Expression of genes of apoptotic pathways during the development

Animal Science Papers and Reports vol. 24 (2006) no. 1, 47-56
Institute of Genetics and Animal Breeding, Jastrzębiec, Poland
Expression of genes of apoptotic pathways during
the development of mammary gland in mice*
Tadeusz Malewski1, Małgorzata Krzyżowska2, Stanisław Kamiński3,
Lech Zwierzchowski1, Zofia Szymańczak1
1
Department of Molecular Biology,
Polish Academy of Sciences Institute of Genetics and Animal Breeding,
Jastrzębiec, 05-552 Wólka Kosowska, Poland
2
Department of Preclinical Sciences, Faculty of Veterinary Medicine,
Warsaw Agricultural University,
Ciszewskiego 8, 02-786 Warsaw, Poland
3
Department of Animal Genetics,
University of Warmia and Mazury in Olsztyn,
Oczapowskiego 5, 10-719 Olsztyn, Poland
(Received December 19, 2005; accepted February 10, 2006)
The Panorama Mouse Apoptosis Gene Array containing 243 genes was used in profiling the gene
expression of mammary gland in mice. In all reports dealing with mammary gland development
that were considered in this study, expression of apoptosis-related genes was identified on both
pathways, i.e. on death receptor and mitochondrial-mediated pathway. The results obtained suggest
that tumour necrosis factor (TNF) receptor pathway prevails during pregnancy. Transition from
pregnancy to lactation alters the expression rate of many apoptosis-related genes, functions of which
in apoptosis are not yet defined precisely.
KEY WORDS: apoptosis / gene expression / macroarray /mammary gland
The removal of superfluous, defective, damaged, or dangerous cells is critical for
normal development and tissue homeostasis in multicellular organisms. The molecular
* Supported by the Ministry of Education and Science grant No 3 P06D 017 23
47
T. Malewski et al.
control of the genetically programmed cell death process has been evolutionarily
conserved in metazoans [Kerr et al. 1980, Majno and Joris 1995]. Apoptosis is driven
by a family of aspartate-specific cysteine proteases, known as caspases, which cleave
many cellular proteins and proteolytically activate enzymes that contribute to cell
destruction [Kerr et al. 1980, Majno and Joris 1995, Robertson et al. 2000].
In mammals there are two principal signalling pathways of apoptosis that converge
at the level of caspase activation [Rathmell and Thompson 1999]. One is a direct
pathway from death receptor to caspase cascade activation and cell death. Death
receptor ligation triggers recruitment of the precursor of caspase 8 to a death-inducing
complex through the Fas-associated protein with death domain (FADD), which leads
to caspase 8 activation. The other pathway is triggered by stimuli such as drugs,
radiation, infectious agents and reactive oxygen and is initiated in mitochondria. After
cytochrome c is released into the cytosol from the mitochondria, it binds to Apaf1 and
ATP, which then activate caspase 9 [Kroemer and Reed 2000]. Activation of initiator
caspase 8 and caspase 9 results in activation of effector caspases, such as caspase 3.
Recently, endoplasmic reticulum has also been shown to execute apoptosis [Kroemer
and Reed 2000]
Apoptosis plays an important role in sculpturing the overall shape and organization
of organs during development and tissue remodelling [Bissell et al. 2003]. The
morphogenesis of organs typically involves the coordination of several cellular
processes, including migration, proliferation, apoptosis and changes in cell shape or
polarity [Bissell et al. 2003]. The morphogenesis of milk ducts in the development
of the mammary gland is dependent on the selective death of epithelial cells to form
mammary acini, in which a hollow tube lined with polarized mammary epithelial cells
is formed. Following lactation, in the mammary gland involution process develops,
culminating in return of the organ to a pre-pregnancy state. In involution stage I
the appearance of apoptotic mammary epithelial cells takes place in the alveoli. In
stage II destruction of the lobular-alveolar architecture of the gland and apoptosis of
mammary epithelial cells follow, as cells loose connections with extracellular matrix
and basement membrane [Lund et al. 1996].
Gene expression profiling of mouse mammary gland during transition from
pregnancy to lactation showed significant changes in expression of numerous
apoptosis-related genes [Malewski et al. 2005, Master et al. 2002]. The aim of the
current study was to profile the expression of apoptosis-related factors, caspases and
mitochondrial-associated proteins involved in signalling pathways during pregnancy,
lactation and subsequent involution of mammary gland in mice.
Material and methods
Animals and tissues
Mammary gland samples of MIIZ mice at four different physiological stages were
used (six samplings), obtained from following groups of animals: 7 weeks-old virgin
48
Apoptotic pathway genes expression during mammary gland development
mice (V), mice on day 6 and day16 of pregnancy (P6 and P16, respectively), mice
on day 1 of lactation (L), and mice on post-weaning day 3 and day 8 (mammary gland
involution, In3 and In8, respectively). Tisue samples were excised immediately after
animal cervical dislocation, cleared from most adjacent muscles, fat and connective
tissues, frozen at -25°C and stored at -75°C until use.
RNA extraction
Total RNA was extracted with TRI Reagent (SIGMA-ALDRICH ) according
to the manufacturer’s protocol. To quantify the amount of total RNA extracted, the
absorbance at 260 nm was measured with DU-68 Spectrophotometer (BECKMAN).
RNA integrity was electrophoretically verified in agarose gels stained with 1.5%
ethidium bromide.
cDNA macroarray
For cDNA synthesis equal volumes of RNA were extracted from five mice at each
of four investigated stages (six samplings), and pooled. To confirm the validity of the
assay, cDNA synthesis was performed in duplicate. The cDNA labelling reactions
were performed in two steps according to the manufacturer’s protocol. Details of
labelled cDNA synthesis and hybridization were those described by Malewski et
al. [2005]. Briefly, Nylon array (Panorama Mouse Apoptosis Gene Array, SIGMAGenosys, The Woodlands, TX) were hybridized with labelled probes at 65°C
overnight. After hybridization, the nylon membranes were washed with 0.5×SSPE
+ 1% sodium dodecyl sulphate (SDS) and 0.1×SSPE + 1% SDS, and then exposed
to Phosphorscreen (KODAK, Japan) for 24 h. The screens were scanned by BioRad
FX Scanner. Results from three independent hybridizations were obtained for each
probe. Images were analysed by Quantity One (BioRad) software. Each image was
overlaid with grids so that signal intensities of individual spots could be assessed.
Local background for each membrane was calculated on the basis of 10 positions
with no DNA-spotted area. Expression levels of individual genes are presented in
arbitrary units after subtracting background. Intensity-based global normalization was
then performed.
Results and discussion
Macroarrays provide a powerful tool for analysing complex biological systems. In
this study we simultaneously analysed 243 transcripts to determine basic expression
pattern of genes associated with apoptosis in adult mouse mammary gland. Examples
of some macroarray analyses are shown in Figure 1. Probes present in the Panorama
Mouse Apoptosis Gene Array cover eight ontologic categories as defined by the
Gene Ontology Consortium (http://www.geneontology.org) - Khatri et al. [2002].
Apoptosis-related factors are represented by 66 probes belonging to those categories.
Results of expression profiling of apoptosis pathways genes during the development
49
T. Malewski et al.
Fig. 1. Examples of microarray analyses. A – electrophoretic image of RNA isolated from mammary
glands of mice: V – virgin mice seven-weeks old; P6 and P16 – mice on day 6 and day 16 of pregnancy;
L – mice on day 1 of lactation; I3 and I8 – mice on day 3 and day 8 of mammary gland involution.
B – DNA array hybridization from lactation day 1 (L); C – DNA hybridization from inhibition day 3
(I3). Hybridizations were performed using radioactive 32P-labelled probes prepared from lactating and
involuting mammary glands of five animals each. Indicated are changes in expression of selected genes
during transition from lactation to involution.
50
Akt1
Bag1
Bak1
Bcl2l1
Bcl2l2
Birc2
Birc3
Birc5
Casp1
Casp2
Cav2
Cd180
Cd47
Cidea
Cldn3
Clu
Ctsd
Cycs
Dap
Dffa
Dnase2
Dnclc1
Dpf2
Fadd
Gpx1
Gsn
Gzmb
Hnrpa1
Lgals
Mcl1
Gene
symbol
NM_009652
NM_009736
NM_007523
NM_009743
NM_007537
NM_007465
NM_007464
NM_009689
NM_009807
NM_007610
AF141322
NM_008533
NM_010581
NM_007702
NM_009902
L08235
NM_009983
NM_007808
AI196645
NM_010044
NM_010062
NM_019682
NM_011262
NM_010175
NM_008160
NM_010354
NM_013542
NM_010447
X16834
NM_008562
Gene Bank
acc. No.
Thymoma viral proto-oncogene 1
Bcl2-associated athanogene 1
Bcl2-antagonist/killer 1
Bcl2-like 1
Bcl2-like 2
Baculoviral IAP repeat-containing 2
Baculoviral IAP repeat-containing 3
Baculoviral IAP repeat-containing 5
Caspase 1
Caspase 2
Caveolin2
CD 180 antigen
CD47 antigen
DNA fragmentation factor, alpha subunit-like effector A
Claudin 3
Clustrin
Cathepsin D
Cytochrome c, somatic
Death-associated protein
DNA fragmentation factor, alpha subunit
Deoxyribonuclease II alpha
Dynein, cytoplasmic, light chain 1
D4, zinc and double PHD fingers family 2
Fas (TNFRSF6)-associated via death domain
Glutathione peroxidase 1
Gelsolin
Granzyme B
Heterogeneous nuclear ribonucleoprotein A1
Lectin, galactose binding, soluble 3
Myeloid cell leukemia sequence 1
Gene name
Table 1. Expression profiling of genes in the murine mammary gland of mice
3.39
R
N
I
16.78
N
N
N
I
N
0.32
N
I
N
I
0.19
0.19
0.51
3.64
1.34
N
R
I
N
0.25
0.12
I
0.10
N
0.79
1.21
I
N
0.63
0.81
N
N
N
N
N
1.12
N
1.41
N
1.57
2.74
1.72
0.79
0.89
1.40
N
I
0.80
N
1.11
0.82
R
2.74
N
0.69
0.78
R
N
1.25
0.90
N
N
I
N
I
R
N
0.58
I
1.05
0.34
0.49
0.68
1.14
1.13
N
R
1.08
N
R
R
N
R
N
1.55
0.97
I
I
0.89
0.85
I
I
R
I
1.01
N
I
1.39
0.16
0.75
2.28
2.72
0.82
0.78
1.77
I
I
1.34
I
I
I
I
I
I
0.82
1.09
6.46
R
R
0.19
R
R
N
R
R
I
R
1.71
6.29
R
1.18
4.43
4.28
0.40
R
R
R
0.98
R
1.39
24.45
R
4.00
R
1.26
Gene mRNA amount at different stages of mammary
gland development
P6/V
P16/P6
L/P16
In3/L
In8/In3
Apoptotic pathway genes expression during mammary gland development
51
52
NM_008594
NM_008771
NM_013614
NM_008798
NM_011107
NM_011113
NM_008898
NM_011163
NM_008960
AB041997
AF117759
AB017337
AW106709
NM_009383
NM_011609
NM_011610
NM_009399
NM_001033161
NM_011660
Mfge8
Mt2
Odc1
Pdcd1
Pla2g1b
Plau
Por
Prkr
Pten
Ptges
Sfrp5
Srebf1
Srebf2
Tial1
Tnfr1a
Tnfrsf1b
Tnfrsf11a
Tradd
Txn
Milk fat globule-EGF factor 8 protein
Metallothionein 2
Ornithine decarboxylase, structural 1
Programmed cell death 1
Phospholipase A2, group IB, pancreas
Plasminogen activator, urokinase
P450 (cytochrome) oxidoreductase
Double standed RNA-dependent protein kinase
Phosphatase and tensin homolog
Prostaglandin E synthase
Secreted frizzled-related protein 5
Sterol regulatory element-binding protein-1
Sterol regulatory element binding factor 2
Cytotoxic granule-associated RNA-binding protein-like 1
Tumor necrosis factor receptor superfamily, member 1a
Tumor necrosis factor receptor superfamily, member 1b
Tumor necrosis factor receptor superfamily, member 11a
TNFRSF1A-associated via death domain
Thioredoxin
Gene name
*Gene mRNA abundance at different stages of mammary gland development.
R - gene repression.
I - gene induction.
N - gene expression not found.
Gene Bank
acc. No.
Gene
symbol
Table 1 continued.
I
I
I
N
N
I
N
N
0.23
N
I
N
0.36
N
I
N
N
N
0.21
0.99
R
3.13
N
I
R
N
I
1.29
I
0.81
I
2.70
I
R
N
I
I
1.28
2.95
I
0.62
I
1.23
N
N
R
R
R
1.19
R
0.61
R
N
N
1.05
R
0.21
0.81
1.18
0.28
0.46
R
I
N
N
I
N
1.31
N
1.05
I
I
I
1.89
N
4.66
1.08
1.64
R
R
N
R
I
N
5.86
N
R
I
1.94
3.50
R
R
R
N
1.60
Gene mRNA amount at different stages of mammary
gland development
P6/V
P16/P6
L/P16
In3/L
In8/In3
T. Malewski et al.
Apoptotic pathway genes expression during mammary gland development
of mouse mammary gland obtained in the present study are shown in Table 1.
Genes involved in death receptor pathways of apoptosis
During early pregnancy (P6) up-regulation of caspase 2 gene was observed,
followed by up-regulation of gene Tnfr1, what indicates activation of TNFR1dependent pathway of apoptosis. On day 16 of pregnancy (P16) and during the
involution (In3 and In8), both caspase 2 and Tnrf1 expression were down-regulated.
The up-regulation of gene Tradd with no simultaneous up-regulation of gene Fadd,
the latter encoding death-inducing protein adaptor, indicates that during pregnancy
both Tnfr1 and Tnfr2 are involved in cell survival and proliferation rather, than in
apoptosis.
Apart from TNFR1, expression of other members of TNF receptor superfamily was
observed in mammary gland [Hsu et al. 1996]. Besides their role in apoptosis, most
of them have other functions, such as participation in proliferation, differentiation,
immune regulation, and gene expression. Receptors with pleiotropic functions include
TNF-R1, TNF-R2, NGF-R and RANK. These receptors are structurally similar
membrane proteins of type I. In its extracellular domain each receptor possesses from
2 to 6 imperfect repeats of about 40 amino acids, each with approximately six Cys
residues. Their cytoplasmic domains generally lack considerable sequence similarity
[Hsu et al. 1995, 1996, Dempsey et al. 2003]. The recently identified TNF superfamily
member – TRANCE (RANKL/TNFSF11) – has been shown to play essential role in
the developmental processes leading to both lymph node formation, and function of
mammary glands during pregnancy and lactation [Walsh and Choi 2003]. TRANCE
interacts with TRANCE-R (RANK/TNFRSF11A) receptors. It has been shown by Fata
et al. [2000] that TRANCE-R is constitutively expressed throughout the mammary
gland and TRANCE-deficient mice fail to undergo mammary gland development
during pregnancy, resulting in dramatic decrease in the amount of the β-casein yielded
during lactation. Indeed, Table 1 demonstrates that expression of gene Tnfrsf11a was
up-regulated on day P16 and down-regulated during involution days In3 and In8.
Death receptor-mediated apoptosis pathway may also be regulated by kinases.
Phosphoinositide 3-kinase (PI3K) possesses a dual-specificity with lipid and protein
activity. One of its targets is the protein kinase B (PKB or AKT). The PI3-K/PKB
pathway has been shown to deliver an anti-apoptotic signal, which is negatively
regulated by the lipid and protein phosphatase Pten [Datta et al. 1999, li et al.
1997]. In the current study, expression of Akt/Pkb gene during pregnancy was upregulated, to become down-regulated during involution, which was accompanied
by Pten up-regulation. Interestingly, down-regulation of apoptosis-inhibiting genes
Birc2 (formerly named cIAP-2) and Birc3 (formerly named cIAP-1) was observed
by Deveraux and Reed [1999] on day In8. Birc2 and Birc3 proteins bind to TNF-α
receptor–associated factor 1 and 2 (Traf1 and Traf2) and thus can be recruited to
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T. Malewski et al.
the activated TNFR complex. X-linked IAP, Birc2, and Birc3 were shown to interact
with and inhibit downstream caspases 3 and 7, thereby inhibiting apoptosis initiated
either by receptor-mediated or by cytochrome c-dependent mechanisms [Deveraux
and Reed 1999, Shiozaki 2004].
Genes involved in mitochondrial pathways of apoptosis
Second pathway of apoptosis induction involves release of cytochrome c from
mitochondrial intermembranous space into the cytoplasm. Cytochrome c, together
with protein Apaf1, interacts with caspase 9. Interaction between Apaf1 and caspase
9 leads to activation of the latter [Rathmell and Thompson 1999, Kim 2005, Kluck
et al. 1997]. Mitochondrial pathway of apoptosis induction is regulated by the family
of proteins named Bcl-2. In mammals, this family consists of both pro- and antiapoptotic proteins, which show both sequential and structural similarity in the BH
(Bcl2 homology) domains [Rathmell and Thompson 1999]. The anti-apoptotic factors
include Bcl-2, Bcl2l2 (formerly named Bcl-w), Bcl2l1 (formerly named Bcl-xL),
Bcl2l10 (formerly named Boo/Diva/Bcl-B), and Mcl1 [Rathmell and Thompson
1999, Boise et al. 1993, Gibson et al. 1996]. These proteins all share three or four
BH domains, and they occur in the cytoplasmic site of intracellular membranes, such
as the outer mitochondrial membrane, the endoplasmic reticulum, and the nuclear
envelope. A subgroup of pro-apoptotic Bcl-2 family members, including Bax, Bak,
Bcl2l1 (former name Bcl-xs) and Bcl-GL, have two or three BH domains and are
structurally very similar to their prosurvival relatives [Rathmell and Thompson 1999,
Kluck et al. 1997, Boise et al. 1993]. The latter proteins act as sensors for apoptotic
stimuli and initiate the apoptotic cascade that can be antagonized by Bcl-2.
In this study, regulation of expression of both pro-apoptotic and anti-apoptotic
mitochondria-related factors was shown (Tab. 1) during pregnancy, lactation and
involution. In early pregnancy (P6) observed was induction of anti-apoptotic genes
Bcl2l1, Bcl2l2 and Mcl1 accompanied by up-regulation of cytochrome c gene encoding
mitochondrial pathway key protein. Later in pregnancy (P16) expressions of Bcl2l1,
Bcl2l2, Mcl1 and cytochrome c were reduced, then remained unchanged during
lactation, and were followed by reduction of expression during the whole involution
phase tested (In3 and In8) Additionally, on day In8, up-regulation of cytochrome c
was observed, indicating possible induction of apoptosis. Bcl-2 protein is the target
of Bag1. Bag1 prevents the release of pro-apoptotic factors such as cytochrome c
from mitochondria, important for activation of caspases in response to many apoptotic
inducers [Takayama and Reed 2001, Townsend et el. 2003]. Expression of Bag1 was
repressed on day P6 and induced by day P16, then repressed during lactation, induced
again on day In3 and increased on day In8. Expression of Bag1 during pregnancy
was inversely regulated in comparison to expression of anti-apoptotic genes Bcl2l1,
Bcl2l2 and Mcl1 and co-regulated with cytochrome c indicating its involvement in
apoptosis suppression.
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Apoptotic pathway genes expression during mammary gland development
Macroarray analysis of mice mammary gland gives outlook of activity of
genes related to apoptosis at different stages of mammary gland development. This
allows choosing the correct set of genes for future research of apoptosis and tissue
remodelling during development. The results presented here suggest that the TNF
receptor pathway prevailes during pregnancy. Transition from pregnancy to lactation
changes the intensity of expression of many apoptosis-related genes, function(s) of
which in apoptotic pathways is (are) not precisely defined.
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Tadeusz Malewski, Małgorzata Krzyżowska, Stanisław Kamiński,
Lech Zwierzchowski, Zofia Szymańczak
Profilowanie ekspresji genów szlaków apoptozy
w gruczole mlekowym myszy
Streszczenie
Dokonano profilowania ekspresji 243 genów w gruczole mlekowym myszy stosując makromacierz
Panorama Mouse Apoptosis Gene Array. Ekspresję genów szlaków receptorowego i mitochondrialnego
stwierdzono w gruczole mlekowym myszy we wszystkich czterech badanych stadiach jego rozwoju – u
siedmiotygodniowych myszy dziewiczych, myszy ciężarnych (w 6 i 16 dniu ciąży), w pierwszym dniu
laktacji i po odsadzeniu młodych (3 i 8 dzień inwolucji).
Uzyskane wyniki wskazują, że szlak receptora TNF jest głównym szlakiem apoptozy podczas ciąży.
Przejściu od ciąży do laktacji towarzyszy zmiana ekspresji wielu genów biorących udział w apoptozie,
których funkcja nie została jeszcze dokładnie określona.
56