From the
In the present report we describe a heretofore unrecognized role
for a Jak/STAT signaling pathway, namely the stimulation of expression
of the aromatase P450 (CYP19) gene, and hence of estrogen biosynthesis,
in human adipose tissue. Expression of this gene in adipose tissue as
well as in adipose stromal cells maintained in the presence of serum
and glucocorticoids is regulated by a distal TATA-less promoter, I.4,
which contains a glucocorticoid response element, an Sp1 binding site,
and an interferon-
Estrogen biosynthesis in humans occurs in a number of tissue
sites of expression including the granulosa cells and corpus luteum of
the ovary(1, 2) , the Leydig cells of the
testis(3) , the syncytiotrophoblast of the placenta, various
sites in the brain including the hypothalamus, amygdala, and
hippocampus(4, 5) , as well as in adipose
tissue(6) . The significance of adipose tissue as a source of
estrogens was first recognized some 20 years ago by MacDonald, Siiteri,
and their colleagues (7, 8, 9, 10) who
determined the fractional conversion of circulating androstenedione to
estrone in male and female human subjects. They found that not only was
there a striking increase with obesity, suggesting that most of the
extragonadal conversion occurred in adipose tissue, but also that there
was an equally striking increase with age for any given body weight.
Estrogen biosynthesis is catalyzed by an enzyme known as aromatase
P450 (11-14) (P450arom, the product of the CYP19
gene(15) ). CYP19 is a member of the P450 superfamily of genes,
which currently contains over 300 members in some 36 gene
families(15) . Typically, these enzymes catalyze the insertion
of oxygen atoms derived from molecular oxygen into organic molecules to
form hydroxyl groups. In the case of P450arom, three such attacks by
molecular oxygen give rise to loss of the C19 angular methyl group of
the steroid substrate as formic acid, with concomitant aromatization of
the A-ring to give the phenolic A-ring characteristic of
estrogens(16) .
A few years ago we and others cloned and
characterized the CYP19 gene, which encodes human
P450arom(17, 18, 19) . The coding region spans 9
exons beginning with exon II. Sequencing of rapid amplification of cDNA
ends-generated cDNA clones derived from P450arom transcripts present in
the various tissue sites of expression revealed that the 5`-termini of
these transcripts differ from one another in a tissue-specific fashion
upstream of a common site in the 5`-untranslated
region(20, 21, 22) . Using these sequences as
probes to screen genomic libraries, it was found that these 5`-termini
correspond to untranslated exons that are spliced into the P450arom
transcripts in a tissue-specific fashion, due to the use of
tissue-specific promoters. Placental transcripts contain at their
5`-ends untranslated exon I.1, which is located at least 40 kilobases
upstream from the start of translation in exon
II(20, 23) . This is because placental expression is
driven from a powerful distal placental promoter, I.1, upstream of
untranslated exon I.1. On the other hand, transcripts in the ovary
contain sequence at their 5`-ends that is immediately upstream of the
start of translation. This is because expression of the gene in the
ovary utilizes a proximal promoter, promoter II. By contrast,
transcripts in adipose tissue contain yet another distal untranslated
exon, I.4, which is located in the gene 20 kilobases downstream from
exon I.1 (24-26). A number of other untranslated exons have been
characterized by ourselves and others(22, 27) ,
including one specific for brain (28). Splicing of these untranslated
exons to form the mature transcripts occurs at a common 3`-splice
junction, which is upstream of the start of translation. This means
that although transcripts in different tissues have different
5`-termini, the protein encoded by these transcripts is always the
same, regardless of the tissue site of expression; thus, there is only
one human P450arom enzyme encoded by a single copy gene.
Using
reverse transcription polymerase chain reaction with an internal
standard, we have studied aromatase expression in samples of adipose
tissue obtained from women of various ages and have found a marked
increase in the specific content of P450arom transcripts in adipose
tissue with increasing age, thus providing a molecular basis for the
previous observation that the fractional conversion of circulating
androstenedione to estrone increases with age(29) . Furthermore,
there are marked regional variations in aromatase expression, with the
highest values being found in adipose from buttocks and thighs as
compared with abdomen and breast(29, 30) .
We also
used this reverse transcription polymerase chain reaction technique to
examine regional variations in aromatase expression in breast adipose
tissue and have found that highest expression occurs in adipose tissue
proximal to a tumor, as compared with that distal to a
tumor(31, 32) . This is in agreement with previous
observations regarding the regional distribution of aromatase activity
within breast adipose (33, 34) as well as an
immunocytochemical study(35) . These results suggest there is
cross-talk between a breast tumor and the surrounding adipose cells in
terms of the ability of the latter to synthesize estrogens and that
factors produced by developing breast tumors may set up local gradients
of estrogen biosynthesis in the surrounding fat via paracrine
mechanisms(36) .
We also found that aromatase expression does
not occur in adipocytes but rather in the stromal cells that surround
the adipocytes, and that may themselves be preadipocytes(37) .
These stromal cells grow in culture as fibroblasts. Consequently we
have employed these cells in primary culture as a model system to study
the regulation of estrogen biosynthesis in adipose tissue(38) .
When serum is present in the culture medium, expression is stimulated
by glucocorticoids including dexamethasone(39) . Under these
conditions P450arom transcripts contain primarily untranslated exon I.4
at their 5`-ends(25) . We subsequently have characterized the
region of the CYP19 gene upstream of exon I.4 and have found it to
contain a TATA-less promoter as well as an upstream glucocorticoid
response element and an Sp1 sequence within the untranslated exon, both
of which are required for expression of reporter gene constructs in the
presence of serum and glucocorticoids (40). Additionally, we found this
region to contain an interferon-
In the present study we
have observed for the first time that the effect of serum to stimulate
aromatase expression in human adipose stromal cells (in the presence of
glucocorticoids) can be mimicked by specific factors, namely members of
the interleukin-11 (IL-11), oncostatin-M (OSM), IL-6, and leukemia
inhibitory factor (LIF) lymphokine family. This stimulation is mediated
by a member of the Jak family of tyrosine kinases as well as a STAT
transcription factor, which binds to the GAS element within promoter
I.4 of the P450arom gene. Thus we have uncovered a regulatory pathway
whereby expression of the P450arom gene, via the distal promoter I.4,
is stimulated by members of the above cytokine family. Since P450arom
transcripts in adipose tissue appear to be derived primarily from
expression of promoter I.4 and since adipose tissue is the major site
of estrogen biosynthesis in men and in postmenopausal
women(44) , this pathway composed of a Jak/STAT signaling
mechanism acting in conjunction with glucocorticoids and Sp1 appears to
be the principal means whereby estrogen biosynthesis is regulated in
the elderly.
Generally, experiments were performed
at least 3 times on separate occasions, and frequently more, to
generate consistent results.
The findings of the present study reveal a hitherto
unrecognized role for a Jak/STAT signaling pathway, namely the
stimulation of expression of the P450arom gene and hence of estrogen
biosynthesis in human adipose tissue. The extracellular ligands that
initiate this response are members of the IL-11/OSM/LIF family of
lymphokines(50, 51) . Ligands that have no effect
include interferon-
Addition of IL-11
resulted in a rapid phosphorylation of Jak1 kinase in a
concentration-dependent fashion, with a maximal effect obtained after
10 min and at a concentration of IL-11 of 10 ng/ml, similar to the
concentration of IL-11 required for maximal stimulation of aromatase
activity. By contrast, Jak3 kinase was not phosphorylated under these
conditions to any significant extent, whereas Jak2 kinase was
phosphorylated to an equal extent both in the presence and absence of
IL-11. A similar action of interferon-
The action of IL-11 also results in the rapid
phosphorylation of STAT3 on tyrosine residues, but this was not the
case for STAT1. Recently it has been shown that STAT3 is the substrate
of choice for the IL-6/LIF/OSM lymphokine receptor family and that the
specificity of STAT phosphorylation is not based upon which Jak kinase
is activated (43, 50, 55) but rather is determined by specific
tyrosine-based motifs in the receptor components, namely gp130 and
LIFR
Activation of this pathway of
expression by these lymphokines is absolutely dependent on the presence
of glucocorticoids. This action of glucocorticoids is mediated by a
glucocorticoid response element downstream of the GAS
element(40) . Additionally, an Sp1-like element present within
untranslated exon I.4 also is required, at least for expression of the
-330/+170 bp construct(40) . These sequences, while
present within a 400-bp region of the gene, are not contiguous, and the
nature of the interaction among STAT3, the glucocorticoid receptor, and
Sp1, to regulate expression of the P450arom gene via the distal
promoter I.4 remains to be determined.
Activation of aromatase
expression by serum in the presence of glucocorticoids is not confined
to cells present in adipose tissue but also has been reported in skin
fibroblasts (57) and in hepatocytes derived from fetal
liver(58) . In each of these cell types the P450arom transcripts
contain exon I.4 as their 5`-terminus(27, 59) ; however,
the factors that mimic the action of serum to stimulate aromatase
expression in these cell types have as yet to be elucidated. On the
other hand, in placenta where the distal promoter I.1 is employed (20) and in ovarian granulosa cells where the proximal promoter
II is employed(20, 23) , this signaling pathway is not
in effect. Thus, aromatase expression in ovarian granulosa cells is
driven primarily by cyclic AMP-dependent mechanisms(60) .
As
indicated previously, adipose tissue is the major site of estrogen
biosynthesis in elderly women and men. The fact that this expression is
confined to the stromal cells rather than the adipocytes themselves is
consistent with the known actions of IL-11 to inhibit the
differentiation of 3T3 L1 fibroblasts into adipocytes(61) .
Since adipose stromal cells are believed to function as preadipocytes
and can be converted to adipocytes under appropriate nutritional
stimuli, a role of these lymphokines may be to maintain these cells in
the preadipocyte state for which aromatase expression is a marker. As
indicated previously, aromatase expression in adipose increases
dramatically with age(6, 29) . There is also a marked
regional distribution, with expression being greatest in buttock and
thigh regions as compared with abdomen and
breast(29, 30) . However, within the breast there is
also a marked regional variation with expression being highest in sites
proximal to a tumor as compared with those distal to a
tumor(31, 32) .
Based on the results presented here
we suggest that aromatase expression in adipose tissue may be under
tonic control by circulating glucocorticoids and that regional and
age-dependent variations may be the consequence of paracrine and
autocrine secretion of lymphokines. Schmidt and Loffler(62) , as
well as ourselves(64) , have shown that conditioned medium from
a number of cell types including adipose stromal cells themselves and
endometrial stromal cells, as well as breast tumor cells lines, can
mimic the actions of serum to stimulate aromatase expression in the
presence of glucocorticoids. Thus numerous cell types including breast
cancer cells produce factors that are stimulatory of aromatase
expression by adipose stromal cells. In preliminary experiments we have
shown that a stimulatory factor present in conditioned medium from T47D
breast cancer cells can be titrated by an anti-IL-11 antibody (data not
shown). Additionally, Reed and colleagues have found that fibroblasts
derived from breast tumors secrete IL-6(34, 63) .
Such local paracrine mechanisms could be important in the
stimulation of breast cancer growth by estrogens. Commonly, breast
tumors produce a desmoplastic reaction whereby there is local
proliferation of stromal cells surrounding the tumor, strongly
indicative of the production of growth factors by the tumor. These
proliferating stromal cells express aromatase, as indicated by
immunocytochemistry(35) . It is possible then to envision a
positive feedback loop whereby adipose stromal cells surrounding a
developing tumor produce estrogens, which stimulate the tumor to
produce a variety of growth factors and cytokines(64) . Some of
these act to stimulate the further growth and development of the tumor
in a paracrine and autocrine fashion. Additionally, these or other
factors act to stimulate proliferation of the surrounding stromal cells
and expression of aromatase within these cells. Thus a positive
feedback loop is established by paracrine and autocrine mechanisms,
which leads to the continuing growth and development of the
tumor(32, 36) . Further insight into the paracrine
mechanisms involved in regulation of estrogen biosynthesis in human
adipose tissue will await the characterization of the particular
cytokines that are being produced as well as their levels of
expression, both of which may vary in a region- and age-dependent
fashion.
We thank Melissa Meister for skilled editorial
assistance, Christy Ice and Carolyn Fisher for skilled technical
assistance, and Dr. Paul C. MacDonald for suggesting IL-11 and the IL-6
soluble receptor. We gratefully acknowledge the generous gifts of Jak1,
Jak2, and Jak3 antibodies from Dr. James Ihle (St. Jude
Children's Research Hospital, Memphis, TN) and of STAT3 antibody
from Dr. Christopher Schindler (Columbia University, New York).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
activation site (GAS) element. The stimulatory
action of serum (in the presence of dexamethasone) can be replaced by
interleukin (IL)-11, leukemia inhibitory factor, and oncostatin-M, as
well as by IL-6, providing the IL-6 soluble receptor is also present.
Stimulation of the cells by these factors led to rapid phosphorylation
of Jak1, but not Jak2 or Jak3, on tyrosine residues. STAT3 but not
STAT1 was also phosphorylated and bound to the GAS element in the I.4
promoter region. When regions of this promoter were fused upstream of
the chloramphenicol acetyltransferase reporter gene and transfected
into the cells, mutagenesis or deletion of the GAS element led to
complete loss of reporter gene expression. Since adipose tissue is the
major site of estrogen biosynthesis in men and in postmenopausal women,
this pathway involving a Jak/STAT signaling mechanism acting together
with glucocorticoids and Sp1 appears to be the principal means where-by
estrogen biosynthesis is regulated in the elderly.
activating sequence (GAS)
(
)element. Such sequences are known to bind
transcription factors of the signal transducers and activators of
transcription (STAT) family (41-43).
Materials
Jak1, Jak2, and Jak3 antisera were the
generous gift of Dr. James Ihle (St. Jude Children's Research
Hospital, Memphis, TN). STAT1 (Cat. number S21120) and ISGR3 (Cat.
number G16930) antisera were purchased from Transduction Laboratories
(Lexington, KY). The latter antiserum is raised against a mixture of
STAT1 and STAT1
. STAT3 antiserum was a generous gift of Dr.
Christopher Schindler (Columbia University, New York). IL-11, OSM, LIF,
IL-6, and IL-6 soluble receptor were purchased from R & D
(Minneapolis, MN). INF
and INF
were purchased from Sigma.
Anti-phosphotyrosine antibody (4 G10) was purchased from UBI (Lake
Placid, NY). Herbimycin, H7, and cycloheximide were purchased from
Sigma.
Cell Culture and Preparation of Nuclear
Extracts
Subcutaneous adipose tissue was obtained from women at
the time of reduction abdominoplasty or reduction mammoplasty. Consent
forms and protocols were approved by the Institutional Review Board,
University of Texas Southwestern Medical Center at Dallas. Adipose
stromal cells were prepared as described (38) and maintained in
primary culture in Waymouth's enriched medium containing 10% Nu
serum (10% v/v) (Collaborative Research Inc.) and allowed to grow to
confluence (5-6 days) before treatment. At this time, serum was
removed for 24 h, and the cells were treated with 250 nM
dexamethasone for 48 h before recombinant human IL-11, OSM, IL-6, and
LIF were added at various concentrations and times depending on the
different requirements for each experiment. Aromatase activity was
determined by the incorporation of tritium into
[H]water from
[1
-
H]androstenedione as described previously
(38). Cytoplasmic and nuclear extracts were prepared as described by
Cooper et al.(45) and Dignam et al.(46) with some modifications. Cells were cooled on ice,
scraped from the plates, washed 3 times with phosphate-buffered saline,
and then lysed in modified RIPA (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM sodium
orthovanadate, 1 mM sodium fluoride, 1% aprotinin, 1 mM phenanthroline, 10 mM pepstein, 0.1% leupeptin, 0.5
mM phenylmethylsulfonyl fluoride, and 15% glycerol) with
protease and phosphatase inhibitors for 30 min on ice. Lysates were
precleared by centrifugation for 30 min at 4 °C. For nuclear
extracts, after cells were swollen in 2 ml of hypotonic buffer
(containing protease and phosphatase inhibitors as in RIPA) they were
homogenized with 12 strokes of an all glass Dounce homogenizer (B type
pestle). Nuclei were centrifuged, and the pellet was then suspended in
500 µl of chilled buffer (20 mM Hepes, pH 7.7, 420 mM NaCl, 1.5 mM MgCl
, 0.2 mM EDTA, 0.5%
dithiothreitol, and 20% glycerol and protease and phosphatase
inhibitors as in RIPA). After centrifugation, the supernatant was
stored at -70 °C until used.
Immunoprecipitation and Western Blotting
Analysis
Immunoprecipitations were performed essentially as
described by Harlow and Lane (47) or following instructions of
the manufacturers of the respective antibodies. Lysates were incubated
with nonimmune serum and Protein A-Sepharose (50 µl of 50% slurry)
for 1 h, followed by centrifugation. Antibodies were incubated with
lysates (100 µg) overnight at 4 °C. Immunoprecipitates were
isolated with Protein A- or Protein G-coupled agarose or Sepharose
(Oncogene Sciences or Sigma) and washed carefully 3 times with the same
lysis buffer mentioned above. Proteins in the immunoprecipitates were
resolved by 8% SDS-PAGE, transferred to polyvinylidene difluoride
membrane (Bio-Rad), and incubated with the appropriate antibody
followed by anti-mouse or anti-rabbit IgG conjugated to horseradish
peroxidase and the enhanced chemiluminescence detection system
(Amersham International, U.K.).
DNA Reporter Gene Constructs Containing Deletion
Mutations, Site-directed Mutagenesis, and Transient
Transfections
Chimeric DNA constructs containing the GAS element
in the genomic region flanking the 5`-end of promoter I.4 of the human
CYP19 gene were prepared using polymerase chain reaction. These
constructs were fused upstream of the chloramphenicol acetyltransferase
(CAT) reporter gene. For mutagenesis, an 800-bp fragment containing the
GAS element was digested by HindIII and EcoRI and
gel-purified to generate the template for mutagenesis. The GAS element
was mutated from TTCCTGTGAA to TTCGACTGAA by polymerase chain reaction.
The mutated fragment also was fused upstream of the CAT reporter gene.
The fidelity of mutagenesis was verified by dideoxy sequencing using a
Sequenase DNA kit (U.S. Biochemical Corp.). The transfection was
performed by means of calcium phosphate coprecipitation with minor
modifications as described(40) . Nearly confluent adipose
stromal cells in primary culture were transfected with 20 µg of
cesium chloride-purified plasmid. Glycerol shock was carried out for 1
min. The cells were allowed to recover overnight in serum-containing
media, serum-starved for 24 h, and then treated with 250 nM dexamethasone for 48 h and 10 ng/ml IL-11 overnight. The
transfected cells were then harvested and lysed by means of freeze/thaw
3 times. Total protein was estimated with a kit as recommended by the
manufacturer (Pierce). The CAT assay was performed using normalized
amounts of cell proteins, which were incubated for 6-8 h at 37
°C. The products of CAT reactions were analyzed by silica gel thin
layer chromatography followed by autoradiography.
Electrophoretic Mobility Shift Assay and Southwestern
Blotting Analysis
A double-stranded oligonucleotide
(5`-GGGTGTTTCCTGTGAAAGTT-3`) was Klenow-labeled using
[-
P]dCTP and then incubated (5000 cpm) with
nuclear extracts (5 µg) on ice for 10 min. For the competition
assay, the unlabeled oligonucleotide used as competitor was added
simultaneously with the labeled fragment at various ratios. The
resulting DNA-protein complexes were analyzed by electrophoresis using
an 8% polyacrylamide gel with 0.5
Tris borate-EDTA as running
buffer. Southwestern blotting analysis was done following the procedure
described by Singh et al.(48) with minor modification.
Nuclear extracts (60 µg) were fractionated on 8% SDS-PAGE. Proteins
were transferred to a nitrocellulose membrane. The membrane was
processed through a denaturation/renaturation cycle with 6 M
guanidine hydrochloride. Then the membrane was hybridized with the
Klenow-labeled probe employed in gel shift analysis, followed by
washing and autoradiography.
IL-11, OSM, and LIF Stimulate Aromatase Activity in
Cultured Adipose Stromal Cells in the Presence of
Dexamethasone
In previous studies we observed that the
stimulatory effect of glucocorticoids on aromatase activity in cultured
human adipose stromal cells was manifest only if serum was present in
the medium(39) . As shown in Fig. 1A, a number of
lymphokines, namely IL-11, OSM, and LIF can mimic the action of serum
to stimulate aromatase activity of adipose stromal cells in the
presence of dexamethasone but not in its absence. In the case of IL-11
and OSM, the effect far exceeded that of serum. Following exposure for
24 h, half-maximal stimulation by IL-11 and OSM was achieved at
concentrations of 2-4 ng/ml (
10
M), although the stimulation by LIF was maximal at 0.5
ng/ml. The time course of stimulation by IL-11 (Fig. 1B)
was biphasic, with an initial rapid phase lasting 12 h followed by a
slower phase extending beyond 48 h of stimulation.
Figure 1:
A, concentration dependence of the
actions of LIF, IL-11, and OSM to stimulate aromatase activity of
adipose stromal cells in the absence (opensymbols)
or presence (solidsymbols) of dexamethasone (250
nM). Confluent adipose stromal cells in primary culture were
maintained for 24 h in the presence or absence of dexamethasone (250
nM) and LIF (circles), IL-11 (squares), or
OSM (triangles) in various concentrations. Control dishes
incubated in the absence or presence of serum are indicated as ± S, with openbars indicating the absence and solidbars the presence of dexamethasone. B,
time course of stimulation of aromatase activity of adipose stromal
cells by IL-11. Confluent adipose stromal cells in primary culture were
maintained for 24 h in the presence (solidsquares)
or absence (opensquares) of dexamethasone (DEX). IL-11 (10 ng/ml) was added to half the dishes, and
incubation continued for an additional 48 h. C, action of IL-6
and its soluble receptor on aromatase activity of adipose stromal
cells. Confluent adipose stromal cells in primary culture were
maintained for 24 h in the presence of dexamethasone (250 nM)
and in the presence or absence of IL-6 (2-20 ng/ml) and its
soluble receptor (SR; 5-20 ng/ml). Solidcircles, no SR; solidsquares, 5 ng/ml
SR; opencircles, 10 ng/ml SR; opensquares, 20 ng/ml SR. Control dishes were incubated in
the absence or presence of serum (±S) or OSM (10 ng/ml)
and in the absence (openbars) or presence (solidbars) of dexamethasone. In each of the experiments shown
in A-C, aromatase activity was measured at the end of
the incubation period as described under ``Experimental
Procedures.'' Data are presented as the mean ± S.E. of
results from triplicate replicate dishes.
As shown in Fig. 1C, IL-6 had no stimulatory activity up to a
concentration of 20 ng/ml, either in the presence or absence of
dexamethasone. However, a stimulatory action of IL-6 was manifest upon
addition of the IL-6 soluble receptor at concentrations ranging from 5
to 20 ng/ml. At concentrations of IL-6 and its receptor of 20 ng/ml
each, stimulation was equal to that achieved by adding OSM at a
saturating concentration of 10 ng/ml. In other experiments,
interferon- and interferon-
were found to have no effect to
stimulate aromatase activity of these cells (data not shown).
IL-11 Rapidly and Specifically Stimulates Tyrosine
Phosphorylation of Jak1 Kinase
To determine whether Jak kinases
are involved in the action of IL-11 to activate estrogen biosynthesis
in adipose stromal cells, we examined their ability to undergo tyrosine
phosphorylation in the presence of IL-11 (Fig. 2). Adipose
stromal cells prepared from mammoplasty and abdominoplasty samples and
maintained in primary culture were treated with dexamethasone for 48 h
with or without IL-11 (10 ng/ml) for 10 min. The cells were lysed, the
Jak kinases were immunoprecipitated with the appropriate antibodies,
and the immunoprecipitates were resolved on SDS-PAGE. The gels were
subsequently blotted to filters and probed with a monoclonal antibody
against phosphotyrosine. IL-11 stimulation resulted in the appearance
of a band of 130 kDa, which was immunoprecipitated using
antibodies to JAK1 and was undetectable in the absence of IL-11. By
contrast, Jak2 kinase was constitutively phosphorylated on tyrosine
since the Jak2 band (
130 kDa) was evident prior to the IL-11
treatment, and the level of tyrosine phosphorylation of Jak2 did not
change even after IL-11 stimulation. By contrast, tyrosine
phosphorylation of Jak3 was not apparent with or without IL-11
treatment. Similar results were obtained using LIF and OSM. Nonimmune
serum and an unrelated immune serum failed to precipitate proteins of
130 kDa (data not shown). Additionally, the 130-kDa bands corresponding
to Jak1 and Jak2 were not observed when the immunoprecipitations were
conducted in the the presence of peptides used to raise these
antibodies (data not shown). Probing with a mixture of Jak1, Jak2, and
Jak3 antisera showed that no change in the amount of kinase protein
occurred within the time frame of the experiment (lowerpanel). It is concluded that in human adipose stromal
cells, Jak2 is constitutively phosphorylated on tyrosine residues, and
only Jak1 is phosphorylated on tyrosine residues in response to IL-11
treatment under the conditions employed here. A similar situation has
been reported to occur in T-lymphocytes stimulated with
interferon-
(49) .
Figure 2:
Effect of IL-11 on tyrosine
phosphorylation of Jak1. Adipose stromal cells in primary culture were
placed in serum-free medium for 24 h. The cells were treated with
dexamethasone (Dex) for 48 h before addition of IL-11 (10
ng/ml) for 10 min. The cells were collected and washed, and extracts
were prepared. Aliquots of extracts (2 10
cells)
from untreated and treated cells were immunoprecipitated with Jak1,
Jak2, or Jak3 antisera as described. The immunoprecipitates were
resolved by means of SDS-PAGE and transferred to filters. The filters
were probed with the 4G10 anti-phosphotyrosine monoclonal antibody (toppanel) or with a mixture of Jak1, Jak2, and Jak3
antisera (lowerpanel).
Characterization of Jak1 Tyrosine
Phosphorylation
The kinetics of tyrosine phosphorylation of Jak1
were examined using the same cell lysates as utilized above, and the
results are shown in Fig. 3A. Phosphorylation was
maximal 10 min after IL-11 addition and then subsequently declined. In
a study of the concentration dependence of Jak1 phosphorylation (Fig. 3C), it was observed that phosphorylation was
undetectable employing IL-11 at a concentration of 1 ng/ml.
Phosphorylation was detectable when 5 ng/ml IL-11 was used (visible on
the original autoradiograph if not on the printed figure) and reached a
maximum at a concentration of IL-11 of 10 ng/ml. ()
Figure 3:
Time course and concentration dependence
of tyrosine phosphorylation of Jak1. Adipose stromal cells in primary
culture were treated with dexamethasone (250 nM) for 48 h;
IL-11 (10 ng/ml) was then added, and incubation continued for 5, 10,
20, 30, and 60 min. Cells were collected at the different time points,
and extracts were prepared and then immunoprecipitated with Jak1
antiserum. The immunoprecipitates were fractionated by SDS-PAGE,
transferred to filters, and probed with anti-phosphotyrosine monoclonal
antibody (panelA) or Jak1 antiserum (panelB). Cells were treated with dexamethasone for 48 h; IL-11
was added at concentrations of 1, 5, 10, and 20 ng/ml; and incubation
was continued for 10 min. Cells were collected, and extracts were
prepared. The extracts were precipitated with Jak1 antiserum, and
precipitates were resolved by SDS-PAGE and transferred to a
polyvinylidene difluoride filter. The transferred proteins were probed
with anti-phosphotyrosine monoclonal antibody (C) or Jak1
antiserum (D). Other details are described under
``Experimental Procedures.''
Since
OSM and LIF also stimulate aromatase activity in adipose stromal cells
in primary culture, their action to stimulate tyrosine phosphorylation
of Jak1 was also examined. Adipose stromal cells were treated with
dexamethasone for 48 h and with FCS (15%), OSM (5 ng/ml), IL-11 (5
ng/ml), and LIF (5 ng/ml) for 10 min. The cells were lysed, the cell
lysates were mixed with Jak1 antibody, and the precipitates were
resolved by SDS-PAGE and probed with anti-phosphotyrosine monoclonal
antibody. The effects of herbimycin A (a tyrosine kinase inhibitor), H7
(a serine/threonine kinase inhibitor), and cycloheximide on Jak1
tyrosine phosphorylation were also examined. As shown in Fig. 4,
OSM and LIF could induce tyrosine phosphorylation of Jak1 to about the
same extent as IL-11. The tyrosine phosphorylation of Jak1 induced by
IL-11 was inhibited by herbimycin A, whereas H7 had no effect. These
results further support the concept that the phosphorylation of Jak1
induced by IL-11 is indeed on tyrosine residues. In addition, the rapid
phosphorylation of Jak1 was not inhibited by cycloheximide, an
inhibitor of protein synthesis, indicative that this is not mediated by
the new synthesis of protein factors.
Figure 4:
Effect of OSM and LIF on tyrosine
phosphorylation of Jak1 in adipose stromal cells in primary culture.
Cells were treated with dexamethasone (250 nM) for 48 h; and
then OSM (5 ng/ml), LIF (5 ng/ml), IL-11 (10 ng/ml), or FCS (15%) were
added, and incubation continued for 10 min. Following treatment with
dexamethasone, cells in other dishes were incubated with herbimycin A
(5.2 µM) for 14 h, H7 (40 µM) for 30 min, or
cycloheximide (CHX) (10 µM) for 6 h as indicated.
Then IL-11 (10 ng/ml) was added, and incubation continued for a further
10 min. Immunoprecipitation and Western blot analysis were carried out
as described under ``Experimental
Procedures.''
STAT3 Phosphorylation and Binding to the GAS Element in
the Upstream Region of Promoter I.4 of the CYP19 Gene
In order
to determine whether STAT transcription factors are involved in signal
transduction in adipose stromal cells in primary culture in response to
IL-11, we examined phosphorylation of STAT1 and STAT3 by
immunoprecipitation employing appropriate antisera and subsequent
probing with anti-phosphotyrosine monoclonal antibody. Adipose stromal
cells prepared from mammoplasty and abdominoplasty samples and
maintained in primary culture were treated with dexamethasone for 48 h
with or without IL-11 (10 ng/ml) for 10 min. The cells were lysed, and
aliquots were mixed with the appropriate antibodies. As can be seen in Fig. 5, a band of 92 kDa was immunoprecipitated by STAT3
antiserum after the cells were treated with IL-11. However, no bands
were detectable following immunoprecipitation with antisera raised
against STAT1. As a positive control, we showed by Western blotting
that the anti-STAT1 antibody reacted with not only recombinant STAT1
but with STAT1 present in adipose stromal cells treated with
dexamethasone in the presence or absence of IL-11 (data not shown).
Although the STAT3 antiserum cross-reacts with STAT1,
the use of the commercial STAT1
antiserum ruled out the
possibility that the STAT3 antiserum was detecting STAT1.
Figure 5:
Effect of IL-11 on tyrosine
phosphorylation of STAT3. Adipose stromal cells were treated with
dexamethasone (Dex; 250 nM) for 48 h. IL-11 (10
ng/ml) was then added, and incubation continued for 10 min. Extracts
were prepared, and aliquots were immunoprecipitated with appropriate
antisera as indicated. Immunoprecipitates were resolved on SDS-PAGE and
transferred to a nylon membrane. The transferred proteins were probed
with anti-phosphotyrosine monoclonal antibody (toppanel) or a mixture of appropriate antisera (lowerpanel) as described under ``Experimental
Procedures.''
To examine
the binding activity of nuclear factors to the GAS element of promoter
I.4, nuclear extracts were prepared from adipose stromal cells treated
with dexamethasone for 48 h and treated with IL-11 for 30 min.
Incubation of nuclear proteins from cells treated with IL-11 plus
dexamethasone or serum plus dexamethasone with the wild-type promoter
I.4 GAS element between -288 and -269 bp as radiolabeled
probe (5`-GGGTGTTTCCTGTGAAAGTT-3`) gave rise to a single band (Fig. 6). The band was barely detectable in cells treated with
dexamethasone alone. A 100-fold excess of nonradiolabeled consensus GAS
sequence resulted in complete competition of the DNA binding (lane5), although use of a mutated sequence (see above)
resulted in no displacement (data not shown). In addition, binding of
the probe to nuclear protein was displaced when anti-STAT3 serum was
added (lane7) but not when anti-STAT1 or control
nonimmune sera were employed (data not shown).
Figure 6:
Gel mobility shift analysis of proteins
binding to the GAS element. Adipose stromal cells in primary culture
were treated with dexamethasone (250 nM) for 48 h and then
with IL-11 (10 ng/ml) or serum for 30 min. Nuclear extracts were
analyzed by gel shift analysis employing the P-labeled
-288/-269 bp fragment as probe. Nuclear extracts prepared
from cells treated with dexamethasone (250 nM) alone (lane2), IL-11 (10 µg/ml) plus dexamethasone (lane3), or 15% FCS plus dexamethasone (lane4) were incubated with the radiolabeled
-288/-269 bp fragment, and the reaction mixtures were
subjected to polyacrylamide gel electrophoresis in an 8% gel. For
competition, a 100-fold molar excess of the nonradiolabeled
-288/-269 bp fragment (lane5) was added
to the incubation mixture. To determine whether STAT3 is a component of
the protein binding to the GAS, anti-STAT3 serum (2.5 µl) was
incubated with the radiolabeled DNA probe in the absence (lane6) or in the presence of nuclear extracts (lane7). Lane1, free probe electrophoresed
in the absence of nuclear extract. Other details are described under
``Experimental Procedures.'' The arrow indicates the
position of the radiolabeled band.
Southwestern blot
analysis also was performed to further examine the binding of the GAS
probe to nuclear proteins prepared from cells treated with IL-11 plus
dexamethasone. Nuclear proteins were fractionated by SDS-PAGE and
transferred to nitrocellulose membrane. The transferred proteins were
subjected to a denaturation/renaturation cycle and hybridized with the
radiolabeled probe, followed by autoradiography. As shown in Fig. 7, the radiolabeled GAS probe hybridized to a 92-kDa
protein; the intensity of the band was greatly increased when nuclear
extracts from cells treated with IL-11 or serum plus dexamethasone were
employed, as compared with those treated with dexamethasone alone. The
apparent molecular mass of the nuclear protein that bound to the probe
(92 kDa) was similar to that of STAT3(43) . In control
experiments, when the mutated GAS sequence was used as probe, no
hybridization was detected. Additionally, hybridization to the native
probe was also conducted in the presence and absence of a 100-fold
excess of native and mutated sequence. Whereas the former resulted in
displacement of the radiolabeled probe, the latter did not (data not
shown).
Figure 7:
Southwestern blot analysis of proteins
binding to the GAS element. Nuclear extracts (60 µg) were separated
by SDS-PAGE on an 8% gel, and proteins were transferred to a
nitrocellulose membrane. The transferred proteins were subjected to a
denaturation/renaturation process and hybridized to the P-radiolabeled -288/-269 bp fragment followed
by autoradiography. Lane1, nuclear extracts prepared
from cells treated with dexamethasone (Dex; 250 nM)
alone; lane2, nuclear extracts prepared from cells
treated with dexamethasone plus IL-11 (10 ng/ml); lane3, nuclear extracts prepared from cells treated with
dexamethasone in the presence of serum (15% FCS). Other details are
described under ``Experimental
Procedures.''
The GAS Element Is Essential for Expression of P450arom
Fusion Gene Constructs in Adipose Stromal Cells Incubated with
IL-11
Genomic constructs containing the wild type GAS, a
deletion mutation, and a site-directed mutation fused upstream of the
CAT reporter gene were transfected into adipose stromal cells by means
of calcium phosphate coprecipitation. Cells were allowed to recover
overnight in medium containing 10% serum and then deprived of serum for
24 h followed by dexamethasone treatment for 48 h and IL-11 or serum
treatment for 16 h. Cytosolic proteins were prepared, and CAT assays
were performed (Fig. 8). CAT reporter activity was undetectable
in cells treated with dexamethasone alone, but it was readily apparent
in extracts of cells treated with IL-11 or serum plus dexamethasone.
CAT reporter gene expression was lost when the GAS element was deleted
or mutated to the sequence TTCGACTGAA. These results indicate that the
intact GAS element is essential for IL-11- and serum-induced expression
driven by promoter I.4.
Figure 8:
Role of the GAS element in transient
expression of -330/+170 bp/CAT fusion gene construct. Fusion
gene constructs containing the wild-type -330/+170 bp
sequence, the sequence in which the GAS element was deleted
(-270/+170 bp), and the mutated GAS sequence (GASmCAT)
linked to CAT, were transfected into adipose stromal cells in primary
culture. Cells were treated with 250 nM dexamethasone (Dex) for 48 h and incubated with or without IL-11 (10 ng/ml)
and serum (15% FCS) for 16 h. Cells were then harvested, and lysates
were prepared for assay of CAT activity. The products of the CAT
reaction were analyzed by TLC followed by autoradiography. RSV-CAT and
pCAT are positive and negative vector controls, respectively. Other
details are described under ``Experimental
Procedures.''
, interferon-
, and IL-6. However,
responsiveness to IL-6 is established upon addition of soluble IL-6
receptor. Members of this lymphokine family employ a receptor system
involving two different Jak-associated components, gp130 and LIFR
,
or a related
-component(52) . However, the IL-6 receptor
complex includes an
component whose cytoplasmic domain is
apparently not involved in signaling (52) and which can exist in
a soluble form(53) . Recently an
-subunit of the IL-11
receptor complex has been cloned(54) , although this does not
apparently exist in a soluble form. The concentration dependence of the
stimulation of aromatase by IL-6, IL-11, LIF, and OSM is indicative of
high affinity receptor binding, since half-maximal stimulation was
achieved at ligand concentrations of approximately 2 ng/ml
(considerably less in the case of LIF), which corresponds to a molar
concentration of
10
M. Of the
stimulatory lymphokines, the response to OSM in the presence of
dexamethasone was the greatest in terms of aromatase induction and far
exceeded the response to serum. Addition of LIF or IL-11 together with
OSM resulted in no further increase in stimulation, suggesting that
aromatase expression was maximally induced in the presence of OSM and
that all of these lymphokines utilized the same signal transduction
pathway (data not shown). It should be noted that a variety of other
growth factors have no action to stimulate aromatase expression of
adipose stromal cells including epidermal growth factor,
platelet-derived growth factor, fibroblast growth factor, growth
hormone, prolactin, and IGF-1 (data not shown).
has been reported in human
T-lymphocytes(49) . As indicated by blotting with an
anti-phosphotyrosine antibody and by inhibition in the presence of
herbimycin A, this phosphorylation occurred on tyrosine residues
present in the Jak1 kinase. Both gp130 and LIFR
can associate with
and activate at least three members of the Jak family, Jak1, Jak2, and
Tyk2, but utilize different combinations of these in different cells
(50). From the results presented here, it is apparent that Jak1 is the
kinase of choice in human adipose stromal cells. Although there was a
rapid phosphorylation of Jak1 on tyrosine residues, Western blot
analysis utilizing an antibody against Jak1 indicated that there was no
change in the absolute levels of Jak1 throughout this time period of
stimulation.
, shared by these lymphokines(56) . Gel shift analysis,
utilizing a double-stranded oligonucleotide corresponding to the
wild-type GAS sequence in the promoter I.4 region of the P450arom gene
as a probe, indicated binding to a single component. This binding was
barely detectable in control cells but was present within 30 min of
addition of IL-11 to the cells. This binding was competed by addition
of excess nonradiolabeled probe and was also competed upon addition of
anti-STAT3 antibody. These results are indicative that STAT3 can
interact with the GAS element present in the promoter I.4 region of the
P450arom gene upon addition of IL-11 to these cells. This interaction
in turn results in activation of expression, as indicated by
transfection experiments employing chimeric constructs in which the
region -330/+170 bp of the I.4 promoter region was fused
upstream of the CAT reporter gene. The results indicate that deletion
of the GAS sequence, as well as mutagenesis of this sequence, resulted
in complete loss of IL-11- and serum-stimulated expression in the
presence of glucocorticoids.
activation site; STAT, signal transducers and
activators of transcription; IL, interleukin; LIF, leukemia inhibitory
factor; OSM, oncostatin-M; RIPA, radioimmune precipitation buffer;
PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol
acetyltransferase; bp, base pair(s); FCS, fetal calf serum.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.