1 Division of Biological Sciences, Molecular Biology Section, University of
California San Diego, La Jolla, CA 92093, USA
2 Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030,
USA
3 University of Colorado Health Science Center, Department of Physiology,
Denver, CO 80262, USA
4 Department of Anatomy, University of California San Francisco, San Francisco,
CA 94143, USA
5 Eppley Institute for Research in Cancer and Allied Diseases, University of
Nebraska Medical Center, Omaha, NE 68198, USA
* Author for correspondence (e-mail: rjohnson{at}biomail.ucsd.edu)
Accepted 15 January 2003
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SUMMARY |
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Key words: Hypoxia, HIF1, Mammary gland, Lactation, Differentiation, Metabolism, Mouse
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INTRODUCTION |
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Under normoxic conditions, HIF1 protein is rapidly degraded through
targeted ubiquitination mediated by direct binding of its oxygen dependent
domain to the ß subunit of the von Hippel Lindau (VHL) tumor suppressor
protein (reviewed by Kondo and Kaelin,
2001
). In response to hypoxia, HIF1
protein accumulates,
owing to decreased interaction with VHL
(Krek, 2000
). An increase in
HIF1
protein is first detectable at partial pressures of oxygen
equivalent to 6% O2, and is maximal between 0.5-1.0% O2
(Stroka et al., 2001
). In a
hypoxic environment, HIF1 activates the hypoxic response elements (HREs) of
target gene regulatory sequences (Huang et
al., 1998
; Salceda and Caro,
1997
), resulting in the transcription of genes implicated in the
control of metabolism and angiogenesis, as well as apoptosis and cellular
stress (reviewed by Giordano and Johnson,
2001
). Some of the direct targets include erythropoietin, the
angiogenic factor vascular endothelial growth factor (VEGF), glucose
transporters and multiple glycolytic enzymes. The connection between the
hypoxic response and angiogenesis is also clear from the study of individuals
with VHL disease, an autosomal, dominantly inherited cancer syndrome.
Individuals heterozygous for one inactivating mutation in VHL are
predisposed to developing a variety of tumor types, including renal clear cell
carcinomas that are massively hypervascular with highly elevated levels of
VEGF expression caused by constitutive HIF1 activity in response to
inactivation of the second VHL allele
(Kondo and Kaelin, 2001
).
Recently, HIF1 has been demonstrated to be upregulated in a variety
of human solid tumors, in particular breast tumors that exhibit high rates of
proliferation (Bos et al.,
2001
; Zhong et al.,
1999
). Zhong et al. reported that HIF1
protein was
overexpressed in breast tumors, as well as bordering `normal' areas adjacent
to tumors, but not in normal breast tissue
(Zhong et al., 1999
). These
observations in breast tumors are consistent with our previous findings that
HIF1
functions as a positive regulator of tumor growth
(Ryan et al., 2000
;
Seagroves and Johnson, 2002
).
In a subsequent study, the level of HIF1
expression in breast tumors
was correlated with other prognostic factors. Specifically, in ductal
carcinoma in situ (DCIS) lesions, relatively high levels of HIF1
expression were associated with increased proliferation, as well as increased
expression of VEGF and the estrogen receptor
(Bos et al., 2001
). However,
HIF1
expression did not correlate with p53, supporting the observations
of our own laboratory that the effects of loss of Hif1a on cell
growth, metabolism or tumorigenesis are independent of p53 expression
(Ryan et al., 2000
). In order
to provide a foundation for understanding the role of HIF1 in mammary
tumorigenesis, the function of HIF1
was investigated during normal
mammary gland development.
Several laboratories have demonstrated that HIF1 is required to
regulate the response to lowered oxygen levels in developing murine tissues
(Iyer et al., 1998
;
Ryan et al., 1998
;
Schipani et al., 2001
;
Yun et al., 2002
). With
respect to known functions of HIF1, there were several compelling reasons to
study HIF1
function in the context of normal mammary gland development.
First, the normal mammary parenchyma undergoes tremendous expansion as it
prepares for lactation during the course of pregnancy
(Matsumoto et al., 1992
),
including formation of new blood vessel networks to provide oxygen and
nutrients to the lactating mammary gland. For example, in the rat, the
vasculature doubles by mid-pregnancy through angiogenesis via sprouting and
intersucception (Djonov et al.,
2001
).
In addition, in preparation for lactation, there is a requirement for
glucose to provide energy as well as to synthesize lactose, the primary
carbohydrate in milk. Notably, the increased activity of several glycolytic
enzymes involved in glucose metabolism has been reported at the transition
from pregnancy to lactation (Mazurek et
al., 1999). The transition from differentiation during pregnancy
to successful milk secretion at lactation is complex, and has been divided
into two stages, recently termed secretory differentiation and secretory
activation (McManaman and Neville,
2003
). Secretory differentiation begins at mid-gestation with the
production of significant quantities of milk protein and lipid. Secretory
activation is coordinated with the birth of pups, and depends on the
completion of secretory differentiation. The increased demands for energy for
synthesis of milk components that begin during pregnancy persist during
lactation, as the gland is actively making and secreting milk. Because the
developing mammary gland is both highly vascularized, and metabolically
active, with a requirement for glucose to produce milk, it serves as an ideal
tissue to determine the in vivo role of HIF1 and its subunit HIF1
in a
developmentally regulated metabolic switch.
The clear increase in demands for energy during lactation, as well as the
striking and extensive angiogenesis that occurs during pregnancy, led us to
hypothesize that during both secretory differentiation and activation,
HIF1 may be required to alleviate transient hypoxia through
angiogenesis, increased dependence on glycolysis and regulation of substrates
for the production of milk. In order to test this hypothesis, we have
specifically removed Hif1a from the mammary epithelium using
previously characterized HIF `floxed' mice
(Ryan et al., 2000
) that
express MMTV-Cre (Wagner et al.,
2001
; Wagner et al.,
1997
). In these mice, multiple facets of the differentiation
process were impaired, culminating in a functional failure of the mammary
gland.
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MATERIALS AND METHODS |
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Quantitation of DNA, RNA and protein
A piece of inguinal mammary gland harvested at day 18 of gestation was
finely ground to a powder under liquid nitrogen and homogenized in a modified
RIPA buffer (50 mM Tris, pH 7.4, 1% NP-40, 0.25% sodium desoxycholate, 400 mM
NaCl, 1 mM EDTA in RNase-free water). DNA was quantitated after Hoechst 33258
staining using a Horiba Micromax fluorometer (excitation, 350 nm; emission 473
nm). To measure RNA, homogenates were first treated for 90 minutes with DNase
I, and the fluorescence intensity quantitated following incubation with
Ribogreen Dye (Molecular Probes). Protein was measured using a Bradford assay
(BioRad).
Milk collection and analysis
At birth pups were removed from their natural mothers, randomized and 10-12
pups placed with each dam. The average pup weight per litter per day was
determined until mid-lactation (day 9-11 lactation), when milk and mammary
tissues were collected. Milk was collected under gentle vacuum into tared
tubes on ice from weaned dams injected with oxytocin (1.5 U per leg, i.m.).
For each sample, the water, fat, nitrogen, lactose, sodium and chloride
contents were measured according to standard protocols
(Jensen, 1995). Briefly, the
percentage of water (% w/w) was measured as weight loss after drying, and
sodium and chloride were measured by inductively coupled plasma spectrometry
using a Spectro-CIROSCCD (Spectro Analytical Instruments). To
compare wet weight of the lactating glands, both inguinal glands were
dissected following milking, flash frozen and weighed immediately.
Primary culture, adenoviral infection and transplantation
Primary mammary epithelial cells (MEC) were isolated from
Hif1af+/f+ pregnant mice according to Pullan and Streuli
(Pullan and Streuli, 1997).
Equal volumes of cells were allowed to spread onto plastic dishes in plating
medium (Ham's F12 containing 10% FBS, 5 µg/ml insulin, 1 µg/ml
hydrocortisone, 20 ng/ml murine epidermal growth factor, 5 ng/ml cholera toxin
and 50 µg/ml gentamicin, 100 U Penincillin/10U Streptomycin) for 48 hours
before replacing this medium with growth medium (same as plating, but no
cholera toxin and 5% FBS). The next day, the cells were infected overnight
with either Adenovirus-ß-galactosidase (Adeno-ßgal) or
Adenovirus-Cre (Adeno-Cre, generously provided by Dr Frank Giordiano) at a
multiplicity of infection of 60-65 particles per cell
(Rijnkels and Rosen, 2001
).
The next day, the cells washed several times with PBS and fresh growth medium
was added. Cells were allowed to recover from infection for 24-48 hours. To
compare mRNA expression of target genes of cultured MEC or to prepare nuclear
extracts for western blotting, the medium was changed to growth medium
containing 25 mM HEPES pH 7.4 at 0 hours. Cells were then left at normoxia or
transferred to a hypoxic incubator (0.5% O2 balanced with
N2) for 24 hours. For transplantation into host mice, cells were
trypsinized, washed and resuspended to 50,000-100,000 cells/µl in HBSS.
Approximately 10-15 µl of cells were injected into the cleared inguinal fat
pads of 3-week old immunocompromised Rag1/
females (Jackson Labs). After a period of MEC outgrowth of at least 10 weeks,
the hosts were then mated and the outgrowths (4R, Adeno-b-gal-infected, wild
type; 4L, Adeno-Cre infected, Hif1a/)
harvested and fixed in 10% NBF.
Nuclear extract preparation and western blotting
Nuclear extracts (NE) were prepared as described previously
(Ryan et al., 1998).
HIF1
protein was detected by western blotting using 60 µg input of
NE resolved by a 6% SDS-PAGE gel and transferred to PVDF membrane. The
membrane was blocked overnight at 4°C in 10% nonfat dry milk, followed by
a 3 hour incubation in a 1:1000 dilution of anti-mouse HIF1
antibody
(Novus, NB100-123). The blot was then incubated in a 1:10,000 dilution of
anti-mouse whole IgG-HRP for 30 minutes followed by incubation in ECLPlus
substrate (Amersham) prior to exposure to Kodak MR film.
Preparation of RNA and DNA
Cells were washed twice with cold PBS before being directly extracted with
RNAzol B for total RNA preparation (Tel-Test) or scraped into buffer
containing proteinase K for preparation of genomic DNA. To prepare total RNA
from tissues, snap-frozen tissue was pulverized with a mortar and pestle
directly in liquid nitrogen and homogenized in chilled RNAzol B and RNA
prepared according to manufacturer's instructions.
Immunostaining
For CD31 staining and Chalkley analysis slides were processed as previously
described (Ryan et al., 2000).
Slides were blinded and ten 10x power fields counted twice independently
(n=5 per genotype). Anti-Cre immunostaining was performed as
previously described (Seagroves and Li,
2002
). For Glut1, antigen retrieval was performed on paraffin wax
sections using 1x citrate buffer (DAKO) followed by overnight incubation
at room temperature of a 1:200 dilution of anti-Glut1 antibody (Alpha
Diagnostics). Staining was visualized via the ABC Elite staining kit (Vector
Laboratories) using DAB as a substrate followed by counterstaining with
Hematoxylin. For double labeling to detect both Glut1 and Cre on the same
paraffin wax sections, Glut1 was first detected as described using DAB
substrate. The sections were then re-blocked with 10% goat serum and Cre
detected using Vector VIP (purple color) substrate followed by counterstaining
with Methyl Green.
Semi quantitative reverse transcription PCR assays
Random-primed reverse transcription was carried out on 30 ng of total RNA.
The cDNA was amplified using primers to mouse Xor,
-lactalbumin, ß-casein, adipophilin (Adfp), butyrophilin
and ß-actin genes. Samples were prepared for loading onto the Applied
Biosystems 310 Genetic Analyzer by mixing 12 µl of formamide, 1 µl of
TAMARA size standard (Perkin Elmer Applied Biosystems) and 2 µl of PCR
product. The size and amount of PCR product was calculated using GeneScan
software (Perkin Elmer Applied Biosystems). Control experiments were performed
to define signal linearity for each probe pair.
Real-time PCR assays
Two micrograms of total RNA was DNase I treated and directly used to
prepare first-strand cDNA from random hexamer primers using the Superscript II
Reverse Transcription Kit (Invitrogen). For real-time detection PCR (RTD-PCR),
5 ng of input cDNA was analyzed in triplicate per primer pair per sample and
the corresponding threshold cycle (Ct) values expressed as the
mean±s.e.m. All reactions were performed using 2x Taq Master Mix
(Perkin Elmer Applied Biosystems), 900 nM each of the forward and reverse PCR
primers and 250 nM of a fluorescently tagged primer pair-specific probe in a
total volume of 25 µl using default cycling parameters on an ABI Prism 7200
Sequence Detector. The following primer and probe sequences were used.
Normalization of real-time PCR assays
In cultured cells, target gene mRNA expression was first normalized to 18S
rRNA and then expressed as a percentage of signal observed in wild-type cells
(Hif1af+/f+, Adeno-ß-gal-infected)
cultured at normoxia (onefold). For mammary gland tissue samples, expression
of each sample was first normalized to cytokeratin 19 (Ck19), a gene
exclusively expressed in epithelial cells
(Nagle et al., 1986), to
correct for any differences in epithelial cell content between genotypes prior
to comparison of gene induction between wild-type
(Hif1af+/f+ only; MMTV-Cre negative) and null
(Hif1af+/f+, MMTV-Cre-positive) glands. Expression of each
target in null glands was determined relative to the signal observed in wild
type samples (onefold) according to standard procedures [ABI Prism 7200 manual
and Muller et al. (Muller et al.,
2002
)]. Each primer set approached 100% amplification efficiency,
allowing direct comparison of Ct values to determine relative gene expression
(Muller et al., 2002
). To
determine the efficiency of deletion of Hif1a, genomic DNA was
prepared and the expression level of Hif1a was compared with that of
a control primer set, Jun.
Vasculature labeling
Fluorescein-conjugated lectin from tomato (Vector Laboratories) diluted to
1mg/ml in PBS was injected into the tail vein of live mice 5 minutes prior to
perfusion. One minute prior to perfusion, mice were anesthetized and 1%
paraformaldehyde (PFA)/0.5% glutaraldehyde was perfused directly in the heart
at a rate of 1 ml per minute followed by clearing with PBS. Mammary tissues
were equilibrated in cold 30% sucrose/PBS for 2 hours before embedding in OCT
compound on dry ice. Thick frozen sections (40-50 µm) were post-fixed in 4%
PFA for 5 minutes, treated with 0.5% Triton-X for 10 minutes, and incubated
with AlexaFluor 595-conjuated phalloidin (Molecular Probes) at a 1:250
dilution in 1% BSA/PBS for several hours prior to mounting. A Zeiss confocal
microscope was used to capture 0.5 µm serial slices at low power, which
were then merged into one plane to visualize the vasculature (green) and actin
filaments of the epithelial network (red).
Statistical analysis
Statistical significance was determined by an unpaired t-test
(P set to <0.05), using StatView 5.0 (SAS). Those samples that
achieved statistical significance, comparing wild-type with
Hif1a/ samples, are indicated with an
asterisk.
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RESULTS |
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Conditional deletion of HIF1 in the mouse mammary gland
To determine whether HIF1 function is required in vivo for normal mammary
gland development, a conditional gene deletion strategy was employed to delete
Hif1a in the mammary epithelium of mice. Mice harboring two `floxed'
alleles of exon 2 of the Hif1a locus
(Hif1af+/f+)
(Ryan et al., 2000) were bred
with Hif1af+/f+ mice that expressed Cre under control of the mouse
mammary tumor virus (MMTV)-LTR, which targets deletion in the mammary
epithelium, but not in the stroma. Hif1af+/f+
progeny negative for MMTV-Cre (referred to as wild type) were compared with
Hif1af+/f+ littermates that expressed MMTV-Cre.
The temporal-spatial pattern of Cre recombinase activity in this line of mice
(line A) has been extensively described
(Wagner et al., 2001
).
Southern blot analysis comparing wild-type and
Hif1a/ mammary tissue indicated that
MMTV-Cre consistently targeted deletion of Hif1
in at least
50% of the epithelial cells (Fig.
1C). However, because the DNA was prepared from whole tissue, the
intensity of recombined allele, which is only present in the epithelial cells,
is actually underestimated by Southern blotting. As described in detail by
Wagner et al. using ROSA reporter mice crossed to individual lines of
MMTV-Cre transgenic mice, by lactation, the majority of the epithelial cells
have been targeted for recombination
(Wagner et al., 2001
).
Therefore, based on these previous observations, as well as the data generated
by Southern blotting, the mammary glands isolated from
Hif1af+/f+, MMTV-Cre-positive mice will be
referred to as Hif1a null (Hif1a/).
Furthermore, in contrast to reports that this line of MMTV-Cre transgenic mice
induced excision in the ovaries of mature mice
(Wagner et al., 2001
), no
recombination of the Hif1a locus could be detected in DNA prepared
from whole ovaries of Hif1af+/f+,
MMTV-Cre-positive females either by Southern blotting
(Fig. 1C) or by RTD-PCR.
During pregnancy deletion of HIF1 impairs secretory
differentiation, but not vascular expansion
In order to pinpoint the stage of mammary gland development at which
HIF1 function may be required, mammary tissue was harvested from mice
over the course of gestation. No differences in ductal morphogenesis were
noted between genotypes in nulliparous mice (data not shown). At day 10 of
gestation, a stage of development prior to differentiation, no defects in
histology were observed in Hif1a/ glands at
either the gross or microscopic level (data not shown), indicating that
HIF1
is not crucial for early rounds of alveolar proliferation.
Similarly, by day 12 of pregnancy, when secretory differentiation typically
begins in most mouse strains, no differences in morphology were noted (data
not shown).
However, by day 15 of pregnancy, well into the period of secretory differentiation, although the glands of both genotypes were indistinguishable at the whole-mount level (data not shown), histological examination revealed significant abnormalities in the Hif1a/ glands (Fig. 2A,B). In particular, the protein and lipid droplets that give the wild-type epithelium a `lacy' appearance were completely absent in the Hif1a/ glands. In addition, null alveoli were smaller, with reduced lumens, and the surrounding connective tissue that normally regresses as the alveoli mature was more prominent than in wild-type glands. These defects resulted from a block in differentiation, rather than proliferation, as the rates of incorporation of bromodeoxyuridine were equivalent at this stage of development (data not shown).
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Expression profiles of HIF1 targets and markers of differentiation in
pregnant mice
The expression of HIF1 targets, normalized to the epithelial cell marker
cytokeratin 19 (Ck19; Krt1-19 Mouse Genome
Informatics), was then analyzed in mammary glands of pregnant mice at day 15
of gestation. In contrast to cultured primary cells exposed to hypoxia,
neither Pgk nor Vegf mRNA expression differed significantly
between genotypes (Fig. 3A).
However, Glut1 expression was decreased by 60%. During secretory
differentiation, transcription of markers associated with milk production
increase sharply; therefore, to further characterize the defects in
differentiation in the epithelial cells, a panel of markers associated with
production of milk components was compared using semi-quantitative RT-PCR
following normalization to ß-actin
(Fig. 3B). Two of these
markers, ß-casein and -lactalbumin (
-lac), are markers of
the casein and whey fraction of milk, respectively. In addition, several
markers were analyzed that are associated with the milk lipid globule (MLG).
These included xanthine oxidoreductase (XOR; XDH Mouse Genome
Informatics), a redox enzyme immunolocalized to the apical plasma membrane of
lactating alveoli (McManaman et al.,
2002
), butyrophilin, a hydrophobic glycoprotein found only in
differentiated mammary epithelial cells
(Banghart et al., 1998
), the
cytoplasmic, lipid-droplet-associated adipophilin protein, also known as ADFP
(Heid et al., 1996
), and
perilipin, a marker of the adipose fraction in the mammary gland. Perilipin is
normally downregulated over the course of pregnancy as the adipose fraction
shrinks (Blanchette-Mackie et al.,
1995
). Interestingly, recently, Adfp has been identified
as a novel hypoxia-inducible gene in MCF-7 cells
(Saarikoski et al., 2002
). In
response to deletion of Hif1a, ß-casein,
-lac,
Adfp and Xor mRNA levels decreased by over 50%
(Fig. 3B), whereas butyrophilin
expression remained fairly constant (data not shown) and there was a failure
to downregulate perilipin. These results indicate that there are severe
deficiencies in expression of markers of milk production.
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Because mammary epithelium-associated angiogenesis is complete by the end
of pregnancy, we next analyzed microvessel density (MVD) in these tissues.
Vessels were visualized by immunostaining with anti-CD31 antibodies, and the
density of the vessels calculated by Chalkley counting as described previously
(Ryan et al., 2000). As
expected, based on the previous results of lectin staining at day 15 of
gestation, there was no significant difference between genotypes in MVD at day
18 of gestation (Fig. 4H).
Finally, although there appeared to be fewer alveoli present per field in
the Hif1a/ glands, it was possible that this
was an artifact resulting from the lack of alveolar cell expansion associated
with differentiation. In order to compare cellularity and secretory activity,
the amount of DNA, RNA and protein produced per gram of tissue was quantified.
It has been previously demonstrated in the mammary gland that DNA content per
gram of tissue correlates with cellularity
(Knight and Peaker, 1982). As
can be seen in Fig. 4I, there
was no significant difference in DNA content between genotypes of mammary
tissue, suggesting that epithelial cell number is equivalent at this stage of
development. In support of this finding, no significant difference in the rate
of proliferation of epithelial cells was observed at day 18 of pregnancy (data
not shown). However, there was a significant decrease in the amount of RNA
produced by Hif1a/ glands. Finally, there
was trend for decreased production of protein in Hif1a null glands,
although because of animal-to-animal variability, this difference did not
reach statistical significance.
HIF1 is required for production and secretion of milk during
lactation
The histology of mammary glands on the date of birth (day 1 of lactation)
was compared without prior weaning of the litter. The
Hif1a/ glands exhibited fewer alveoli, which
contained fewer milk granules, and increased evidence of trapping of lipid
droplets within the epithelial cells (Fig.
5A,B). In contrast to day 18 of gestation, when expression of Cre
was non-uniform, almost 100% of the epithelial cells expressed Cre recombinase
by the onset of lactation (data not shown).
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Together, these defects resulted in reduced pup growth and viability. Although the pups contained milk in their stomachs, confirming normal suckling behavior, all of the pups were runted compared with wild-type controls, and a majority died within 15 days of birth. In order to more fully characterize the differences in pup growth, pups were weighed every day after birth. As evident in Fig. 5F, the differences in weight were observed as early as day 3 of lactation, and were maintained as lactation progressed. The decrease in growth could be reversed if litters that began nursing from Hif1a/ glands were fostered to a wild-type dam instead (data not shown), showing that the failure of the pups to grow resided in defects in the mother. Furthermore, to control for potential deleterious effects of Cre expression upon mammary gland development, lactating mice that expressed only the MMTV-Cre transgene were also analyzed. No defects in pup weight gain or mammary gland histology were noted in these dams (n=4), confirming that expression of Cre alone does not impair mammary gland development (data not shown).
Milk volume is reduced and milk composition is altered as a result of
deletion of HIF1
To determine if milk quality was affected by deletion of Hif1a,
milk was collected from mid-lactation dams and analyzed for percentage of
nitrogen, fat, water and lactose, as well as sodium and chloride ion
concentrations. Several trends were noted in collection of milk from
Hif1a/ glands. First, the milk was more
difficult to collect, was more viscous and was more difficult to dissolve into
the water at collection, suggesting a high fat content. Furthermore, less
total volume could be collected from the
Hif1a/ glands
(Fig. 6A). No statistical
differences were observed in protein content, whereas the amount of lactose
and fat varied widely (data not shown). However, highly significant
differences in water content and ion content were observed
(Fig. 6A). Water content was
decreased and the [Na+] and [Cl] were greatly
elevated in milk from Hif1a/ glands relative
to milk of wild-type glands. These changes reflected an ionic concentration
closer to that observed in plasma, and indicated a fundamental failure to
regulate mammary secretion properly.
|
A requirement for HIF1 in the mammary epithelium
To confirm that the defects observed in Hif1a null glands were
epithelial cell-autonomous, primary Hif1af+/f+ MEC were
infected with an adenoviral vector expressing either ß-galactosidase
(control, wild type MEC) or Cre recombinase (generating
Hifa/ MEC). Infection with Adeno-Cre induced
deletion of Hif1a in over 99% of cells (data not shown). The
wild-type and Hifa/ MEC were then
transplanted into the right and left cleared, inguinal fat pads, respectively,
of 3-week-old female immunocompromised Rag1/
host mice, using a technique previously described by Rijnkels and Rosen
(Rijnkels and Rosen, 2001).
After outgrowth of the transplanted cells into the Hif1a wild-type
stroma for a period of 12 weeks, the hosts were then mated and the outgrowths
harvested from hosts on the date of birth of pups. Transplanted wild-type
cells successfully differentiated and secreted milk
(Fig. 7A, purple granules).
However, the Hif1a/ outgrowths contained
small, poorly differentiated alveoli with collapsed lumens, and retained lipid
droplets in the cytoplasm (Fig.
7B). In addition, the alveoli were surrounded by increased
connective tissue (stained blue). Therefore, the histology of the
Hif1a/ alveoli, which regenerated in the
presence of Hif1a wild-type stroma, recapitulated the phenotypes
observed in intact Hif1a-null glands, confirming that HIF1
acts in an epithelial cell-autonomous manner to control mammary gland
development. Glut1 immunostaining was also performed on paraffin wax-embedded
sections from the transplanted outgrowths. As shown in
Fig. 7C, expression of Glut1
was uniformly detected in the wild type alveoli. By contrast, in the
Hif1a null alveoli (Fig.
7D), expression of Glut1 was decreased and patchy. Therefore, the
defects in mammary gland development and physiology resulting from deletion of
Hif1a via MMTV-Cre were not due to defects in the stroma or deletion
in other tissues.
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DISCUSSION |
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Deletion of Hif1a did not impact ductal morphogenesis in
nulliparous mice or the proliferation of alveoli during pregnancy. Instead,
defects in differentiation were observed by histology beginning at day 15 of
gestation. Loss of HIF1a inhibited the expression of markers critical to
secretory function, including several milk protein and milk fat globule
markers. For example, expression of Xor mRNA was dramatically reduced
at day 15 of gestation in Hif1a/ glands.
This result is intriguing in light of a recent report that mice heterozygous
for Xor fail to properly secrete lipid into milk at lactation,
ultimately resulting in pup death (Vorbach
et al., 2002), a phenotype similar to that observed in response to
deletion of HIF1
. In addition, expression of Adfp mRNA, which
has recently been shown to be induced up to 70-fold by hypoxia in MCF-7 cells
(Saarikoski et al., 2002
), was
also reduced by 50% in glands null for Hif1a.
Furthermore, as observed at day 18 of gestation, the failure of the alveoli
to differentiate and to produce milk components corresponded completely with
the expression pattern of Cre. Hence, the pronounced block in differentiation
is due to loss of Hif1a. Consistent with this finding,
transcriptional activity, as measured by RNA content per gram of tissue, was
reduced by 50%. Because of the tight association observed between the presence
or absence of Hif1a and the production of milk components in
preparation for lactation, HIF1 is a critical regulator of the process
of secretory differentiation in the mammary gland.
In addition, it is important to note that mammary gland DNA content was similar between genotypes at the end of pregnancy, and that no differences in rates of epithelial cell proliferation were noted at either day 15 or day 18 of gestation (data not shown). Therefore, the observed differences in histology of pregnant mice must have resulted from a failure to accumulate milk products in preparation for lactation. Of note, this mouse model is the first to date to describe defects in differentiation during pregnancy without accompanying changes in mammary epithelial cell proliferation.
With respect to loss of HIF1 function, the selective decrease of Glut1 mRNA
expression by 60% at day 15 of gestation, as well as the decreased levels of
Glut1 protein noted at day 18 of gestation, may explain both the defects in
differentiation and lipid metabolism observed during pregnancy in
Hif1a/ mice. Normally, Glut1, the exclusive
glucose transporter used in the mammary epithelium at lactation, is
considerably upregulated during secretory differentiation in order to increase
glucose availability (Camps et al.,
1994). This is crucial because glucose is a required substrate for
the production of lactose, the primary carbohydrate in milk. Furthermore,
glucose transport has been proposed to be a rate-limiting factor in glucose
use in the mammary gland (Threadgold and
Kuhn, 1984
). And, in rodents and other animals lacking the
acetate-based fatty acid synthetic pathway, glucose is also used for the
production of fatty acid precursors. Therefore, loss of HIF1
function
during pregnancy may deprive the gland of the glucose it needs to
differentiate.
The severity of the defects observed during pregnancy was demonstrated
during lactation since dams lacking HIF1 in the mammary epithelium were
unable to support nursing pups. Normally, secretory activity peaks at
mid-lactation, but, as evident by histology, the HIF1
null alveoli
contained relatively few milk granules, and large lipid droplets that are
normally secreted into milk as micro-droplets remained trapped within the
epithelial cells. As a consequence of defective secretion, the glands yielded
less milk volume and milk nutrition was poor. In addition, the sodium and
chloride content of milk was elevated, resembling concentrations observed in
plasma. These changes are hallmarks of mastitis, in which the normally closed
tight junctions become permeable (Nguyen
and Neville, 1998
). As the claudin proteins are implicated in
tight junction strand regulation (Furuse
et al., 2002
), and claudin 8 expression was downregulated by 50%,
HIF1 may also play a role in tight junction closure.
Although it is possible that the severe block in differentiation observed
in the HIF1 null mammary gland prevented the transition to secretory
activation, based on the previously described functions of HIF1
(Semenza, 1999
), it is more
likely that loss of HIF1
impaired metabolic activity at the time of
highest demand: lactation. In support of this hypothesis, at mid-lactation,
the normalized expression of PGK was reduced by over 67% in the HIF1
null glands.
During peak lactation, HIF1 mediation of glycolytic activity may be
necessary to supplement energy production since synthesis and transport of
milk components, as well as tight junction closure, are energy-dependent
processes. This proposed function for HIF1 in the mammary epithelium is also
supported by previous observations that increases in glycolytic enzyme
activities occur at lactation (Mazurek et
al., 1999). Therefore, glycolysis is likely to be necessary to
maintain energy production at lactation. Interestingly, previous studies have
shown that inhibitors of glucose metabolism interfere with lactation.
Administration of 2-deoxyglucose, which inhibits glucose-6-phosphate
metabolism in the lactating rat mammary gland, reduced lactose synthesis, as
well as protein synthesis and secretion
(Sasaki and Keenan, 1978
).
Thus, HIF1-dependent regulation of glucose metabolism may be necessary for
achieving differentiation during pregnancy as well as the high metabolic rate
in the mammary gland at lactation. Based on our observations of HIF1
transcriptional activity, it is possible that during pregnancy, when energy
demands are lower than for lactation, successful differentiation may depend
more upon Glut1 function than that of PGK. Conversely, during lactation,
regulation of PGK may become more critical. The mechanisms underlying the
differential impact of deletion of HIF1
on Glut1 and PGK mRNA
expression during pregnancy versus mid-lactation are not clear, and will
require further investigation.
In Hif1a/ epithelial cell cultures
exposed to hypoxia, VEGF mRNA decreased by 50%. However, we were unable
to detect any changes in MVD in the intact mammary gland in vivo, either by
qualitative analysis of the vasculature or by Chalkley counts after CD31
immunostaining. In addition, there was no significant decrease in VEGF mRNA
expression in Hif1a/ glands at day 15 of
gestation. As loss of HIF1
did not impact vasculature expansion, the
mechanisms regulating angiogenesis during normal mammary gland development
must be HIF1
-independent. The absence of an effect on angiogenesis in
response to deletion of HIF1
is in agreement with previous results
obtained by our laboratory, which showed that the MVD of both developing bone
as well as fibrosarcomas remain equivalent to wild type when HIF1
is
conditionally deleted (Ryan et al.,
2000
; Schipani et al.,
2001
).
Low levels of HIF1 were detectable in primary mammary epithelial
cells cultured at normoxia. HIF1
stability was dramatically increased
by hypoxic treatment and loss of HIF1
diminished HIF1 transcriptional
activity; therefore, the hypoxic response is intact in normal mammary
epithelium. Interestingly, it has been shown that HIF2
, which is
structurally related to HIF1
, may also induce expression of HRE-based
reporter constructs in a hypoxia-dependent manner
(Wiesener et al., 1998
);
therefore, it is possible that HIF2
may play some role in mammary gland
development. However, although both Hif1a and Hif2a
transcripts are expressed throughout mammary gland development, and neither
gene shows marked fluctuation in expression levels, Hif2a mRNA is
expressed at levels approximately one-tenth of those of Hif1a (M.N.,
unpublished observations). In addition, we argue that as expression of HIF1
targets was reduced to basal levels in hypoxically stimulated primary cells
lacking HIF1
, HIF2
plays little, if any, role in transcriptional
regulation of HIF target genes in the mammary gland. Furthermore, because
HIF2
was unable to compensate for loss of HIF1
during pregnancy
and lactation in vivo, it is not likely that HIF2
is a critical
mediator of mammary gland development.
Although hypoxia is the classic inducer of HIF1 stability, we have
been unable to document the presence of hypoxia in Hif1a wild-type
and null mammary tissue using reagents known to detect DNA adducts created by
hypoxia, such nitroamidzole (EF5) (Evans et
al., 1996
). Although waves of hypoxia were readily detectable in
mammary tumors, no areas of hypoxia could be detected in the normal mammary
gland at any stage of development (data not shown). However, detection of
adducts created by hypoxia via immunostaining may not be as sensitive in
normal tissue, which can more readily adapt to hypoxia than a rapidly dividing
tumor with a necrotic center.
Alternatively, hypoxia per se may not be a stimulus of HIF1 activity
in the mammary gland. Several laboratories have reported that in vitro
HIF1
protein is also stabilized upon treatment with insulin,
insulin-like growth factor 1 (IGF1), IGF2 or activation of HER2/neu receptor
upon addition of heregulin all potent cell survival factors/mitogens
for normal and breast cancer cell lines
(Feldser et al., 1999
;
Laughner et al., 2001
;
Zettl et al., 1992
).
Additionally, a positive feedback loop between HIF1
and Igf2
transcription has been reported in human 293 cells and in mouse embryonic
fibroblasts (Feldser et al.,
1999
). These observations are noteworthy, as IGF2 may function as
a local, paracrine mitogen in developing alveoli
(Wood et al., 2000
).
Furthermore, it has been demonstrated that the end products of glycolysis
itself, pyruvate and lactate, can induce HIF1
protein stability even
under aerobic conditions (Lu et al.,
2002
).
Recently, Le Provost et al. have deleted the partner of Hif1a,
Arnt, in the mammary gland (Le
Provost et al., 2002). Deletion of Arnt blocked early
alveolar development and impaired fertility
(Le Provost et al., 2002
).
Based on these results, as well as transplantation of transgenic tissues into
the cleared fat pads of host mice, it was argued that deletion of
Arnt affects mammary gland development through uncharacterized,
indirect effects in the ovary, although there were no differences in
circulating estrogen and progesterone levels
(Le Provost et al., 2002
). We
have not noted any differences in ovarian histology or detected recombination
of the Hif1a locus in the ovaries of transgenic mice. Furthermore, in
comparison with deletion of Arnt, loss of Hif1a impacted
relatively late stages of mammary gland development. These differences are
perplexing, as HIF1
partnering with ARNT is required for HIF1 activity.
It is possible that Arnt function may be compensated for by other
family members that complex with HIF1
, such as ARNT2
(Keith et al., 2001
) or ARNT3
(ARNTL Mouse Genome Informatics)
(Takahata et al., 1998
).
Nevertheless, transplantation of Hif1a/
epithelium into Rag1/ female hosts revealed
that the defects associated with deletion of Hif1a were mammary
epithelial cell autonomous. Therefore, even if low, but undetectable, levels
of recombination of the Hif1a locus were present in the ovaries in
this line of transgenic mice, they have no impact on the phenotype in the
mammary gland.
Because deletion of Hif1a specifically inhibited the synthesis of milk components during pregnancy and milk production and secretion at lactation, we argue that HIF1 activity is essential for the transition from pregnancy to functional lactation, and is also required for the maintenance of normal lactation and production of milk. Furthermore, these defects are manifested independently of regulation of angiogenesis through VEGF.
Although overexpression of HIF1 has been documented in breast tumors
compared with normal tissues (Bos et al.,
2001
; Zhong et al.,
1999
), it is not clear if this contributes to tumorigenesis or is
an effect of hypoxia induced by rapid proliferation. Future experiments to
compare the gene expression profiles of normal mammary tissue versus mammary
tumors will be useful in determining the complexity of HIF1
regulation
of epithelial cell biology and secretion. As there are significant differences
in how primary cells and tumor cells respond to hypoxia
(Brown and Giaccia, 1998
), the
differential pathways that regulate these processes may prove to be excellent
targets for tumor-specific, hypoxia-responsive therapeutic drugs.
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ACKNOWLEDGMENTS |
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