Involvement of SOCS-1, the Suppressor of Cytokine Signaling, in the Prevention of Prolactin-Responsive Gene Expression in Decidual Cells
Uriel Barkai,
Anne Prigent-Tessier,
Christian Tessier,
Gil B. Gibori and
Geula Gibori
Department of Physiology and Biophysics College of Medicine
University of Illinois Chicago, Illinois 60612
 |
ABSTRACT
|
---|
The cells forming the rat decidua produce PRL and
PRL-related proteins and express both the long and short forms of the
PRL receptor. Yet, only a defined subpopulation, the mesometrial cells,
express the PRL-dependent
2-macroglobulin
gene. This gene is silenced in vivo in the antimesometrial
cells and in the GG-AD cell line, derived from antimesometrial cells.
To examine whether the lack of
2-macroglobulin expression is due to
defective components in the PRL signaling pathway, we compared the
relative expression of Janus kinase 2 (Jak2), signal transducer and
activator of transcription 5 a and b (Stat5 a and b), suppressor
of cytokine signaling-1 (SOCS-1), and the tyrosine phosphatase SHP-2
mRNA in mesometrial and antimesometrial decidua on days 12 and 13 of
pseudopregnancy, the time of maximal
2-macroglobulin expression. We found no
significant differences in the relative expression of either Jak2,
Stat5 (a and b), or SHP-2 in the two cell populations. However, we
discovered a profound difference in the expression of SOCS-1, an
inhibitor of the Jak/Stat pathway. This gene was highly expressed in
the antimesometrial cells and in the GG-AD cells, which do not produce
2-macroglobulin. Immunoprecipitation
experiments with GG-AD cells revealed that although Jak2 and Stat5
coprecipitate in response to PRL stimulation, no phosphorylation of
Jak2 and Stat5 could be observed. To examine whether SOCS-1 plays a
role in silencing the
2-macroglobulin gene,
we cultured GG-AD cells in the presence of either a SOCS-1 antisense
oligonucleotide or an irrelevant oligonucleotide for 4, 12, and 28
h. Cells were also treated with PRL. Within 4 h of SOCS-1
antisense treatment,
2-macroglobulin mRNA
expression was initiated. After 28 h, only cells treated with PRL
and SOCS-1 antisense oligonucleotide retained the ability to express
the
2-macroglobulin gene. In summary,
results of this study reveal that constitutive expression of SOCS-1 can
prevent PRL signaling and that the lack of PRL-induced expression of
2-macroglobulin in a defined decidual cell
population is largely due to SOCS-1 expression in these cells.
 |
INTRODUCTION
|
---|
In the rat, decidualization of uterine stromal cells, induced by
either implantation or artificial stimuli, produces two regions of
transformed cells, the mesometrial and antimesometrial decidua, which
differ in cellular morphology, physiological characterization, and
protein production (1, 2, 3, 4). The major protein produced by the
mesometrial cells is
2-macroglobulin
(
2-MG) (5, 6), a protease inhibitor, which
appears to play an important role in limiting trophoblast invasion
(7, 8, 9, 10, 11) and whose expression is induced in several target tissues,
including the decidua, by PRL and PRL-related hormones (6, 12, 13, 14). Yet
despite the fact that the antimesometrial cells produce PRL and
PRL-related hormones and that the PRL-receptor (PRL-R) is present
in both cell types (15), only the mesometrial cells express the
2-MG gene. The reason why
2-MG expression is silenced in the
antimesometrial cells is not clear. One key event governing the
transduction of the PRL signaling is well characterized: the presence
of the effector induces membrane receptor dimerization which leads to
transphosphorylation of the associated tyrosine kinase, Janus kinase 2
(Jak2), followed by activation of the signal transducer and activator
of transcription 5 (Stat5) pathway (16, 17, 18), leading to Stat5
translocation to the nucleus and its binding to specific promoter
sequences (19, 20). PRL was shown to up-regulate
2-MG expression via activation of Stat5 in
ovarian primary granulosa cells (13, 21) and whole tissue (14, 22).
Although the paradigm of PRL signaling through the Stat pathway is well
established, very little is known as to how the signal is switched off.
Some evidence suggests the involvement of the protein tyrosine
phosphatase SHP-2 (SH2-containing protein tyrosine phosphatase-2) in
PRL signaling, although this phosphatase acts as a positive rather than
negative regulator (23).
Recently a new family of SH2-containing proteins, named SOCS
(suppressor of cytokine signaling) was discovered and shown to block
cytokine signaling (24, 25, 26, 27). Structurally, this family is linked by the
presence of a central SH2 domain and a conserved carboxy-terminal
domain termed the SOCS box. SOCS genes are differentially induced by
different cytokines (28, 29, 30). At least eight members are presently
grouped in this category (SOCS-17 and CIS) (31). SOCS-1 and 3 have
been shown to block the activation of gene transcription by PRL and GH,
SOCS-1 being a more potent inhibitor (29, 32, 33, 34). SOCS-1 has been
independently discovered by three groups and named either SOCS-1 (24),
JAB (JAK binding protein) (25), or SSI-1 (Stat-inducible Stat
inhibitor) (26). The mouse and rat SOCS-1 genes encode proteins of 212
amino acids, whereas the human gene encodes a protein of 211 amino
acids. Mouse, rat, and human SOCS-1 proteins share 9599% amino acid
homology (24). SOCS-1 interacts with the catalytic region of Jak
kinase, suppresses its tyrosine kinase activity, and thus prevents the
phosphorylation of Stat5 (32). It was shown recently to suppress PRL
signaling at low levels of expression (32, 33). We have recently found
that the rat decidua express SOCS-1. This prompted us to examine the
level of SOCS-1 expression in the different decidual cell populations
and in PRL-producing and uterine-derived cell lines (35, 36) that
either express or do not express the
2-MG gene
and to examine whether inhibition of SOCS-1 expression with an
antisense oligonucleotide can lead to
2-MG
expression in cells where this gene is usually silenced.
 |
RESULTS
|
---|
2-Macroglobulin mRNA Expression in the
Rat Mesometrial and Antimesometrial Decidua and in Two Uterine-Derived
Cell Lines
The results shown in Fig. 1
confirm
our previous results indicating that
2-MG mRNA
is expressed principally in the mesometrial decidual cells and very
little, if any, in the antimesometrial decidual cells. The results also
revealed that the
2-MG gene is not expressed
in the GG-AD cells that were derived from antimesometrial cells (36)
but is expressed in UIII cell line. These cells,
which originate from endometrial stroma, behave similarly to decidual
cells in culture (35). Both cell lines were shown to produce PRL by
immunocytochemistry, Western blotting, and RT-PCR (37, 38).

View larger version (68K):
[in this window]
[in a new window]
|
Figure 1. Expression of 2-MG in
Mesometrial and Antimesometrial Decidual Tissue
Total RNA was isolated from day 9 mesometrial (M) and antimesometrial
(A) decidual tissue after separation by dissection or from
UIII and GG-AD cells cultured as described in
Materials and Methods. Touch-down PCR was performed
using specific primer sets for 2-MG and L-19 as an
internal control.
|
|
Expression of PRL Signaling Components in the Antimesometrial and
Mesometrial Decidua and in the Two Uterine-Derived Cell Lines
If PRL is responsible for
2-MG
regulation, any impediment with the normal signaling pathway, such as a
deficiency in the expression of an essential transducing component, may
prevent the expression of target genes. To test this possibility, we
examined the expression of several components known to participate in
PRL signaling to the
2-MG gene in the two
different tissues forming the decidua and in the two uterine-derived
cell lines. Since maximal expression of
2-MG
in the mesometrial decidua occurs on days 1213 of pseudopregnancy,
decidual tissue was collected at these days of pseudopregnancy and
separated into mesometrial and antimesometrial decidua. Total RNA was
subjected to RT-PCR analysis with L19 as an internal control, and we
looked at the expression of the positive regulators Jak2, Stat5 (a and
b variants), the putative modulator SHP-2, and the negative controller
SOCS-1 with that of the target gene. No significant differences in the
expression of Jak2 (Fig. 2A
) and Stat5
(Fig. 2B
) or that of SHP-2 (Fig. 2C
) were observed between mesometrial
and antimesometrial tissue. In sharp contrast, levels of SOCS-1
expression were vastly different in mesometrial and antimesometrial
decidual tissue (Fig. 2D
). SOCS-1 mRNA was highly expressed only in the
antimesometrial decidua, which does not produce
2-MG.

View larger version (55K):
[in this window]
[in a new window]
|
Figure 2. PRL Signaling Components in Pseudopregnant
Mesometrial (M) and Antimesometrial (A) Decidua
Total RNA (1 µg) was reverse transcribed and 40 ng of the cDNA were
amplified in a touch-down PCR protocol using specific primers for Jak2
and Stat5 (a and b variants), SHP-2, SOCS-1, and L19, which served as
an internal control. Lower panels show the densitometric
analysis and are mean ± SEM from three independent
experiments.
|
|
To further examine whether the same differential expression of SOCS-1
exists in the two uterine cell lines, we performed similar experiments
in the GG-AD and the UIII cell lines. As shown in
Fig. 3
, both cell lines express the PRL-R
long form (PRL-RL) mRNA. They also express Jak2 and Stat5 (a and b
variants) and SHP-2. SOCS-1 was found to be expressed at much lower
levels in the
2-MG expressing
UIII cells than the
2-MG
silent GG-AD cell line.

View larger version (63K):
[in this window]
[in a new window]
|
Figure 3. PRL Signaling Components in UIII and
GG-AD Cells
Touch-down PCR analysis was performed using specific primer sets for
PRL-RL, Jaks (1 2 ), Stat5 (a and b), SHP-2, SOCS-1, and L-19 as an
internal control. Total RNA was isolated from UIII and
GG-AD cells as described in Materials and Methods.
|
|
To examine whether PRL is able to transduce its signal in GG-AD cells
that express high levels of SOCS-1, these cells were cultured in the
presence of PRL between 045 min. Cell extracts were then subjected to
immunoprecipitation and Western analysis. Results shown in Figs. 4
, 5
, and 6
indicate that Jak2 and Stat5 exist as translated products in
GG-AD cells (Figs. 4A
and 6A
). Moreover,
PRL is not able to induce Jak2 phosphorylation (Fig. 4B
). However, a
short 2-min exposure to PRL is sufficient to induce the association of
Jak2 with Stat5 (Fig. 5
), indicating that
Jak/Stat association can occur even between nonphosphorylated Jak2 and
Stat5. This association did not lead to Stat5 phosphorylation (Fig. 6B
), confirming that Jak2 is inactive, in spite of its transient
association with Stat5.

View larger version (84K):
[in this window]
[in a new window]
|
Figure 4. Lack of Jak2 Phosphorylation upon PRL Treatment in
GG-AD Cells
GG-AD cells were treated with 1 µg/ml PRL for the indicated periods
of time. Cellular extracts were precipitated with the monoclonal
anti-Jak2 antibody and processed for Western blotting analysis as
described in Materials and Methods. The blot was first
incubated with a monoclonal antibody specific to phosphotyrosine
( Py) to examine for phosphorylated Jak2 (panel B). The same blot was
reprobed, after stripping, with the monoclonal anti-Jak2 antibody
(panel A).
|
|

View larger version (70K):
[in this window]
[in a new window]
|
Figure 5. Coprecipitation of Jak2 and Stat5 after PRL
Treatment
GG-AD cells were treated with 1 µg/ml PRL for the indicated periods
of time. Cellular extracts were precipitated with the monoclonal
anti-Stat5 antibody separated on 10% denaturing polyacrylamide gel and
transferred to nitrocellulose membrane. Blot was incubated
simultaneously with the anti-Stat5 and anti-Jak2 antibodies.
|
|

View larger version (68K):
[in this window]
[in a new window]
|
Figure 6. Effect of PRL Treatment on Stat5 Phosphorylation in
GG-AD Cells
Cells were treated with 1 µg/ml PRL for the indicated periods of
time. Cellular extracts were precipitated with the monoclonal
anti-Stat5 antibody, separated on 10% denaturing polyacrylamide gel,
and transferred to nitrocellulose membrane. Blot was incubated first
with the monoclonal antibody specific to phosphotyrosine ( Py) to
examine for phosphorylated Stat (panel B). After stripping, the same
blot was reprobed with the monoclonal anti-Stat5 antibody (panel A).
ECL reaction was carried out for 30 sec (A) and for 3 min (B).
|
|
Effect of SOCS-1 Antisense on
2-MG mRNA
Levels in GG-AD Cells
To examine whether SOCS-1 is involved in preventing PRL signaling
to the
2-MG gene, we challenged the cells with
a chimeric phosphorothioate antisense, directed against the 5'-terminus
of SOCS-1. An irrelevant chimeric phosphorothioate antisense, which was
shown to be devoid of any homology with any known gene product, served
as a negative control. As shown in Fig. 7
, this oligomer prevented the expression
of SOCS-1 mRNA in a PRL-independent manner. Moreover, the addition of
the SOCS-1 antisense oligonucleotide was able to induce the
2-MG gene expression. As shown in Fig. 8
,
2-MG mRNA
became clearly detectable in GG-AD cells after 4 h exposure to the
antisense in serum-free medium (lane 2). The
2-MG mRNA levels were further increased after
8 h of culture in the presence of serum (Fig. 8
, lanes 5 and 6). During this
period of time, exogenous PRL had no effect on
2-MG gene expression. The lower expression of
2-MG in the absence of antisense may be
related to decreased endogenous levels of SOCS-1 due to the first
period of culture in the absence of serum. However, 24 h later,
2-MG expression was observed only in cells
transfected with the phosphorothioate anti-SOCS-1 and further treated
with PRL (Fig. 8
, lane 10). This appears to be due to the reduced
ability of the GG-AD cells to secrete PRL at this stage of culture (A.
Prigent-Tessier and G. Gibori, unpublished) causing the cells to be
more responsive to exogenous PRL.

View larger version (43K):
[in this window]
[in a new window]
|
Figure 7. Effect of SOCS-1 Oligonucleotide Antisense on
SOCS-1 Gene Expression
GG-AD cells were cultured at 33 C to 75% confluence and transferred to
39 C for an additional period of 12 h. They were challenged with
lipofectamine coated 250 nM SOCS-1 antisense oligomer for
4 h in Opti-Mem or with the vehicle only. Cells were then washed
and complete medium containing either PRL (1 µg/ml) or vehicle was
added for 20 h. RNA isolation and RT-PCR were as described in
Materials and Methods.
|
|

View larger version (48K):
[in this window]
[in a new window]
|
Figure 8. Effect of SOCS-1 Oligonucleotide Antisense on
2-MG Gene Expression
GG-AD cells were cultured at 33 C to 75% confluence and transferred to
39 C for an additional period of 12 h. They were then challenged
with lipofectamine-coated 250 nM SOCS-1 antisense oligomer
or with the irrelevant oligomer for 4 h (lanes 1 and 2) in
serum-free medium (Opti-MemI, Life Technologies). Cells
were then washed and cultured in RPMI 164010% FBS containing either
PRL (1 µg/ml) or vehicle for another 8 (lanes 36) or 24 h
(lanes 710). RNA isolation and RT-PCR were performed as described in
Materials and Methods.
|
|
 |
DISCUSSION
|
---|
PRL signal transduction through the activation of the Jak2/Stat5
pathway is well defined. However, the mechanisms by which signaling is
prevented are just beginning to be understood. The SOCS protein appears
to play an important role in blocking GH- and PRL-induced
transactivation of responsive gene promoters (32, 33, 34). Whereas in some
tissues, little or no SOCS-1 expression is detectable in the absence of
stimulation, constitutive expression was observed in others (24).
Results of this investigation revealed for the first time that
constitutive expression of SOCS-1 can prevent PRL signaling to a
PRL-regulated gene in cells producing PRL. The results also revealed
that the presence of the PRL-R, Jak2, and Stat5, while being mandatory
for PRL signaling, is not a sufficient requirement and that inhibitors
of the Jak2/Stat5 signaling pathways in a defined cell population are
powerful molecules that can silence the expression of genes normally
up-regulated by PRL.
In addition to SOCS-1, SOCS-3 was shown to inhibit the activation of
gene transcription by PRL in human mammary cancer cells while SOCS-2
was able to restore PRL signaling (33). Whether SOCS-2 and SOCS-3 play
an important role in PRL signal transduction in the decidua needs to be
determined. The recent generation of SOCS-1 -/- mice does not allow
investigation as to the role of this protein during pregnancy since
SOCS-1-deficient mice die before weaning with fatty degeneration of the
liver (39).
In many cells that do not express the SOCS proteins, cytokines and PRL
first activate the Jak/Stat pathway and thereafter stimulate the
expression of the SOCS protein that acts to switch off the signaling
pathway. In cells that constitutively express SOCS-1, PRL signaling
appears to be shut off and PRL-regulated gene expression silenced. This
appears to be the reason for which one defined population in the rat
decidua expresses the PRL-regulated gene
2-MG
and another population does not, although both cell types express the
PRL-R and are subjected not only to PRL produced by the decidua (38)
but also to pituitary PRL and rat placental lactogens produced by the
trophoblast. We first thought that the cells that do not express
2-MG may lack a critical component in PRL
signal transduction. Our results indicate that this is not the case but
that Stat5 in these cells is phosphorylated neither before PRL
treatment nor in response to PRL stimulation, most probably due to the
high levels of SOCS-1 expression. Expression of SOCS-1 and
2-MG are inversely related, and blocking
SOCS-1 expression leads within 4 h to the appearance of
2-MG mRNA in GG-AD cells. Thus, the
combination of an endogenously generated effector and inhibition of
SOCS-1 expression leads to
2-MG
expression.
The constitutive expression of SOCS-1 in the antimesometrial cells and
the lack of expression in the mesometrial cells may be of great
physiological importance in regard to which cells express
2-MG, leading to differential differentiation
of decidual cells and allowing for limited trophoblast invasion.
Indeed, it is the mesometrial decidua, which lacks SOCS-1 and expresses
2-MG, that is the site of trophoblast
invasion. These cells are much less differentiated than the
antimesometrial cells (1, 2) and remain loosely connected, allowing
trophoblast cells to invade without causing massive cell destruction.
The limited differentiation of these cells may well be due to
2-MG, which binds and prevents the activity of
several growth factors involved in cell differentiation (40, 41, 42, 43, 44, 45, 46, 47, 48). In
addition, the invasive nature of the trophoblast cells is related to
the secretion of proteolytic enzymes (49, 50). These trophoblast cells
invade without restraint any tissue other than the mesometrial decidua.
The abundant secretion of
2-MG, a potent
protease inhibitor known to limit trophoblast invasion (50), may be of
critical importance for the prevention of mesometrial tissue damage
during placentation.
The question as to why SOCS-1 is constitutively expressed in one cell
type and not the other, causing differential responsiveness to PRL
stimulation, remains a subject of further study. Nevertheless, the
results of this investigation suggest that the constitutive expression
of SOCS, or the lack of it in defined cells of the decidua, may play an
important role in the normal development of the placenta.
 |
MATERIALS AND METHODS
|
---|
Chemicals and Biochemicals
Tissue culture media M199 and RPMI-1640, antibiotic-antimycotic
solution, nonessential amino acids, and sodium pyruvate were obtained
from Mediatech (Washington, DC). FBS was purchased from HyClone Laboratories, Inc. (Logan, UT). ExTaq was purchased from Panvera
(Madison, WI). The unmodified and phosphorothioate oligonucleotides
were obtained from Life Technologies (Gaithersburg, MD).
Ovine PRL (APF 10677 C) was provided by the NIDDK (Bethesda, MD). The
monoclonal anti-Stat5 (G-2) and anti-p-Tyr (PY99) antibodies and the
polyclonal anti-Jak2 (HR-758) antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other reagents
were of analytical grade.
Animals and Surgical Procedures
Pseudopregnant Holztman rats were obtained from Harlan Sprague Dawley, Inc. (Madison, WI). They were kept under
controlled temperature (2224 C) and light conditions of 14 h
light, 10 h dark with free access to standard rat chow and water.
Pseudopregnancy was induced by mating females with vasectomized males.
The day of vaginal plug was designated as day 1 of pseudopregnancy.
Decidualization of the uterine endometrium was induced, under ether
anesthesia, on day 5 of pseudopregnancy by scratching the
antimesometrial side with a hooked needle. Rats were killed at days 12
and 13 of pseudopregnancy, and uteri were isolated, trimmed of adherent
tissue, and washed thoroughly in ice-cold PBS. The mesometrial and
antimesometrial decidual tissues were separated as described by Martel
et al., (51). Tissue was kept at -80 C until used for RNA
isolation.
Cell Culture
Both the UIII and the GG-AD cell lines were stably
transfected with the PRL-R long form (34, 35) and shown to produce PRL
(36, 37) as previously described. The rat endometrial stromal cell
line, UIII, is derived from adult Sprague Dawley female
rats (34). They express the vimentin filament and have retained several
characteristics of uterine stromal cells including progesterone and PRL
receptors. These cells also have the ability to differentiate
spontaneously in culture, giving rise to large cells that express the
desmin intermediate filament and consequently behave as decidual cells.
GG-AD are temperature sensitive cells derived from pure rat
antimesometrial decidual cells (35). They have retained morphological
characteristics of antimesometrial cells: they are polynucleated,
large, and have a cytoplasm filled with lipids droplets. They also
express the same mRNAs as antimesometrial cells such as activin ßA
and decidual PRL-related protein (dPRP). They were grown in
media containing nonessential amino acids (1x), antibiotic-antimycotic
solution (2x), sodium pyruvate (1x), D-glucose (0.45%),
and FBS (10%). M199 culture medium was used for UIII cells
and RPMI-1640 for GG-AD cells. UIII cells were cultured at
37 C, whereas the temperature-sensitive GG-AD cells were first cultured
at 33 C to allow cell growth and then transferred to 39 C before
treatment as previously described (35). Culture media were replaced
every 48 h and cells were harvested at 7090% confluence.
RNA Isolation and RT-PCR
Total RNA was extracted from cells and tissue using
guanidium isothiocyanate and phenol in a commercial kit (RNA-NOW,
Biogentex, Houston, TX) according to the manufacturers protocol. One
microgram of total RNA was reverse transcribed using Advantage RT for
PCR (CLONTECH Laboratories, Inc. Palo Alto, CA), and the
final volume was adjusted to 100 µl. Diluted RT product (34 µl,
representing 3040 ng of total RNA) was amplified. The reaction
mixture consisted of 1xPCR buffer (ExTaq buffer, Panvera, Madison,
WI), 150 µM deoxynucleoside triphosphates, 4.5%
dimethylsulfoxide, 20 pmol specific oligonucleotide primers, and
0.8 U ExTaq in a final volume of 40 µl. Two sets of amplification
cycles were used. In the first five cycles, the annealing and extension
temperature of 68 C for 5 min was followed by a denaturation
temperature of 93 C for 1 min. In the second set, the annealing
temperature of 63 C for 25 sec was followed by a 30-sec extension at 71
C and another 25-sec denaturation at 92 C. Cycle number varied for each
of the amplified products and was in the range of 2535 cycles. The
conditions were such that amplification of the product was in the
exponential phase, and the assay was linear with respect to the amount
of input cDNA. The ribosomal L19 protein was used as internal control
to normalize the data. L19 and the specific gene were amplified
separately, PCR products were mixed in 1:1 ratio, and 820 µl of the
mix resolved on 2.5% Metaphore agarose gel (FMC Corp.
BioProducts, Rockland ME) containing 0.5 µg/ml ethidium
bromide in 0.75xTris-borate-EDTA. The resulting gels were
photographed using UV transilluminator and a digital camera
(Electrophoresis Documentation and Analysis System 120, Eastman Kodak Co., New Haven, CT).
For the detection of Stat5a and Stat5b, a common sense primer
5'-GGGCATCACCATTGCTTGGAAG-3' was combined with a specific Stat5a
antisense 5'-GGAGCTTCTGGCAGAAGTGAAG-3' or with a specific Stat5b
antisense 5'-CACGACTAGTATTAACACTTCAC-3' based on the sequences of rat
Stat5a (Ref. 52 ; GenBank accession no. U24175) or 5b (Ref. 53 ; GenBank
accession no. X97541). The sizes of the coamplified cDNA products were
498 and 610 bp for Stat5a and Stat5b, respectively. The other primers
were as follows: PRL-RL (54), 5'-AAAGTATCTTGTCCAGACTCGCTG-3' and
5'-AGCAGTT-CTTCAGACTTGCCCTT-3' (279 bp cDNA fragment);
2-MG (55) 5'-GTAATCCTTCTAACTGTTCGGCGA-3' and
5'-CCAATGAAGATCGTTTCATACGGA-3' (343 bp cDNA fragment); Jak-1 (Ref. 56 ;
GenBank accession no. AJ000556), 5'-CTATGAG-CCAGCTGAGTTTCGATC-3'
and 5'- CATCTCGGACACAGA-CGCCGTA-3' (275 bp cDNA fragment); Jak-2
(Ref. 57 ; GenBank accession no. U13396),
5'-GTTCTTACCGAAGTGCGTG-CGA-3' and 5'-GGTAATGGTGTGCATCCGCAGTT-3'
(523 bp cDNA fragment); SHP-2 (Ref. 58 ; GenBank accession no. U09307),
5'- CGGGAGTTAAGCAAGCTAGCCG-3' and 5'-CCTCACACGCATGACGCCATAC-3' (465 bp
cDNA fragment); and SOCS-1, 5'-GCAGCTCGAAGAGGCAGTCGAA-3' and 5'-
GCTCCCACTCTGATTACCGGCG-3' (273 bp cDNA fragment). No rat SOCS-1 mRNA
was ever published in the GenBank database. Thus, we employed BLAST to
search for rat homologs to a published mouse sequence (GenBank
accession no. U88325). A 13.2-kb rat genomic sequence (GenBank
accession no. Z46939) was found that includes, in addition to other
genes, the rat SOCS-1 sequence (start site at position 12119, end of
last amino acid at position 13155). PCR primers were designed to a
piece of this cDNA sequence. A cDNA sequence from a SOCS-1
amplification experiment was sequenced and found to match the rat
SOCS-1 sequence. The primers for ribosomal protein L19 were as follows:
5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-CGTTCACCTTGATGAGCCCATT-3' (59).
Antisense Experiments
Chimeric oligonucleotides were designed for the antisense
experiments. The SOCS-1 antisense was designed as a 26-bp
single-stranded oligonucleotide, covering the ATG start site of the
gene with each of the four external nucleotides on both the 5'- and the
3'-ends carrying a modified phosphorothioate backbone:
5'-CACCTGGTTACGTGCTACCATCCTAC-3'. The control antisense oligomer had an
identical type of structure, 5'-CAGTGCATACGCTGTACGTCATGTAC-3'. Cells
were grown in RPMI-1640 medium. At 75% confluence, the cultures were
transferred to 39 C for 12 h and then washed twice with PBS. The
antisense oligomer, precoated with lipofectamine (Life Technologies) at a ratio of 1:26, was added to the cultures at a
final concentration of 250 nM in 2 ml Opti-Mem (Life Technologies). After 4 h, cells were washed with PBS and cultured
with RPMI-164010% FBS supplemented with or without 1 µg/ml
PRL.
Western and Immunoprecipitation Analysis
Cells were grown to 80% confluency, washed twice with cold PBS,
and lysed at 4 C for 1 h in ice-cold lysis buffer (PBS containing
2% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride and 2 µg/ml of aprotinin, leupeptin, and pepstatin). Cells
were scraped, sonicated, and cleared by centrifugation. Protein
concentrations were determined using a Protein Assay Dye Reagent kit
(Bio-Rad Laboratories, Inc., Hercules, CA). For
immunoprecipitation analysis, 800 µg protein extracts were incubated
overnight at 4 C with monoclonal anti-Stat5 (G-2) or anti-Jak2
antibodies. Complexes were then precipitated with Protein A/G Sepharose
(Santa Cruz Technology, Inc.) and boiled for 5 min in
sample buffer: 62.5 mM, Tris-HCl, pH 6.8, 5%
ß-mercaptoethanol, 2% SDS, 20% glycerol, and 0.1% bromophenol
blue. Proteins were resolved on 10% denaturing polyacrylamide gels
according to the method described by Laemmli (60). After gel
electrophoresis, proteins were electrophoretically transferred to
nitrocellulose filters (Protran, Schleicher & Schuell, Inc., Keene, NH). The blots were incubated 1 h at room
temperature with 5% nonfat dry milk in Tris-buffered saline (TBS, pH
7.6) containing 0.1% Tween 20. Blots were washed and incubated
overnight at 4 C with the primary antibody (1:2000) and then washed and
incubated with a horseradish peroxidase-conjugated antirabbit IgG
(1:5000) for 1 h at room temperature. Complexes were visualized
using the enhanced chemiluminescence Western blotting detection kit
(Western Luminol Reagent; Santa Cruz Biotechnology, Inc.).
Statistics
Data were examined by one-way ANOVA, followed by Duncans
multiple-range test. A level of P < 0.05 was accepted
as statistically significant.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to the NIDDK and the National Hormone and
Pituitary Program (NIH) for the oPRL. We thank Dr. Hélène
Cohen for providing the UIII stromal cell line.
We thank Rose Clepper for animal care and Vivian Rogala for preparation
of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois, 835 South Wolcott Avenue, Chicago, Illinois 60612-7342.
This work was supported by NIH Grant HD-12356 (to G.G.) and Ernst
Schering Research Foundation (to C.T.).
Received for publication October 15, 1999.
Revision received December 22, 1999.
Accepted for publication December 28, 1999.
 |
REFERENCES
|
---|
-
Bell SC 1983 Decidualization: regional differentiation
and associated function. Oxf Rev Reprod Biol 5:220271
-
OShea JD, Kleinfeld RG, Morrow HA 1983 Ultrastructure of
decidualization in the pseudopregnant rat. Am J Anat 166:271298[Medline]
-
Gibori G 1994 The decidual hormones and their role in
pregnancy recognition. In: Glasser SR, Mulholland S, Psychoyos A (eds)
Endocrinology of Embryo Endometrium Interactions. Plenum Press, New
York, pp 217222
-
Gibori G, Gu Y, Srivastava RK 1995 Differential gene
expression and programmed cell death. In: Dey SK (ed) Molecular and
Cellular Aspects of Periimplantation Processes. Springer-Verlag Press,
New York, pp 6783
-
Jayatilak PG, Puryear TK, Herz Z, Fazleabas A, Gibori G 1989 Protein secretion by mesometrial and antimesometrial rat decidua
tissue: evidence for differential gene expression. Endocrinology 125:659666[Abstract]
-
Gu Y, Jayatilak, PG, Parmer TG, Gauldie J, Fey GO, Gibori G 1992
2-Macroglobulin expression in the
mesometrial decidua and its regulation by decidual luteotropin and
prolactin. Endocrinology 131:13211328[Abstract]
-
Bell SC 1979 Immunochemical identity of
"decidualization-associated protein" and acute-phase macroglobulin
in the pregnant rat. J Reprod Immunol 1:193206[CrossRef][Medline]
-
Hayashidah K, Tsuchiya Y, Kuroka WS, Hattoe N,
Ishiboshi H, Okuba H, Sakai Y 1986 Expression of rat
2-macroglobulin gene during pregnancy. J
Biochem 100:989993[Abstract]
-
Fletcher S, Thomas T, Shreiber G, Heinrich P, Yeah GCT 1988 The development of rat
2-macroglobulin.
Studies in vivo and in cultured fetal rat hepatocytes. Eur
J Biochem 171:703709[Abstract]
-
Behrendtsen O, Alexander CM, Werb Z 1992 Metalloproteinases
mediates extra- cellular matrix degradation by cell from mouse
blastocyst outgrowths. Development 114:447456[Abstract]
-
da Silva GC, Teixeira N, Pringle JH, Bell SC 1996 Expression
of mRNA encoding decidualization-associated protein, a variant of
acute-phase
2-macroglobulin, by rat uterine
tissues during pregnancy and pseudopregnancy. J Reprod Fertil 108:289298[Abstract]
-
Gaddy-Kurten D, Richards JS 1991 Regulation of
2-macroglobulin by luteinizing hormone and
prolactin during cell differentiation in the rat ovary. Mol Endocrinol 5:12801291[Abstract]
-
Dajee M, Kazansky AV, Raught B, Hocke GM, Fey GH, Richards JS 1996 Prolactin induction of the
2-macroglobulin gene in rat ovarian granulosa
cells: Stat5 activation and binding to the interleukin-6 response
element. Mol Endocrinol 10:171184[Abstract]
-
Russell DL, Norman RL, Dajee M, Liu X, Henninghausen L,
Richards JS 1996 Prolactin-induced activation and binding of Stat
proteins to the IL-6RE of the
2-macroglobulin
(
2M) promoter: relation to the expression of
2M in the rat
ovary. Biol Reprod 55:10291038[Abstract]
-
Gu Y, Srivastava RK, Clarke DL, Linzer DI, Gibori G 1996 The
decidual prolactin receptor and its regulation by decidua-derived
factors. Endocrinology 137:48784885[Abstract]
-
Finidori J, Kelly PA 1995 Cytokine receptor signaling through
two novel families of transducer molecules: Janus kinases, and signal
transducers and activators of transcription. J Endocrinol 147:1123[Medline]
-
Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Thierfelder WE,
Kreider B, Silvennoinen O 1994 Signaling by the cytokine receptor
superfamily: JAKs and STATs. Trends Biochem Sci 19:222227[CrossRef][Medline]
-
Bole-Feysot C, Goffin V, Edering V, Edering M, Binard N, Kelly
PA 1998 Prolactin (PRL) and its receptor: actions, signal transduction
pathways and phenotypes observed in PRL receptor knockout mice. Endocr
Rev 19:225268[Abstract/Free Full Text]
-
Standke GJ, Meier VS, Groner B 1994 Mammary gland factor
activated by prolactin on mammary epithelial cells and acute-phase
response factor activated by interleukin-6 in liver cells share DNA
binding and transactivation potential. Mol Endocrinol 8:469477[Abstract]
-
Jahn GA, Daniel N, Jolivet G, Belair L, Bole-Feysot C, Kelly
PA, Djiane J 1997 In vivo study of prolactin (PRL)
intracellular signaling during lactogenesis in the rat: JAK/STAT
pathway is activated by PRL in the mammary gland but not in the liver.
Biol Reprod 57:894900[Abstract]
-
Dajee M, Fey GH, Richards JS 1998 Stat 5b and the orphan
nuclear receptors regulate expression of the
2-macroglobulin (
2-M) gene in rat ovarian
granulosa cells. Mol Endocrinol 12:13931409[Abstract/Free Full Text]
-
Ruff SJ, Leers-Sucheta S, Melner MH, Cohen S 1996 Induction
and activation of Stat 5 in the ovaries of pseudopregnant rats.
Endocrinology 137:40954099[Abstract]
-
Ali S, Chen Z, Lebrun JJ, Vogel W, Kharitonenkov A, Kelly PA,
Ullrich A 1996 PTP-1D is a positive regulator of the prolactin signal
leading to ß-casein promoter activation. EMBO J 15:135142[Abstract]
-
Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins
BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ 1997 A
family of cytokine-inducible inhibitors of signaling. Nature 387:917921[CrossRef][Medline]
-
Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitui
K, Matsumoto A, Tanimiura S, Ohtsubo M, Misawa H, Miyazaki T, Leonor N,
Taniguchi T, Fujita T, Kanakura Y, Komiya S, Yoshimura A 1997 A new
protein containing an SH2 domain that inhibits Jak2 kinases. Nature 387:921924[CrossRef][Medline]
-
Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A,
Nishimoto N, Kajita T, Taga T, Yoshizaki K, Akira S, Kishimoto T 1997 Structure and function of a new STAT-induced STAT inhibitor. Nature 387:924929[CrossRef][Medline]
-
Faure H, Benhamov A, Finidori S, Kelly PA, Edery M 1999 Dual
effects of suppressor of cytokine signaling (SOCS-2) on growth hormone
signal transduction. FEBS Lett 453:6366[CrossRef][Medline]
-
Sakamoto H, Yasukawa H, Masuhara M, Tanimura S, Sasaki A, Yuge
K, Ohtsubo M, Ohtsuka A, Fujita T, Ohta T, Furukawa Y, Iwase S, Yamada
H, Yoshimura A 1998 A Janus kinase inhibitor, JAB, is an interferon-
inducible gene and confers resistance to interferons. Blood 92:16681676[Abstract/Free Full Text]
-
Suzuki R, Sakamoto H, Yasukawa H, Masuhara M, Wakioka T,
Sasaki A, Yuge K, Komiya S, Inoue A, Yoshimura A 1998 CIS3 and JAB have
different regulatory roles in interleukin-6 mediated differentiation
and STAT3 activation in M1 leukemia cells. Oncogene 17:27712278[CrossRef][Medline]
-
Song MM, Shuai K 1998 The suppressor of cytokine signaling
SOCS1 and SOCS3 but not SOCS2 proteins inhibit interferon mediated
antiviral and antiproliferative activities. J Biol Chem 273:3505635062[Abstract/Free Full Text]
-
Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA,
Sprigg NS, Starr R, Nicholson SE, Metcalf D, Nicola NA 1998 Twenty
proteins containing a C-terminal SOCS box form five structural classes.
Proc Natl Acad Sci USA 95:114119[Abstract/Free Full Text]
-
Pezet A, Favre H, Kelly PA, Edery M 1999 Inhibition and
restoration of prolactin signal transduction by suppressors of cytokine
signaling. J Biol Chem 274:2449724502[Abstract/Free Full Text]
-
Helman D, Sandowski Y, Cohen Y, Matsumoto A, Yoshimura A,
Merchav S, Gertler A 1998 Cytokine-inducible SH2 protein (CIS3) and
JAK2 binding protein (JAB) abolish prolactin receptor-mediated Stat5
signaling. FEBS Lett 441:287291[CrossRef][Medline]
-
Adams TE, Hansen JA, Starr R, Nichols NA, Hilton DJ,
Billestrup N 1998 Growth hormone preferentially induces the rapid,
transient expression of SOCS-3, a novel inhibitor of cytokine receptor
signaling. J Biol Chem 273:12851287[Abstract/Free Full Text]
-
Cohen H, Pageaux JF, Melinand C, Fayard JM, Laugier C 1993 Normal rat uterine stromal cells in continuous culture:
characterization and progestin regulation of growth. Eur J Cell
Biol 61:116125[Medline]
-
Srivastava RK, Gu Y, Zilberstein M, Ou JS, Mayo KE, Chou JY,
Gibori G 1995 Development and characterization of a simian virus
40-transformed, temperature-sensitive rat antimesometrial decidual
cell line. Endocrinology 136:19131919[Abstract]
-
Prigent-Tessier A, Barkai U, Tessier C, Cohen H, Gibori
G, Prolactin expression and endogenous regulation of
prolactin-dependent genes, estradiol receptor-ß,
2-macroglobulin, involving Jak 2, Stat5 in the
UIII rat uterine stromal cell line. Program of
the 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999,
p 390 (Abstract)
-
Prigent-Tessier A, Tessier C, Hirosawa-Tokamori N,
Ferguson-Gottschall S, Gibori G 1999 Rat decidual prolactin.
Identification, molecular cloning, and characterization. J Biol
Chem 274:3798237989[Abstract/Free Full Text]
-
Starr R, Metcalf D, Elefanty AG, Brysha M, Willson TA, Nicola
NA, Hilton DJ, Alexander WS 1998 Liver degeneration and lymphoid
deficiencies in mice lacking suppressor of cytokine signaling-1. Proc
Natl Acad Sci USA 95:1439514399[Abstract/Free Full Text]
-
Huang JS, Huang SS, Duel TF 1984 Specific covalent binding of
platelet-derived growth factor to human
2-macroglobulin. Proc Natl Acad Sci USA 81:342346[Abstract]
-
Borth W, Lugar TA 1989 Identification of
2-macroglobulin as a cytokine-binding plasma
protein. J Biol Chem 264:58185825[Abstract/Free Full Text]
-
Danielpour D, Sporn MB 1990 Differential inhibition of
transforming growth factor 1 and 2 by
2-macroglobulin. J Biol Chem 265:69736977[Abstract/Free Full Text]
-
Matsuda T, Hirano T, Nagasawa S, Kishimoto T 1989 Identification of
2-macroglobulin as a carrier
protein for interleukin-6. J Immunol 142:148152[Abstract/Free Full Text]
-
Dennis PA, Saksela O, Harpel P, Rifkin DG 1989
2-Macroglobulin is a binding protein for basic
fibroblast growth factor. J Biol Chem 264:72107216[Abstract/Free Full Text]
-
Southard JN, Tallamentes F 1989 High molecular weight forms of
placental lactogen: evidence for
lactogen-
2-MG complexes in rodent and
human. Endocrinology 125:791800[Abstract]
-
Vaughan JM, Vale WE 1994
2-Macroglobulin is a binding protein of
inhibin and activin. Endocrinology 132:20382050[Abstract]
-
Koo PH, Liebl DJ 1993 Serotonin-activated
2-macroglobulin
inhibits neurite outgrowth and survival of embryonic sensory and
cerebral cortical neurons. J Neurosci Res 35:170182[Medline]
-
da Silva GC, Teixeira N, Bell SC 1996 Major secretory product
of the mesometrial decidua in the rat, a variant of
2-macroglobulin, binds insulin-like growth
factor I via a protease-dependent mechanism. Mol Reprod Dev 44:103110[CrossRef][Medline]
-
Owers NO, Blandau RJ 1971 Proteolytic activity of the rat and
guinea pig blastocyst in vitro. In: Blandau RJ (ed) Biology
of the Blastocyst. University of Chicago Press, Chicago, pp 207294
-
Cross JC, Werb Z, Fisher SJ 1994 Implantation and the
placenta: key pieces of the developmental puzzle. Science 266:15081518[Medline]
-
Martel D, Monier MN, Psychoyos A, DeFeo VJ 1984 Estrogen and
progesterone receptors in the endometrium, myometrium and metrial gland
of the rat during the decidualization process. Endocrinology 114:16271634[Abstract]
-
Kazansky AV, Raught B, Lindsey SM, Wang YF, Rosen JM 1995 Regulation of mammary gland factor/Stat5a during mammary gland
development. Mol Endocrinol 9:15981609[Abstract]
-
Luo G, Yu-Lee L 1997 Transcriptional inhibition by
Stat5b: differential activities at growth-related versus
differentiation-specific promoters. J Biol Chem 272:2692626934[Abstract/Free Full Text]
-
Clarke DL, Arey BJ, Linzer DIH 1993 Prolactin receptor
messenger ribonucleic acid expression in the ovary during the rat
estrous cycle. Endocrinology 133:25942603[Abstract]
-
Gehring MR, Shiels BR, Northemann W, de Bruijin MHL, Kan CC,
Chain AC, Nooman DJ, Fey GH 1987 Sequence of rat liver
2-macroglobulin and acute phase control of
messenger RNA. J Biol Chem 262:446454[Abstract/Free Full Text]
-
Appel K, Gebicke-Haeter PJ 1997 Cloning and expression of JAK1
mRNA in cultured rat microglia. GenBank accession no.
AJ000556
-
Duhe RJ, Rui H, Greenwood JD, Garvey K, Farrar WL 1995 Cloning
of the gene encoding rat Jak2, a protein tyrosine kinase. Gene 158:281285[CrossRef][Medline]
-
Ding W, Zhang WR, Sullivan K, Hashimoto N, Goldstein BJ 1994 Identification of protein-tyrosine phosphatases prevalent in adipocytes
by molecular cloning. Biochem Biophys Res Commun 202:902907[CrossRef][Medline]
-
Chan YL, Lin A, McNally J, Peleg D, Meyuhas O, Wool IG 1987 The primary structure of rat ribosomal protein L19. A determination
from the sequence of nucleotides in a cDNA and from the sequence of
amino acids in the protein. J Biol Chem 262:11111115[Abstract/Free Full Text]
-
Laemnli UK 1970 Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680685[Medline]