Department of Obstetrics and Gynecology, New York University School of Medicine, New York, New York 10016, USA
Address for correspondence: Dr. Seth Guller, Department of OB/GYN, NYU School of Medicine, 550 First Ave., New York, NY 10016. Voice: 212-263-8594; fax: 212-263-5742.
gulles01{at}med.nyu.edu
The goal of the current study was to examine the role of the
ubiquitin-proteasome system (UPS), a pathway of intracellular
degradation, in the regulation of fetal fibronectin (FFN) expression
in human placenta. Primary cultures of cytotrophoblasts (CTs)
and placental mesenchymal cells (PMCs) were isolated from human
term placentas and were maintained in serum-free medium (SFM)
in the presence of inhibitors of proteasome-mediated degradation
(e.g., MG132) as well as inhibitors of other proteases. Levels
of secreted FFN and interleukin (IL)-8 in culture media were
quantitated by enzyme-linked immunosorbent assay (ELISA), and
cell viability was assessed by trypan blue exclusion. Intracellular
levels of FFN and ubiquinated proteins were measured by Western
blotting, and levels of fibronectin mRNA were determined following
Northern blotting. We found that proteasome inhibitors (MG132,
MG262, and PSI) potently suppressed levels of secreted FFN in
cultures of CTs, but they not did affect levels of IL-8. Lysosomal,
calpain, and serine protease inhibitors as well as the anti-inflammatory
compound sulfasalazine did not markedly affect levels of secreted
FFN in CT cultures. Proteasome inhibitors did not compromise
cell viability during the initial 16-18 hours of treatment and
did not affect intracellular levels of FFN protein or fibronectin
mRNA. The efficacy of suppression of FFN in CT culture media
by proteasome inhibitors reflected their effects on intracellular
accumulation of ubiquinated proteins. By contrast, the presence
of proteasome inhibitors did not alter levels of secreted FFN
in cultures of PMCs. We conclude that inhibitors of proteasome-mediated
degradation potently and specifically suppressed extracellular
expression of FFN in CTs through a cell type-specific pathway
that did not involve alterations in FFN synthesis. This suggests
that accumulation of ubiquinated proteins in the presence of
proteasome inhibitors blocks FFN secretion or promotes the extracellular
degradation of FFN. This experimental paradigm will be useful
for dissecting the role of the UPS in regulating CT function.
