Cell Tissue Res (2011) 346:175189

DOI 10.1007/s00441-011-1253-z

REGULAR ARTICLE

Distribution of prolactin receptors suggests an intraductal

role for prolactin in the mouse and human mammary gland,
a finding supported by analysis of signaling in polarized
monolayer cultures
Eric K. Ueda & KuangTzu Huang & Virginia Nguyen &
Marco Ferreira & Saudade Andre & Ameae M. Walker

Received: 10 June 2011 / Accepted: 15 September 2011

# Springer-Verlag 2011

Abstract Despite the important role of prolactin (PRL)
in mammary gland development and function, little is
known about the distribution of the different forms of the
prolactin receptor (PRLR) under various physiological
circumstances. Here, the distribution of the long (LF) and
the short (S3 in mouse) receptor common to both mice
and rats was determined by immunofluorescence on
frozen sections of virgin, pregnant and lactating mouse
mammary gland. Myoepithelial cells were consistently
and intensely stained for both receptors. For luminal cells
at all stages (ducts and alveoli), a large proportion of
PRLR staining was unexpectedly present on the apical
face. In the non-lactating state, no basal staining of
luminal cells was detectable. During lactation, a proportion of
both receptors moved to the basolateral surface. In vitro,
HC11 cells showed constitutive expression of LF but
This work was supported by a grant from the California Breast Cancer
Research Program, 10 PB-0127 and a grant from the Susan Love
Foundation.
This work was presented in part at the Annual Meeting of the
Endocrine Society, 2008.
E. K. Ueda : K. Huang : V. Nguyen : A. M. Walker (*)
Division of Biomedical Sciences, University of California,
Riverside CA 92521, USA
e-mail: ameae.walker@ucr.edu
M. Ferreira
Servio de Anatomia Patolgica,
CHLN Hospital de Santa Maria, E.P.E.,
Lisbon, Portugal
S. Andre
Servio de Anatomia Patolgica, Insituto Portugus de Oncologia
de Lisboa Francisco Gentil, E.P.E.,
Lisbon, Portugal
expression of S3 only upon the formation of adherent
junctions. Tight junction formation was accelerated by
incubation in pseudo-phosphorylated PRL, as measured by
transepithelial resistance and the expression and placement
of the tight junction protein, zonula occludens-1. Once
an intact monolayer had formed, all LF and S3 receptors
were apical (akin to the non-lactating state) and only
apical application of PRL activated the Jak2-STAT5 and
ERK pathways. By contrast, basolateral application of PRL
resulted in a reduction in basal ERK phosphorylation,
suggesting an involvement of a dual specificity protein
phosphatase. Normal human breast samples also showed
apical PRLRs. These results demonstrate important
contextual aspects of PRL-PRLR interactions with implica-
tions for the analysis of the role of PRL in breast cancer.
Keywords Apical . Ductal prolactin . Long and short
prolactin receptors . Immunofluorescence . Human . Mouse
Introduction
In newborn mice, the mammary gland has a rudimentary
ductal system. This grows rapidly during puberty, continuing
to develop with each estrus cycle until an arborizing structure
fills the mammary fat pad. During pregnancy, these original
ducts gain additional side branches and secretory alveoli
develop (Richert et al. 2000). Many hormones/growth
factors are involved in the regulation of full mammary
gland development and function, including estrogen,
progesterone and prolactin (PRL; Neville et al. 2002).
Here, our focus is on PRL and on what the distribution of
PRL receptors (PRLRs) can tell us about the way that PRL
achieves its known functions.
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Alveolar structures do not develop in the PRLR
knockout mouse, even when mammary epithelium from
such animals is transplanted into the fat pads of normal
mice during pregnancy (Ormandy et al. 1997). This
illustrates an absolute requirement for some form of
mammary epithelial PRLR for alveolar development.
PRL also contributes to ductal branching and to the
expansion of existing ducts (Hovey et al. 2001; Kuo et al.
2002), although significant branching still occurs in the
absence of PRLR (Ormandy et al. 1997). Both ductal
branching and the production of alveoli involve epithelial
cell proliferation, epithelial invasion of the basement
membrane and remodeling of the basement membrane
and myoepithelial architecture (Gudjonsson et al. 2005).
Since these processes are central to the progression of
breast cancer, a more complete examination of their
regulation by PRL is important for a better understanding
of the disease process (Clevenger et al. 2003) and of the
normal physiology of the mammary gland.
A number of PRLR isoforms have been found, most
of which are formed by alternative splicing of a single
gene transcript. In the mouse and human, four trans-
membrane forms have been described, a long form (LF)
and three short forms (called S1-3 in the mouse),
differing from one another in the length and sequence
of their cytoplasmic regions (Kelly et al. 1991; Ormandy
et al. 1998; Hu et al. 2001; Trott et al. 2003; Pujianto et al.
2010). To date, no immunohistochemical study has been
undertaken comparing the overall and specific PRLR
localization in an animal model in which it is possible
to examine this aspect under various physiological
circumstances. Since the rat expresses only one short
form of the receptor (Kelly et al. 1991) and since this is
homologous to the mouse S3 receptor, we have focused on
the LF and S3 receptors on the assumption that anything
learned would be at least applicable to both rodent
species. We present results that have provided some new
and unexpected insights into contextual aspects of the
action of PRL in vivo. These include evidence that
myoepithelial cells have PRLRs and hence, that they
might be direct targets of this hormone. In addition, we
show that, under all physiological circumstances, a large
proportion of LF and S3 PRLRs is apically located on
luminal epithelial cells and that we can only detect these
PRLR on the basolateral surface during lactation. A
similar apical localization of PRLRs has been found in
samples of normal female human breast. Together with
other supporting in vitro signaling data, these results
suggest that the duct lumen in the non-lactational state
constitutes the normal microenvironment in which PRL
interacts with receptors capable of activating luminal
epithelial Jak2-STAT5 and extracellular signal-regulated
kinase (ERK) pathways.
Cell Tissue Res (2011) 346:175189
Materials and methods
Human and animal tissues
Samples of normal female, non-pregnant and non-lactating
breast tissue were obtained from the archives of the
Pathology Department of Instituto Portugus de Oncologia
de Lisboa Francisco GentilE.P.E. (Lisbon, Portugal). The
review board, Comisso de tica, did not require approval
for the use of these archival materials.
The care and handling of the animals were in accordance
with procedures approved by the University of California,
Riverside Institutional Animal Care and Use Committee
and were in accordance with guidelines from the American
Association for Laboratory Animal Care, the United States
Department of Agriculture and the National Institutes of
Health. Mice (C57BL/6) were bred in house and given food
and water ad libitum. Animals were observed daily and the
observation of a vaginal plug was counted as day 1 of
pregnancy. Litter sizes were between six and eight in all
animals used for the lactation part of the study.
Antibody analysis
The main PRLR antibodies were originally a gift from
Dr. Patricia M. Ingleton and, after her retirement, were a
gift from Dr. Michael Symonds (University of Nottingham,
UK). The antibodies were produced against specific peptide
regions of rat PRLRs. Antiserum R122 was raised against
residues 309325 specific to the intracellular region of the LF
receptor (100% identity exists between the rat and mouse
receptors for this region). R133 was raised against
residues 281296 specific to the intracellular domain of
the short rat receptor, which has 87% identity (100% for
residues 281290) to the mouse S3 short receptor. There
is no recognition of the other two mouse short forms.
None of these sequences shares significant homology
with other members of the cytokine receptor family or
any other protein in the National Center for Biotechnology
Information database. For example, for the 281296
sequence, homology to the S3 mouse receptor is more
than 100-fold greater than for any part of another protein
(determined by Basic Local Alignment Search Tool
analysis). These antibodies have been used by a variety
of laboratories examining PRLRs in diverse species
(Nevalainen et al. 1996, 1997; Bispham et al. 1999; Coss
et al. 1999; Gregory et al. 2000; Pearce et al. 2005).
To create positive controls for the Western blot analysis
of the anti-receptor antibodies, the mouse LF and S3
PRLRs were cloned: total RNA from mouse liver was
extracted with Trizol (Invitrogen, Carlsbad, Caif., USA)
according to manufacturers protocol. First-strand cDNA
was reverse-transcribed. The cDNA for mouse PRLR LF
Cell Tissue Res (2011) 346:175189
was amplified by the polymerase chain reaction PCR: the
N- and C-terminal transcripts were first synthesized by
using Accuprime pfx polymerase (Invitrogen). During the
process and as an added control for their later positive
identification, the receptors were tagged with the Myc
epitope (EQKLISEEDL). The primer sequences were: 5-
atgccatctgcacttgct-3 (N1, forward), 5-ctcccactcatctgctt
tcttca-3 (N2, reverse), 5-catcatcacagtaaatgccacg-3 (C1,
forward) and 5-tcacagatcctcttcagagatgagtttctgctcgtgaaag
gagtgcat-3 (C2, reverse); an S3-specific C2 reverse primer
(5-tcacagatcctcttcagagatgagtttctgctcgtagtcaagttcccc-3) was
used for synthesizing the S3 cDNA (nucleotides coding for
the Myc epitope plus the stop codon are underlined). PCR
products were resolved on a 1% agarose gel. DNA
fragments were excised and extracted by using a Qiagen
extraction kit (Qiagen, Valencia, Calif., USA). These
DNA fragments were used as templates for a second
PCR by using N1 and C2 primers. Extra nucleotides
corresponding to EcoRI (GAATTC) and XhoI (CTCGAG)
were designed to be upstream of the initiation codon and
downstream of the stop codon. PCR amplicons were
extracted from the gel, digested with EcoRI and XhoI
and inserted into the expression vector, pcDNA3.1(+)
(Invitrogen), which was restriction-digested with the same
enzymes. Complete sequence analysis was performed on
the acquired plasmids.
Transient transfections of the plasmids containing the
receptors were performed by using Fugene HD transfection
reagent (Roche Diagnostics, Indianapolis, Minn., USA),
according to the manufacturers protocol. Briefly, 2106 HEK
293 cells were seeded in each 6-cm dish. The next day,
5 g DNA (per dish) was first incubated with 250 l
serum-free RPMI and then 10 l Fugene HD reagent.
This mixture was incubated for 15 min and then added to
the cells. Cells were harvested and lysed for protein
extraction 48 h after transfection. These lysates served as
positive controls in the Western blots of mammary tissue.
Processing of mammary tissue for Western blot
Mouse mammary glands were collected at lactation day 7
(L7) and L18, snap-frozen in liquid nitrogen and stored
at 80C until use. About 0.05 g frozen tissue was
ground with a mortar and pestle in liquid nitrogen.
Extraction was carried out in a glass homogenizer in
0.5 ml cell lysis buffer (0.14 M NaCl, 20 mM TRIS base,
10 mM Na2P2O7, 10 mM NaF, 1 mM Na3VO4, 1% NP-40,
0.02% NaN3, 0.5 mM EDTA) supplemented with Complete
Mini protease inhibitor cocktail (Roche Diagnostics,
Indianapolis, Iind., USA), followed by two freeze-and-thaw
cycles to help release membrane contents. Transfected or
non-transfected HEK 293 cell lysates were similarly
extracted but without the mortar and pestle step.
177
For Western immunodetection, equal quantities of whole
cell lysates (50 g protein) were resolved on a reducing
10% SDS polyacrylamide gel and the proteins were
transferred onto a nitrocellulose membrane (Bio-Rad,
Hercules, Calif., USA). Membranes were blocked with
5% skim milk and then probed with rabbit anti-PRLR
(R122, or R133, 1:10,000) overnight at 4C. Antigen-antibody
interactions were detected by using horseradish-peroxidase-
conjugated goat anti-rabbit antibodies (1:25,000; Jackson
ImmunoResearch Laboratories, Bar Harbor, Me., USA).
Immunofluorescent localization of PRLRs
Mammary glands (4 and 5 inguinal glands) from nulliparous
18-day-pregnant and from 2-, 7-, 12- and 18-day lactating
animals were fixed in phosphate-buffered formalin, infiltrated
with 30% sucrose for 23 days at 4C and then frozen in
Tissue-Tek Optimal Cutting Temperature (OCT) medium
(Miles Scientific, Naperville, Ill., USA). Frozen blocks were
stored at 80C until sectioning. Frozen sections of 7 m
thickness were cut on a cryotome (HM 500 OM, Microm
Lamborgeraete) set at 30C. Most immunolocalization was
performed on freshly cut sections.
Frozen sections were allowed to thaw in 50% ethanol.
This removed OCT before dehydration through graded
ethanol and then fixation in methanol at 20C for 6 min.
The sections were then rinsed in phosphate-buffered saline
(PBS) before being blocked in PBS containing 3% bovine
serum albumin (BSA). This was followed by overnight
incubation in the anti-PRLR antibodies diluted 1:50 in
PBS, with or without mouse anti-smooth muscle actin
(1:100, Abcam, Cambridge, Mass., USA). Sections were
then washed (35 min) in PBS before incubation in
fluorophor-conjugated secondary antibodies: Alexa 555
donkey anti-rabbit IgG (1:400 dilution; Invitrogen) and/or
Alexa-488 goat anti-mouse IgG (1:400 dilution; Invitrogen).
The sections were then washed (410 min) with PBS
and mounted in ProLong Gold Antifade Reagent with
4,6-diamidino-2-phenylindole (DAPI; Invitrogen).
For HC11 cells, fixation was in 4% paraformaldehyde
in PBS for 15 min at 4C. This was followed by
permeabilization with 1% Triton X-100 in PBS for
10 min at room temperature. Autofluorescence from
paraformaldehyde fixation was quenched with 20 mM
glycine in PBS. The cells were then washed (35 min)
with PBS before being blocked with PBS containing 5%
fetal bovine serum (FBS) and 0.1% Triton X-100 for
30 min. This was followed by incubation in anti-receptor
antibodies as above and/or rabbit anti-zonula occludens-1
(ZO-1; 1:1000 dilution; Zymed, San Francisco, Calif., USA),
mouse anti-E-cadherin (1:1000 dilution; BD Biosciences,
San Jose, Calif., USA), mouse anti-occludin (1:2000
dilution; Zymed), or mouse anti-smooth muscle actin
178
(1:100) in PBS/0.1% Triton-X 100 at 4C overnight.
After three 10-min washes with PBS, cells were
incubated for 1 h at room temperature with fluorophor-
conjugated secondary antibodies, washed and mounted,
as above. A minimum of four different animals were
examined for each physiological state and stage and
where appropriate, ducts and alveoli were separately
assessed. Serial sections throughout inguinal glands 4
and 5 were examined.
Controls included the use of isotype-matched non-specific
antibodies or a non-specific antiserum (as appropriate) in
place of the first antibody, analysis for second antibody
cross-reactivity with dual labeling and analysis for
spectral bleeding between channels on the confocal
microscope. In the hopes of producing an additional test
of antibody specificity, paraffin-embedded mammary
tissue from PRLR knockout and wild-type littermates
was obtained from Dr. Chris Ormandy (Garvan Institute,
Sydney, Australia). However, since the tissue processing
and embedding prevented staining of the wild-type
control, we could not use the absence of staining of the
PRLR knockout tissue to support the specificity of the
antibodies.
Confocal images were obtained by using a Leica TCS
SP2 confocal laser-scanning microscope. Fluorophor Alexa
488 was excited with a 488-nm laser line and imaged at
500530 nm. Fluorophor Alexa 555 was excited at 543 nm
and imaged at 560600 nm. DAPI was excited at 351 nm
with an Ar-UV laser source and imaged at 458 nm. Images
were analyzed by using Leica confocal software, version
2.5 and produced by using Adobe Photoshop version 7.0
software. Pinhole values were set to obtain 1-m optical
sections. Images (512512 pixels) were taken at 12-bit
resolution with pixel times of 6.4 s and 23 line-averaging.
All multichannel images were obtained in multitrack
imaging mode to avoid crosstalk with other channels.
Human tissue
After the archived sections had been dewaxed with xylene,
endogenous peroxidase was blocked with 2% hydrogen
peroxide in methanol and antigen retrieval consisted in
1 min pressure cooking in a buffered 0.01 M sodium citrate
solution, pH 6. An anti-PRLR monoclonal antibody raised
against purified human PRLRs (NeoMarkers, Fremont,
Calif., USA; clone B6.2, diluted 1:1500) with overnight
incubation at 4C was used. The bound primary antibody
was detected by using the labeled streptavidinbiotin
method (Dako K 5001; Dako, Glostrup, Denmark), with
3-3-diaminobenzidine tetrahydrochloride as the chromo-
gen. Counterstaining was achieved with Mayer's hematox-
ylin. Controls involved substitution with an isotype-
matched non-specific primary antibody.
Cell Tissue Res (2011) 346:175189
HC11 cell culture
This normal mouse mammary epithelial cell line was
grown on 24-mm Transwell clear polyester 8-m pore
filter inserts (Corning, N.Y., USA) in RPMI-1640
medium supplemented with 10% heat-inactivated FBS,
1% penicillinstreptomycin, 5 g/ml insulin and 10 ng/ml
epidermal growth factor at 37C in a 5% CO2/95% air
atmosphere. The cultures were kept for 2 days at confluency
before the medium was changed to RPMI-1640 medium
containing 10% FBS and 1% penicillinstreptomycin and
either insulin (10 g/ml) and dexamethasone (1 M) or
these plus PRL, or S179D PRL (both at 1 g/ml and
produced as described previously, e.g., by Coss et al. (1999).
After addition of hormones, the cells were cultured for an
additional 9-day period and the medium was replaced every
3 days. Only low passage number cells were used.
Transepithelial resistance measurements
Transepithelial resistance (TER) was measured on Transwell
filter-grown cells. These were transferred into Advanced
RPMI-1640 and allowed to cool to room temperature
before use of the Epithelial Voltohmmeter (World Precision
Instruments). TER measurements were taken after electrode
(chopstick-like) sterilization with ethanol. Calculations for
ohms.cm2 were made by subtracting a blank filter and
multiplying by the area of the monolayer [TER (cm2)=
(Total resistance Blank resistance) ()Area (cm2)]
(4.5 cm2 for the 24-mm filter inserts). Statistical analysis
was by an analysis of variance with post tests and corrections
for multiple comparisons.
Signal generation after engagement of the receptor
To detect the activation of signaling molecules, cells
were incubated in Advanced RPMI-1640 containing
dexamethasone and insulin (as above) and were then
treated with PRL (1 g/ml) for the times indicated. Cells were
extracted in lysate buffer (0.14 M NaCl, 20 mM TRIS base,
10 mM Na2P2O7, 10 mM NaF, 1 mM Na3VO4, 1% NP-40,
0.02% NaN3, 0.5 mM EDTA) supplemented with Complete
Mini protease inhibitor cocktail, as above. Lysate proteins
were resolved by reducing and denaturing polyacrylamide
gel electrophoresis and subsequently transferred to a
polyvinylidene fluoride membrane. After being blocked with
5% skim milk proteins, the membrane was incubated with
anti-phosphoERK (recognizing tyrosine phosphorylation)
to assess ERK activation (dilution 1:1000; Santa Cruz
Biotechnology, Santa Cruz, Calif., USA). The blots were
stripped and re-probed with anti-total ERK (dilution 1:1000;
Santa Cruz Biotechnology). Antigenantibody interactions
were detected by using horseradish-peroxidase-coupled
Cell Tissue Res (2011) 346:175189
secondary antibodies (1:2000 for 1 h) and enhanced
chemiluminescence (Amersham Biosciences, Piscataway,
N.J., USA).
For Stat5 activation, cell lysates were pre-cleared by
incubation with protein ASepharose beads (Santa Cruz
Biotechnology) at 4C for 1 h. The sample was then
incubated with the beads pre-coupled with anti-Stat5A/B
antibody or a rabbit nonspecific -globulin (Santa Cruz
Biotechnology) for 18 h at 4C. After being washed,
bound components were eluted by boiling in gel sample
buffer for 5 min. Blots were probed with antibodies
against phospho-tyrosine, stripped and re-probed with
anti-total-Stat5 (1:1000; Santa Cruz Biotechnology).
Results
Characterization of the antisera
We have used immunofluorescence on frozen sections to
examine PRLR expression in sexually mature nulliparous,
pregnant and lactating mouse mammary glands. Although
the antisera employed have been previously characterized
by Western blot for studies of the rat and human prostate
and of sheep adipose tissue (Nevalainen et al. 1996, 1997;
Bispham et al. 1999; Coss et al. 1999; Gregory et al. 2000;
Pearce et al. 2005), they have not to our knowledge been
previously characterized for the localization of mouse
receptors or for localization in mammary tissue. The rat
has an LF and only one short form of the PRLR, the latter
being homologous to the mouse S3 receptor. One might
predict therefore that focus on the LF and S3 receptors
should provide insight into aspects of PRLR function in the
mammary gland, aspects that are at least common to both
these species. The use of these two antibodies is also
anticipated to shed light on the importance of the LF versus
the short form and on the overall PRLR localization.
Figure 1a, b shows Western blots of tissue/cell extracts
by using the anti-S3 or anti-LF antibodies, respectively.
Samples of lactating mammary gland were obtained on
days 7 and 18 of lactation. In addition, extracts of HEK 293
cells lacking endogenous PRLR and of HEK 293 cells
transfected with LF or S3 mouse receptors served as
negative and positive controls. Bands were seen at the
correct molecular weight for the receptors (Fig. 1, solid
arrows). A non-specific band was present in whole cell
lysates from non-transfected HEK 293 cells (Fig. 1, open
arrows). Controls with non-specific primary antibody and
development without any primary or secondary antibody
indicated that this was an endogenous cellular peroxidase.
In other words, although producing a band on the Western
blot because the methodology employed peroxidase detec-
tion, this was not a cross-reacting protein that would cause
179
Fig. 1 Western blots with antibodies against the short (S3) or long
form (LF) of prolactin receptor (PRLR): anti-S3 (a) or anti-LF (b)
antibodies. Extracts of lactating glands on lactation days 7 and 18 (L7,
L18) were compared with extracts of HEK 293 cells. HEK 293 cells,
which do not have endogenous PRLR, were used as a negative control
or as a positive control when transfected with LF (293LF) or S3
(293S3) receptor (solid arrows bands of the correct molecular weight,
open arrow non-specific band, numbers right location of molecular
weight markers)
non-specific immunofluorescence in our immunolocaliza-
tion studies. However, to be certain that not more than one
component was present in the band, we conducted an
immunofluorescence study on HEK 293 cells to determine
whether any component at the same molecular weight as
the peroxidase might have contributed to apparent
receptor staining when used on mammary tissue. No
immunofluorescence staining was observed by confocal
microscopy or fluorescence-activated cell sorting (FACS)
analysis on a FACScan, with or without cell permeabilization
(data not shown as all negative).
PRLR immunoreactivity in mouse
The presented figures are described in terms of the
particular antibody used but a similar localization was
obtained with each antiserum, unless otherwise described.
Preliminary studies gave the initial impression that most
PRLRs were localized at the base of luminal epithelial cells,
a plausible location in order for them to respond to
circulating PRL. However, a more detailed examination
showed that the basal staining was largely coincident with
structures having the morphology of myoepithelial cells.
Co-staining with antibodies to smooth muscle actin was
therefore initiated.
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Figure 2a, b shows the use of the anti-S3 antibody to
localize PRLRs (red) in the nulliparous mammary gland.
The epithelial component of these glands is only ductular.
In the same sections, myoepithelial cells have been
Fig. 2 Immunohistochemical demonstration of PRLRs in ducts. a, b
S3 PRLRs (red) and smooth muscle actin (green) in a duct from a
nulliparous animal (white arrow apical PRLR staining, yellow arrows
in a, b same regions of the myoepithelial layer illustrating that red
staining is not the result of bleeding from one channel to the next).
Some bright green spots in a are not present in red in b and vice versa
(white asterisk lumenin; here and in subsequent figures). Bar100 m.
c Region of duct taken from an 18-day lactating animal. Tissue was
stained with anti-LF (red), anti-smooth muscle actin (green) and
4,6-diamidino-2-phenylindole (DAPI) for nuclei (blue). The images
from the various fluorescence channels have been combined.
Colocalization of red and green fluorescence gives yellow, as seen
in large portions of the myoepithelial cells. Bars40 m
Cell Tissue Res (2011) 346:175189
identified by staining with anti-smooth muscle actin
(green). In Fig. 2, one can clearly see the apical localization
of PRLR immunoreactivity on the luminal epithelial cells
(white arrow). In these glands, we also noted a large
amount of staining of material in the actual duct lumen,
regardless of the PRLR antibody used. This was a
consistent finding not seen, to any comparable degree, in
the pregnant or lactating state and might be the result of
accumulated shed epithelial cells, PRLR-positive immune
cells, or shed receptors. The co-localization of smooth
muscle actin and PRLRs could also be found in the
myoepithelial cells. Non-specific staining with a control
rabbit antibody (control for anti-PRLR) was faint (see
Fig. 3 in which the use of only one primary antibody
makes the negative result easier to appreciate). The
yellow arrows in Fig. 2a, b draw attention to an area that
is brightly green but not extremely red, thereby illustrating
that colocalization in the myoepithelial cells is not attributable
to fluorescence channel bleed-through or cross-reactivity of
the second antibodies used. Likewise, the region illustrated by
the white arrow shows that the red fluorescence is not
bleeding into the green channel and again that the second
antibodies are not cross-reactive.
Apical luminal epithelial and myoepithelial PRLR
staining of the ducts was found at all stages of gland
development. Figure 2c, for example, shows a duct from a
lactating animal. Having demonstrated that co-localization
of PRLR and smooth muscle actin was not attributable to
cross-reactivity or bleed-through, this part of the figure
presents a merged image in which co-localization in
myoepithelial cells produces yellow staining. In this
example, the antibody specific to the LF receptor was used
and nuclei were illustrated with DAPI staining.
Apical luminal (white arrow) and myoepithelial (yellow
arrow) staining was also seen in alveoli (Fig. 3). This
example shows part of a pregnant mammary gland, this
time only stained with an anti-PRLR antiserum (anti-S3; no
anti-smooth muscle actin) together with a control section.
The control section was cut and processed concurrently by
using non-specific rabbit antiserum. The control and
specifically-labeled sections were visualized with the same
microscope settings and the images were identically processed.
At day 2 of lactation, localization of the PRLR was the
same as in the pregnant animals shown in Fig. 3 but by day
7, several differences had occurred. First, one could
appreciate heterogeneity at the level of PRLR expression
(white arrows in Fig. 4a, c indicating cells with a high level
of expression, stained with anti-LF and anti-S3, respectively).
The degree of heterogeneity differed among animals and was
regional within one gland. Heterogeneity in the level of
expression of PRLR has been described previously (Hovey et
al. 2001; Rosen 2003). Based on experience gained from the
current study, the heterogeneity was not only regional within
Cell Tissue Res (2011) 346:175189
Fig. 3 Immunohistochemical demonstration of PRLRs in a pregnant
animal. a In this instance, staining was with anti-S3 (white arrow
apical staining, yellow arrow myoepithelial staining). b Background
with the control antibody with the same microscope settings and
image processing technique. Bar80 m
the gland at 7 days but also disappeared as lactation
continued. At higher magnification in more homogeneously
labeled areas, labeling was observed within the luminal cells.
With anti-LF, labeling was of intracellular vesicular structures
with an apparent diameter of 23 m (Fig. 4b, white
arrowhead). These structures were not stained with the anti-
S3 antibody (Fig. 4d). With anti-S3, intracellular labeling did
appear punctate but the labeled structures were much smaller.
181
In some images, where favorable sections of alveolar
samples from 7-day lactation caught the myoepithelial
cell away from the base of the luminal epithelial cell,
one could see anti-PRLR staining on regions of the
luminal cell that extended basally; staining with both
anti-LF and anti-S3 was observed (Figs. 4b, 5a, b, green
arrowheads). This was not seen in the pregnant animals or
at day 2 of lactation, despite careful analysis of similar
images.
With both antibodies, staining of luminal alveolar
cells was concentrated in apical depressions (seen most
clearly at white arrowheads in Fig. 5b, which is stained
with anti-S3 and in Fig. 5c, which is stained with anti-LF)
about the size of lipid droplets (58 m) and confirmed as
such by oil red O staining (data not shown). At days 12
and 18 of lactation, the situation was similar. At 18 days of
lactation, overall staining is best appreciated in Fig. 6a and
concentration in the depressions in Fig. 6b, which also
shows a region that lies at the base of the luminal
epithelial cell and that is stained with anti-LF PRLR
(green arrowhead).
Human tissue
To ensure that the results obtained by analyzing mouse
tissues were of relevance to the human condition, samples
of normal, non-pregnant and non-lactating female breast
tissue were examined. Figure 7 shows substantial apical
antibody staining of the ductal epithelium and staining of
components in the duct, just as was observed in the
nulliparous mice (Fig. 7a, b, larger and smaller duct,
respectively). However, myoepithelial cell staining with
this antibody was minimal.
In vitro modeling
Having demonstrated the presence of apical PRLR immuno-
reactivity on the luminal cells, our next question was
whether this could be modeled in vitro to assess
functionality by using mouse HC11 cells. Figure 8a, b
shows the immunostaining of HC11 cells with either
anti-LF or anti-S3 and co-staining with anti-E-cadherin
during the development of confluency. All cells stained with
the anti-LF antibody but only those bounded by E-cadherin
stained with the anti-S3 antibody. This result and the
previous differential localization of intracellular PRLRs
further demonstrated that the two antisera recognized
different proteins.
When cells were grown to confluency in growth medium
(containing 10 g/ml insulin, 10 ng/ml EGF) on a semi-
permeable membrane in a Transwell system and then the
medium was changed to one without either supplement for
9 days, the cells formed junctions containing E-cadherin,
182 Cell Tissue Res (2011) 346:175189

Fig. 4 Immunohistochemical
demonstration of PRLR in
day-7 lactating animals.
Staining was with anti-LF
(a, b) or anti-S3 (c, d).
The heterogeneity of staining
is easier to appreciate at low
power (white arrows in a, c),
whereas parts of the
discontinuous myoepithelial
cell layer are easier to see in
b (yellow arrowheads).
The larger PRLR-containing
vesicles are indicated by a
white arrowhead in b. Note
the finer punctate labeling
throughout the cells in d.
The green arrowhead in
b indicates a region at the base
of a cell showing receptor
positivity in the absence of a
myoepithelial cell. Bars
80 m (a, c),40 m (b, d)

claudin, occludin and ZO-1. Occludin is illustrated in
Fig. 8d and ZO-1 in Fig. 9 (others not shown). TER was
consistently about 70 ohms/cm2 with no hormone supple-
mentation (Fig. 9a). Supplementation with dexamethasone
(1 M) and insulin (10 g/ml) increased junction formation
such that, on day 9, TER was 400 ohms/cm2. In other
words, tighter junctions formed but were not extensive,
similar to the situation in the non-lactating gland in vivo
(Nguyen and Neville 1998). Addition of unmodified PRL
to the mix (1 g/ml) did not change the rate or final extent
of junction formation. However, when a molecular mimic
of phosphorylated PRL, namely S179D PRL, was used
instead of unmodified PRL, an acceleration of tight
junction formation occurred. This was assessed by three
measurements: (1) immunofluorescent staining for ZO-1
(Fig. 9c-e), (2) Western blot analysis of ZO-1 expression
(Fig. 9b) and (3) TER (Fig. 9a). Acceleration was obvious
at day 7 of treatment. However, by day 9, all hormone
combinations produced equivalently sealed junctions.
Confocal serial sectioning through the entire cell layer
and three-dimensional reconstruction were used to deter-
mine the distribution of receptors in 9-day post-confluent
cultures. Figure 8g shows an example co-stained for
occludin. As presented in the reconstructed image viewed
from the side, all detectable PRLR staining was apical. The
example shown was immunostained with anti-S3 but the
same result was obtained with anti-LF.
Signaling from PRLRs
Similar polarized cell preparations were then used to
examine signaling with PRL placed apically or basally
with respect to the cells. When applied apically, PRL
stimulated the phosphorylation of both ERK and Stat5
(Fig. 10a, b). When applied basally, similarly exposed
Western blots showed no detectable increase in the
phosphorylation of these molecules (not shown). When
the blot was exposed to film long enough to produce a
substantial band for phosphorylated ERK in the absence of
added PRL (0 min), the time course showed that basally
applied PRL actually caused the dephosphorylation of
ERK (Fig. 10c). No similar effect was seen when
examining Stat5 phosphorylation in response to basal
application (data not shown).
Cell Tissue Res (2011) 346:175189
Fig. 5 Receptor localization on day 7 of lactation. a Portion of a
myoepithelial cell stained with anti-LF and showing luminal epithelial
cell portions left and right (white arrow). Bar 20 m. b Region stained
with anti-S3 (white arrowheads two apical depressions). Bar40 m.
c Staining with anti-LF, again illustrating LF-positive larger intracellular
vesicular structures and apical depression. Green arrowheads in a,
b indicate regions of the cell base positive for PRLR. Bars40 m
Discussion
The mammary gland has two major forms of epithelial cell,
namely luminal cells and myoepithelial cells, each derived
from a single stem cell precursor (Visvader 2009). Ducts
are bilayered, with luminal epithelial cells sitting above
an apparently complete myoepithelial cell layer. This
myoepithelial layer in turn sits on the basement membrane
(Hayward et al. 1996; Pitelka et al. 2009). Alveoli, by
contrast, have an intermittent myoepithelium such that
Fig. 6 LF receptor localization at day 18 of lactation. Tissue is
stained with anti-receptor (red), anti-smooth muscle actin (green) and
DAPI (blue, nuclei). The higher magnification in b illustrates an area
where PRLRs are seen basally (green arrowhead) below a dendritic
arm belonging to a myoepithelial cell (yellow). Bars 40 m
183
Fig. 7 PRLR localization in normal human breast tissue. Note the
apical staining in the form of the brown peroxidase reaction product in
both the large (a) and small (b) ducts. Bars40 m
luminal epithelial cells have large regions in direct contact
with the basement membrane (Hayward et al. 1996; Pitelka
et al. 2009).
Our expectation at the outset of this study was that
PRLRs on luminal epithelial cells would be basally
oriented, being placed to receive ligand from the circula-
tion, which for ductal cells would be by passage between
myoepithelial cells. However, we observed that at least a
large proportion of receptors were apically oriented both
in ducts and alveoli. Because of this unexpected result,
we closely examined previous studies of PRLR localiza-
tion in human tissue. Our finding was the same as that
described by Gill et al. (2001). Reynolds et al. (1997)
using an entirely different antibody described both apical
and basolateral staining in normal epithelium. In addition,
we examined normal human female breast tissue by using
a commercially available monoclonal antibody to the
human PRLR that had been used previously in several
studies of breast cancer (Mertani et al. 1998; Bhatavdekar
et al. 2000; Gill et al. 2001; Glasow et al. 2001). Once
again, we observed luminal staining. This agrees with a
previous report by some of us (Ferreira et al. 2008), which
similarly showed that the vast majority of staining was
apical on the luminal epithelial cells in a large duct of a
male with gynecomastia. In toto, this means that the use of
four different antibodies in mouse and human tissues has
given the same result. Whereas one of the antibodies used
for analyses in human tissue might not recognize the
PRLR per se (Galsgaard et al. 2009), there is agreement
that at the very least it recognizes a PRLR-associated
molecule (Galsgaard et al. 2009). Thus, our findings, with
regard to PRLR localization, are likely not to be specific to
the mouse and are not a function of the particular antibodies
that we used. Moreover, in addition to immunolocalization,
we have demonstrated functionality of apically oriented
receptors by using the polarized HC11 cells.
An apical orientation of receptors suggests an important
role for luminal (ductal) PRL in mammary gland function.
Luminal PRL might derive from the mammary epithelium
184 Cell Tissue Res (2011) 346:175189

Fig. 8 PRLR localization

on growing (a, b) and
polarized (c-g) HC11 cells.
PRLR are stained red
throughout. a, b E-cadherin
is immunostained green.
d, f, g Occludin is
immunostained green.
c Nuclei are stained with
DAPI (blue). g Cells have
been serially optically sectioned,
reconstructed and turned so
that the image represents
the cells from the side. One can
appreciate that all detectable
receptors lie above the level of
the nuclei. None is detectable
basally (images obtained
with the S3 antibody but the
same localization was obtained
with the antibody against the
LF receptor). Bars40 m (a),
60 m (c)

itself (autocrine) or be circulatory in origin. The importance PRLR in intracellular locations

of autocrine PRL in mammary development is illustrated
by a study from the Ormandy and Horseman laboratories
showing that when PRL expression is absent from
mammary epithelium, a 2.8-fold decrease occurs in
bromodeoxyuridine incorporation into mammary epithelium
when measured at the end of pregnancy (Naylor et al. 2003).
However, alveoli, whose formation is absolutely dependent
on PRL, do indeed form. Since the intercellular junctions
in pregnancy are permeable to molecules twice the size
of PRL (Nguyen and Neville 1998), PRL from the
circulation probably gains access to the luminal receptors
by passing between cells.
Tight junctions between mammary epithelial cells do not
become well-developed until lactation (Nguyen and Neville
1998). By day 7 of lactation in our study, sufficient receptors
(both LF and S3) have been found on the basolateral
surface of the luminal alveolar cells for detection by
immunofluorescent staining. Thus, the receptors appear to
redistribute at a time when paracellular passage of PRL might
no longer be possible and in order for the cells to be receptive
to suckling-stimulated circulating PRL.
In many of our images, the LF-PRLR-positive vesicular
structures of 23 m have been seen to accumulate
around forming lipid droplets. Others have described a
peripheral set of vesicular structures, derived from the
endoplasmic reticulum and associated with forming lipid
droplets (Wu et al. 2000). These are thought to contribute
membrane during the apocrine secretion of lipid droplets.
The concentration of PRLR in the lipid-droplet-sized
apical membrane depressions further contributes to the
impression that the LF-PRLR-positive structures are
associated with lipid droplet secretion. The finding of no
anti-S3 immunostaining of these 2 to 3-m vesicular
structures suggests some specificity to their formation
rather than the random acquisition of membrane compo-
nents from the endoplasmic reticulum where receptors are
synthesized. Although no anti-S3 staining of the 2 to 3-
m structures has been detected, anti-S3 does stain the
apical membrane depressions, suggesting that the smaller
S3-containing vesicles join the nidus of secretion at the
membrane.
Cell Tissue Res (2011) 346:175189 185

Fig. 9 Formation of junctions

in post-confluent HC11 cultures.
a Development of transepithelial
resistance (TER) as a function
of time and medium
supplementation: , control;
dexamethasone plus insulin;
, dexamethasone plus insulin
plus PRL; , dexamethasone
plus insulin plus S179D
PRL, a molecular mimic of
phosphorylated PRL. Analysis
was of triplicate samples from
each of three separate occasions.
*P<0.05, different from day 1;
#
P<0.05, different from *.
b Development of tight junctions
as measured by Western blot
analysis of zonula occludens-1
(ZO-1) expression (C control,
P PRL, S S179D PRL).
c-e Relative expression of ZO-1
on day-7 post-confluency in
control versus PRL-treated or
S179D PRL-treated cultures.
Bar30 m

Some unevenness of PRLR staining among cells was
observed on day 7 of lactation. Similar unevenness of
expression of PRLR at the mRNA level has been
previously described (Hovey et al. 2001; Rosen 2003).
In these reports, PRLR mRNA was co-localized with
estrogen and progesterone receptors. These triple-positive
cells were not proliferating and hence proliferation was
dependent on paracrine factors generated by the receptor-
positive cells. However, as lactation progressed, cells in
the current study were observed to be more uniformly
stained, a finding that also seemed to correspond to that
seen at the mRNA level (Hovey et al. 2001). Thus, more
luminal cells appear to become directly responsive to PRL
as lactation progresses.
In vitro modeling
Using HC11 cells, we were able to demonstrate that all cells
in a growing culture stained positively with the anti-LF
antiserum but that cells only stained positively with the
anti-S3 antiserum once surrounded by E-cadherin. Thus,
proliferating cells expressed the LF receptor but the S3
receptor was not expressed until cells were approaching
confluency and junctions began to form. The role of
the S3 receptor under these circumstances is consistent
with a dominant-negative effect on autocrine PRL- and
Stat5-mediated cell proliferation (Berlanga et al. 1997;
Brockman and Schuler 2005; Schroeder et al. 2003).
Once a sealed monolayer was formed, all immuno-
fluorescently detectable receptors were apical (the equivalent
of facing the ductal lumen). In other words, under these
conditions, the HC11 cells modeled the nulliparous and
pregnant state in terms of LF and S3 receptor localization.
Apical application of PRL produced normal PRLR activation
of the Stat5 and ERK pathways (Ben-Jonathan et al. 2008),
thereby demonstrating that the immunolocalized apical
receptors were functional in this location. Basal application
of PRL on the other hand produced no activation of
either of these signaling pathways and actually reduced
ERK phosphorylation below that seen in the absence of
added PRL (some PRL is present from autocrine
sources). A response (albeit reduced phosphorylation)
demonstrates the presence of a PRLR of some kind, being
present either at levels not detectable by immunofluorescence
or not recognized by the antisera that we used, such as the S1
or S2 mouse PRLR. This kind of rapid dephosphorylation of
ERK in response to a ligand can be observed when ERK
phosphatases are themselves stabilized by phosphorylation or
are rapidly induced (Liu et al. 2007; Patterson et al. 2009).
A variety of such phosphatases exists, collectively now
usually referred to as DUSPs (dual specificity phosphatases),
a name derived from their ability to dephosphorylate
186
Fig. 10 Western analysis
of signals generated by the
application of PRL either
apically (a, b) or basally (c)
(ERK extracellular signal-
regulated kinase, P-ERK phos-
phorylated ERK, IP immuno-
precipitation, -stat5 anti-Stat5,
WB Western blot, -pY anti-
phosphotyrosine)
both the threonine and tyrosine of activated ERK
(Patterson et al. 2009). DUSP regulation of signaling
through the mitogen-activated protein kinases can be
regional within a cell (Patterson et al. 2009), as is also
implied by our results.
Our in vitro model achieved a receptor distribution and
junctional competency akin to the pre-lactational state. By
contrast, when EpH4 mouse mammary epithelial cells were
grown as polarized monolayers or mammospheres, Xu et al.
(2009) found what we would now describe as a lactational
PRLR distribution (substantially basolateral). The reason
for this difference might be related to their EpH4 cells
being selected for their continued high level of milk protein
production (much higher than HC11 cells) and hence a
lactational phenotype with tighter junctions.
Other work has shown that the transfection of HC11
cells with the human short receptors accelerates the
production of junctions, as measured by TER (Huang et
al. 2008). In the present study, we re-tested the effect of
hormone supplements, routinely used to differentiate
mammary epithelial cells, on tight junction formation. This
was in order to differentiate between unmodified and
phosphorylated PRL (Huang et al. 2008) in an era when
most investigators are turning to the use of recombinant
PRL and therefore to the use of only unmodified PRL.
Given sufficient time, junctions formed equally well in the
absence or presence of PRL under the conditions used.
However, the molecular mimic of phosphorylated PRL
accelerated junction formation. In previous studies of tight
junction formation in response to PRL, preparations
extracted from pituitaries containing both unmodified and
phosphorylated PRL were used (Armburo et al. 1992;
Stelwagen et al. 1999). Others have reported that 80%-100%
of milk PRL is phosphorylated (Kacsh et al. 1991, 1993;
Cell Tissue Res (2011) 346:175189
Ellis and Picciano 1993, 1995). Combined with our result,
this suggests that early phosphorylated PRL in the duct
lumen promotes the closure of intercellular junctions at the
beginning of lactation. The observed ability of the mimic
of phosphorylated PRL to promote junction formation is
also consistent with its previously documented ability to
upregulate short PRLR expression in mouse and human
cells (Wu et al. 2003, 2005) and with the association of
S3 expression with junction formation observed here.
Myoepithelial cells
The studies presented provide immunolabeling evidence
that myoepithelial cells are targets of PRL. Given that
myoepithelial cells are long and thin or dendritic, depending
on the physiological state of the gland, difficulties would have
been experienced in distinguishing the labeling of these
versus luminal cells by in situ hybridization for PRLR mRNA
(Hovey et al. 2001; Rosen 2003). Moreover, the general
presumption that most PRLR would be basally oriented in
order to respond to a circulating hormone might have
contributed to a lack of discernment of myoepithelial
labeling with low magnification immunohistochemistry in
previous studies. The double-labeling with anti-smooth
muscle actin and anti-PRLR in the mouse tissues clearly
demonstrated that PRLRs (both LF and S3) were present on
myoepithelial cells. By analyzing microarray data available
in the literature, we have also found supporting evidence of
PRLR mRNA expression in mouse mammary myoepithelial
cells in a paper by Kendrick et al. (2008). At the mRNA
level, expression was lower for myoepithelial than for
luminal epithelial cells. At the protein level, according to
our results, staining appears to be more intense for
myoepithelial cells. This might reflect a regional concentra-
Cell Tissue Res (2011) 346:175189
tion of receptors on myoepithelial cells, a suggestion
consistent with some green and some yellow staining on
merged images (e.g., Fig. 6a). Alternatively, it may reflect
differential turnover rates for the receptor protein in the
two cell types. The lack of significant myoepithelial
staining of the human tissues is probably a function of
antibody epitope recognition but this needs to be further
examined before any conclusions can be drawn about
differences between species or the types of receptors
expressed in the two cell types.
There are two potential sources of PRL for activation of
myoepithelial PRLR: the circulation and PRL made locally
by luminal epithelial cells (Ben-Jonathan et al. 1996;
Reynolds et al. 1997). PRL from the circulation might
affect myoepithelial cells in various ways, which in turn
might then affect luminal epithelial cells. For example, on
the basis of gene expression, notch signaling is probably
directional from myoepithelial to luminal epithelial cells
(Kendrick et al. 2008). In the opposite direction, the ability
of increased luminal epithelial production of PRL to
influence myoepithelial biology is illustrated in recent in
vivo work from the Schuler laboratory. Thus, increased
production of PRL in ductal cells created myoepithelial
abnormalities (Arendt et al. 2009).
The localization of PRLR on myoepithelial cells raises
the interesting new concept that myoepithelial cells are a
primary intended target of circulating PRL during
pregnancy in order to initiate myoepithelial remodeling.
Moreover, only when levels of PRL are elevated might it
be possible for PRL from the circulation to make it past
the PRLRs on the myoepithelial cells to travel on into
the ductal lumen. Under these circumstances, the PRL
that passes through might add to the level of luminal cell
proliferation governed by autocrine PRL.
Although remodeling of the myoepithelial layer begins
in pregnancy, we could not detect any basal PRLRs on
luminal epithelial cells at this time, suggesting that all
PRL/placental lactogen effects on luminal cells via the LF
and S3 receptors during pregnancy are the result of ductal
PRL-PRLR interactions. By contrast, during lactation, a
proportion of these PRLRs on the luminal epithelial cells
moves to the basal surface at a time when paracellular access
of circulating PRL to the luminal receptors is no longer
possible. Furthermore, once the myoepithelial layer is
remodeled, luminal cells are in direct contact with the
basement membrane and luminal epithelial cell-basement
membrane interactions are crucial for the optimal production
of milk proteins in response to PRL (Akhtar and Streuli 2006;
Xu et al. 2007).
Collectively, the results obtained from this study
suggest that PRLR signaling is more contextual than
previously appreciated and that myoepithelial cells might be
previously unrecognized targets of PRL. The unexpected
187
PRLR localization and differential signaling at the apical and
basolateral faces of the luminal cells, plus the presence of
PRLR on myoepithelial cells, lead to the important new
concept that the roles of ductal versus circulating PRL in
the non-lactating gland are highly different: PRL-PRLR
interactions that generate the classically recognized
proliferative signaling appear to take place within the
duct lumen in which the microenvironment (e.g., binding
proteins and fluid dynamics) might be dramatically
different from that at the basal surface. PRL is found in
human breast fluids (Rose et al. 1987) and rat (Kacsh et al.
1991; Kacsh et al. 1993) and human (Ellis and Picciano
1993, 1995; Healy et al. 1980) colostrum and milk at
concentrations reported to range from six- to 100-fold
those in the circulation. In virgin and pregnant animals
(i.e., before the intercellular junctions are tight), higher
concentrations in ductal fluids could be accomplished by
the sequestration of either autocrine or circulating PRL
(or both) by some component of the ductal fluid.
Acknowledgements The authors thank Dr. Christian Lytle for
advice on the TER measurements and access to his equipment,
Dr. Christopher Ormandy for provision of the PRLR knockout
tissue blocks, Dr. Michael Symonds for provision of the antibodies
and Dr. Mary Lorenson for critical reading of the manuscript.
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