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First published online 11 February 2009
doi: 10.1242/dev.028423
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1 Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT,
UK.
2 Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261,
USA.
3 British Columbia Cancer Research Centre, Vancouver, BC V5Z 1L3, Canada.
¶ Author for correspondence (e-mail: charles.streuli{at}manchester.ac.uk)
Accepted 4 December 2008
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MATERIALS AND METHODS
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DISCUSSION
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Key words: Adhesome, Breast, Differentiation, Focal adhesion kinase, Glandular development, Integrin, Integrin-linked kinase, Mammary, Mouse
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MATERIALS AND METHODS
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DISCUSSION
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). An emerging concept is that integrins monitor the cellular
ECM environment, thereby equipping cells with context-dependent checkpoints
for growth factor and hormone signalling
(Giancotti and Tarone, 2003;
Katz and Streuli, 2007).
However, a full understanding of normal developmental mechanisms, and
abnormalities in epithelial diseases such as carcinoma, depends upon learning
how cell-ECM interactions link with downstream endpoints, such as
differentiation, and dissecting the specific intermediates in this process
remains an important challenge.
To determine the molecular mechanisms by which integrins control the
development and tissue-specific functions of epithelia, our laboratory is
using the mammary gland system where the spatial (i.e. adhesive) signals that
permit differentiation integrate with temporal (i.e. hormonal) developmental
cues (Naylor and Streuli,
2006). We have previously shown that post-pregnancy
differentiation of the luminal epithelial cells that synthesise milk proteins
is influenced by converging signals derived from the ECM and the endocrine
hormone prolactin. Indeed, β1-integrin is crucial for mammary gland
development and epithelial cell function
(Faraldo et al., 1998;
Klinowska et al., 1999;
Li et al., 2005;
Streuli et al., 1991).
Furthermore, genetic approaches have indicated that β1-integrin is
required for differentiation, and that it cooperates via RAC1 (RAS-related C3
botulinum substrate 1) with prolactin signalling to control STAT5, a major
regulator for mammary cell fate (Akhtar and
Streuli, 2006; Naylor et al.,
2005). The aim of the current work was to extend these previous
studies and identify proximal β1-integrin effectors that control cellular
differentiation.
Integrins bind ECM proteins when in an extended active conformation, and
simultaneously promote the formation of multiprotein adhesion complexes
(adhesomes) at the plasma membrane, thus acting as both focal centres for
cytoskeletal assembly and as signalling platforms
(Humphries et al., 2003;
Zaidel-Bar et al., 2007).
Despite the importance of integrins in determining cell fates, it is not well
understood which proximal integrin adaptor and signalling proteins are
involved in controlling specific phenotypes. Two key mediators of
β1-integrin signal transduction are integrin-linked kinase (ILK) and
focal adhesion kinase (FAK), and these proteins have different downstream
effectors (Hannigan et al.,
2005; Schatzmann et al.,
2003). Integrin signalling through focal adhesions has not widely
been studied in the context of a multicellular 3D environment, and here we
have used a genetic analysis to determine whether these proteins are involved
in glandular differentiation, both in vivo as well as in a 3D culture model of
primary mammary epithelial cells (MECs).
We demonstrate that ILK and FAK are located within the same adhesion complexes; however, only one of these proteins, ILK, but not FAK, is required for the tissue-specific function of MECs. This indicates for the first time that one specific adhesome component mediates this key role for integrin, i.e. the control of cellular differentiation. Moreover, ILK has not previously been studied in any glandular epithelium in vivo, and our study also provides new insights into its involvement in tissue development, as well as pointing to a novel differentiation function for this integrator of adhesion signals.
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Naylor et al., 2005;
Selbert et al., 1998;
Terpstra et al., 2003;
Wintermantel et al., 2002).
For the in vivo analysis, Cre-mediated specific Ilk or Fak
gene deletion in luminal mammary epithelial cells was as described for the
β1-integrin gene (Naylor et al.,
2005). Cre-mediated specific Ilk gene deletion in luminal
mammary epithelial cells was achieved by crossing Ilkfx/fx
mice with CreTg/· mice to produce
Ilkfx/+;CreTg/· mice. Double
heterozygous Ilkfx/+;CreTg/· mice were
then crossed with Ilkfx/fx mice to produce offspring that
carried Ilkfx/+, Ilkfx/+;CreTg/·,
Ilkfx/fx or
Ilkfx/fx;CreTg/· alleles. To avoid
problems in nursing pups that were caused by mothers with potentially
defective mammary glands, only the male mice of the breeding pairs carried the
Cre transgene. The genotypes of offspring were determined by PCR
amplification of ear genome, and
Ilkfx/fx;CreTg/· females were used.
Among the controls, Ilkfx/+,
Ilkfx/+;CreTg/· and
Ilkfx/fx were apparently indistinguishable from wild type,
so we used Ilkfx/fx. An identical strategy was employed to
generate FAK-null mammary epithelium. Cre expression is usually highly
penetrant, although there is some degree of variability, and in control mice
with the Cre transgene crossed to Rosa26 mice, X-gal stained most of the
epithelial component of the glands blue. To confirm gene deletion in the
Ilkfx/fx and Fakfx/fx crosses, PCR was
carried out on DNA isolated from glands of each mouse used in the in vivo
study. Female mice were mated between 8 and 12 weeks of age. Growth rate analysis of their pups was performed by standardising litter sizes to eight pups/litter, and weighing pups at least every second day through lactation. Mouse lines were maintained by repeated backcrossing with C57Bl/6 mice in a specific pathogen-free environment. Mice were housed and maintained according to the University of Manchester and UK Home Office guidelines for animal research.
Primary cell culture and virus infection
Primary MECs were isolated and cultured from pregnant mice as described
(Pullan and Streuli, 1996).
Cells were cultured in growth media containing 5 µg/ml insulin, 1 µg/ml
hydrocortisone (Sigma), 3 ng/ml epidermal growth factor (EGF), 10% foetal calf
serum (Biowest), 50 U/ml penicillin/streptomycin, 0.25 µg/ml fungizone and
50 µg/ml gentamycin in Ham's F12 medium (Gibco). Differentiation medium,
DMEM/F12 (Gibco) containing insulin and hydrocortisone with or without 3
µg/ml prolactin (Sigma), was added for 24 hours before harvest.
For viral infection in situ, the required amount of virus was diluted into a minimal volume of low-Ca2+ Ham's F12 and incubated on cells cultured in monolayer for 2 hours. Low Ca2+ disruption of intercellular junctions in confluent cultures aided viral infection by allowing viruses to penetrate all surfaces of the cells. The same volume of growth media containing twice the concentrations of additives and Ca2+ as above was then added onto the virus-containing medium and incubated overnight for maximal infection.
For Ilk or Fak gene deletion, primary MECs from 15.5- to
17.5-day pregnant were infected with adenovirus to enable up to 90% infection
rates. This strategy was used because the logistics of obtaining sufficient
Ilkfx/fx;CreTg/· females precluded
primary culture experiments from Ilk–/–
glands. Ilkfx/fx or FAKfx/fx cells
were infected with either Ad-Cre (Ad5 viruses expressing Cre recombinase under
the control of mouse CMV promoter; Ad-CreM1 from Microbix Biosystems) or
Ad-lacZ (Ad5 viruses expressing β-galactosidase from Dr Hazel
Weir and Dr Anne Ridley) and plated onto reconstituted BM-matrix (Matrigel, BD
Biosciences) as described previously
(Watkin and Streuli, 2002). In
control experiments, infection of primary cultures of mammary cells isolated
from wild-type mice with Ad-Cre or Ad-lacZ had no effect on the
levels of ILK or β-casein (see Fig. S2 in the supplementary
material).
For rescue experiments, cells were first infected in situ in monolayer with
control Ad-lacZ or with Ad-Cre in order to allow sufficient time for
endogenous ILK protein to be removed. Cells were trypsinised and re-infected
in suspension for 1 hour with either (1) Ad-ILK-GFP encoding a wild-type ILK
fused to GFP containing a farnesylation sequence
(Boulter et al., 2006) and
control Ad-GFP (both Ad5 viruses made by Vector Biolabs, see below); or (2)
Ad-V12Rac and control Ad-lacZ (Ad5 viruses derived from pJM17)
(Wojciak-Stothard and Ridley,
2002). It was not possible to use co-infection because the
kinetics of de novo protein expression by Ad-virus was not compatible with the
time required for ILK protein turnover.
In each case, cells were plated onto BM-matrix (Matrigel) in growth media. After 48 hours, medium was changed to differentiation medium with or without prolactin and incubated for a further 24 hours before harvest for immunoblotting and immunofluorescence.
Generation of ILK-GFP adenovirus
Recombinant adenoviruses (serotype5 dE1/E3) expressing ILK-GFP and GFP were
constructed using the pDUAL-CCM adenoviral cloning vector (Vector Biolabs,
PA). The first-generation viral stock was generated using the Ad-HQ system by
Vector Biolabs. Membrane-targeted ILK-GFP was subcloned from
pcdna3.1-ILK-GFP-F (Boulter et al.,
2006) into pDUAL-CCM using HindIII restriction sites to
create pDUAL-CCM-ILK-GFP. To generate the GFP vector, GFP was amplified by PCR
from pCDNA-3GFP (Akhtar et al.,
2000) with a quadropeptide linker using following primers:
forward, 5'-CCCAAGCTTCCCACCCATGAGTAAAGGAGAAGAACTTT-3'; and
reverse, 5'-ACCGGTACCCCCGGGTTTGTATAGTTCATCCATGCC-3'. The product
was cloned into pDUAL-CCM using HindIII and KpnI restriction
sites to create pDUAL-CCM-GFP. In each case, the localisation of the expressed
proteins was checked by transfection into 293 cells prior to generating virus.
Large-scale adenoviral stocks were amplified in E1-competent 293 human
embryonic kidney cells and purified on a caesium chloride gradient as
previously described (Watkin and Streuli,
2002).
Morphological and histological analysis
Whole-mount analysis was performed by spreading inguinal mammary glands on
polysine slides, fixing in 10% neutral buffered formalin overnight, defatting
in acetone before staining with carmine alum (0.2% carmine, 0.5% aluminium
sulphate) overnight. The whole mount was dehydrated using a graded ethanol
series followed by immersion in Slide Bright (Genta Medical) for 1 hour and
stored in methyl salicylate before photography using a Leica dissecting
microscope. Whole mounts were subsequently paraffin embedded before sectioning
(5 µm) and subjected to standard Haematoxylin and Eosin staining. Histology
was imaged with a Zeiss Axioscop2 microscope using PlanNeoFluar 40x lens
(numerical aperture 0.75) fitted with a Zeiss AxioCam colour camera, and
analysed with Openlab (3.1.7) software (Improvision).
ORO staining was performed by staining 6 µm mammary cryosections in freshly diluted ORO solution [six parts 0.5% ORO stock solution (Sigma) and four parts water] for 15 minutes. Sections were rinsed twice with 60% isopropanol, once with water and then counterstained with Mayer's Haematoxylin for 1 minute before photography as above.
Protein analysis
Proteins were extracted using 1x NP-40 lysis buffer (10% w/v
glycerol, 50 mM Tris-HCl, 100 mM NaCl, 1% w/v Nonidet-P40, 2 mM
MgCl2 and fresh protease/phosphatase inhibitors; pH 7.5) for cells
in monolayer or 2x NP-40 buffer for cells on BM-Matrix. Equal amounts of
proteins were used and equivalent loading assessed by referral to controls,
such as Calnexin (Bioquote SPA-860). Immunoblotting was as described
(Zoubiane et al., 2004) and
specific binding was detected using horseradish peroxidase secondary
antibodies (Jackson ImmunoResearch) with SuperSignal West Pico
Chemiluminescent substrate (Pierce) and Biomax Light film (Kodak).
Primary antibodies were β-casein
(Streuli and Bissell, 1991),
β-galactosidase (Promega #z3783), Cre (Chemicon #MAB3120), ILK (BD
Transduction #611802), FAK (a gift from A. Ziemiecki, University of Bern,
Bern, Switzerland), pT202/Y204-p42/44-MAPKinase (Cell Signaling #9101), ERK2
(Santa Cruz #sc-154), pS473-PKB (Cell Signaling #9271), pT308-PKB (Cell
Signaling #9275), PKB (Cell Signaling #9272), pS9-GSK3β (Cell Signaling
#9336), GSK3β (BD Transduction #610201), RAC1 (23A8; Upstate #05-389),
Myc (9E10, Roche #1-667-203), Actin (Sigma #A2066), STAT5A (Santa Cruz
#sc-1081), pY694/Y699-Stat5 (Upstate), Adipophilin (Progen Biotechnik
#GP40).
RAC activity assay was performed as previously described using Pak1-PBD
agarose (Akhtar and Streuli,
2006; Benard and Bokoch,
2002).
Immunostaining
Cells cultured in monolayer or BM-Matrix were fixed with 4% formaldehyde
(Polysciences) in PBS, stained as described and imaged after mounting with
Prolong Anti-Fade (Molecular Probes) by conventional or confocal microscopy
(Akhtar and Streuli, 2006).
Conventional microscopy was on a Zeiss Axioplan2 microscope using
PlanApochromat 100x, 63x and 40x lenses (numerical aperture
1.40) with Immersol 518F oil. Z sections (0.2 µm) were captured
with a Hannamatsu Orca ER camera and denconvolved with Volocity (3.5.1)
software. In some cases, acini were visualised by confocal imaging using Leica
SP2 AOBS confocal microscope, with the pinhole set to 1 Airy Unit.
Immunofluorescence of mammary tissue was imaged by confocal microscopy and
analysed with Volocity. Immunohistochemistry of mammary gland was performed on
paraffin-embedded tissue using DakoCytomation EnVision + System-HRP (DAB) kit
for immunohistochemistry and protein expression was detected with a Zeiss
Axioscop2 microscope.
STAT5 nuclear translocation was performed by infecting primary mammary
epithelial cells grown in monolayer with viruses for 24 hours, using the EHS
overlay assay (Streuli et al.,
1995b). After this time, virus containing media was replaced with
differentiation medium containing BM-Matrix (1:50 dilution) without prolactin
for a further 24 hours. Fifteen minutes before harvest, cells were stimulated
with prolactin, fixed and immunostained with STAT5A, β-galactosidase and
Cre (Naylor et al., 2005).
Antibodies different to those used in immunoblotting were: β1-integrin
(Klinowska et al., 1999),
mouse-ILK (Upstate Biotechnology #05-575), rabbit-ILK (Cell Signaling
Technology), phospho-FAK Y397 (Biosource International #44-624G), rhodamine-RX
conjugated phalloidin (Molecular Probes), Alexa Flour 488-conjugated wheat
germ agglutinin (Molecular Probes #W11261) and β-catenin (BD Biosciences
#C19220). Secondary antibodies were conjugated to Cy2, rhodamine-RX and Cy5
(Jackson ImmunoResearch).
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). β1-Integrin is required for milk protein
expression, suggesting that either ILK or FAK might mediate the
differentiation signals. We therefore tested this hypothesis using the
Cre-LoxP system. Initially, we examined the role of ILK in mammary development and differentiation in vivo by deleting its (LoxP-flanked) gene with Cre driven by promoters for the milk proteins β-lactoglobulin (BLG) or whey acidic protein (WAP), both of which are specific for luminal MECs. ILK gene deletion was analysed in glands of Ilkfx/fx;Blg-CreTg/· or Ilkfx/fx;Wap-CreTg/· (hereafter called Ilk–/–) mice, and the corresponding wild-type littermates (Ilkfx/fx). PCR analysis identified the recombined 230 bp product in Ilk–/– glands and the floxed Ilk allele (fx, 2.1 kb) in both wild-type and Ilk–/– tissue (Fig. 1B; the Ilk gene is still intact in myoepithelial and stromal cells of Ilk–/– glands). Consistent with excision of Ilkfx/fx alleles, the ILK protein level was lower in the Ilk–/– glands than those of wild-type littermates (Fig. 1C). ILK was not expressed in the luminal epithelial cells within Ilk–/– glands (Fig. 1D) but was retained in myoepithelial cells localised at the periphery of alveoli (see Fig. S1 in the supplementary material), demonstrating that its deletion was specific to luminal MECs.
To determine whether loss of ILK affects mammary gland function, we examined whether the dams were able to nurse their pups efficiently. The pups from Ilk–/– dams gained weight slower than those suckling on wild-type mothers (Fig. 1E), suggesting that ILK is required for normal post-pregnancy mammary gland development.
To ascertain the mechanism of the nursing deficiency, we performed
whole-mount and histological analyses. Normal ductal elongation, side
branching and lobuloalveolar development occurred in
Ilk–/– glands in pregnancy and at lactation
(Fig. 1F). Mammary alveoli
formed in both Ilkfx/fx;Blg-CreTg/· or
Ilkfx/fx;Wap-CreTg/· mice, indicating
that ILK is not required for the cell fate decision leading to
alveolargenesis. However, histological examination revealed two major defects
in alveolar development in Ilk–/– transgenics
(Fig. 1G). First, the alveoli
displayed morphological imperfections. In late pregnancy, the lumina were
smaller, and by lactation day 2 the epithelial cells protruded into the lumen,
with continued abnormalities visible at lactation day 8. This is similar to
the phenotype of β1-integrin-null mammary glands. Second, there was an
absence of cytoplasmic lipid droplets during mid/late pregnancy (as in the
β1-integrin-null mammary glands), and the alveolar lumina were smaller
throughout lactation, suggesting a lactation defect. The basal distribution of
laminin-1, 
6-, β1- and β4-integrins was normal (not shown),
indicating that the morphological and differentiation deficiency occurred
downstream of integrins.
These data show that the phenotype of the Ilk–/– and Itgβ1–/– mammary glands is similar, although the nursing defect is not as marked as following integrin deletion. In order to identify proximal β1-integrin effectors for differentiation, we focused on the role of ILK in the functional differentiation of MECs.
ILK is required for the functional differentiation of epithelia
To investigate whether ILK is necessary for MEC differentiation in vivo, we
examined the ability of Ilk–/– alveoli to make
milk products including fat and protein. ILK ablation had a severe effect on
synthesis and secretion of milk fat, shown by small quantities of
intra-luminal fat droplets (Fig.
1G) and Oil Red O staining
(Fig. 2A). The size and numbers
of adipophilin-coated cytoplasmic lipid droplets were also reduced in
Ilk–/– alveoli
(Fig. 2B), concomitant with
less adipophilin protein (Fig.
2C). The levels of WAP were lower in the absence of ILK,
indicating that milk protein synthesis was defective in vivo
(Fig. 2D).
To confirm that ILK is required for milk synthesis, we examined 3D cultures of cells isolated from Ilkfx/fx mammary glands, where the Ilk gene was deleted using Cre-expressing adenovirus (Ad-Cre). This resulted in loss of ILK, but no changes in the levels of FAK (Fig. 2E) or the basal location of β1-integrin. MECs resemble in vivo alveoli when plated on basement membrane (BM)-matrix, and differentiate in the presence of prolactin. Neither the infection procedure nor Cre-expression in control MECs adversely affected milk protein expression (see Fig. S2 in the supplementary material). Prolactin treatment led to β-casein synthesis in the mock-infected Ilkfx/fx cells (Fig. 2F); similarly, control Ilkfx/fx acini infected with Ad-lacZ synthesised milk proteins (Fig. 2G). However, deletion of ILK prevented milk protein synthesis (Fig. 2F,G), indicating that the defect observed in vivo is a direct consequence of ILK-loss in the luminal MECs.
We also examined whether expression of a wild-type ILK fused to
farnesylated-GFP (hereafter called ILK-GFP, see Materials and methods) could
rescue the milk protein defect after ILK deletion
(Boulter et al., 2006). ILK-GFP
localised to focal adhesions of monolayer-grown wild-type MECs and the basal
surface of acini in 3D culture (not shown). ILK was first deleted in
Ilkfx/fx MECs with Ad-Cre and then those cells were
re-infected with either control or ILK-GFP-expressing viruses. β-Casein
was synthesised in the controls (Fig.
3F, lane 2) and cultures infected with either Ad-GFP or Ad-ILK-GFP
(Fig. 3A,B,F, lanes 3,4). As
above, ILK deletion prevented β-casein synthesis, and the expression of
GFP was unable to restore the defects (Fig.
3C,D,F, lanes 5,6). However, the expression of ILK-GFP in the
ILK-null cells restored basally located ILK
(Fig. 3E) and rescued the
ability of cells to synthesise milk proteins
(Fig. 3E,F, lane 7).
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Specific integrin signalling enzymes are required for the functional differentiation of epithelia
ILK is a key mediator of integrin signalling; thus, its requirement for
mammary differentiation might just reflect the essential role of
β1-integrin in this process. To test the specificity for ILK in linking
integrins with differentiation, we examined the role of an equally important
integrator of integrin signalling, FAK. We employed a similar strategy to that
described above, using Cre recombinase to ablate the Fak gene from
mammary epithelia both in vivo and in the culture model.
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Thus, two key integrators of integrin signalling that are located within the same supra-molecular adhesion complexes have distinct functions, and only one of them, ILK, is required to deliver signals that lead to MEC differentiation.
Signals downstream of ILK for mammary differentiation
To explore the mechanism underlying the requirement for ILK in mammary
differentiation, we examined the potential involvement of signalling pathways
downstream of ILK. One pathway activated downstream of ILK is that of the
small GTPase RAC (Boulter et al.,
2006). Indeed, ILK-null MECs had reduced levels of active RAC
(Fig. 5A). RAC1 integrates
integrin-mediated adhesion with lactational differentiation, and we therefore
reasoned that it might be an intermediate linking ILK with the differentiation
response (Akhtar and Streuli,
2006). To test this hypothesis, we examined whether an activated
V12Rac1 could rescue differentiation in Ilk-null cells. We first
generated Ilk–/– cells by infecting primary
MECs from Ilkfx/fx mice with Ad-Cre virus. When these
cells were re-infected with Ad-V12Rac1 or a control Ad-lacZ
adenovirus and plated in 3D culture to form acini, we found that V12Rac1 was
able to restore the lactation defect, leading to β-casein expression
(Fig. 5B, compare lanes 5 and
6). These results suggest that a RAC1 pathway transmits the ILK-mediated
signals required for milk protein expression.
ILK also has serine/threonine kinase activity in vitro, and can direct the
phosphorylation of GSK3β at S9 and PKB/Akt at S473 in certain cultured
cell types (Hannigan et al.,
2005). We found that ILK deletion in mammary acini did not alter
steady-state phosphorylation of GSK3β or PKB/Akt, or their transient
phosphorylation after insulin stimulation (see Fig. S3 in the supplementary
material). The loss of ILK also did not alter ERK phosphorylation in the
steady state or after acute stimulation with EGF. These data suggest that, at
least in primary MECs, ILK does not influence Akt or Erk signalling.
As β1-integrin is required for prolactin signalling, we also examined
whether ILK is necessary for the phosphorylation and nuclear translocation of
STAT5, a key transcription factor for mammary differentiation
(Oakes et al., 2006).
Immunofluorescence analysis revealed nuclear STAT5 in the alveoli of wild-type
mice in vivo, but this was diminished significantly within
Ilk–/– alveoli
(Fig. 5C). In primary MECs,
prolactin triggered STAT5 nuclear translocation and phosphorylation within 15
minutes in Ad-lacZ infected Ilkfx/fx cells, but
was unable to do so in the majority of Ad-Cre-infected cells
(Fig. 5D,E). Moreover, the
levels of STAT5-dependent transcripts encoding β-casein and WAP were
lower in Ad-lacZ-infected Ilkfx/fx cells than in
wild-type controls (Fig.
5F).
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); (2) there are numerous studies examining how integrins
control for example migration, but virtually nothing is known about how they
regulate cellular differentiation (DeMali
et al., 2003; Giancotti and
Tarone, 2003). Here, we show that in both in vivo and in 3D culture models of mammary gland behaviour, ILK, but not FAK, acts downstream of integrins to control lactational differentiation. Thus, we have (1) identified a high degree of specificity within the integrin-based adhesome that links cell-matrix interactions with the function of epithelia, and (2) determined a key downstream component of integrin signalling involved in differentiation.
Specific integrin adhesome proteins control lactational capacity and tissue function
β1-integrins control the terminal differentiation of MECs through a
permissive interaction with endocrine signalling
(Faraldo et al., 1998;
Naylor et al., 2005). This
indicates a direct influence of integrins on pathways driving transcriptional
endpoints (Streuli et al.,
1995a). Here, we extend this model by identifying one proximal
integrin-containing adhesion complex protein, ILK, that is required for this
differentiation pathway. Our results show that the ability of dams to nurse is
diminished in the absence of ILK. This is not due to a delay in mammary
development, but rather to the inability to synthesise and secrete adequate
quantities of milk proteins and lipids. Interestingly, ILK is not required for
specification of alveoli, because lobuloalveolar development occurs even when
Cre is expressed prior to the initiation of pregnancy using the
β-lactoglobulin promoter.
A novel aspect of the integrin control on differentiation regards the
synthesis of milk lipids. We had previously observed that β1-integrins
are required for efficient lipid production in mammary epithelium, as judged
by ORO staining, and here we extend those data by showing the involvement of
ILK. One possible mechanism may be through transcriptional or
post-transcriptional control of adipophilin, which stabilises the triglyceride
core in cytoplasmic lipid droplets, and we observed that both were decreased
in Ilk–/– glands. An alternative might be the
defective expression or regulation of key lipid metabolic proteins, e.g.
Glut-1 transporter, fatty acid desaturase, fatty acid elongase and sterol
regulatory element binding protein (SREBP1), which are normally upregulated
during pregnancy and lactation (Russell et
al., 2007). We are currently examining both these as targets for
integrin/ILK signalling.
Although the loss of ILK and β1-integrin result in very similar phenotypes in terms of STAT5 activation, milk protein synthesis and altered morphology in 3D cultures of MECs, these phenotypes are not as severe in the Ilk–/– glands in vivo as the β1-integrin-null glands. In mammary tissue, the morphology of the alveoli is altered similarly in the absence of ILK and β1-integrin, and there is also a similar decrease in milk fat. However, the reduction in STAT5 activation and milk protein synthesis is less marked than in the β1-integrin-null glands, and the pups are underweight, whereas most do not survive when nursing from β1-integrin-null dams. This suggests that factors other than ILK are involved in permitting full lactation to occur in vivo.
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).
This phenotype was ascribed to severe hypoplasia, and our in vitro data extend
those results by demonstrating that FAK deletion does not compromise the
ability of MECs to differentiate.
Importantly, our data provide insights into the way that adhesion complexes
work. They show that signalling platforms within the adhesion complex have
completely different functions, because FAK, which resides within the same
adhesion complexes as ILK as determined by immunostaining, is dispensable for
milk protein expression both in vivo and in 3D culture. There are distinct
effectors downstream of ILK and FAK, and our data suggest that those specific
to ILK control differentiation. For example, The FAK/Src complex is a crucial
tyrosine kinase that activates several effectors through phosphorylation, and
it is also an adaptor for other adhesion complex proteins such as paxillin and
CAS (Schatzmann et al., 2003).
By contrast, ILK binds numerous other proteins, including pinch and parvins,
and is a serine/threonine kinase in some cell types
(Hannigan et al., 2005). We are
currently screening for specific effectors using genetic approaches.
Possible mechanisms for ILK to control differentiation
There are several possible mechanisms underlying the requirement for ILK in
differentiation. One relates to the altered morphology acquired by the
ILK-null glands and 3D cell cultures (Fig.
1G, Fig. 3C). We
are currently investigating the mechanism of this disruption, although it is
unlikely to be due to disturbed cell-ECM interactions because laminin 1 in the
basement membrane surrounding alveoli and the β1-integrins are both
located basally, in vivo and in culture (data not shown). We have previously
shown that the morphology of mammary acini is not linked to their ability to
express milk proteins, suggesting that altered cellular organisation of
ILK-null acini does not directly compromise differentiation but rather that it
may be due to modified signalling downstream of ILK
(Streuli et al., 1991;
Zoubiane et al., 2004).
In the context of ILK-mediated signalling pathways, we found no evidence to associate ILK with PKB signalling in mammary epithelia, indicating that it most probably functions as an adaptor protein to recruit other signalling moieties rather than as a kinase itself.
Instead, our evidence indicates that RAC1 acts downstream of ILK in the
differentiation response. In previous studies we have shown that RAC is
involved with mammary differentiation by rescuing the lactation defect of
integrin-deficient cells with an activated V12Rac1
(Akhtar and Streuli, 2006). A
similar strategy employed here also rescued milk protein expression after ILK
ablation, thus confirming that the tissue-specific differentiation response is
under the control of small GTPases. It seems likely that a function of
adhesion is to activate RAC1 in a spatially restricted context, e.g. via an
ILK-signalling complex, which is in turn required for prolactin signalling, at
least in MECs. In addition, the appropriate RAC effectors might not be
recruited in the absence of ILK, compounding the effect on differentiation
(Kawashima et al., 2006).
Thus, ILK may provide a structural platform for localised activation of GEFs,
as well as the recruitment of RAC effectors, which together contribute to
downstream signals.
A current focus of our attention is to identify the ILK-binding partners
required to activate RAC1, and thereby influence differentiation. A candidate
is parvin, which binds the guanine nucleotide exchange factor PIX in
some cell types, and in doing so it regulates the activity of RAC1
(Rosenberger and Kutsche,
2006; Sepulveda and Wu,
2006). Other potential links from ILK to RAC include associations
with the Arf GAP, PKL/GIT2 and βPIX, or with pinch, NCK2 and DOCK180
(Boulter and Van Obberghen-Schilling,
2006; Legate et al.,
2006). Future studies knocking down each of these components in
MECs, in combination with gene transfer and rescue approaches will elucidate
the details of how the ILK-scaffold controls epithelial polarity and
morphogenesis.
Prolactin is a key player in post-pubertal mammary development, as it is
required for the specification of alveoli, their growth and proliferation
(Oakes et al., 2006). The
JAK-STAT pathway is an effector of prolactin signalling, culminating in the
phosphorylation, dimerisation and nuclear translocation of STAT5. In terms of
the alveolar differentiation response, it is known that β1-integrins
exert their control through the prolactin-STAT5 pathway. Our analysis now
extends this conclusion by demonstrating that ILK has a novel role in
licensing signalling through this cytokine receptor, because STAT5 activation
is diminished in both the mammary gland in vivo and the primary culture model
in its absence. A possible consequence of reduced lactational differentiation
in ILK-null mammary epithelia might be altered acinar morphology, because
there are not enough milk products to expand the lumens to their maximum
extent. We are currently examining whether there is a direct effect of ILK
deletion on ion channels and thereby fluid movement. Interestingly, both
prolactin receptor-null and STAT5-null mammary epithelia show a similar
phenotype with disorganised alveolar structures and only small lumina
(Miyoshi et al., 2001).
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/6/1019/DC1
* Present address: Department of Molecular, Cell and Developmental Biology,
University of California, Santa Cruz, CA 95064, USA 
Present address: UMR CNRS 6237, Laboratory of Signalling and Matrix
Receptors (SiRMa), UFR Sciences of Reims, 51687 Reims, Cedex 2, France
Present address: Pharmazentrum/Biozentrum, University of Basel,
Klingelbergstrasse, 50/70 CH-4056 Basel, Switzerland
Present address: Garvan Institute of Medical Research, Darlinghurst, NSW
2010, Australia
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Akhtar, N. and Streuli, C. H. (2006). Rac1
links integrin-mediated adhesion to the control of lactational differentiation
in mammary epithelia. J. Cell Biol.
173,781
-793.
Akhtar, N., Hudson, K. R. and Hotchin, N. A.
(2000). Co-localization of Rac1 and E-cadherin in human epidermal
keratinocytes. Cell Adhes. Commun.
7, 465-476.[Medline]
Benard, V. and Bokoch, G. M. (2002). Assay of
Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods
Enzymol. 345,349
-359.[CrossRef][Medline]
Boulter, E. and Van Obberghen-Schilling, E.
(2006). Integrin-linked kinase and its partners: a modular
platform regulating cell-matrix adhesion dynamics and cytoskeletal
organization. Eur. J. Cell Biol.
85,255
-263.[CrossRef][Medline]
Boulter, E., Grall, D., Cagnol, S. and Van Obberghen-Schilling,
E. (2006). Regulation of cell-matrix adhesion dynamics and
Rac-1 by integrin linked kinase. FASEB J.
20,1489
-1491.
DeMali, K. A., Wennerberg, K. and Burridge, K.
(2003). Integrin signaling to the actin cytoskeleton.
Curr. Opin. Cell Biol.
15,572
-582.[CrossRef][Medline]
Faraldo, M. M., Deugnier, M. A., Lukashev, M., Thiery, J. P. and
Glukhova, M. A. (1998). Perturbation of beta1-integrin
function alters the development of murine mammary gland. EMBO
J. 17,2139
-2147.[CrossRef][Medline]
Giancotti, F. G. and Tarone, G. (2003).
Positional control of cell fate through joint integrin/receptor protein kinase
signaling. Annu. Rev. Cell Dev. Biol.
19,173
-206.[CrossRef][Medline]
Hannigan, G., Troussard, A. A. and Dedhar, S.
(2005). Integrin-linked kinase: a cancer therapeutic target
unique among its ILK. Nat. Rev. Cancer
5, 51-63.[CrossRef][Medline]
Humphries, M. J., McEwan, P. A., Barton, S. J., Buckley, P. A.,
Bella, J. and Mould, A. P. (2003). Integrin structure: heady
advances in ligand binding, but activation still makes the knees wobble.
Trends Biochem. Sci. 28,313
-320.[CrossRef][Medline]
Katz, E. and Streuli, C. H. (2007). The
extracellular matrix as an adhesion checkpoint for mammary epithelial
function. Int. J. Biochem. Cell Biol.
39,715
-726.[CrossRef][Medline]
Kawashima, T., Bao, Y. C., Nomura, Y., Moon, Y., Tonozuka, Y.,
Minoshima, Y., Hatori, T., Tsuchiya, A., Kiyono, M., Nosaka, T. et al.
(2006). Rac1 and a GTPase-activating protein, MgcRacGAP, are
required for nuclear translocation of STAT transcription factors.
J. Cell Biol. 175,937
-946.
Klinowska, T. C., Soriano, J. V., Edwards, G. M., Oliver, J. M.,
Valentijn, A. J., Montesano, R. and Streuli, C. H. (1999).
Laminin and beta1 integrins are crucial for normal mammary gland development
in the mouse. Dev. Biol.
215, 13-32.[CrossRef][Medline]
Legate, K. R., Montanez, E., Kudlacek, O. and Fassler, R.
(2006). ILK, PINCH and parvin: the tIPP of integrin signalling.
Nat. Rev. Mol. Cell Biol.
7, 20-31.[Medline]
Li, N., Zhang, Y., Naylor, M. J., Schatzmann, F., Maurer, F.,
Wintermantel, T., Schuetz, G., Mueller, U., Streuli, C. H. and Hynes, N.
E. (2005). Beta1 integrins regulate mammary gland
proliferation and maintain the integrity of mammary alveoli. EMBO
J. 24,1942
-1953.[CrossRef][Medline]
McLean, G. W., Komiyama, N. H., Serrels, B., Asano, H.,
Reynolds, L., Conti, F., Hodivala-Dilke, K., Metzger, D., Chambon, P., Grant,
S. G. et al. (2004). Specific deletion of focal adhesion
kinase suppresses tumor formation and blocks malignant progression.
Genes Dev. 18,2998
-3003.
Miyoshi, K., Shillingford, J. M., Smith, G. H., Grimm, S. L.,
Wagner, K. U., Oka, T., Rosen, J. M., Robinson, G. W. and Hennighausen, L.
(2001). Signal transducer and activator of transcription (Stat) 5
controls the proliferation and differentiation of mammary alveolar epithelium.
J. Cell Biol. 155,531
-542.
Nagy, T., Wei, H., Shen, T. L., Peng, X., Liang, C. C., Gan, B.
and Guan, J. L. (2007). Mammary epithelial-specific deletion
of the focal adhesion kinase gene leads to severe lobulo-alveolar hypoplasia
and secretory immaturity of the murine mammary gland. J. Biol.
Chem. 282,31766
-31776.
Naylor, M. J. and Streuli, C. H. (2006).
Integrin regulation of mammary gland development. In Integrins and
Development (ed. E. Danen), pp. 176-185.
Georgetown, TX: Landes Bioscience.
Naylor, M. J., Li, N., Cheung, J., Lowe, E. T., Lambert, E.,
Marlow, R., Wang, P., Schatzmann, F., Wintermantel, T., Schuetz, G. et al.
(2005). Ablation of {beta}1 integrin in mammary epithelium
reveals a key role for integrin in glandular morphogenesis and
differentiation. J. Cell Biol.
171,717
-728.
Oakes, S. R., Hilton, H. N. and Ormandy, C. J.
(2006). The alveolar switch: coordinating the proliferative cues
and cell fate decisions that drive the formation of lobuloalveoli from ductal
epithelium. Breast Cancer Res.
8, 207.[CrossRef][Medline]
Pullan, S. and Streuli, C. H. (1996). The
mammary gland epithelial cell. In Epithelial Cell
Culture (ed. A. Harris), pp. 97-121.
Cambridge, UK: Cambridge University Press.
Rosenberger, G. and Kutsche, K. (2006).
AlphaPIX and betaPIX and their role in focal adhesion formation.
Eur. J. Cell Biol. 85,265
-274.[CrossRef][Medline]
Russell, T. D., Palmer, C. A., Orlicky, D. J., Fischer, A.,
Rudolph, M. C., Neville, M. C. and McManaman, J. L. (2007).
Cytoplasmic lipid droplet accumulation in developing mammary epithelial cells:
roles of adipophilin and lipid metabolism. J. Lipid
Res. 48,1463
-1475.
Schatzmann, F., Marlow, R. and Streuli, C. H.
(2003). Integrin signaling and mammary cell function.
J. Mamm. Gland Biol. Neoplasia
8, 395-408.[CrossRef][Medline]
Selbert, S., Bentley, D. J., Melton, D. W., Rannie, D.,
Lourenco, P., Watson, C. J. and Clarke, A. R. (1998).
Efficient BLG-Cre mediated gene deletion in the mammary gland.
Transgenic Res. 7,387
-396.[CrossRef][Medline]
Sepulveda, J. L. and Wu, C. (2006). The
parvins. Cell Mol. Life Sci.
63, 25-35.[CrossRef][Medline]
Streuli, C. H. (2009). Integrins and cell-fate
determination. J. Cell Sci.
122,171
-177.
Streuli, C. H. and Bissell, M. J. (1991).
Mammary epithelial cells, extracellular matrix, and gene expression.
Cancer Treat. Res. 53,365
-381.[Medline]
Streuli, C. H., Bailey, N. and Bissell, M. J.
(1991). Control of mammary epithelial differentiation: basement
membrane induces tissue-specific gene expression in the absence of cell-cell
interaction and morphological polarity. J. Cell Biol.
115,1383
-1395.
Streuli, C. H., Edwards, G. M., Delcommenne, M., Whitelaw, C.
B., Burdon, T. G., Schindler, C. and Watson, C. J. (1995a).
Stat5 as a target for regulation by extracellular matrix. J. Biol.
Chem. 270,21639
-21644.
Streuli, C. H., Schmidhauser, C., Bailey, N., Yurchenco, P.,
Skubitz, A. P., Roskelley, C. and Bissell, M. J. (1995b).
Laminin mediates tissue-specific gene expression in mammary epithelia.
J. Cell Biol. 129,591
-603.
Terpstra, L., Prud'homme, J., Arabian, A., Takeda, S., Karsenty,
G., Dedhar, S. and St-Arnaud, R. (2003). Reduced chondrocyte
proliferation and chondrodysplasia in mice lacking the integrin-linked kinase
in chondrocytes. J. Cell Biol.
162,139
-148.
Watkin, H. and Streuli, C. H. (2002).
Adenoviral-mediated gene transfer in two-dimensional and three-dimensional
cultures of mammary epithelial cells. Methods Cell
Biol. 69,403
-423.[Medline]
Wintermantel, T. M., Mayer, A. K., Schutz, G. and Greiner, E.
F. (2002). Targeting mammary epithelial cells using a
bacterial artificial chromosome. Genesis
33,125
-130.[CrossRef][Medline]
Wojciak-Stothard, B. and Ridley, A. J. (2002).
Rho GTPases and the regulation of endothelial permeability. Vascul.
Pharmacol. 39,187
-199.[CrossRef][Medline]
Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R. and
Geiger, B. (2007). Functional atlas of the integrin adhesome.
Nat. Cell Biol. 9,858
-867.[CrossRef][Medline]
Zoubiane, G. S., Valentijn, A., Lowe, E. T., Akhtar, N., Bagley,
S., Gilmore, A. P. and Streuli, C. H. (2004). A role for the
cytoskeleton in prolactin-dependent mammary epithelial cell differentiation.
J. Cell Sci. 117,271
-280.
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  N. Akhtar, R. Marlow, E. Lambert, F. Schatzmann, E. T. Lowe, J. Cheung, E. Katz, W. Li, C. Wu, S. Dedhar, et al. Molecular dissection of integrin signalling proteins in the control of mammary epithelial development and differentiation J. Cell Sci., March 15, 2009; 122(6): e607 - e607. [Full Text]
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10% in the absence of ILK (n=4 independent experiments). Error
bars are standard deviation from mean, and a pair of asterisks indicates
significance at P<0.01 (Student's paired t-test). A
representative example of immunofluorescence staining for β-galactosidase
or Cre (green), STAT-5 (red) and DAPI (blue) is on the right; double-headed
arrows show either Ad-lacZ-infected (top panels) or Cre-negative
cells (bottom panels), where STAT5 translocates to nuclei; the single arrows
show Ad-Cre-infected cells, where there is no STAT5 translocation. Scale bars:
32 µm. (E) Immunoblots of primary Ilkfx/fx MECs
show that Ad-Cre infection prevents efficient STAT5 tyrosine phosphorylation
(Y694/699) by prolactin. (F) RT-PCR of primary
Ilkfx/fx MECs shows that Ad-Cre infection results in lower
levels of STAT5 transcriptional activity as determined by levels of
β-casein and WAP transcripts.