Estrogen targets genes involved in protein processing ... · results demonstrate that estrogens...

Estrogen responsiveness of uterine genes in ERα(-/-) mice

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Estrogen targets genes involved in protein processing, calcium homeostasis and Wnt

signaling in the mouse uterus independent of ER and ER *

Sanjoy K. Das‡§, Jian Tan‡, Shefali Raja‡, Jyotsnabaran Halder§, Bibhash C. Paria§¶

and Sudhansu K. Dey§

From the ‡Department of Obstetrics & Gynecology, §Department of Molecular & Integrative

Physiology, ¶Department of Pediatrics, Ralph L. Smith Research Center, University of Kansas

Medical Center, Kansas City, KS 66160

*This work was supported in part by NIH grants (ES-07814 to S. K. Das and HD-12304

and HD-29968 to S. K. Dey). Center grants in Reproductive Biology (HD-33994) and Mental

Retardation (HD-02528) at the University of Kansas Medical Center provided access to various

core facilities.

‡To whom Correspondence should be addressed: Departments of Obstetrics &

Gynecology, MRRC 37/3004, University of Kansas Medical Center, 3901 Rainbow Boulevard,

Kansas City, Kansas 66160-7338. Telephone: 913-588-7379; Fax: 913-588-5677; E-mail:

[email protected].

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 11, 2000 as Manuscript M003827200 by guest on February 3, 2019

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1The abbreviations used are: LF, lactoferrin; ERα, estrogen receptor-α; ERβ, estrogen

receptor-β; Bip, immunoglobulin heavy chain binding protein; GRP78, glucose regulated

protein 78 kDa; CalP, calpactin I; CalM, calmodulin; Sik-SP, sik-similar protein; SFRP-2,

secreted frizzled related protein-2; E2, estradiol-17β; 4OHE2, 4-hydroxyestradiol-17β; ICI, ICI

182,780; P4, progesterone; Cyhx, cycloheximide; snoRNP, small nucleolar ribonucleoprotein;

SARP-1, secreted apoptosis related protein-1; rpL7, ribosomal protein L-7.

Running Title: Estrogen responsiveness of uterine genes in ER (-/-) mice

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Estrogen actions in target organs are normally mediated via activation of nuclear

estrogen receptors (ERs). Using mRNA differential display technique, we show, herein,

that estradiol-17 (E2) and its catechol metabolite 4-hydroxy-E2

(4OHE2) can modulate

uterine gene expression in ER (-/-) mice. While administration of E2 or 4OHE2 rapidly

upregulated (4-8 fold) the expression of immunoglobulin heavy chain binding protein

(Bip), calpactin I (CalP), calmodulin (CalM) and Sik similar protein (Sik-SP) genes in

ovariectomized wild-type or ER (-/-) mice, the expression of secreted frizzled related

protein-2 (SFRP-2) gene was downregulated (4 fold). Bip, CalP and CalM are calcium-

binding proteins and implicated in calcium homeostasis, while SFRP-2 is a negative

regulator of Wnt signaling. Bip and Sik-SP also possess chaperone-like functions.

Administration of ICI-182,780 or cycloheximide failed to influence these estrogenic

responses, demonstrating that these effects occur independent of ER , ER or protein

synthesis. In situ hybridization showed differential cell-specific expression of these genes

in wild-type and ER (-/-) uteri. Although progesterone can antagonize or synergize

estrogen actions, it had minimal effects on these estrogenic responses. Collectively, the

results demonstrate that estrogens have a unique ability to influence specific genes in the

uterus not involving classical nuclear ERs.

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Estrogens regulate diverse physiological responses including normal functioning of the

reproductive and cardiovascular systems and bone metabolism (1-3). The uterus is a primary

target for various estrogenic responses during the cycle and pregnancy. In the mouse, estrogen

induces uterine epithelial cell proliferation, while together with progesterone (P4)1 it directs

stromal cell proliferation and epithelial cell differentiation. These coordinated estrogen and P4

interactions prepare the uterus to the receptive state for implantation (reviewed in 4). The

mechanism by which estrogen renders the P4-primed uterus receptive for implantation is not

clearly understood.

Estrogen actions are primarily executed by its binding to nuclear estrogen receptors, ERα

and/or ERβ, which are ligand-inducible transcription factors (5,6). They modulate transcription

of genes by virtue of their binding as hormone receptor complexes to specific DNA sequences

(hormone response elements) in target promoters (5,6). Despite the classical estrogenic actions,

there is increasing evidence that gene transactivation or modulation of cell functions by

estrogens is also mediated independent of nuclear ERs (7-10). In many cells, a myriad of

estrogenic effects occurs rapidly within seconds or minutes. These responses do not require

RNA or protein synthesis and are considered to be mediated by estrogen binding to the plasma

membrane (10-12). For example, increases in intracellular cAMP, calcium influx, inositol

triphosphate and release of prolactin are all attributed to membrane mediated estrogen actions

(10-12). Although the presence of membrane ER has been claimed for more than two decades

(13), the subject is still controversial. However, the identity of a membrane ER has recently

been addressed by transfection studies in Chinese hamster ovary cells using cDNAs for ERα or

ERβ (14). It was shown that functionally active ERα or ERβ is localized in the plasma

membrane and in the nucleus originating from the same mRNA transcript. Furthermore, the

existence of a membrane estrogen binding protein, maxi-K channel, has also been reported


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(15). This channel consists of a regulatory subunit α that confers higher Ca++ sensitivity and

binds to estrogen for channel activation in the presence of a pore-forming β-subunit.

There is a general consensus that rapid actions of estrogens, especially in tissues lacking

nuclear ERs, are the result of a novel mechanism that involves estrogen interaction with a yet

unidentified receptor (7). Using ERα deficient mice and an ER antagonist, we have previously

demonstrated that 4-hydroxy-estradiol-17β (4OHE2), a catechol metabolite of estradiol-17β

(E2), can induce the expression of lactoferrin (LF, an estrogen-responsive gene) in uteri of

ERα(-/-) mice (7). The result suggested that the response is not mediated by ERα or ERβ and

points toward a novel pathway of estrogen actions in the mouse uterus. To better understand the

estrogen actions independent of ERα and/or ERβ, we sought to identify genes that are targets

of E2 and/or 4OHE2. We used mRNA differential display to identify estrogen responsive genes

in ERα(-/-) uteri. Upon identification, the expression patterns of these genes were analyzed in

wild-type and ERα(-/-) uteri exposed to estrogens in the presence or absence of an ER

antagonist. While mRNA expression of four of the genes, glucose-regulated protein-78 kDa

(GRP78)/immunoglobulin heavy chain binding protein (Bip), calpactin I (CalP), calmodulin

(CalM) and SIK-similar protein (Sik-SP) was upregulated, the expression of the secreted

frizzled-related protein-2 (SFRP-2) was downregulated in the uterus by E2 or 4OHE2 in both

the wild-type and ERα(-/-) mice. We also observed that these estrogenic responses are not

influenced by an ER-antagonist ICI-182,780 (ICI), a protein synthesis inhibitor cycloheximide

(Cyhx) or progesterone, suggesting that these effects occur independent of ERα, ERβ, protein

synthesis or progesterone receptor (PR).

Bip, a member of the HSP70 (chaperone) family and a major protein of the endoplasmic

reticulum lumen, is induced under a variety of stress situations (16). It is involved in the

storage of rapidly exchanging Ca++ pool, and correct folding of the newly synthesized proteins


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(17-19). CalP and CalM, two calcium binding proteins, are expressed ubiquitously in

eukaryotic cells, and participate in the modulation of several Ca++-dependent functions

including protein kinase, adenylate cyclase and cyclic nucleotide phosphodiesterase activities

(20-22). CalM can also regulate ER transcriptional activity by its direct association with ER

and interact with myosin light chain kinase to control uterine muscle contraction (23-26). CalP,

a member of the annexin family, plays a role in immunomodulation (27). Sik-SP is conserved

with a gene family nop5/sik1 that encodes components of small nucleolar ribonucleoprotein

complexes. They have an essential role in rRNA processing, and may also be involved in

chaperone-like function (28). SFRP-2 is a modulator of Wnt signaling (29) which is involved in

cell proliferation, differentiation, migration, polarity and cell fate determination during

development (29-31). Wnts interact with cell surface frizzled receptors and were originally

identified as regulators of tissue polarity in Drosophila (30-32). SFRP-2 is a secreted frizzled,

lacking the seven transmembrane and intracellular signaling domains (29,33). Secreted frizzled

proteins are expressed in many cell types during embryogenesis (34,35), and participate in

modulating Wnt-frizzled signaling (29) and apoptosis (36). Our present results showing

estrogen’s influence on uterine expression of genes that are involved in three fundamental

cellular functions such as protein processing, calcium homeostasis and Wnt signaling

independent of the classical ER or PR pathway, suggests diverse mode of estrogen actions.

MATERIALS AND METHODS

Animals—Litter-mate wild-type and ERα(-/-) mice of the same genetic background

(129/J/C57BL/6J) were produced by crossing heterozygous females and males (37). Litter-mate

wild-type and PR(-/-) mice of the same genetic background (129SvEv/C57BL/6) were

produced by crossing homozygous males with heterozygous females (38). In all comparison


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studies, litter-mate wild-type mice were analyzed under similar conditions against ERα(-/-) or

PR(-/-) mice. ERα and PR mutant mice were originally obtained from Dennis B. Lubahn

(University of Missouri, Columbia, MO) and Bert O’Malley (Baylor College of Medicine,

Houston, Texas), respectively. They were housed in the animal care facility at the University of

Kansas Medical Center according to the NIH and institutional guidelines for the care and use of

laboratory animals. Mice were genotyped by PCR analysis of tail DNA. Adult (8-10 weeks old)

mice were ovariectomized and rested for one week before they received any injections.

Injection Schedule--ERα(-/-) and litter-mate wild-type mice were given an injection of oil

(0.1 ml), E2 (250 ng/mouse), 4OHE2 (250 ng/mouse), ICI-182,780 (ICI, 500 µg/mouse) or the

same dose of ICI 30 min prior to steroid injections. Mice were killed 6 h after the last injection.

For temporal studies, mice were killed at 0.5, 1, 2, 6, and 24 h after steroid injections. For

protein synthesis inhibition studies, cycloheximide (Cyhx,100 µg/mouse) was used 30 min

prior to the injection of steroids. PR(-/-) and litter-mate wild-type mice were given an injection

of E2 (250 ng/mouse) and/or P4 (2 mg/mouse). They were killed 6 h after the last injection. All

of the test agents were dissolved in sesame oil and injected (0.1 ml/mouse) subcutaneously.

Differential Display of mRNA--To examine the estrogenic responses on uterine gene

expression independent of ERα, we utilized ERα(-/-) mice. Using differential display

technique, we compared mRNA profiles of uterine samples obtained 6 h after single injections

of either E2 or 4OHE2 with those of oil-treated controls. Differential display technique

followed the protocol as previously described with some modifications (39,40). In brief, 1.0 µg

of DNA-free total RNA was used for reverse transcription (RT) reactions using three different

one-base anchored primers as described earlier (39) with the following modification: LHT11C

(5’-TGCCGAAGCTTTTTTTTTTTC-3’), LHT11G (5’-TGCCGAAGCTTTTTTTTTTTG-3’)

and LHT11A (5’-TGCCGAAGCTTTTTTTTTTTA-3’). The polymerase chain reaction (PCR)


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was performed in a reaction mixture containing 100 µl of the RT product, 1X PCR buffer (10

mM Tris-HCl, pH 8.3; 2.5 mM MgCl2 and 50 mM KCl), 600 µM each of dATP, dTTP, dGTP,

dCTP, 500 µCi/mL 35S-dATP (1200 Ci/mmol, New England Nuclear, Boston, MA), 0.5 µM

of the respective primers: LHT11C, LHT11G or LHT11A, 0.5 µM of the arbitrary primer and

20 units/mL Ampli TaqTM DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). The

arbitrary primers were as described (39), but with the following modification: LHAP1 (5’-

TGCCGAAGCTTGATTGCC-3’), LHAP2 (5’TGCCGAAGCCTTCGACTGT-3’) or LHAP3

(5’-TGCCGAAGCTTTGGTCAG-3’). PCR was performed in a Perkin-Elmer 480

thermocycler using the following cycling parameters: first cycle at 94 oC for 1 min, 40 oC for 4

min and 72 oC for 1 min followed by 35 cycles at 94 oC for 45 sec, 60 oC for 2 min and 72 oC

for 1 min. The amplified cDNAs were separated on a 6% DNA sequencing gel. Differentially

displayed bands of interest were re-amplified by PCR using the appropriate primers and the

reaction conditions as described above. The products were then cloned into the pCR-ScriptTM

SK (+) vector (Stratagene cloning systems, Stratagene La Jolla, CA).

Sequencing of cDNA subclones of the PCR fragments--Double-stranded DNA sequencing

was carried out with either T7 or T3 primers using the SequiTherm long-read cycle sequencing

kit LC (Epicenter Technologies, Madison, Wisconsin). The nucleotide sequences were

analyzed by the BLAST Sequence Similarity Searching Program (blastn) using the GenBank

sequence database of the National Center for Biotechnology Information, National Institute of

Health.

Hybridization Probes--For Northern hybridization, 32P-labeled antisense cRNA probes

were generated using either T7, T3 or SP6 RNA polymerases. For in situ hybridization, sense

and antisense 35S-labeled cRNA probes were generated. A 1.8 kb cDNA fragment


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(EcoRI/SacI) of a mouse cDNA clone for c-fos (41) was subcloned in pSP64 vector at

EcoRI/SacI sites. The clone description for ribosomal protein L-7 (rpL7) cDNA was reported

earlier (42).

Northern blot hybridization--For Northern blot hybridization, total RNA (6.0 µg) was

denatured and separated by formaldehyde/agarose gel electrophoresis, transferred to nylon

membranes and UV cross-linked. Northern blots were prehybridized, hybridized and washed as

described by us (40,42). Quantitation of hybridized bands was analyzed by densitometric

scanning.

In Situ Hybridization--In situ hybridization was performed as previously described (42).

Frozen uterine sections (10 µm) were fixed in 4% paraformaldehyde in PBS for 15 min at 4oC.

Following fixation, sections were prehybridized and hybridized to 35S-labeled antisense cRNA

probes for 4 h at 45 oC. As negative controls, uterine sections were hybridized with the 35S-

labeled sense probes. RNase-A resistant hybrids were detected within 3-7 days of

autoradiography. The slides were post-stained with hematoxylin and eosin.

Competitive PCR--The quantitation of mRNAs by competitive PCR was previously

described by us (7). In brief, the competitor templates were generated by introducing a

nonspecific DNA fragment into a mouse target cDNA clone. Specifically, a 185 bp blunt-

ended fragment (SspI), obtained from pGEM7Zf(+) vector, was inserted into the cDNA clones

for CalP at the SmaI site, for CalM and SFRP-2 at the StuI site, and for Bip at the SspI site.

These modified cDNA templates were used as competitors to carry out the competitive PCR for

the respective genes. The following primers were used for RT-PCR: 5’-GCG CTG AAG TCA

GCC TTA TC-3’ (nts 263-282, sense) and 5’-GGT CCC CTT TGA CCT CTT TC-3’ (nts 742-

761, antisense) for CalP mRNA (GenBank Accession # M14044); 5’-GCA CCA TTG ACT

TCC CAG AG-3’ (nts 211-230, sense) and 5’-GGG CTT CTG ACA TCA GCT TC-3’ (nts


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597-616, antisense) for CalM mRNA (GenBank Accession # X61432); 5’-TTG GCT TAT

ACC GTG CAC TT-3’ (nts 1487-1506, sense) and 5’-TAT TTG AGG GCA TCA TGC AA-3’

(nts 1764-1783, antisense) for SFRP-2 mRNA (GenBank Accession # U88567); 5’-CCG AGT

GAC AGC TGA AGA CA-3’(nts 1622-1641, sense) and 5’-GCC ACT TGG GCT ATA GCA

TT-3’ (nts 2184-2203, antisense) for Bip mRNA (GenBank Accession # D78645). The internal

primers: 5’-CTG GGG ACT GAC GAG GAC TC-3’ (nts 368-387, sense) for CalP; 5’-AGT

GCG GCA GAA CTG CGC CA-3’ (nts 330-349, sense) for CalM; 5’-GCC CTC ATG AGC

TCT GAC CA-3’ (nts 1681-1700, sense) for SFRP-2; 5’-GGC TGG AAA GCC ACC AGG

AT-3’ (nts 1906-1925, sense) for Bip were used for Southern blot hybridization of the RT-PCR

amplified products. For rpL7, primers used for RT-PCR and Southern hybridization were same

as described earlier (7). Quantitation of band intensity on the autoradiogram was achieved by

densitometric analysis. The ratio of band intensities for the competitor and the target cDNA

was calculated for each sample and plotted against the amounts of the competitor. The

efficiency of the RT reaction was controlled by measuring the levels of rpL7 mRNA in each

sample, and were similar in all samples (~4.0 X 107 copies/µg of total RNA).

RESULTS

Estradiol and catecholestradiol regulate gene expression in ER (-/-) uteri--We

previously demonstrated that the expression of the LF gene, normally induced by E2 in the

mouse uterus, is upregulated within 6 h after an injection of 4OHE2, but not E2, in ERα(-/-)

uteri. Further, this response was not inhibited by prior administration of an ER antagonist, ICI,

and was accompanied by early estrogenic responses, such as uterine water imbibition and

macromolecular uptake (7). These results suggested that estrogens execute some uterine effects

that are independent of ERα and/or ERβ (7). To examine whether estrogen can also modulate


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other genes in the uterus in the absence of ERα, we investigated the effects of E2 or 4OHE2 on

uterine gene expression in ERα(-/-) mice using the mRNA differential technique. We analyzed

twenty-six PCR amplified products that were displayed differentially in uterine RNA samples

obtained from ovariectomized ERα(-/-) mice 6 h after an injection of oil, E2 or 4OHE2.

Cloning, sequencing and expression studies led to the identification of five authentic cDNA

clones whose corresponding genes showed either upregulation or downregulation after estrogen

treatments (Fig. 1). Among the five genes, the expression of Bip, CalP, CalM and Sik-SP was

upregulated, while that of SFRP-2 was downregulated by E2 or 4OHE2. It is to be noted that

after 4OHE2 treatment, a band was detected on the differential display gel within close

proximity but not of the same size of the SFRP-2 band as observed in the oil-treated

sample. However, cloning, sequencing and Northern blot hybridization revealed this band

to be an artifact.

Differentially displayed genes are rapidly modulated by estrogen in wild-type or ER (-/-)

uteri and are unresponsive to antiestrogen treatment--Although several genes were

differentially displayed by uterine RNA samples of ovariectomized ERα(-/-) mice after

estrogen treatment, we wanted to confirm their authenticity, differential responses to estrogens

and an ER antagonist ICI. We examined the levels of Bip, CalP, CalM, Sik-SP and SFRP-2

mRNAs in ovariectomized wild-type mice 6 after an injection of oil, E2 or 4OHE2 with or

without ICI by Northern hybridization (Fig. 2). We observed very low levels of uterine Bip,

CalP, CalM and Sik-SP mRNAs after an injection of oil. However, an injection of E2 or 4OHE2

increased the levels of these mRNAs 4-8 fold by 6 h. Treatment of mice with ICI prior to the

injections of estrogens failed to show any effects. In contrast, high levels of SFRP-2 mRNA


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were detected in oil-treated uterine samples, and these high levels were readily downregulated

(4 fold) by estrogen treatments. Again, ICI did not antagonize these effects.

To examine the temporal expression patterns of these genes by E2 or 4OHE2, Northern

blot hybridization was performed using uterine RNA samples isolated at different times (0.5, 1,

2, 6 and 24 h) after an injection of E2 or 4OHE2 in ovariectomized wild-type mice (Fig. 3).

RNA samples from oil-treated uterine samples at 6 h served as controls. The effects of

estrogens on the expression of these five genes were compared with that of c-fos, a known

estrogen dependent early-inducible gene in the rodent uterus (43). As expected, the levels of

Bip, CalP, CalM and Sik-SP mRNAs remained low in vehicle-treated uterine samples.

However, an injection of E2 (Fig. 3A) or 4OHE2 (Fig. 3B) rapidly upregulated the expression of

these four genes and c-fos within 1-2 h. The levels of BIP mRNA peaked at 1 h and remained

high through 6 h, while those of CalP and CalM reached highest levels at 6 h. As observed

previously (43), the induction of c-fos mRNA by estrogen was very rapid and transient,

reaching its peak at 1 h followed by a rapid decline. In general, the induction level of these

genes by E2 or 4OHE2 was 4-8 fold at 6 h. In sharp contrast, the levels of SFRP-2 mRNA were

high in oil-treated ovariectomized uteri, but declined rapidly after an E2 or 4OHE2 injection,

reaching its lowest levels by 1 h.

Although the results of differential display suggested estrogen modulation of these five

genes in the ERα(-/-) uterus with very low levels of ERβ (44), the extent of their

responsiveness to estrogen or the participation of ERβ in these estrogenic responses could not

be ascertained. We used a quantitative RT-PCR technique to address these questions, because

of the limited availability of uterine RNA from ERα(-/-) mice. This technique uses gene

specific competitive templates to measure mRNA levels of choice, and was used to measure the

mRNA levels of differentially displayed genes in ERα(-/-) uteri after exposure to estrogens. As


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shown in Tables I-III, an injection of E2 or 4OHE2 significantly increased the uterine levels of

Bip (≈3-16 fold), CalP (≈5-7 fold) and CalM (≈4-6 fold) mRNAs in ovariectomized ERα(-/-)

mice within 6 h. In contrast, similar treatments with estrogens drastically reduced the levels (8-

10 fold) of uterine SFRP-2 mRNA (Table IV). ICI, which neutralizes ERα and ERβ functions

(45), failed to antagonize these estrogenic responses (Tables I-IV), suggesting that ERβ is also

not involved in these responses. These results suggest that estrogens can modulate expression

of certain genes in the mouse uterus independent of the classical ERs.

Effects of E2 or 4OHE2 on uterine gene expression are independent of protein synthesis--

The rapid responses of these genes to estrogens independent of ERα and ERβ led us to examine

whether these responses required new protein synthesis. Uterine RNA was analyzed by

Northern hybridization. As shown in Fig 4, the levels of Bip, CalP, CalM and Sik-SP mRNAs

remained low after an injection of oil or Cyhx alone. Although a single injection of E2, as

expected, up-regulated the mRNA levels of these genes, a prior treatment with Cyhx failed to

alter the estrogen induced responses. Similarly, the downregulation of SFRP-2 mRNA levels by

estrogen was also not affected by Cyhx pretreatment. The effects of Cyhx on 4OHE2-induced

modulation of these various genes were similar to those of E2 (data not shown). An

administration of the same dose of Cyhx (100 g) 30 min prior to an injection of estrogen

was shown to block uterine amino acid incorporation into protein in the rat (46), or

uterine c-myc expression in the mouse during the early phase (47).

Differentially displayed genes are spatially expressed by E2 and 4OHE2 in wild-type and

ER (-/-) uteri--To determine whether estrogen modulates uterine gene expression in a cell-type

specific manner, in situ hybridization was performed on uterine sections obtained from


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ovariectomized wild-type or ERα(-/-) mice 6 h after receiving an injection of oil, E2 or 4OHE2

with or without ICI. The accumulation of Bip, CalP, CalM and Sik-SP mRNAs was low to

undetectable in wild-type or ERα(-/-) uteri after an injection of oil (Figs. 5 and 6). However, an

injection of E2 or 4OHE2 in wild-type mice showed increased accumulation of these mRNAs

predominantly in luminal and glandular epithelia with low levels in the stroma (Figs. 5 and 6).

In contrast, similar treatments induced these genes differentially in ERα(-/-) uteri (Figs. 5 and

6). For example, E2 or 4OHE2 primarily induced the expression of Bip mRNA in stromal cells

(Fig. 5), CalP in epithelial cells (Fig. 5) and CalM in both epithelial and stromal cells (Fig. 6).

Interestingly, Sik-SP mRNA accumulation was primarily detected in epithelial cells by E2, but

in both stromal and epithelial cells by 4OHE2 (Fig. 6). In contrast, distinct accumulation of

SFRP-2 mRNA was observed in stromal cells of ovariectomized oil-treated wild-type and

ERα(-/-) uteri (Fig. 7), while an injection of E2 or 4OHE2 dramatically downregulated its

expression in these cells (Fig. 7). Pretreatment of mice with ICI did not influence the levels or

the pattern of expression for all of these genes in response to estrogens either in wild-type or

ERα(-/-) mice (Figs. 5, 6 and 7). Furthermore, the responses to ICI alone in the wild-type and

ER(-/-) mice were similar to those of vehicle-treated controls (Figs. 5-7). The expression of

these genes was specific, since hybridization with corresponding sense cRNA probes did not

show any positive signals (data not shown).

Es tr ogen dependent modulation of uter ine gene expres sion is not alter ed by pr ogester one--

Since estrogen interacts with P4 s ynergis tically or antagonis tically, w e s ur mis ed that the estrogenic

ef fects on these genes could be modulated by P4. Thus, w e compar ed the eff ects of P4 on uter ine

expr ess ion of these genes in wild- type mice with those in PR(- /-) mice. Ovariectomized wild- type

or P R(- /- ) mice r eceived an injection of oil, P 4 or P4 plus E2. M ice w ere killed 6 h later and uterine


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RN A was analyzed by Norther n hybridization. As shown in F ig. 8, levels of Bip and Sik- SP

mRNA s w er e low in vehicle treated uteri, w hile those of SFRP-2 w as high in both the wild- type

and PR(- /-) mice. As expected, an injection of E2 upr egulated the levels of Bip and Sik- SP mRN As

and dow nr egulated the levels of SF RP- 2 mRN A in these mice. In contr as t, tr eatment with P4 alone

or w ith E2 f ailed to s how any noticeable eff ects on the levels of Sik- SP and SFRP-2 mRN As in

wild-type or PR(- /-) uter i. However , uterine Bip expres sion was modestly upregulated by P4 alone

in w ild-type, but not in PR(- /-) mice, although P 4 did not antagonize or s ynergize E2-induced Bip

expr ess ion. Thes e res ults suggest that P4 alone can inf luence this gene via activation of PR, but

does not inf luence its r esponsiveness to estrogen. O ur initial s tudies als o s howed that P 4 is

inef fective in influencing the expres sion of CalP or CalM ( data not s hown) .

DISCUSSION

Many of the diverse biological functions of estrogens are the result of their direct

interactions with nuclear ERs. There is now evidence for specific functions and gene

expression in the target organs elicited by estrogens independent of ERα and/or ERβ (7,44).

For example, 4OHE2, but not E2, can induce LF expression, water imbibition and

macromolecular uptake in ERα(-/-) uteri, and these responses are not neutralized by ICI (7).

The signaling system involved in these responses is not yet understood. The present

investigation demonstrates that not only 4OHE2, but also E2 can modulate a group of genes in

the uterus that are involved in protein processing, calcium homeostasis and Wnt signaling

without involving classical ERs, PR and nascent protein synthesis. These unique uterine

estrogenic responses point toward the concept that certain fundamental estrogenic functions,

such as protein processing, calcium homeostasis and Wnt signaling in the target organ are

retained in the absence of classical ERs. Whether orphan or yet unidentified nuclear receptors


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are involved in these responses remains unknown. Nonetheless, our present results are

intriguing and likely to stimulate further research in identifying the signaling mechanism for

these responses.

Although there are numerous examples of estrogen upregulation of various genes in the

uterus, very few reports of estrogen induced downregulation of uterine gene expression are

available. Our present results showing upregulation of Bip, CalP, CalM and Sik-SP mRNAs,

and downregulation of SFRP-2 mRNA in the uterus by estrogens independent of classical ERs,

PR or protein synthesis are unique and suggest that estrogen actions are more complex than

currently recognized. Although estrogen induction of these genes is independent of ERs, their

differential cell-specific expression between the wild-type and ERα(-/-) uteri suggests an

interaction between this novel pathway and classical ERs in specifying cell-specific expression.

Epithelial-mesenchymal “cross-talk” is important for normal uterine functions and gene

expression (48). It is possible that this “cross-talk” is impaired or absent in ERα(-/-) uteri

causing differential cell-specific gene expression.

In adult mice, estrogens produce a biphasic uterine response (49,50). The immediate early

responses occur within 6 h of estrogen administration, and water imbibition and

macromolecular uptake are two predominant characteristics. The late or growth responses

occur by 18-30 h and are characterized by hyperplasia and hypertrophy. Our present

observation of rapid modulation of genes after injection of estrogens suggests that specific

early estrogenic responses are independent of classical ERs or new protein synthesis. However,

these early responses could be important for the onset of the late growth phase that is ERα

dependent. The manifestation of these early responses with the absence of the growth phase in

ERα(-/-) mice suggests the lack of the machinery for the growth phase. The induction of

immediate early genes (c-fos, c-jun and c-myc) by short-acting estrogens is not adequate to


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stimulate DNA synthesis in the rodent uterus (51). Thus, it appears that the mitogenic

stimulation requires further changes that depend on prolonged estrogen action. A “cross-talk”

between the non-classical and classical actions of steroid hormones is described by

Katzenellenbogen (52). For example, protein kinase activators enhance the ERα transcriptional

activity. There is also evidence that IGF-1 and agents that raise intracellular cAMP also

stimulate ER phosphorylation and activation (53). Estrogen activation of the traditional

“genomic” pathway involves mRNA and protein synthesis, whereas rapid estrogenic responses

occuring via a non-traditional pathway are believed to be mediated via membrane receptor and

do not require new protein synthesis. However, the identity of the putative membrane receptor

is still controversial. Our observation of rapid estrogenic modulation of uterine gene expression

independent of protein synthesis and classical ERs are also characteristics of an early response.

Defining the signaling mechanism of the early estrogenic responses may have clinical

significance in distinguishing the beneficial effects (cardiovascular and neurological

protections) of estrogens from their long-term detrimental (carcinogenic consequences) effects.

Rapid responsiveness of uterine Bip and Sik-SP to estrogens could be physiologically

important. The late estrogen action primarily involves uterine growth which requires correct

folding and functioning of a variety of newly synthesized proteins. Because of Bip’s

involvement in folding and translocation of nascent proteins within the endoplasmic reticulum,

one of the early functions of estrogen could be to prepare the uterine environment for protein

processing for the late phase. A chaperone-like role for Bip was recently reported in the rat

uterus during decidualization (54). Sik-SP could also be involved in similar functions, because

of its chaperone-like functions. We suggest that genes regulated by estrogen independent of

nuclear ERs could be linked with the ER dependent late estrogenic effects.


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Calcium plays a major role in mediating estrogen signaling (55,56) and it can act as a

second messenger to induce Bip in monocytes (57). Cellular calcium homeostasis depends on

the concerted efforts of calcium binding proteins. Since Bip, CalP and CalM all bind calcium

and are regulated in the uterus by estrogen, it is possible that calcium is involved in regulating

these genes. The spatiotemporal regulation of uterine Bip, CalP and CalM by estrogen suggests

that these genes function in a coordinated manner. In rodents, uterine CalM levels increase

during pregnancy and after estrogen treatment (58). Furthermore, an intrauterine injection of

CalM antagonist (chlorpromazine) inhibits implantation in the rat (59), suggesting its role in

this process. Since estrogen is an absolute requirement for implantation in mice, it is possible

that one of estrogen’s action in implantation is to induce CalM via a non-ER pathway. CalP is

localized in the syncytiotrophoblast cells in the developing human placenta and possesses Fc

gamma receptor activity, suggesting its role in immunomodulation (60). Uterine CalP

expression by estrogen may have a role in local immunomodulation.

The uterine regulation of SFRP-2 is an interesting observation, since very few genes are

known to be downregulated by estrogen (61-65). To our knowledge, this is a gene that is

abundantly expressed in quiescent uterine stromal cells, but is downregulated by estrogens.

Since SFRP-2 negatively regulates Wnt functions, it is envisioned that its downregulation by

estrogen allows Wnt-frizzled signaling to execute specific uterine functions. Wnt ligands

participate in mesenchymal-epithelial interactions (66), and uterine expression of Wnt ligands

(Wnt-4, Wnt-5a and Wnt-7) is tightly regulated during the estrous cycle by estrogen and/or P4

(67,68). Since Wnts regulate cellular proliferation, differentiation and/or reorganization, we

suggest that they act as estrogen mediated transducers of these events in the uterus. Bip could

also be a part of this system, since Wnt-1 interacts with Bip for its secretion from the cell (69).

Wnts are involved in two signaling pathways. They can activate β-catenin which modulates


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transcription of specific target genes in the nucleus. They can also stimulate increases in

intracellular Ca++ or PKC activity via activation of pertussis toxin sensitive G-proteins.

Whether these Wnt signaling pathways are operative in the uterus remains to be examined.


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FIGURE LEGENDS

Fig. 1. Differential display of uterine mRNAs in ER (-/-) mice after injections of oil, E2 or

4OHE2. Three different uterine total RNA samples isolated from ovariectomized ERα(-/-) mice

6 h after injections of oil, E2 (250 ng/mouse) or 4OHE2 (250 ng/mouse) were compared by

differential display. Reverse transcription reaction was performed using 5.0 µg of total RNA in

the presence of one base anchored modified primers: LHT11G, LHT11C or LHT11A. Each

primer-driven RT products were PCR amplified using the corresponding RT primer together

with an arbitrary primer: LHAP1, LHAP2 or LHAP3 (Liang et al . 1994). The PCR amplified

cDNA fragments were obtained by a pair of primers: (a) LHAP3/LHT11G, (b)

LHAP3/LHT11A, (c) LHAP2/LHT11C and (d) LHAP3/LHT11A. Arrows indicate cDNA

bands displayed differentially. These experiments were repeated twice with independent RNA

samples and similar results were obtained.

Fig. 2. E2 or 4OHE2 regulates uterine expression of Bip, CalP, CalM, Sik-SP and SFRP-2

mRNAs in ovariectomized wild-type mice and this expression is unresponsive to ICI.

Adult ovariectomized mice were given an injection of oil, E2 (250ng/mouse), 4-OH-E2

(250ng/mouse), ICI (500 µg/kg) or ICI 30 min before an injection of E2 or 4OHE2 and killed 6

h after the last injection. Total uterine RNA (6 µg) pooled from 5-7 mice was used for each

group. Autoradiographic exposures were 6 h for SFRP-2 and Sik-SP, 3 h for Bip, CalP and

CalM, and 2 h for rpL7. These experiments were repeated twice with independent RNA

samples and average values with range of responses from two experiments are shown in

histograms. Fold changes in mRNA levels were calculated with respect to oil after normal-

ization with rpL7 mRNA levels.


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Fig. 3. Temporal effects of E2 (A) or 4OHE2 (B) on uterine expression of Bip, CalP, CalM,

SFRP-2, Sik-SP, c-fos and rpL7 mRNAs in ovariectomized wild-type mice. Adult ovariecto-

mized mice were given a single injection of E2 (250ng/mouse) or 4OHE2 (250 ng/mouse) and

killed at the times indicated. Mice injected with oil and killed 6 h later served as a control.

Total uterine RNA (6 µg) pooled from 5-7 mice was used for each group. Autoradiographic

exposures were 6 h for SFRP-2, Sik-SP and c-fos, 3 h for Bip, CalP and CalM and 2 h for rpL7.

These experiments were repeated two times with independent RNA samples and average values

with range of responses from two experiments are shown in histograms. Fold changes in

mRNA levels were calculated with respect to oil and were normalized against rpL7 mRNA

levels.

Fig. 4. Effects of Cycloheximide (Cyhx) on uterine expression of Bip, CalP, CalM, Sik-SP

and SFRP-2 mRNAs in response to E2 or 4OHE2 in ovariectomized wild-type mice. Adult

ovariectomized mice were given a single injection of oil, E2 (250ng/mouse), 4-OH-E2

(250ng/mouse), Cyhx (100 µg/mouse), or the same dose of Cyhx 30 min before the injection of

E2 or 4OHE2 and killed 6 h after the last injection. Total uterine RNA (6 µg) pooled from 5-7

mice was used for each group. Autoradiographic exposures were 6 h for SFRP-2 and Sik-SP, 3

h for Bip, CalP and CalM, and 2 h for rpL7. These experiments were repeated twice with

independent RNA samples and similar results were obtained.

Fig. 5. In situ hybridization of Bip and CalP genes in uteri of ovariectomized wild-type

and ER (-/-) mice after exposure to E2, E2 plus ICI, 4OHE2 or 4OHE2 plus ICI. Adult

ovariectomized mice rested for two weeks were used. Mice were given a single injection with

oil, ICI (20 mg/kg), E2 (250 ng/mouse), 4OHE2 (250 ng/mouse), or the same dose of ICI 30


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min before the injection of E2 or 4OHE2 and they were killed 6 h after the last injection. Frozen

sections (10 µm), fixed in paraformaldehyde, were mounted onto glass slides, prehybridized

and hybridized with 35S-labeled sense (data not shown) or antisense cRNA probes.

RNase-A-resistant hybrids were detected after 2-7 days of autoradiography. Dark-field

photomicrographs of uterine sections are shown at 100X. le, luminal epithelium; ge, glandular

epithelium; s, stroma; and myo, myometrium. These experiments were repeated three times

with 3-4 mice in each group and similar results were obtained.

Fig. 6. In situ hybridization of CalM and Sik-SP genes in uteri of ovariectomized wild-

type and ER (-/-) mice after exposure to E2, E2 plus ICI, 4OHE2 or 4OHE2 plus ICI.

Injection schedules and the doses of various agents were same as described in the figure legend

5. Frozen sections (10 µm), fixed in paraformaldehyde, were mounted onto glass slides,

prehybridized and hybridized with 35S-labeled sense (data not shown) or antisense cRNA

probes. RNase-A-resistant hybrids were detected after 2-7 days of autoradiography. Dark-field




Fig. 7. In situ hybridization of SFRP-2 gene in uteri of ovariectomized wild-type and

ER (-/-) mice after exposure to E2, E2 plus ICI, 4OHE2 or 4OHE2 plus ICI. Injection

schedules and the doses of various agents were same as described in the figure legend 5. Frozen

sections (10 µm), fixed in paraformaldehyde, were mounted onto glass slides, prehybridized

and hybridized with 35S-labeled sense (data not shown) or antisense cRNA probes.

RNase-A-resistant hybrids were detected after 2-7 days of autoradiography. Dark-field


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Fig. 8. Effects of P4 on estrogen-induced uterine expression of Bip, Sik-SP and SFRP-2 in

wild-type and PR(-/-) mice. Adult ovariectomized mice were given an injection of oil, E2

(250ng/mouse), P4 (2 mg/mouse) or the same doses of E2 plus P4. Total uterine RNA (6 µg)

pooled from 5-7 mice was used for each group. Autoradiographic exposures were 6 h for

SFRP-2 and Sik-SP, 3 h for Bip, and 2 h for rpL7. These experiments were repeated twice with

independent RNA samples and similar results were obtained.


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TABLE ILevels of uterine Bip mRNA in ovariectomized ER (-/-) mice after treatment with estrogens

and/or ICI at 6 h

Injection schedules and the doses of various agents were same as described in theMaterials and Methods. Data were calculated from triplicate set of experiments. Fold increaseswere calculated comparing against the values obtained with those treated with oil. Values withdifferent superscript letters are statistically different (p<0.05, ANOVA followed by Newman-Keul's multiple range test).___________________________________________________________________________Treatments Levels of mRNA mRNA copies Fold

(fg/µg total RNA) (molecules/µg total RNA) Increase____________________________________________________________________________

Oil 32.9 ± 2.9a 8423 ± 145a 1

ICI 182,780 29.6 ± 3.3a 7580 ± 139a 0.9

E2 440.9 ± 19.3b 112868 ± 10131 13.4

E2 + ICI 496.8 ± 22.6b 127187 ± 9278b 15.1

4OHE2 523.1 ± 33.1b 133925 ± 11313b 15.9

4OHE2 + ICI 460.6 ± 36.1b 117922 ± 11379b 14.0

____________________________________________________________________________


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TABLE IILevels of uterine CalP mRNA in ovariectomized ER (-/-) mice after treatment with estrogens

and/or ICI at 6 h

See the legend to the TABLE I for details. Values with different superscript letters arestatistically different (p<0.05, ANOVA followed by Newman-Keul's multiple range test).____________________________________________________________________________Treatments Levels of mRNA mRNA copies Fold


Oil 29.3 ± 4.6a 6015 ± 156a 1

ICI 182,780 33.2 ± 3.2a 6616 ± 140a 1.1

E2 161.2 ± 25.2b 33082 ± 1331b 5.5

E2 + ICI 181.7 ± 39.0b 37293 ± 1399b 6.2

4OHE2 205.1 ± 37.5b 42105 ± 1415b 7.0

4OHE2 + ICI 199.2 ± 41.0b 40902 ± 1510b 6.8

____________________________________________________________________________


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TABLE IIILevels of uterine CalM mRNA in ovariectomized ER (-/-) mice after treatment with estrogens

and/or ICI at 6 h



Oil 38.9 ± 5.6a 7979 ± 156a 1

ICI 182,780 46.7 ± 7.2a 9574 ± 140a 1.2

E2 175.1 ± 12.2b 35905 ± 1082b 4.5

E2 + ICI 202.3 ± 39.0b 41490 ± 1052b 5.2

4OHE2 233.4 ± 19.5b 47874 ± 1215b 6.0

4OHE2 + ICI 221.7 ± 41.0b 45480 ± 1051b 5.7

____________________________________________________________________________


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TABLE IVLevels of uterine SFRP-2 mRNA in ovariectomized ER (-/-) mice after treatment with

estrogens and/or ICI at 6 h


(fg/µg total RNA) (molecules/µg total RNA) Decrease____________________________________________________________________________

Oil 232.5 ± 5.3a 33415 ± 1316a 1

ICI 182,780 211.4 ± 9.6a 30377 ± 1140a 1.1

E2 22.1 ± 1.1b 3182 ± 134b 10.5

E2 + ICI 24.5 ± 2.9b 3517 ± 118b 9.5

4OHE2 26.7 ± 1.3b 3840 ± 120b 8.7

4OHE2 + ICI 25.3 ± 2.3b 3632 ± 131b 9.2

_________________________________________________________________________

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K. DeySanjoy K. Das, Jian Tan, Shefali Raja, Jyotsnabaran Halder, Bibhash C. Paria and Sudhansu

β and ERαsignaling in the mouse uterus independent of EREstrogen targets genes involved in protein processing, calcium homeostasis and Wnt

published online July 11, 2000J. Biol. Chem.

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Estrogen targets genes involved in protein processing ... · results demonstrate that estrogens...

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