Integrin Upregulation and Localization to Focal Adhesion Sites in Pregnant Human Myometrium

Abstract

Focal adhesions are integrin-rich microdomains that structurally link the cytoskeleton to the extracellular matrix and transmit mechanical signals. In the pregnant uterus, increases in integrin expression and activation are thought to be critical for the formation of the mechanical syncytium required for labor. The aim of this study was to determine which integrins are upregulated and localized to focal adhesions in pregnant human myometrium. We used quantitative polymerase chain reaction, Western blotting, and confocal microscopy to determine the expression levels and colocalization with focal adhesion proteins. We observed increases in several integrin transcripts in pregnant myometrium. At the protein level, integrins such as α(5)-integrin (ITGA5), ITGA7, ITGAV, and ITGB3 were significantly increased during pregnancy. The integrins ITGA3, ITGA5, ITGA7, and ITGB1 colocalized with focal adhesion proteins in term human myometrium. These data suggest that integrins α3β1, α5β1, and α7β1 are the most likely candidates to transmit mechanical signals from the extracellular matrix through focal adhesions in pregnant human myometrium.

Full text

Original Article
Integrin Upregulation and Localization
to Focal Adhesion Sites in Pregnant
Human Myometrium
Heather R. Burkin, PhD
1
, Monica Rice, BS
1
, Apurva Sarathy, MS
1
,
Sara Thompson, BS
1
, Cherie A. Singer, PhD
1
, and
Iain L. O. Buxton, PharmD
1
Abstract
Focal adhesions are integrin-rich microdomains that structurally link the cytoskeleton to the extracellular matrix and transmit
mechanical signals. In the pregnant uterus, increases in integrin expression and activation are thought to be critical for the
formation of the mechanical syncytium required for labor. The aim of this study was to determine which integrins are upregulated
and localized to focal adhesions in pregnant human myometrium. We used quantitative polymerase chain reaction, Western
blotting, and confocal microscopy to determine the expression levels and colocalization with focal adhesion proteins. We
observed increases in several integrin transcripts in pregnant myometrium. At the protein level, integrins such as a
5
-integrin
(ITGA5), ITGA7, ITGAV, and ITGB3 were significantly increased during pregnancy. The integrins ITGA3, ITGA5, ITGA7, and
ITGB1 colocalized with focal adhesion proteins in term human myometrium. These data suggest that integrins a3b1, a5b1, and
a7b1 are the most likely candidates to transmit mechanical signals from the extracellular matrix through focal adhesions in
pregnant human myometrium.
Keywords
myometrium, integrin, pregnancy
Introduction
The switch of uterine myometrium from a quiescent to a
contractile state at the end of pregnancy is a highly regulated
process that requires activation of at least 2 signal transduction
pathways initiating from the fetus.
1
The first pathway is caused
by mechanical distension of the uterus due to rapid fetal
growth. Mechanical stretch leads to upregulation of
myometrial contraction-associated
2
and mechanosensitivity
proteins.
3
Contraction-associated protein expression is
believed to prime or activate the uterus to effectively respond
to a second endocrine-based signal transduction pathway
originating from the fetal hypothalamic–pituitary–adrenal–
placental axis.
1
In a mouse model, increased expression of
contractile-associated proteins appears to be responsible for
increased uterine contractility at birth.
4
The switch to increased myometrial contractility is associated
with increased expression of proteins associated with focal adhe-
sion complexes.
3
Focal adhesions are membrane domains that
structurally link the cell cytoskeleton to the extracellular matrix
and transmit mechanical signals mediating a variety of pro-
cesses, such as adhesion-dependent migration, survival, and pro-
liferation.
5
Focal adhesion sites are rich in integrins, a family of
heterodimeric transmembrane cell surface receptors that act as
mechanosensors.
6
Integrin engagement at focal adhesion sites
results in activation and recruitment of focal adhesion kinase
(FAK) to these sites.
6,7
FAK then interacts with a host of regu-
latory and signaling proteins to affect changes in the actin
cytoskeleton organization, protease activation, and focal adhe-
sion stability.
7
Research in animal models indicates change in myometrial
integrin expression patterns during pregnancy. At the transcript
level, expression of ITGA1, ITGA3, and ITGB1 increases dur-
ing pregnancy, although changes in the corresponding protein
levels were not observed.
8
ITGA5 transcript and protein levels
increase in the rat myometrium during pregnancy
9
and these
increases appear to be due to mechanical stretch of the tissue.
10
In addition, ITGA5 localizes to focal adhesion complexes in
the myometrium of the pregnant rat and ewe.
9,11
However, lit-
tle is known about integrin expression and localization in the
pregnant human myometrium.
1
Department of Pharmacology, University of Nevada School of Medicine,
Center for Molecular Medicine, Reno, NV, USA
Corresponding Author:
Iain L. O. Buxton, Department of Pharmacology, University of Nevada School
of Medicine, 1664 North Virginia St., Reno, NV 89557, USA.
Email: ibuxton@medicine.nevada.edu
Reproductive Sciences
20(7) 804-812
ªThe Author(s) 2013
Reprints and permission:
sagepub.com/journalsPermissions.nav
DOI: 10.1177/1933719112466303
rs.sagepub.com

Since increases in integrin receptors and their corresponding
extracellular matrix adhesion molecules are thought to be critical
for focal adhesion development and formation of a mechanical
syncytium,
12
we conducted a series of experiments to determine
which integrins are upregulated and localized to focal adhesions
in term human myometrium. The ITGA5 has been reported to be
upregulated in laboring human myometrium.
13
Although addi-
tional integrins have been identified in the uterus, their expres-
sion in term and preterm human myometrium remains largely
uncharacterized. The objective of the experiments described
here is to characterize integrin expression and localization in the
term human myometrium.
Materials and Methods
Tissue Collection
All research was reviewed and approved by the University of
Nevada Biomedical Review Committee (institutional review
board) for the protection of human participants. Human uterine
myometrial biopsies were obtained with written informed
consent from women undergoing hysterectomy when
premenopausal and without pathology involving the uterine
muscle or from mothers undergoing elective Cesarean section
either in labor at term or at term not in labor. Tissues were
transported to the laboratory immediately by suspension in cold
physiological buffer, dissected to isolate smooth muscle, snap
frozen in liquid nitrogen, and stored at 80C. The average age
of disease-free nonpregnant patients was 41 +13 years. The
average age for patients in the pregnant laboring and nonlabor-
ing groups was 28 +11 and 30 +9 years, respectively. Preg-
nant patients ranged from 37 to 40 weeks of gestation, with the
mean at 38.9 weeks for both laboring and nonlaboring groups.
Parity ranged from 1 to 4, with the average at 2.5 for nonlabor-
ing patients and 2.3 for laboring patients. Patients represented a
range of ethnicities and were 48%Caucasian, 41%Hispanic,
7.3%African American, and 3.7%Pacific Islander. All sam-
ples were from singleton pregnancies.
Quantitative Polymerase Chain Reaction
For quantitative polymerase chain reaction PCR (qPCR), total
RNA was extracted from the frozen human myometrial tissue
using Trizol reagent (Invitrogen, Carlsbad, California) as
described.
14
The RNA was converted to complementary DNA
(cDNA) using SuperScript III reverse transcriptase (Invitrogen)
and random hexamers. The cDNA from 6 myometrial samples
per group was pooled and subjected to qPCR on a human
extracellular matrix and adhesion molecules PCR array
(PAHS-013C, SABiosciences, Frederick, Maryland). The
qPCR reactions were carried out in an ABI StepOne Plus
machine according to manufacturer’s protocol. To confirm the
transcript level changes, qPCR was performed in triplicate on
6 individual human myometrial cDNA samples per group using
gene-specific TaqMan gene expression assays (900 nmol/L pri-
mers, 250 nmol/L 6-carboxyfluorescein labeled TaqMan
dihydrocyclopyrroloindole tripeptide minor groove binder
probe) and 1TaqMan Fast mix (Applied Biosystems, Foster
City, California) in an ABI 2720 real-time thermocycler accord-
ing to the recommended cycling parameters. Quantity means
were normalized to 18S ribosomal RNA, whose expression is
known to be stable during human pregnancy and labor.
14
Western Blotting
For Western blotting, tissue from 9 to 15 patients per group was
extracted in radioimmunoprecipitation assay buffer containing
150 mmol/L sodium chloride, 1.0%NP-40, 1 mmol/L EDTA,
and 50 mmol/L Tris, pH 7.4 with Halt protease inhibitors
(Thermo Scientific, Rockford, Illinois) and quantified by
bicinchionic acid assay (ThermoScientific). Equal amounts of
total protein were separated on 7.5%or 10%sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gels and transferred
to nitrocellulose membranes. Primary antibodies were added to
blots in Odyssey blocking (Licor Biosciences, Lincoln,
Nebraska) buffer and incubated overnight. Primary antibodies
to ITGA1 (MAB1973Z, 1:500), ITGA3 (AB1920, 1:500), and
ITGAV (AB1930, 1:5000) were purchased from Millipore
(Billerica, Massachusetts). Primary antibodies to ITGB3
(PM6/13, 1:200), ITGB1 (1:2000), and ITGB5 (D01P, 1:500)
were purchased from ABD Serotec (Raleigh, North Carolina),
Abnova (Taipei City, Taiwan), and Abcam (Cambridge,
Massachusetts), respectively. Antibody to ITGA7 (1:1000) was
a gift from Stephen Kaufman, University of Illinois, Urbana,
Illinois. Antibody to ITGA5 (1:1000) was a gift from Dr Maria
Valencik, University of Nevada, Reno, Nevada. Primary
antibodies were detected by incubating blots with
Alexafluor680 (Molecular Probes, Eugene, Oregon) or
IRDye800 (Rockland Immunochemicals, Gilbertsville,
Pennsylvania) fluorescently conjugated secondary antibodies.
Bands were detected and band intensities quantified with an
Odyssey Infrared Imaging System (LiCor Biosciences).
Confocal Microscopy
Human uterine myometrial biopsies dissected to reveal smooth
muscle were flash frozen in Tissue-TEK O.C.T. compound in
liquid nitrogen-cooled isopentane and stored at 80C.
Samples were cut into 10 mm sections with a cryostat and placed
on coated slides (Surgipath, Buffalo Grove, Illinois). For ITGA3,
ITGA5, ITGA7, ITGB1, ITGB3, and ITGB5, sections were
fixed in 4%paraformaldehyde and permeabilized with 0.5%Tri-
ton X-100. For ITGAV and ITGA5 sections were fixed in
20C acetone. The sections were blocked with 5%bovine
serum albumin and incubated with polyclonal anti-ITGA3
(Millipore), anti-ITGA5 (M. Valencik, University of Nevada),
anti-ITGA7 (S. Kaufman, University of Illinois), anti-ITGB1
(AbCam, Cambridge, Massachusetts), anti-ITGB3 (ABD Sero-
tec, Raleigh, North Carolina), or anti-ITGB5 (Abnova). A fluor-
escein isothiocyanate-goat anti-rabbit immunoglobulin G (IgG)
secondary antibody (Santa Cruz Biotech Santa Cruz, California)
was used for integrin detection. Sections were counterlabeled
Burkin et al 805

mouse anti-vinculin (Sigma-Aldrich, St. Louis, Missouri)
detected with TRITC-donkey anti-mouse IgG (Santa Cruz Bio-
tech). Sections were mounted in Vectashield plus DAPI (Vector-
Labs, Burlingame, California), and viewed using an Olympus
Fluoview confocal microscope.
Data Analysis
Relative gene expression was determined using SABiosciences
PCR array software and the DDCt was method normalized to a
set of 6 control genes. TaqMan quantity means were
normalized to 18S ribosomal transcript and ratios between the
groups were compared by Welch corrected ttest. Integrin
protein expression was normalized to glyceraldehyde
3-phosphate dehydrogenase (GAPDH) and ratios between the
groups were compared by 1-way analysis of variance followed
by the Tukey-Kramer multiple comparisons test with P<.05
considered significant. Some data sets were log transformed
in order to meet statistical test assumptions. For colocalization
experiments, Pearson correlation coefficients were calculated
with Fluoview software (Olympus America, Center Valley,
Pennsylvania). We defined a colocalization coefficient >0.7
as a highly positive correlation, >0.5 as a positive correlation,
and 0.5 or less as no colocalization.
15
Results
Integrin Transcript Levels in Pregnant Human
Myometrium
It has been proposed that uterine smooth muscle cells undergo a
transitionto a more contractile phenotype at the end of pregnancy
and that this transition is associated with major changes in
the expression of extracellular matrix proteins and their
associated receptors.
1
To test this hypothesis, total RNA
was extracted from nonpregnant, pregnant nonlaboring, and
pregnant laboring human myometrial samples. The RNA
was converted to cDNA and each group of samples was
pooled and subjected to qPCR on an array of primers to
human extracellular matrix and adhesion molecules. Tran-
scripts for ITGA1,ITGA3,ITGA5,ITGA7,ITGAV,ITGB1,
ITGB2,ITGB3, and ITGB5 were significantly higher in term
nonlaboring human myometrial samples than in nonpregnant
control samples (Table 1). The ITGA1,ITGA3, and ITGAV
transcripts were approximately doubled in pregnant, nonla-
boring myometrium compared to nonpregnant controls. Tran-
scripts for ITGA7,ITGB3, and ITGB5 were increased 3- to 4-
fold. We observed a 21-fold increase in ITGA5 in pregnant,
nonlaboring myometrium compared to nonpregnant controls.
In laboring myometrium, ITGA1,ITGA3,ITGAV,ITGA7,
ITGB3, and ITGB5 transcripts decreased from nonlaboring
levels, but were still higher than in the nonpregnant myome-
trial samples. The ITGB1 transcript was slightly increased in
nonlaboring samples and increased to 2-fold in laboring tis-
sue. Transcript levels of ITGA2,ITGA4,ITGAL, and ITGB4
were significantly lower in pregnant myometrium compared
to control samples. Transcript levels for ITGA6,ITGA8, and
ITGAM were not significantly different in the term nonlabor-
ing pregnant uterus compared to the nonpregnant controls.
In order to confirm the accuracy of the array data, TaqMan
qPCR was performed in individual patient samples with
primers and probes to 3 different integrin chains. Quantity
means were normalized to 18S ribosomal transcript and
confirmed a 3.7-fold increase in ITGA7, a 21-fold increase in
ITGA5, and a 2.0-fold increase in ITGAV transcripts in
pregnant human myometrial samples compared to nonpregnant
controls (Figure 1).
Integrin Protein Levels in Pregnant Human Myometrium
In order to determine whether the observed transcript level
changes corresponded to changes in expression at the protein
Table 1. Transcripts for Integrins A1, A3, A5, A7, AV, B1, B2, B3, and B5 Are Significantly Increased in Both Nonlaboring and Laboring Pregnant
Human Uterine Myometrium Compared to Nonpregnant Myometrium.
NL:NP Fold change PValue L:NP Fold Change PValue
ITGA1 1.97 .008 1.33 .036
ITGA2 0.37 .007 0.26 .002
ITGA3 1.8 .019 0.67 .116
ITGA4 0.55 .019 0.61 .011
ITGA5 21.12 .001 10.47 <.001
ITGA6 0.92 .909 0.4 <.001
ITGA7 3.46 .004 2.75 <.001
ITGA8 1.91 .056 1.06 .697
ITGAL 0.26 <.001 0.17 <.001
ITGAM 1.41 .132 0.99 .944
ITGAV 1.99 .020 1.78 .001
ITGB1 1.65 .019 2.1 .002
ITGB2 1.51 .040 1.79 .002
ITGB3 4.49 <.001 2.39 <.001
ITGB4 0.67 .088 0.35 .006
ITGB5 3.66 .004 1.98 .001
Abbreviations: ITG, integrin; L, pregnant laboring; NL, nonlaboring; NP, pregnant nonpregnant.
806 Reproductive Sciences 20(7)

level, we performed semiquantitative Western blots (Figure 2).
After normalization to GAPDH intensity, ITGA5, ITGA7,
ITGB1, and ITGB3 protein levels were significantly higher
in pregnant nonlaboring and laboring myometrial samples
compared to the nonpregnant controls. The ITGAV protein was
higher in pregnant myometrium compared to controls, but this
was only significant in the nonlaboring group. The ITGA1,
ITGA3, and ITGB5 protein levels did not increase during
pregnancy (Figure 3).
Integrin Colocalization With Focal Adhesion Proteins in
Pregnant Human Myometrium
We used confocal microscopy to determine which of the
upregulated integrins localized to focal adhesions in term
myometrium. All of the integrin chains tested could be detected
in myometrium except ITGA1 and ITGB3, which were
predominantly found in cells of the vasculature. The ITGB5
was predominantly expressed in vascular and interstitial cells.
We observed colocalization of ITGA3, ITGA5, ITGA7, and
ITGB1 with the focal adhesion-associated protein vinculin
(Figure 4), with Pearson coefficients averaging R¼.52,
R¼.8, R¼.58, and R¼.66, respectively (Figure 5). The
ITGA5 also showed high levels of colocalization with another
focal adhesion protein (talin R¼.85, Figure 6). We did not
observe colocalization between ITGB3 (R¼.25) or ITGB5
(R¼.45) and vinculin in our pregnant myometrial samples
(Figures 4 and 5). Because a different fixative was required for
ITGAV, this integrin was observed in combination with talin as
another focal adhesion protein and high levels of colocalization
were not observed (R ¼0.45, Figure 6). No significant
differences in localization were observed between nonlaboring
and laboring samples. Finally, although integrin expression
levels were lower in nonpregnant myometrial samples,
colocalization values were not significantly different between
pregnant and nonpregnant myometrial samples (not shown).
The discrete, punctate labeling pattern we observed around the
myometrial cell membranes with anti-ITGA3, -ITGA5,
-ITGA7, and -vinculin at term (Figure 4, arrows) was not
detected in the nonpregnant samples.
Discussion
Integrin-rich membrane domains known as focal adhesions
(or dense plaques) transmit mechanical signals from the
extracellular matrix to activate signaling pathways inside the
cell.
16
Signaling through integrins and focal adhesions has
been implicated in regulating cell morphology and contracti-
lity.
16,17
During pregnancy, myometrial extracellular matrix
and focal adhesions undergo substantial reorganization and
this may contribute to development of the contractile state
of this tissue at term.
18
At term, focal adhesion sites may func-
tion as intercellular connection sites to coordinate contraction
0
1
2
3
4
5
6
Normalized Gene
Expression
ITGA7/18S
NP
NL
0
5
10
15
20
25
30
Normalized Gene
Expression
ITGA5/18S
NP
NL
0
0.5
1
1.5
2
2.5
Normalized Gene
Expression
ITGAV/18S
NP
NL
*"
**"
**"
Figure 1. TaqMan quantitative polymerase chain reaction (qPCR)
performed on individual patient samples confirmed the observed
changes in integrin transcript level in the pregnant uterus. Quantity
means normalized to 18S ribosomal transcript confirmed a 3.7-fold
increase in a
7
-integrin (ITGA7), a 21-fold increase in ITGA5, and a
2.0-fold increase in ITGAV in pregnant human uterine samples. Graphs
represent average values +standard error of the mean ([SEM]
*P< .05, **P< .01). n ¼6 samples per group.
Figure 2. Representative Western blots depicting the expression of
integrin protein subunits in nonpregnant (NP), pregnant laboring (L),
and pregnant nonlaboring (NL) human uterine myometrial samples.
Burkin et al 807

throughout the myometrium.
3
We hypothesized the myome-
trial integrin expression profile would change during preg-
nancy to complement the changes occurring in the
extracellular matrix. We performed a series of experiments
to compare integrin chain expression in nonpregnant and term
human myometrium.
Transcripts for ITGA1,ITGA3,ITGA5,ITGA7,ITGAV,
ITGB1,ITGB2,ITGB3, and ITGB5 were significantly higher
in term myometrial samples than in nonpregnant control
samples. Changes in gene expression were highly reproducible,
as evidenced by highly consistent results obtained between the
SABiosciences array and Invitrogen Taqman qPCR assays. In
0.00
0.50
1.00
1.50
Normalized Protein
Expression
ITGA1/GAPDH
NP
NL
L
0.00
0.50
1.00
1.50
2.00
Normalized Protein
Expression
ITGA3/GAPDH
NP
NL
L
0.00
2.00
4.00
6.00
8.00
Normalized Protein
Expression
ITGA5/GAPDH
NP
NL
L
0.00
0.50
1.00
1.50
Normalized Protein
Expression
ITGB1/GAPDH
NP
NL
L
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Normalized Protein
Expression
ITGB3/GAPDH
NP
NL
L
0.00
0.50
1.00
1.50
2.00
Normalized Protein
Expression
ITGB5/GAPDH
NP
NL
L
0.00
0.50
1.00
1.50
2.00
2.50
Normalized Protein
Expression
ITGAV/GAPDH
NP
NL
L
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Normalized Protein
Expression
ITGA7/GAPDH
NP
NL
L
*** ***
**
*
***
***
**
*
Figure 3. Western blot quantitation indicates some integrin chains are more abundant in pregnant human myometrial samples compared to
nonpregnant myometrium. The a
5
-integrin (ITGA5), ITGA7, ITGAV, and ITGB3 are significantly increased in the pregnant laboring (L) and
nonlaboring (NL) human uterus compared to nonpregnant controls (NP), n ¼9 to 15 per group, P< .05. Graphs represent average values
+standard error of the mean (SEM; *P< .05, **P< .01, and ***P< .001). n ¼9 to 15 samples per group.
808 Reproductive Sciences 20(7)

addition, we observed significant increases in transcript level in
term myometrium of many genes encoding extracellular matrix
proteins (collagens, laminins, and fibronectin; Supplemental
Table 1) that have previously shown to increase during preg-
nancy in other species.
10,12,19,20
We observed downregulation of a large number of cell
adhesion molecule transcripts in the laboring myometrium
compared to nonlaboring myometrium, with only a single
molecule exhibiting increased transcript levels. It is possible
this reflects a general transcriptional shutdown of genes
encoding extracellular matrix proteins that occurs with the
onset of labor in preparation for remodeling associated with
uterine involution.
The ITGA1 pairs with ITGB1 and the heterodimer binds col-
lagens I, IV, and IX and laminin. Williams et al observed large
increases in ITGA1 transcript in the pregnant rat uterus through-
out pregnancy and levels decreased again postpartum. However,
uterine ITGA1 protein levels did not change in the pregnant ver-
sus nonpregnant rat uterus.
8
Consistent with these observations,
we observed a modest increase in ITGA1 at the transcript level,
but no corresponding increases were observed at the protein
level. In our immunofluorescence experiments, ITGA1 localized
predominantly to uterine blood vessels (not shown).
The ITGA3 pairs with ITGB1 to bind collagen and
laminins.
21
The ITGA3 transcript is increased in the rat uterus
during pregnancy and then decreased to nonpregnant levels
after delivery.
8
The ITGA3 protein tended to decrease in
abundance during pregnancy in the rat, although this decrease
was only significant on day 22 at the very end of pregnancy.
8
We observed statistically significant increases in
ITGA3 transcript, but no corresponding increase in ITGA3
protein in our pregnant human myometrial samples. The
ITGA3 was detected at human myometrial focal adhesion
sites, as determined by colocalization with an antibody
against vinculin.
The ITGA5 chain pairs exclusively with the ITGB1 chain to
act as a fibronectin receptor. We observed a significant 14-fold
increase in fibronectin transcript in the pregnant uterus in
qPCR array experiment (Supplemental Table 1), consistent
with the previous reports.
10,19,20
In addition, we detected a
dramatic 21-fold increase in ITGA5 transcript in our term non-
laboring uterine samples compared to nonpregnant controls and
a corresponding 6-fold increase in ITGA5 protein. Both ITGA5
transcript and protein levels were lower in laboring samples
compared to term, nonlaboring myometrium. These results are
consistent with the previous observations that ITGA5 transcript
Figure 4. a
3
-Integrin (ITGA3), ITGA5, ITGA7, and ITGB1 (green)
colocalize with the focal adhesion complex protein vinculin (red) in
term, nonlaboring human myometrium by confocal microscopy.
Regions of overlapping signal appear yellow. Arrows highlight regions
of integrin/vinculin localization to discrete membrane regions of
myometrial cells. The ITGB3 localized to cells surrounding blood
vessels (BV) and not myometrial cells (M).
Figure 5. Average Pearson coefficients were R¼.52 for a
3
-integrin
(ITGA3), R¼.8 for ITGA5, R¼.56 for ITGA7, and R¼.66 for ITGB1
(B). Correlation coefficient bars represent average values +standard
error of the mean (SEM).
Burkin et al 809

and protein levels are lower in laboring human myometrium
compared to nonlaboring myometrium.
13
Regulation of ITGA5
expression has been extensively studied in the rat model and
both ITGA5 transcript and protein are upregulated in rat uterus
during pregnancy.
9
In the rat, ITGA5 expression is regulated at
least in part by mechanical stretch of uterine tissue. Shynlova
et al observed a 4- to 5-fold increase in ITGA5 transcript and
a modest increase in ITGA5 protein in the gravid horn versus
the empty horn. In this animal model, ITGA5 transcript doubled
in response to the RU486, suggesting that mechanical tension
rather than rising progesterone levels of pregnancy are likely
to be responsible for the observed expression changes.
10
We
observed high levels of colocalization between ITGA5 and the
focal adhesion complex proteins vinculin and talin in our term
pregnant samples, in agreement with reports in other
species.
9,11
The ITGA7 pairs with ITGB1 to form a laminin receptor.
22
The ITGA7 expression has been shown to increase in
differentiated skeletal muscle
23
and is required for expression
and development of the contractile phenotype in airway smooth
muscle cells.
24
We observed significant increases in both
ITGA7 transcript and protein in both laboring and nonlaboring
term human myometria compared to nonpregnant controls. We
also observed colocalization between ITGA7 and the focal
Figure 6. a
5
-Integrin (ITGA5; green) colocalizes with the focal adhesion complex protein talin (red) in term human myometrium by confocal
microscopy. In contrast, little colocalization was observed between ITGAV (green) and talin (red) (A). Regions of overlapping signal appear yel-
low. Average Pearson coefficients were R¼.85 for ITGA5 and talin and R¼.45 for ITGV and talin (B). Correlation coefficient bars represent
average values +standard error of the mean (SEM).
810 Reproductive Sciences 20(7)

adhesion protein vinculin in our term human myometrial
samples.
The ITGAV can pair with several bchains to form integrins
avb1, avb3, avb5, avb6, and avb8. These integrin heterodimers
bind to fibronectin, vitronectin, fibrinogen, or tumor necrosis
factor b–latency-associated protein, many at RGD (Arg-Gly-
Asp) sites. We observed a 2-fold increase in ITGAV transcript
and ITGAV protein in the term nonlaboring uterus compared to
nonpregnant uterine controls. Although present in uterine
myometrium, we did not observe high levels of colocalization
between ITGAV and the focal adhesion protein talin.
The ITGB3 pairs with ITGAV and is a marker for uterine
receptivity during the implantation window of early pregnancy.
In uterine endometrium and Ishikawa cells (uterine epithelial),
ITGB3 transcription is increased by epidermal growth factor
and inhibited by estrogen and progesterone.
25,26
We observed
4-fold more ITGB3 protein in the term, nonlaboring pregnant
uterine tissue (when circulating progesterone levels are high
160) compared to the nonpregnant uterus (when circulating
progesterone levels are low) suggesting other factors are
important for the upregulation of ITGB3 in the pregnant
myometrium. However, it is important to note that ITGB3 was
predominantly expressed in the uterine vasculature and not the
myometrial cells.
The ITGB1 transcript is increased (*10) in the pregnant
mouse uterus throughout pregnancy and returns to nonpregnant
levels by 4 days postpartum. Although Williams et al did not
observe changes in ITGB1 protein abundance by Western blot,
they did observe increased membrane localization.
8
We
observed a modest (1.65-fold) increase in ITGB1 transcript in
the pregnant, nonlaboring uterus compared to nonpregnant
controls, but did not detect changes in total ITGB1 protein
during pregnancy. The differences in the magnitude of
transcript change between our observations and those of
Williams et al may represent genuine species differences or
differences in primers used. We observed colocalization
between ITGB1 and the focal adhesion protein vinculin,
consistent with the role of ITGB1 as a binding partner to
ITGA3, ITGA5, and ITGA7.
It is interesting to note that the increased transcript levels of
ITGA5, ITGA7, ITGAV, and ITGB3 corresponded to
increased protein levels, while increases in ITGA1, ITGA3,
ITGB1, and ITGB5 did not correspond to increased protein
levels. Cells can control molecular expression at both the levels
of transcription and translation. Indeed, for many genes tran-
script levels display little correlation with the final protein lev-
els.
27
Possible explanations for these observations might
include variations in posttranscriptional processing and
differences in protein stability.
27
We observed a discrete, punctate integrin (ITGA3, ITGA5,
ITGA7, and ITGB1) and vinculin labeling pattern around
myometrial cells in our term human samples, but not in our
nonpregnant myometrial samples. In the pregnant rat
myometrium, organized focal adhesion complexes form
relatively late in pregnancy in response to both hormonal and
mechanical cues.
9
In contrast, in the sheep model, the ITGA5
integrin chain localizes to myometrial focal adhesion
complexes that begin to form during the first trimester.
11
Since
focal adhesion signaling has been implicated in myometrial
contractility and in coordinating contraction throughout the
tissue,
3,28
it will be interesting to examine preterm human
samples to determine whether myometrial integrins localize
to discrete focal adhesions in early or late pregnancy and
whether their presence at these sites is associated with
preterm labor.
Among the myometrial genes that were downregulated
during pregnancy are E-cadherin (CDH1cadherin) and
catenin/cadherin-associated protein delta 2 (CTNND2;
Supplmental Table 1). The CDH1 is a cell–cell adhesion mole-
cule that regulates uterine formation, cell proliferation, and
apoptosis.
29
Loss of Cadh1 leads to loss of adherens and tight
junctions, abnormal cell proliferation, and apoptosis in murine
uterine epithelia, but does not cause obvious myometrial
defects.
29
When CTNND2 is low, cadherin is targeted for
degradation and junctions are destabilized.
30
Together with our
observation that myometrial focal adhesions form during preg-
nancy, these data suggest the possibility that myometrial cell
adhesion structures may undergo a switch from cadherin-rich
adhesions junctions in the nonpregnant state to integrin-rich
focal adhesions at term.
In summary, we observed increases in several integrin
chains at both the messenger RNA and protein levels in
human myometrium during pregnancy. Colocalization of the
ITGA3, ITGA5, ITGA7, and ITGB1 chains with focal
adhesion proteins at term suggests a potential role for a5b1,
a3b1, and a7b1 in regulating myometrial contractility.
Crosstalk between these integrins may also play a role in
mechanotransduction and coordination of myometrial
contraction during pregnancy.
Acknowledgments
We would like to thank the Renown Medical Center Labor and
Delivery staff and the following obstetricians for their help in
collecting the human myometrial samples which made this study
possible: Dr Martin E. Dennis, Dr Bruce Farringer, Dr Staci Paul,
Dr Ricardo A. Garcia, Dr Myron W. Bethel, Dr Sheila Sta. Maria,
Dr Alison Westfall, Dr Leah Najima, Dr Karen E. Dearmont, Dr
Randall Jack, Dr Stacey Mellum, Dr Peter Dekay, Dr Susan Perry,
Dr Scott Jacobs, Dr Susan Hsu, Dr Holly Ashley, Dr Vickie Tippett,
Dr Ralph Narinedat, Dr Rafaela Hernandez, Dr Corine Capurro, Dr
Nathan Slotnick, Dr Earl Oki, Dr Larry Klaich, Dr Mark Schumacher,
Dr Harold Chotiner, Dr Laura Thompson, Dr Amoli-Neda Etezadi, Dr
Kathy Jo Cantrell, Dr Craig Klose, Dr John Paas, Dr Stanton Allen,
and Dr Charles Johnson.
Authors’ Note
Supplemental Table 1 is available online at http://rsx.sagepub.com/
supplmental.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Burkin et al 811

Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: the National
Institutes of Health grants R01-HD053028 to ILOB and K99067342 to
HRB, the March of Dimes Foundation and a Gates Grand Challenges
grant to ILOB.
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