EXPRESSION OF sFLT-1 AND JMJD6 IN CULTURED HUMAN TROPHOBLASTS
Joe Hensing , Ronald Torry (Mentor).
College of Pharmacy and Health Sciences, Drake University, Des Moines, Iowa.
METHODS
ABSTRACT
Placenta Growth Factor (PlGF) plays an important role in the formation
and growth of the placenta. This protein binds two receptors, a membrane bound
Flt-1 (mFlt-1) and a soluble Flt-1 (sFlt-1), the latter of which lacks intracellular
signaling. These receptors are splice variants of each other and differ in their
intracellular region. Although we have shown that hypoxia increases the expression
of sFlt-1 in trophoblast, the mechanism for this is not known. Silencing Jumoji
domain-containing protein 6 (Jmjd6) expression in endothelial cells increases
soluble Flt-1 expression and hypoxia lowers the expression of Jmjd6 in endothelial
cells. However, it is not known if Jmjd6 is found in trophoblasts or if it functions in
determining the splicing of Flt-1 in these cells. This study sought to optimize an
assay to assess Jmjd6 expression in trophoblast, how its expression is affected by
hypoxia, and how this correlates to sFlt-1 expression. Trophoblast were isolated
from three different normal placentae and cultured for 24 hours under two
conditions: normoxia (21% oxygen) and hypoxia (1-2% oxygen). The RNA was
harvested, reversed transcribed into cDNA and sFlt-1/Jmjd6 expression was
analyzed in duplicate using real-time or end-point PCR. The fold changes in sFlt-1
between the normoxic and hypoxic groups were 1.98, 0.75, and 0.88, for the
samples. We optimized the PCR of Jmjd6 by changing annealing temperature and
concentrations of primers and cDNA. The fold changes in Jmjd6 are still being
determined. In conclusion we determined Jmjd6 is expressed in trophoblast but its
influence on sFlt-1 expression is unknown.
RESULTS
Cell Culture:
Trophoblast were isolated from normal placenta at Southern Illinois University
School of Medicine. The cells were cultured under two conditions: Normoxia
(21% oxygen) and Hypoxia (1-2% oxygen) for 24 hours. Total RNA was then
harvested and reverse transcribed to produce 150ng of complimentary DNA
(cDNA).
sFlt-1 Expression:
Soluble Flt-1 expression was analyzed using real-time polymerase chain reaction
(q-PCR). The forward and reverse primers used to amplify the 314 bp product
were: F: 5’-TGG CCA TCA CTA AGG AGC ACT-3’ R: 5’-TCC GAG CCT
GAA AGT TAG CAA-3’. The annealing temperature of these primers was 62o C.
Samples were run in duplicate and a negative control (primers with no cDNA)
was used.
Jmjd6 Expression:
Jmjd6 expression was analyzed using polymerase chain reaction (PCR) and gel
electrophoresis. The forward and reverse primers used to amplify the 387 bp
product were: F: 5’–GGC ACA ACT ACT ACG AGA GC-3’ R: 5’–TTT GGC
ACC TTG TAG TCT TCC–3’. An annealing temperature for the primers was
determined to be 53oC using gradient PCR. Samples were run in duplicate and a
negative control (primers with no cDNA) was used.
RPL32:
RPL32 was used as a housekeeping gene to normalize the PCR reactions. The
quantity of this gene remains constant between normoxic and hypoxic treatment
groups and was used to compare fold change in sFlt-1 and Jmjd6 between the
two treatment groups.
Gel Electrophoresis:
Amplicons were separated on a 2% agarose gel and stained with ethidium
bromide to visualize the PCR products.
sFlt-1 qPCR Data
Placenta #
P240
P241
P243
sFlt-1 expression in trophoblast
P240
sFlt-1 (H/N)
1.98
0.75
0.88
P241
P243
100bp
sFlt-1
(314bp)
Jmjd6 Primer Optimization
58.0 55.7 53.4 51.1 48.8 46.5 44.2 42.0 (oC)
Jmjd6
(387bp)
Jmjd6 in human heart, kidney, trophoblast
100bp Kidney Heart P241
Jmjd6
(387bp)
RPL32
(98bp)
Jmjd6 expression in trophoblast
P240
P241
P243
100bp JN RN JH RH JN RN JH RH JN JH
Kidney Heart P241
SUMMARY AND FUTURE STUDIES
PlGF, mFlt-1, and sFlt-1 in Preeclampsia
PlGF
PlGF
PlGF
Jmjd6
(387bp)
RPL32
(98bp)
BACKGROUND
Summary:
•
Jmjd6 mRNA is variably expressed in human trophoblast.
•
Jmjd6 mRNA is also expressed in human kidney and heart tissue.
•
sFlt-1 mRNA expression is affected by hypoxia. However, results among the trophoblast samples were not
consistent, so a statistically significant change could not be determined.
•
Jmjd6 mRNA expression in trophoblast could be affected by hypoxia but this could not be determined with
certainty because only one sample appeared to express Jmjd6.
Future Studies:
•
Improve consistent upregulation of sFlt-1 in trophoblast with dbcAMP
•
Develop new Jmjd6 primers
•
Determine new techniques to quantify Jmjd6 expression
•
Silence Jmjd6 expression with siRNA and determine the effects on sFlt-1 expression
sFlt-1 (ng/ml/mg protein)
Preeclampsia is a condition that affects 7-10% of pregnancies. It is characterized
by high blood pressure (greater than 160/90 mmHg) and high levels of protein in
the urine ( >200mg/24 hours) (1). It generally occurs in the third trimester and
can lead to intravascular coagulation or bleeding, organ failure due to poor
perfusion, and fetus under nutrition. The exact cause is not known, but placenta
growth factor (PlGF) and soluble Flt-1 (sFlt-1) are thought to be involved (2).
PlGF is a key protein in angiogenesis and binds the membrane bound Flt-1
(mFlt-1) and sFlt-1 receptors. When PlGF binds mFlt-1, it produces biological
effects including blood vessel formation and increases perfusion which leads to
increased survival of trophoblast (3). When PlGF binds to sFlt-1, no biological
effect is produced, which inhibits the function of PlGF. During preeclampsia,
there is an increase in plasma levels of sFlt-1 and a decrease in plasma levels of
PlGF(4). This produces an environment in which there is a decrease of proangiogenic PlGF and an increase in anti-angiogenic sFlt-1, taken together, these
two cause a greater reduction in blood vessel formation. The mFlt-1 receptor
structure contains 30 exons. Exons 1 through 15 make up the extracellular
region, exon 16 is the trans-membrane exon, and exons 17 through 30 make up
the intracellular region. sFlt-1 and mFlt-1 differ by their intracellular region.
During RNA splicing, there is a premature termination of the process at exon 13,
which causes the protein transcription to stop and results in the production of
sFlt-1(5). Jumonji domain-containing protein 6 (Jmjd6) has been shown to alter
RNA splicing for some genes (6). Previous studies have shown that inhibition of
Jmjd6 decreases angiogenic sprouting and significantly reduces angiogenesis in
endothelial cells by interfering in the splicing of mFlt-1 causing the production
of sFlt-1(7). It is not known if trophoblast express Jmjd6. The goals for this
project were to determine if trophoblast express Jmjd6 and to determine if sFlt1 expression increases and expression of Jmjd6 decreases in trophoblast under
hypoxia.
100bp
REFERENCES
1.
2.
3.
4.
5.
6.
7.
Sattar N, Greer I. BMJ 2002;325: 157-160.
Ahmad and Ahmed. Circ Res 95: 884-891, 2004.
Ahmed and Cudmore. Biochemical Society Transactions (2009) 37, 1237-1242.
Tidwell, et al., Am J Obstet Gynecol. 184(6):1267-72, 2001.
Sela, S. et al. Circ Res 2008;102:1566-1574.
Webby et.al. Science 2009;325: 90-93.
Boeckel et. al. PNAS Early Ed. 2010;1: 1-6.
Acknowledgements:
Dr. Donald Torry and Kathy Groesch; Southern Illinois University, School of Medicine, Springfield, IL