First published online 11 January 2006
doi: 10.1242/dev.02237
Development 133, 751-759 (2006)
Published by The Company of Biologists 2006
Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor
D. Randall Armant1,2,*,
Brian A. Kilburn2,
Anelia Petkova2,
Samuel S. Edwin3,
Zophia M. Duniec-Dmuchowski2,
Holly J. Edwards2,
Roberto Romero3 and
Richard E. Leach4
1 Department of Anatomy and Cell Biology, C. S. Mott Center for Human Growth and
Development, Wayne State University School of Medicine, Detroit, MI,
USA.
2 Department of Obstetrics and Gynecology, C. S. Mott Center for Human Growth
and Development, Wayne State University School of Medicine, Detroit, MI,
USA.
3 Perinatology Research Branch, National Institute of Child Health and Human
Development, NIH, DHHS, Bethesda, MD, USA.
4 Departments of Obstetrics and Gynecology, and Physiology and Biophysics,
University of Illinois School of Medicine, Chicago, IL, USA.
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Fig. 1. Regulation of EGF family expression by O2. (A)
Immunohistochemical labeling of EGF family members in cytotrophoblast cells.
Antibodies against EGF, TGF (TGF-a), HBEGF, amphiregulin (AR),
betacellulin (BC) and epiregulin (ER) were used, as well as a non-immune goat
IgG control. (B) Antibody labeling was quantified by image analysis in
cells cultured for 24 hours at either ambient O2 levels (black
bars) or 2% O2 (gray bars). *P<0.05;
**P<0.001, compared with ambient culture.
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Fig. 2. Regulation of HBEGF accumulation by O2. (A)
Western blot of HBEGF. Recombinant human HBEGF (20 ng in lane 1) contains the
same sequence as sHBEGF. HBEGF was undetectable in conditioned medium (lane 2)
or in cytotrophoblast cell lysate (lane 3) during culture at ambient
O2 levels. After 24 hours at 2% O2, a 9.7 kDa sHBEGF
band was detected in the medium (lane 4), and an 18.7 kDa pro-HBEGF band
appeared in cell lysates in addition to the sHBEGF band (lane 5). (B)
Cellular and secreted pools of HBEGF were quantified by ELISA in cell lysates
and conditioned medium after shifting cells to 2% O2.
*P<0.05, compared with control (0 hours). (C)
Northern blot of the 2.5 kb HBEGF transcript in cytotrophoblast RNA
0-24 hours after shifting cells to 2% O2. (D) Real time
RT-PCR was used to quantify HBEGF mRNA 0-24 hours after shifting
cells to 2% O2. ANOVA indicated no significant differences.
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Fig. 3. Contribution of the apoptotic pathway to cytotrophoblast cell death.
All cells were cultured for 8 hours at 2% O2 in medium supplemented
as described below and in the Materials and methods. (A)
Internucleosomal cleavage of DNA was assessed by agarose gel electrophoresis
and ethidium bromide staining of genomic DNA. Cells were cultured in medium
containing vehicle (lane 1), CRM197 (lane 2), anti-HBEGF (lane 3), or
antibodies against both HER1 and HER4 (lane 4). (B) Cells exposed to
CRM197 were dually stained using DAPI and the TUNEL method. Pyknotic nuclei
visualized by fluorescence microscopy in the upper panel were also positive
for TUNEL (arrows). (C) Annexin V binding to phosphatidylserine exposed
on the surface of apoptotic cells is nearly absent in control cells (lower
left panel) compared with cells treated with CRM197 (lower right panel). The
upper panels show the same fields labeled with DAPI to indicate the relative
cell densities. (D) Cell death was quantified by TUNEL assay after
culture in medium containing CRM197. The indicated caspase inhibitors (2
µg/ml) or an inactive analog (200 µg/ml) were also added to the medium.
Specificity of each compound is shown in parentheses. The dashed line
indicates the level of TUNEL in cells cultured without CRM197.
*P<0.05, compared with vehicle control.
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Fig. 4. Role of HBEGF shedding at 2% O2. (A) Cellular and
secreted HBEGF concentrations were measured by ELISA after culture for 8 hours
at 2% O2 in medium containing the indicated concentrations of
GM6001. HBEGF values were normalized to vehicle, as in
Table 1.
*P<0.05 compared with vehicle. (B)
Cytotrophoblast cells were cultured for 8 hours at ambient O2
levels (black bars) or 2% O2 (gray bars) in medium supplemented
with vehicle, 10 µg/ml GM6001 or 10 µg/ml of an inactive structural
analog (Control) and TUNEL was determined. *P<0.05
compared with vehicle treatment. (C) Cells were cultured for 8 hours at
2% O2 with 0 µg/ml (x) or 10 µg/ml (all other symbols)
GM6001 and apoptosis was determined by TUNEL. The indicated concentrations of
recombinant human HBEGF (black squares) were added to GM6001-treated cells.
Co-treatment was also conducted with 10 nM of other recombinant EGF family
growth factors, including epiregulin (open circle), betacellulin (open
diamond), amphiregulin (open triangle), EGF (open square) or TGF (solid
triangle). The 0 µg/ml GM6001 control was different (P<0.05)
from all other treatments except those containing 10 µg/ml GM6001 plus 1-10
nM HBEGF.
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Fig. 5. Proposed mechanism for cytotrophoblast survival at 2% O2.
Activated metalloproteinases (MP) at the cell surface cleave the extracellular
domain of proHBEGF (1). The released sHBEGF binds to HER1 or HER4 through its
EGF-like domain and to heparan sulfate proteoglycans (HSPG) through its
heparin-binding domain (2), and this is followed by receptor homo- or
heterodimerization with other members of the HER family. Subsequent
transphosphorylation of HER cytoplasmic domains at key tyrosine residues (Y-P)
initiates downstream signaling that increases proHBEGF accumulation (3) and
inhibits apoptosis (4). This positive feedback loop upregulates HBEGF
secretion to achieve extracellular HBEGF levels sufficient to maintain cell
survival at 2% O2.
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© The Company of Biologists Ltd 2006
