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: m.muller@ulg.ac.be
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.
pituitary-type mRNA lymphocyte-type
mRNA about 150 nucleotides more at 5’ non coding end
2 3 4 5
1b 1a
1b 2 3 4 5
pituitary promoter coding region 1a 5 ’ 1b decidual/lymphoid promoter
A
ZR-75-1 BT-474 MCF10A MDA-MB-231 MDA-MB-453 T-47-D SK-BR-3cell lines
endogenous mRNA transient expression
lymphocyte-type lymphocyte-type lymphocyte-type pituitary-type () pituitary-type (and) pituitary-type () lymphoid lymphoid lymphoid lymphoid lymphoid similar activity (): from Shaw-Bruha, 1997
B
similar activity0 1•104 2•104 3•104
promoter activity
-Pit-1
0 2 4 6 8 10 12
