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Tumor Necrosis Factor-

(TNF-) Inhibits Insulin-like


Growth Factor-I (IGF-I)
Activities in Human
Trophoblast Cell
Cultures Through IGFI/Insulin Hybrid Receptors
By : Herman Budi
S

Introduction
Trophoblast cells invade to the
decidual tissue,and remodel uterine
spiral arteries in human early placenta
Many endocrine factors and cytokines
have been involved in placental
development, including tumor
necrosis factor- (TNF-) and insulinlike growth factors (IGFs).

Introduction
Tumor necrosis factor- (TNF-) is a cytotoxic
molecule produced predominantly by
macrophages
which has been reported to cause insulin
resistance invivo and in vitro.
TNF- is expressed in normal placenta and
increased TNF- levels in mid-trimester
amniotic fluid are associated with impaired
fetal growth

Introduction
Overproduction of TNF- and other
inflammatory cytokines in the
placenta might mediate in part the
response to hypoxia
and therefore plays a role in
pathogenesis of hypoxia induced
intrauterine growth restriction

Introduction
Insulin-like growth factors (IGF-I and IGFII) and their binding proteins (IGFBPs) are
crucial for fetal and placental growth
The biological actions of IGF-I are
mediated through its cell surface receptors:
IGF-I receptor (IGF-IR), insulin receptor
(IR) and IGF-I/insulin hybrid receptor (HR)

Introduction
It is known that an -subunit of the
insulin receptor can form a tetramer
with an -subunit of the IGF-I
receptor and this is termed the IGFI/insulin hybrid receptor (HR)
HR is widely expressed in
manymammalian tissues and in
several cell lines including the
placenta

Introduction
The current studies were undertaken
to analyze whether TNF- inhibits
IGF-I action on the placenta and
whether the inhibitory effect of TNF-
on IGF-I action is mediated by IGF-R
or HR

Materials and Methods


Materials
Tissue culture media, penicillin, and streptomycin
Polyvinylidene difluoride (PVDF) membranes
(Immobilon-P).
Anti-phosphotyrosine antibody (PY99),
Antibody for the subunit of IGF-I receptor
(IGF-IR)
Anti-insulin receptor substrate-1 (IRS-1)
Antibody for the subunit of the insulin receptor
(-IR)
Antibody for the subunit of the insulin receptor
(-IR)

Materials and Methods


Primary Culture of Villous
Trophoblast Cells
Placental tissue between 6 and 10
weeks of gestation was obtained
during legal, elective termination of
pregnancy.

Materials and Methods


Cell Proliferation Assay
Cell proliferation was measured using the Cell
Proliferation ELISA System
Cell Migration Assay
Cells were plated at 1.5 x 104/cm2 in medium
199 containing 10% FBS and allowed to grow
to confluence over 3 days. Wounding was
performed using a single razor blade to scrape
cells off the culture plate, leaving a denuded
area and a sharp visible demarcation line at
the wound edge, as described in previous
reports

Materials and Methods


Immunoprecipitation,
Immunodepletion, and
Immunoblotting
To separate HR and IGF-IR
homodimers, the methods of
immunodepletion and
immunoprecipitation were utilized
previously described Trophoblast
cells were grown to 80% confluency
on10-cm tissue culture dishes.

Results
The Effects of TNF- on IGF-I and
Insulinstimulated Trophoblast Cell
Proliferation
TNF- by itself showed no significant
effect on the cell proliferation.

Results
The Effects of TNF- on IGF-Istimulated Trophoblast Cell
Migration
TNF- suppressed IGF-Istimulated
migration of trophoblast cells in a
dose-dependent

Results
The Effects of TNF- on IGF-Istimulated IGF-IR Phosphorylation
There was marked inhibition of IGF-IR
phosphorylation when concentrations of
either
10 or 50 ng/mL TNF- were added
There was less change in the
abundance of the subunit of IGF-IR
with any concentration of TNF- that
was tested

Results
The Effects of TNF- on IGFI/insulin HR Phosphorylation
Stimulated by IGF-I
TNF- had less effect on
phosphorylation and abundance of
IGF-IR homodimer in trophoblasts.

Results
The Effects of TNF- on IRS-1
Phosphorylation and Its
Association with PI3-kinase
Stimulation by IGF-I
TNF- had no effect on IRS-1
abundance.

Discussion

The aim of the current study was to


assess the effects of TNF-, which
causes insulin resistance, on IGF-I
activity in human trophoblast cells in vitro.
Our results showed that TNF- induced a
loss of sensitivity to stimulation of IGF-I,
or stimulation of IGF-I/insulin HR tyrosine
kinase activity by IGF-I.

Discussion
Multiple studies have demonstrated that
exposure of cells to TNF- results in
impairment of insulin sensitivity
IGF-I and insulin are considered to play
important roles in fetal growth and
placental development.
Human placenta expresses high
concentrations of IGFIR and IR that are
located on microvillous membranes of
syncytiotrophoblasts .

Discussion
Elevated TNF- levels in maternal serum
in the first-trimester in threatened abortion
patients correlates with a poor outcome.
In contrast, TNF- levels are lower in
uncomplicated pregnancy and threatened
abortion patients with a good outcome,
indicating that elevated TNF- levels may
be related to an adverse obstetric
outcome

Discussion
The present study demonstrated that
a high concentration of TNF-
reduced IGFI-stimulated trophoblast
cell proliferation and migration
mediated through IGF-I/insulin HR

Conclusion
TNF-, an important inflammatory
mediator, induced loss of sensitivity to
insulin, and also induced resistance
to IGF-I by attenuation of IGF-I/insulin
HR tyrosine kinase activity.