From the School of Biological Sciences, University of
Manchester, 3.239 Stopford Building, Oxford Road, Manchester, M13 9PT,
United Kingdom, § NIDDK, Laboratory of Biochemistry and
Metabolism, National Institutes of Health, Bethesda, Maryland 20892, and the ¶ Unite d'Endocrinologie Moleculaire, Institute National
de la Researche Agronomique, 78352 Jouy-en-Josas Cedex, France
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ABSTRACT |
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Extracellular matrix and growth factors cooperate
to regulate signaling pathways and gene transcription in adherent
cells. However, the mechanism of extracellular matrix signaling is
poorly defined. In mammary gland, the expression of milk protein genes is controlled by cross-talk between signals derived from the basement membrane protein, laminin, and the lactogenic hormone, prolactin. Signals from basement membrane are transduced by 1
integrins and are required for prolactin to activate DNA binding of the milk protein gene transcription factor, Stat5. Here we show that basement membrane is necessary for tyrosine phosphorylation of the
prolactin receptor and thus directly affects cytokine signaling and
differentiation at the level of the plasma membrane. Prolactin does not
induce tyrosine phosphorylation of its receptor, Jak2, or Stat5 in
nondifferentiated breast epithelia cultured on collagen I, and we show
that this is due to a vanadate-sensitive activity that inhibits the
prolactin pathway. We suggest that protein-tyrosine phosphatases are
novel targets for regulation by extracellular matrix and in mammary
cells represent an additional control to the requirement of integrins
for milk protein production.
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INTRODUCTION |
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Cell behavior is controlled by a network of signals derived from growth and differentiation factors as well as from the local cellular environment. These signals are interpreted by appropriate receptors and converted into intracellular pathways that modulate transcriptional or post-transcriptional events. Migration, proliferation, survival, and differentiation are strongly influenced by cell interactions with the extracellular matrix (ECM)1 (1). For example, integrins determine the activity of both Ras-mitogen-activated protein kinase and phosphatidylinositol 3'-kinase mediated growth factor responses, and cell-ECM interactions contribute to cyclin activation thereby regulating cell cycle entry (2-6). However, the mechanism for this ligand-induced signaling cross-talk has not been established. Many signal transduction pathways, including those conveyed by ECM-integrin interactions are controlled by protein-tyrosine kinases (PTKs) (7). Homeostasis of signaling requires negative regulation through protein-tyrosine phosphatases (PTPs) (8, 9), and these enzymes are therefore of potential significance in the control of ECM signaling.
In the breast, epithelial cell interactions with basement membrane (BM)
control the prolactin-dependent expression of milk protein
genes (10, 11). Both laminin-1 and 1 integrins are required for differentiation of mammary cells (12, 13) and, together
with prolactin, direct milk protein gene transcription. The molecular
details of the prolactin signaling pathway were first described in the
rat lymphoma cell line, Nb2, which requires prolactin for
proliferation. Ligation of the prolactin receptor (PrlR) results in
induction of the associated PTK activity, Jak2 (14-16). This leads to
activation of Stat transcription factors that recognize specific DNA
sequence motifs in the promoters of early response genes implicated in
proliferation (17, 18). Prolactin triggers differentiation through a
similar pathway, and in cells transfected with PrlR, Stat5, and
-casein reporter vectors, prolactin induces transcription from the
-casein promoter via activation of endogenous Jak2 and ectopically
expressed Stat5 (19).
Mammary epithelial cells are adherent and, in contrast to Nb2 cells which grow in suspension, require cell-BM interactions to propagate prolactin signals. Using primary cultures of mouse mammary epithelial cells, we recently demonstrated that BM contributes to prolactin-dependent transcription of milk protein genes by regulating the DNA binding activity of Stat5 (20). Only cells cultured on BM and stimulated with prolactin were able to induce Stat5 activity and produce milk proteins, whereas cells on collagen I did not respond to hormone because they showed no Stat5 activity or milk protein synthesis after prolonged culture with prolactin. These results showed that mammary cells exhibit a strong dependence on both prolactin and BM for Stat 5 DNA binding activity, and we argued that this provided a molecular mechanism to explain the regulation of milk protein gene transcription by the ECM (20).
In this paper we examine whether BM regulates Stat5 DNA binding activity directly or through a control on its upstream signaling components Jak2 and PrlR. In short term experiments where Stat5 activity was induced after 15 min of hormone treatment, we found that BM was required for prolactin signaling both at the level of its receptor as well as Jak2. Moreover, the lack of differentiation in mammary cells cultured on collagen I was additionally due to a vanadate-sensitive activity that inhibited phosphorylation of PrlR, Jak2, and Stat5. Thus, in the absence of correct ECM signals PTPs appear to inhibit cytokine-triggered second messengers, providing a novel paradigm for the mechanism of ECM signaling.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- Mammary epithelial cells were isolated from 14.5-18.5-day pregnant ICR mice and established in culture at a density of 2.5-5 × 105 cells/cm as described (21). Cells were plated on dishes coated with either collagen I or laminin-rich BM matrix (12) and cultured for 48-72 h in Ham's F-12 medium (Sigma-Aldrich Co. Ltd., Poole, UK) containing 10% heat-inactivated fetal calf serum (Advanced Protein Products, Brierley Hill, UK), 10 ng/ml epidermal growth factor (Promega Corp., Southampton, UK), 1 mg/ml fetuin, 880 nM insulin, and 2.8 nM hydrocortisone. The cultures were washed extensively, and the medium was changed to differentiation medium (Dulbecco's modified Eagle's medium/Ham's F-12 medium (Life Technologies Ltd., Paisley, Scotland) containing 880 nM insulin, 2.3 nM hydrocortisone) for a further 24-72 h before stimulating with 150 nM prolactin for the prescribed times.
Protein Analysis--
Cells were washed in ice-cold
phosphate-buffered saline containing 1 mM sodium
orthovanadate and then extracted into lysis buffer (50 mM
Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1%
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1.5 mM pepstatin A, 10 mM leupeptin and aprotinin,
1 mM sodium orthovanadate, 50 mM sodium
fluoride, 5 mM sodium pyrophosphate, 20 mM
-glycerophosphate, and 10 µM ammonium molybdate).
After clearing the detergent-insoluble proteins by centrifugation,
lysates from equal numbers of cells from the collagen I or BM cultures
were immunoprecipitated with anti-Jak2 antibody (Upstate Biotechnology
Inc., Lake Placid, NY), anti-prolactin receptor antibody 46 (22), or
anti-Stat5a and anti-Stat5b antibodies (23) followed by protein
A-Sepharose (Zymed Laboratories Inc., South San
Francisco, CA) before separation by 6.25% SDS-polyacrylamide gel
electrophoresis. After transfer to Immobilon P membrane (Millipore
Ltd., Watford, UK), phosphorylated proteins were revealed with the
anti-phosphotyrosine antibody, 4G10 (Upstate Biotechnology Inc.)
followed by enhanced chemiluminescence using an ECL kit (Amersham
International plc, Little Chalfont, UK). Blots were stripped according
to the Amersham protocol and reprobed with precipitating antibody.
Electrophoretic Mobility Shift Assays (EMSA)-- Primary cultures of mammary epithelial cells from pregnant ICR mice were harvested by trypsinization. Cell pellets were snap frozen in liquid N2, and nuclear extracts were prepared as described (24). The total protein concentration was estimated by the Pierce BCA protein assay (Pierce and Warriner (UK) Ltd., Chester, UK), and 4 µg extract was incubated with 0.5 ng of end-labeled double-stranded oligodeoxynucleotide (i.e. the Stat5 STM site, 5'-GATTCCGGGAACCGCGT; Ref. 25) as described (26) excluding the addition of single-stranded competitor oligodeoxynucleotide. The nuclear extract was added as the final component of the reaction. In supershift assays, nuclear extracts were incubated for 30 min at room temperature with antibodies to Stat1 (M22), Stat 3 (C20), Stat5a (L20), or Stat5b (C17; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) before addition of DNA.
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RESULTS |
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To determine the point of intersection of the BM stimulus with the prolactin cassette, we used a short term assay where the prolactin signaling proteins were rapidly activated in primary cultures of mouse mammary epithelial cells. In this assay, the transcription factor Stat5 was activated 15 min after hormone treatment in cells contacting a reconstituted BM but not in those on the stromal matrix, collagen I (Fig. 1, A and B). The extent of tyrosine phosphorylation in the prolactin signaling proteins was examined, and all the components were found to be phosphorylated in response to hormone in cells cultured on BM but not in cells on collagen (Fig. 1, C-F). In addition, prolactin treatment resulted in an apparent increase in the molecular weight of Stat5b (27). Thus, in adherent mammary cells, there is an ECM-specific component of prolactin signaling that acts at the level of the PrlR. Integrins are required for prolactin-dependent milk protein synthesis (13), indicating that the differentiation signal from BM positively influences the prolactin cassette (Fig. 1G), either directly through receptor clustering (28) or indirectly through an additional pathway.
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The lack of signaling in mammary cells cultured on collagen I was not simply due to a delayed prolactin response, because Jak2 phosphorylation and Stat5 DNA binding activity were not detected in cells treated for up to 2 days with prolactin.2 However, in cultures on BM, we noted that the early signaling events were transient. Tyrosine phosphorylation of Jak2 (Fig. 2A) and co-immunoprecipitated PrlR (Fig. 2, A and B) and of Stat5a and Stat5b (Fig. 2, C and D) as well as Stat5 DNA binding activity (Fig. 2E) occurred rapidly before diminishing to basal levels within 60-120 min. To determine whether this down-regulation of the prolactin cassette shortly after its initial activation was controlled by a PTP activity (29), mammary epithelial cells cultured on BM were incubated with low concentrations (25 µM) of the PTP inhibitor sodium pervanadate (30). After treating cells with prolactin for 120 min, Stat5a and Stat5b retained the maximum level of phosphorylation that had been achieved in vanadate-free cultures (Fig. 2, C and D). The protein levels remained constant throughout the experimental period, indicating that they were first phosphorylated in response to hormone but were subsequently dephosphorylated. Thus, the prolactin-induced phosphorylation of Jak2, PrlR, and Stat5 is transient, and once activated the pathway subsequently comes under negative control by a PTP that reduces the signal 30-60 min after initiation.
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We then asked whether the initial events leading to activation of Stat5 were also influenced by vanadate-sensitive mechanism. Mammary cells cultured on BM were treated with sodium pervanadate for 30 min and then incubated for an additional 15 min with or without prolactin. PTP inhibitor treatment led to hormone-independent activation of the prolactin signaling cassette including the phosphorylation of Jak2, PrlR, Stat5a, and Stat5b (Fig. 3A) as well as the induction of Stat5 DNA binding activity (Fig. 3B), which was confirmed by supershift assays (Fig. 3C). Prolactin triggers Jak-Stat signaling through receptor dimerization, which leads to phosphorylation of Jak2 (14-16), but our data now show that PTP inhibition also contributes to Jak2 activation. This suggests that a PTP and a kinase are normally functionally associated to suppress prolactin signaling, but this can be overcome by either the natural ligand, prolactin, or by artificial inhibitors of PTPs (Fig. 3D). Although vanadate resulted in ligand-independent Stat5 DNA binding, milk proteins were not synthesized even in the presence of prolactin3; an additional PTP may therefore be required (31) for Stat5 to induce milk protein gene transcription (19), or alternatively, Stat1 and Stat3, which were also activated under these conditions (Fig. 3C), may compete for Stat5 DNA binding sites.
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Our observation that PTPs may be involved with initial events of prolactin signaling suggested that PTPs might also be implicated in the lack of response to prolactin in mammary cells cultured on collagen I (Fig. 1). Cells on collagen were treated similarly with sodium pervanadate. Now hormone stimulation led to the phosphorylation of Jak2, PrlR, and Stat5 proteins (Fig. 4A). Thus, in contrast to cells on BM, prolactin signaling is inhibited in cells on collagen I by a PTP that cannot be inactivated by the hormone alone. We noted that the pathway could not be stimulated fully by pervanadate in cells cultured on collagen I. Although Stat5b was tyrosine phosphorylated, it was only partially altered in molecular weight (Fig. 4A), and no Stat5 DNA binding activity (Fig. 4B) or milk protein synthesis3 was observed even in the presence of prolactin. This indicates that additional factors, for example cell geometry (32, 33), contribute to differentiation. Together the data show that one role for BM in mammary differentiation is to regulate the PTP/PTK balance, thereby allowing prolactin to trigger its signaling cassette (Fig. 4C).
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DISCUSSION |
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Two important conclusions can be derived from this study. First, one mechanism by which BM regulates milk protein gene transcription is by permitting prolactin to trigger the intracellular phosphorylation cascade, which leads to Stat5 transcription factor activation. Second, there is a vanadate-sensitive inhibition of prolactin signaling when mammary cells are cultured on collagen, indicating an involvement of PTPs in cell regulation by ECM.
Cross-talk between BM and Prolactin Signaling-- In earlier studies we demonstrated that Stat5 DNA binding activity could only be detected in mammary cells cultured on BM, not in cells on collagen (20), and therefore argued that the previously established role for BM on milk protein synthesis (12, 34) was exerted at the level of transcription factor activation. We now show that BM signals interact with the prolactin pathway upstream of Stat5 at the plasma membrane.
These results indicate that in adherent mammary epithelial cells, the prolactin pathway is not controlled simply by the presence of an endogenous cytokine as it is in hematopoietic cells (14-18), but rather it is under positive regulation from both the cytokine and a specific type of ECM. BecausePTPs Regulate Activation of Prolactin Signaling-- Regulation of homeostasis within the cell by PTKs is mediated by PTPs (8, 9). The involvement of PTPs in cell signaling pathways has frequently been assessed by the use of vanadate (30, 40-42), because there are virtually no inhibitory reagents currently available for specific members of the PTP family. Our results indicate that there are at least three vanadate-sensitive steps in prolactin signaling in mammary cells.
First, the down-regulation of Stat5 phosphorylation 120 min after prolactin treatment was inhibited by vanadate. The PTP, SHP-1, inactivates the homologous erythropoietin signaling pathway by binding to the erythropoietin receptor shortly after initial activation (29). In addition, SHP-1 binds tyrosine phosphorylated Stat5 after growth hormone activation and mediates its dephosphorylation (43). Thus, our results may reflect a similar involvement of PTP in attenuating prolactin-triggered signaling in mammary cells. Second, the initial phosphorylation events in prolactin signaling were independent of ligand following short treatments with low concentrations of vanadate. Given that prolactin is currently perceived to activate its signaling pathway through the PTK, Jak2 (44), our results suggest that Jak2 may normally be under negative regulation by a PTP. One action of prolactin-mediated ligation of its receptor may therefore be to alter the balance of a PTP-Jak2 cycle rather than merely activate the kinase. Third, in mammary cells cultured on collagen, vanadate enabled prolactin to trigger the early phosphorylation events in its signaling pathway. The implication is that in the absence of correct ECM signals, a PTP may inhibit cytokine-dependent signaling. This represents a novel aspect of ECM control on cell differentiation. We do not yet know the identity of the putative PTP(s) involved with ECM signaling in mammary cells or indeed whether the regulation is through PTP-interacting proteins such as signal-regulatory proteins (45, 46). However, the results suggest that PTPs may contribute to selectivity of integrin responses for controlling differentiation. For example, the laminin-binding integrinsSummary-- Our work offers a new paradigm to explain cell regulation by ECM, in which signals from correct integrin-ECM interactions positively influence cytokine-induced differentiation, whereas PTP-mediated inhibition of cytokine signaling occurs when cells are in an inappropriate ECM environment.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Paul Clarke, David Garrod, Andrew Gilmore, and Martin Humphries for critical review of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Wellcome Senior Fellow in Basic Biomedical Science. To whom
correspondence should be addressed: School of Biological Sciences, University of Manchester, 3.239 Stopford Bldg., Oxford Rd., Manchester, M13 9PT, UK. Fax: 44-161-275-3915; E-mail: charles.streuli{at}man.ac.uk.
1 The abbreviations used are: ECM, extracellular matrix; BM, basement membrane; EMSA, electromobility shift assay; PrlR, prolactin receptor; PTP, protein-tyrosine phosphatase; PTK, protein-tyrosine kinase.
2 G. M. Edwards and C. H. Streuli, unpublished data.
3 F. H. Wilford and C. H. Streuli, unpublished data.
4 G. M. Edwards, G. Zoubiane, and C. H. Streuli, unpublished data.
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REFERENCES |
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