mammary tumor cell line SK-BR-3. RT–PCR experiments suggest that a
pituitary-type mRNA is produced in these cells (Fig.1). In transient expression experiments,
the transfected pituitary and the decidual/lymphoid promoter displayed similar transcriptional
activities (Fig.2A). No endogenous Pit-1 mRNA was detected in these cells, however
expression of exogenous Pit-1 dramatically stimulated the transfected pituitary promoter
(Fig.2B). Treatment with epidermal growth factor (EGF) activated only the transfected
pituitary (Fig. 2C), but not the transfected decidual/lymphoid promoter. Both Pit-1 expression
and EGF treatment also stimulated the endogenous hPRL gene transcription. Taken together,
our results strongly suggest that the hPRL pituitary promoter is used in the SK-BR-3
mammary tumor cells.
DISCUSSION
Expression of hPRL in mammary gland is particularly interesting, as this tissue also is the
major target site for PRL in mammals. This study represents the first attempt to investigate
the molecular mechanisms involved in expression and regulation of the hPRL gene in
mammary cells. Analysis of several mammary tumor cell lines suggests that the
lymphoid/decidual promoter is more often used in these tissues and that the genomic hPRL
locus is intact in these cells.
A more precise analysis of the regulatory regions involved in transcription of the hPRL
gene was performed in SK-BR-3 mammary tumor cells. Surprisingly, in transient expression
experiments the basal activity of the lymphoid/decidual promoter is similar to that of the
pituitary promoter, while only the endogenous pituitary promoter is clearly active. This
discrepancy probably means that the chromosomal lymphoid/decidual promoter is kept silent
by a repressive chromatin structure, unlike the transfected naked version. In striking contrast,
the chromosomal pituitary promoter region in SK-BR-3 cells appears to be in an open
configuration, as expression of hPit-1 and EGF treatment clearly enhances hPRL expression
of the endogenous gene. Taken together, these results strongly indicate that the hPRL pituitary
promoter is used in SK-BR-3 cells.
It is interesting to note that EGF stimulates the endogenous hPRL pituitary promoter.
EGF is an important regulator of mammary gland development as well as breast tumor growth
(12), activation of its tyrosine kinase receptor EGFR (ErbB1) leads to receptor
heterodimerization with ErbB2, phosphorylation of both receptors and activation of
downstream pathways. Overexpression of EGFR and ErbB2 is observed in 25-50% of human
breast cancers, also in SK-BR-3 cells, and often correlates with the lack of steroid hormone
receptors and poor prognosis. Recently, Yamauchi et al. (13) demonstrated that PRL secreted
by SK-BR-3 cells is able to activate ErbB2 and the MAPK pathway via autocrine binding to
PRLR followed by Jak2 activation. Our results show that hPRL synthesis is induced by
EGFR/ErbB2, suggesting the existence of a positive PRL/EGFR feedback loop leading to a
strong stimulation of proliferation in mammary tumor cells such as SK-BR-3. Further studies
will be necessary to characterise hPRL gene expression in mammary cells. The understanding
of the regulatory network underlying hPRL expression, EGF regulation and AP-1 activity in
breast cancer cells could provide a framework for developing future cancer therapies.
ACKNOWLEDGMENTS
A.B. holds a F.R.I.A. fellowship. M.M. is a "chercheur qualifié" at the F.N.R.S., Belgium.
This work is supported by grants from "Télévie".
REFERENCES
1. Muller, M. et al. 1998. Transcriptional regulation of the human prolactin gene.
Médecine/Sciences 14: 580-587.
2. Llovera, M. et al. 2000. Involvement of prolactin in breast cancer: redefining the
molecular targets. Exp. Gerontol. 35: 41-51.
3. Fuh, G. & J. A. Wells. 1995. Prolactin receptor antagonists that inhibit the growth of
breast cancer cell lines. J. Biol. Chem. 270: 13133-13137.
4. Ginsburg, E. & B. K. Vonderhaar. 1995. Prolactin synthesis and secretion by human
breast cancer cells. Cancer Res. 55: 2591-2595.
5. Anderson, E. et al. 1993. Serum immunoreactive and bioactive lactogenic hormones in
advanced breast cancer patients treated with bromocriptine and octreotide. Eur. J. Cancer.
29: 209-217.
6. Clevenger, C. V. et al. 1995. Expression of prolactin and prolactin receptor in human
breast carcinoma. Evidence for an autocrine/paracrine loop. Am. J. Pathol. 146: 695-705.
7. Ben-Jonathan N. et al. 1996. Extrapituitary prolactin: distribution, regulation, functions,
and clinical aspects. Endocr Rev. 17: 639-669.
8. Shaw-Bruha, C. M. et al. 1997. Expression of the prolactin gene in normal and neoplastic
human breast tissues and human mammary cell lines: promoter usage and alternative
mRNA splicing. Breast Cancer Res. Treat. 44: 243-253.
9. Manfroid, I. et al. 2001. Inhibition of protein phosphatase PP1 in GH3B6, but not in GH3
cells, activates the MEK/ERK/c-fos pathway and the human prolactin promoter, involving
the coactivator CPB/p300. Mol. Endocrinol. 15: 625-637.
10. Pohnke, Y. et al. 1999. CCAAT/enhancer-binding proteins are mediators in the protein
kinase A-dependent activation of the decidual prolactin promoter. J. Biol. Chem. 274:
24808-24818.
11. Le Provost, F. et al. 1994. Prolactin gene expression in ovine and caprine mammary
gland. Neuroendocrinology 60: 305-313.
12. Kim, H. & W. J. Muller. 1999. The role of the epidermal growth factor receptor family in
mammary tumorigenesis and metastasis. Exp. Cell Res. 253: 78-87.
13. Yamauchi, T. et al. 2000. Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by
autocrine secretion of prolactin in human breast cancer. J. Biol. Chem. 275: 33937-33944.
FIGURE LEGENDS
Figure 1. A) schematic representation of the hPRL gene. The two promoters are mentioned
and the regions which were fused to the luciferase reporter gene for the transient expression
experiments are indicated by the black boxes. The two different types of hPRL mRNA are
also depicted. B) endogenous hPRL mRNA detected by RT-PCR and preferential promoter
activity observed by transient expression in the indicated mammary cell lines.
Figure 2. Transient expression experiments in SK-BR-3 cells. Reporter constructs containing
the luciferase gene driven by the hPRL lymphoid/decidual (lym/decPRL-Luc), the pituitary
(pitPRL-Luc) or a minimal promoter (40P-Luc) were transfected into SK-BR-3 cells. A) Both
promoters display a similar basal activity in transient expression. B) Expression of the
pituitary-specific factor Pit-1 strongly induces the transcriptional activity of the transfected
pituitary promoter. C) Epidermal growth factor (EGF) stimulates the transfected pituitary
promoter, but not the lymphoid/decidual promoter.
A
5’
decidual/lymphoid promoter
1a
pituitary promoter
1b
coding
region
1b
lymphocyte-type
mRNA
B
cell lines
1a
1b
2
3
2 3 4
pituitary-type
mRNA
4 5
about 150 nucleotides more at 5’ non coding end
endogenous mRNA transient expression
BT-474
lymphocyte-type
lymphoid
MCF10A
lymphocyte-type
similar activity
MDA-MB-231
lymphocyte-type
pituitary-type ()
lymphoid
lymphoid
MDA-MB-453
SK-BR-3
pituitary-type (and)
similar activity
T-47-D
pituitary-type ()
lymphoid
ZR-75-1
(): from Shaw-Bruha, 1997
lymphoid
5
A
lym/decPRL-Luc
pitPRL-Luc
40P-Luc
1•10 4
0
2•10 4
3•10 4
promoter activity
B
Pit-1
pitPRL-Luc
5•10
5
1•10
6
promoter activity
C
lym/decPRL-Luc
pitPRL-Luc
40P-Luc
0
2
4
6
8
10
EGF fold induction
12