Archives of Medical Research 32 (2001) 263–267

0188-4409/01 $–see front matter. Copyright © 2001 IMSS. Published by Elsevier Science Inc.PII S0188-4409(01)00289-2

ORIGINAL ARTICLE

Expression of TCF, TPF/YY1, and the Sp Family Transcription Factors in Rabbit Endometrium Throughout Pregnancy

Joel Arias, Antonia Hernández, Arturo Barrón and Ivone Castro

Departamento de Bioquímica y Biología Molecular, Instituto Nacional de Perinatología, Mexico City, Mexico

Received for publication September 29, 2000; accepted March 26, 2001 (00/143).

Background.

TCF, TPF/YY1, and the Sp family are specific transcription factors thatbind sequences found within the uteroglobin (UG) gene promoter region that are neces-sary for transcription. To date, UG gene expression and regulation

in vivo

are not fully un-derstood. The purpose of this study was to assess the expression patterns of these factorsin the rabbit endometrium throughout pregnancy.

Methods.

Endometrial nuclear extracts were obtained from female rabbits on days 0, 3, 5,7, 15, and 28 after mating. Transcription factor expression was assessed by DNA-proteinbinding assays using endometrial nuclear proteins and specific oligonucleotides. Bandshifts were observed on 4% acrylamide gels and analyzed by densitometry.

Results.

The expression patterns of the transcription factors analyzed here differed, asTPF/YY1 and Sp3/SpR-2 were expressed constitutively while TCF and Sp1 showed vari-able expression patterns throughout pregnancy.

Conclusions.

Our results suggest that UG gene expression in the intact pregnant rabbit iscontrolled by two constitutive and two regulated factors, and that the DNA-binding sitesare located at the TATA box and the GT1 sites within the gene promoter. © 2001 IMSS.Published by Elsevier Science Inc.

Key Words:

Transcription factors, Uteroglobin, Rabbit.

Introduction

Uteroglobin (UG) is a small 16 kD protein. Although itsphysiologic function remains unknown (1), several studieshave evidenced that this protein is involved in several func-tions such as the following: a) enhancement of blastocystgrowth before implantation (2); b) progesterone transporta-tion and protection for the developing embryo from proges-terone toxicity (UG shows high affinity for this steroid)(3,4); c) immunomodulation during the implantation periodin rabbits (5); d) chemotaxis and phagocytosis inhibition inmonocytes and neutrophils (6,7), and e) being a phospholi-pase A2 (PLA2) inhibitor (8–9), it may play a role in main-taining uterus quiescence during pregnancy (10). A UG-like

secretory protein has been found in Clara cells of the lung,which also inhibits PLA2 activity and may function as asurfactant hydrolysis regulator (11).

The UG gene has been cloned, sequenced, and character-ized (12,13) and different steroid hormones regulate its ex-pression in a tissue-specific manner. UG expression is regu-lated by progesterone in the endometrium, by estrogens inthe oviduct, by glucocorticoids in the lung, and by andro-gens in the epididymis (14).

Tissue expression is regulated by different nucleotide se-quences within the gene, such as steroid response elements(SRE) and specific transcription factor binding sites locatedwithin or distant from the promoter that are essential fortranscription. Different researchers have characterized thesesites by footprinting techniques, electrophoretic mobilityshift assay (EMSA), and by reporter gene transfections intransient expression experiments (15).

Previous EMSA experiments using the OcTATA oligo-nucleotide (that contains the rabbit [

Oryctolagus cuniculus

]UG TATA box region sequence) and Ishikawa nuclear ex-

Address reprint requests to: Ivone Castro, M.D., Ph.D., Departamentode Bioquímica y Biología Molecular, Instituto Nacional de Perinatología,Montes Urales 800, Col. Lomas Virreyes, 11000 México, D.F., México.Tel.: (

�

525) 520-9900, ext. 341; FAX: (

�

525) 520-0034; E-mail: jicastro@mail.ssa.gob.mx

264

Arias et al./ Archives of Medical Research 32 (2001) 263–267

tract cultures (human endometrium adenocarcinoma) re-vealed the presence of two transcription factors called TCF(TATA core factor) and TPF (TATA palindrome factor)(16). The TPF sequence was determined 2 years later byKlug (1996) (17), who found that it corresponded to tran-scription initiation factor YY1, referred to as TPF/YY1 inthis article. Sp1 and SP3/SpR-2 are other characterized tran-scription factors involved in activating or repressing UGgene transcription by interaction with specific GT1 (regionVI of the UG promoter) sequences (18–20).

The majority of transcription factor studies have beenperformed

in vitro

on mammalian and

Drosophila

cell lines,while very few studies have been performed

in vivo

in whichthe cells are in their natural hormonal environment. In 1996,Pérez et al. (21) demonstrated the presence of two UG geneTATA box binding factors called TRBP1 and TRBP2 in en-dometrial cell nuclear extracts from pseudo-pregnant rabbits.The authors suggested that in the endometrium, progester-one-induced UG gene expression is mediated by both gen-eral and tissue-specific factors. The molecular weights ofthese transcription factors differed from those of TPF/YY1and TCF in Ishikawa cells. However, it was not establishedwhether TRBPs and TPF/TCF are related proteins.

Uterine fluid UG concentrations increase importantlyduring early pregnancy (implantation period) and decreasedramatically thereafter. There are no reports in the medicalliterature describing the expression patterns of these tran-scription factors in rabbit endometrium under normal hor-monal conditions throughout pregnancy. For this reason, weassessed the expression patterns of transcription factors thatbind specifically to the TATA box and GT1 sequences lo-cated within the UG gene promoter regions by EMSA, us-ing nuclear extracts from endometrial cells sampled directlyfrom pregnant rabbits.

Materials and Methods

Preparation of Ishikawa cell nuclear extracts.

A total of 80culture plates (100-mm diameter) (Falcon, Becton DickinsonLabware, Franklin Lakes, NJ, USA) containing Ishikawacells were left to grow to confluence in D-MEM culture me-dium supplemented with 10% fetal calf serum, 200 mML-glutamine, 40 IU/mL insulin, and antibiotics. All reagentswere purchased from GIBCO-BRL (Rockville, MD, USA).

Cell monolayers were recovered mechanically usingbuffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, and 1mM EDTA (Sigma Chemical Co., St. Louis, MO, USA).Nuclei were extracted using the technique described by Kluget al. (16) with minor modifications. A protease inhibitorcocktail (0.2 mM PMSF, 0.5 mM spermidine, 0.15 mMspermine, 0.1 mM aprotinine, and 0.2 mg/m trypsin inhibi-tor) (GIBCO-BRL) was added to the hypotonic buffer. Ali-quots were obtained from the total nuclear extract and storedat

�

70

�

C until analyzed.

Extraction of nuclei from endometrial cells of pregnant rab-bits.

Adult New Zealand nonpregnant female rabbitsweighing 3–4 kg were used for the experiment, kept underadequate temperature, light, and humidity conditions, andfed

ad libitum

with Purina for rabbits. The animals weremated with males previously tested for fertilization capabil-ity. Female rabbits were sacrificed in a CO

2

injection cham-ber on day 0 (unmated rabbits) and on days 3, 5, 7, 15, and28 after mating to extract the uterus. A total of three rabbitswere sacrificed at a time. Both uterine horns were scraped toobtain endometrium, which was washed in saline solution at4

�

C. The cells were immediately lysed in a DOUNCE ho-mogenizer for nucleus extraction using the technique de-scribed by Andreus et al. (22). All extracts were dialyzedwith a solution containing 0.05 M HEPES, 20% glycerol,0.5 mM EDTA, 200 mM KCl, 20 mM EGTA, and 0.5 mMDTT pH 7.6 (Sigma Chemical Co.) Nuclear extract aliquotswere stored at

�

70

�

C until analyzed.

Oligonucleotides.

Drs. Jörg Klug and Guntram Suske ofthe University of Marburg, Germany kindly provided alloligonucleotides used for the present study. The sequencesare described in Table 1 and are located within the UG genepromoter region (Figure 1).

For oligonucleotide annealing, the mixture of both com-plementary sequences (10 nmol each) were heated to 90

�

Cin 100

�

L annealing buffer (10 mM Tris-HCl, pH 8; 1 mMEDTA, 30 mM KCl) (Sigma Chemical Co.) and left to coolat room temperature for 2 h. Double-stranded DNA was la-beled with [

�

-32

P] ATP (Amersham Pharmacia Biotech,Barra Funda, São Paulo, Brasil) using the T

4

polynucleotidekinase enzyme (GIBCO). Nonaligned bands were separatedin a native 6% polyacrylamide (Sigma Chemical Co.) gel.

Electrophoretic mobility shift assay (EMSA).

Band shift gelswere prepared as described by Klug et al. (16): 5–10

�

g nuclearextract was pre-incubated for 10 min in ice with 100 ng un-specific poly(dI-dC) competitor, 100 mM KCl, 0.5 mMMgCl

2

, and 2

�

L 2XD (20 mM HEPES, 2 mM DTT, 0.2mM EDTA, 17.5% glycerol) (Sigma Chemical Co.). Imme-diately after pre-incubation, 0.5 ng of labeled oligonucle-otides (14,000 cpm) was added and the solution was incu-bated for an additional 15 min at room temperature. For thecompetitive assays, unlabeled TATA or Sp1 or random se-quences were added in excess during pre-incubation on ice.Reaction mixtures (final volume

�

25

�

L) were analyzedon a 4% polyacrylamide gel and electrophoresed with spe-cific running buffer (0.05 M Tris-base, 0.045 M boric acid,and 0.5 mM EDTA) (Sigma Chemical Co.) at 60 V for 90min. The gel was placed on a gel drier (Model 583, BIO-RAD, Hercules, CA, USA) at 80

�

C and then exposed on anX-OMAT film (Kodak, Rochester, NY, USA) for 24 h at roomtemperature. Shifted bands were analyzed by densitometry.

Arias et al. / Archives of Medical Research 32 (2001) 263–267

265

Results

Expression patterns of transcription factors that bind to theTATA box within the UG gene promoter region.

Figure 2A showsEMSA analysis of rabbit endometrium nuclear extracts in un-mated rabbits (time 0) and on days 3, 5, 7, 15, and 28 post-coitus. Extracts obtained from Ishikawa cell cultures were in-cluded as controls. Two shifted bands such as those reportedin Ishikawa cells are observed. Densitometric analysis (Fig-ure 2B) shows differences in TPF/YY1 and TCF expressionpatterns. TPF is strongly expressed and does not vary through-out pregnancy, while TCF is weakly expressed, showinggreatest intensity on days 3 and 5 and then declined (most evi-dently on days 7 and 15) and increased on day 28. The highestlevels observed at day 28 of pregnancy are similar to day 0(unmated animals).

Expression patterns of transcription factors binding to GT1sequences within the UG gene promoter.

Figure 3A showsthat both Ishikawa and endometrial cell extracts produce thesame two shifted bands, but with different expression pat-terns. Densitometric analysis (Figure 3B) revealed that,similar to TPF/YY1 expression, the band corresponding to

Sp3/SpR-2 binding shows greater density and is constantthroughout pregnancy. However, the density of the bandproduced by transcription factor Sp1 varied widely through-out pregnancy, showing a peak on day 3, decreasing on day5, increasing once again on day 7, reaching maximum inten-sity on day 15, and declining again on day 28. The highestlevels were observed at day 0, corresponding to unmatedanimals.

Discussion

We demonstrate here differences in the expression patternsof certain transcription factors that bind to the UG gene pro-moter TATA box, which suggest that transcription factorssuch as TPF/YY1 and TCF could play an important role in

Table 1.

Oligonucleotides used for EMSA

Oligonucleotide Sequence

ocTATA up 5

�

AGC TTC TTG CCG GAG AAT ACA AAA AGGCAC CTC GAG G 3

�

Sp up 5

�

CAC CCC TT GCC ACA CCC CTG CAC AAG 3

�

Random 5

�

CAG CGA CTA ACA TCG ATC GC-3

�

Figure 1. Uteroglobin gene promoter sequence. Underlined letters repre-sent the sequences used for the Sp and ocTATA oligonucleotides design.

Figure 2. EMSA experiments using the UG promoter TATA box regionand nuclear extracts obtained on different days of pregnancy. A) 32P-labeled TATA oligonucleotide incubated alone (oligo), incubated in thepresence of Ishikawa cell nuclear extracts (Ishikawa), and incubated in thepresence of endometrial cell nuclear extracts on days 0, 3, 5, 7, 15, and 28of pregnancy. The endometrial nuclear extracts were incubated in theabsence (�) or presence of excess (200-fold) unlabeled specific competitor(sc) and unspecific competitor (uc). B) Densitometric analysis of bandshifts corresponding to reactions incubated without competitors.

266

Arias et al./ Archives of Medical Research 32 (2001) 263–267

UG expression throughout pregnancy in the rabbit. WhileTPF/YY1 was expressed constitutively, TCF expressionfluctuated throughout pregnancy. We found that TCFshowed highest expression levels on day 5. Because UGconcentrations are highest between days 5 and 7 post-coitus(the exact time of blastocyst implantation in the rabbit) andboth UG mRNA and UG concentrations remain constantduring the remainder of pregnancy, several authors havesuggested that UG is involved in the implantation process(23–25). The physiologic role of these transcription factorsis not understood; however, because TCF is considered atranscription activator, TCF may function as a regulator ofUG gene expression at critical moments, such as at blasto-cyst implantation.

On the other hand, the fact that TPF/YY1 is constitu-tively expressed may be related with UG gene basal expres-

sion during the remainder of pregnancy, retaining low pros-taglandin concentrations to prevent uterine contractions andpremature labor. A clear negative correlation between UGand prostaglandin levels was demonstrated by Mukherjee(26), who found a sharp decline of UG concentrations and asharp increase of prostaglandins 1 day before parturition.

It was interesting to see that TCF diminished while ges-tation increased, at least until day 15; additionally, it in-creased importantly at day 28, when a low content of UGhas been demonstrated. However, Peri et al. in 1995 (10)demonstrated a similar pattern expression of both UG andmRNA UG on days 18, 22, 26, and 29 of gestation in rab-bits and showed an important decline at day 31. Unfortu-nately, they do not report results of previous days (betweendays 15 and 17) on which TCF diminution was observed.For that reason, we are to date unable to state clearlywhether TCF is truly involved in UG expression during thisperiod, and more research must be done in this regard.

The Sp family represents another group of UG gene ex-pression regulating transcription factors (Sp1, Sp3/SpR-2,and Sp4) that activate or inactivate the gene binding specifi-cally to GC and CT sites within the UG promoter (19). Ourresults show that the Sp3/SpR-2 expression pattern is simi-lar to that of TPF/YY1, as it was expressed constitutively.However, Sp1 expression varied throughout pregnancy,suggesting as in the case of TCF that this factor may be atranscription regulator.

Several factors are known to regulate UG gene transcrip-tion

in vitro

. Sp1 and Sp3 are expressed at similar levelsin insects, Sp1 and Sp4 act as transcription activators, andSp3/SpR-2 functions as repressor of Sp1-mediated UG tran-scription in eukaryotic cell (27). Studies in endometrial rab-bit tissue cultures have demonstrated the influence of someof these factors on UG gene transcription activation

in vivo

.Hormone-induced recruitment of Sp1 mediates estrogen ac-tivation of the rabbit UG gene in endometrial epithelium,suggesting that this factor participates in estrogen-depen-dent activation of this gene (28).

We did not find ubiquitous Sp1 expression, which de-clined on pregnancy days 3 and 5. Further experiments areneeded to establish whether a repressor effect of Sp3 uponSp1 could explain this finding. Moreover, as estrogen re-ceptors and Sp1 synergies function to induce UG gene ex-pression after an estrogenic stimulus (28), it is possible thatthe levels of Sp1 (on special days of gestation) may play animportant role in expression of this gene; however, this re-mains to be demonstrated in other experiments.

On the other hand, there may be other unknown Sp1 regu-lating factors expressed in the intact pregnant rabbit (in con-trast to pseudo-pregnancy) in which the effects of several ste-roid hormones or other factors are observed. It was recentlydescribed that factors TBRP1 and TBRP2 are expressed inpseudo-pregnant rabbits on day 5. Unfortunately, the authorsstudied TRBP1 and TRBP2 expression only on day 5 (21).We have demonstrated here that TCF and TPF/YY1 initially

Figure 3. EMSA experiments using the specific GT1 UG promotersequence and nuclear extracts obtained on different days of pregnancy. A)32P-labeled GT1 oligonucleotide incubated alone (oligo), incubated in thepresence of Ishikawa cell nuclear extracts (Ishikawa), and incubated in thepresence of endometrial cell nuclear extracts on days 0, 3, 5, 7, 15, and 28of pregnancy. The endometrial nuclear extracts were incubated in theabsence (�) or presence of excess (200-fold) unlabeled specific competitor(sc) and unspecific competitor (uc). B) Densitometric analysis of bandshifts corresponding to the reactions incubated without competitors.

Arias et al. / Archives of Medical Research 32 (2001) 263–267

267

described in Ishikawa cells were also found expressed in theintact pregnant rabbit on day 5. As TCF levels begin to declineafter day 5, perhaps TRBP1 or TRBP2 could be involved inTCF expression regulation, but whether and how these tran-scription factors (TRBP1, TRBP2, TCF, and TPF/YY1) inter-act to regulate UG expression remain to be clarified.

Our results show that the transcription factors found inIshikawa cells are also expressed in the endometrium of thepregnant rabbit and that apparently UG gene expression wascontrolled throughout pregnancy by two constitutive (TPF/YY1 and Sp3/SpR-2) and two regulated (TCF and Sp1) fac-tors. We solely analyzed factors that bind to the TATA boxand GT1 sites (region VI) localized within the UG pro-moter; however, other factors such as TRBP1 and TRBP2or perhaps other yet unknown factors may be involved inregulation of this gene

in vivo

.

Acknowledgments

This work was supported by the National Institute of Perinatology,Mexico City, Reg. #212250/07061. The authors wish to thank Drs.Jörg Klug and Guntram Suske for providing the oligonucleotidesused in our experiments, as well as Susana González for her helpin keeping and caring for the rabbits.

References

1. Mukherjee AB, Kundu GC, Matilde-Selvaggi G, Yuan CJ, MandalAK, Chattopadhyay S, Sheng F, Pattabiraman N, Zhang Z. Uteroglo-bin: a novel cytokine? Cell Mol Life Sci 1999;55:71.

2. Krishnan RS, Daniel JC. Blastokinin inducer and regulator of blasto-cyst development in the rabbit uterus. Science 1967;158:490.

3. Bochskanl R, Kichner C. Uteroglobin and the accumulation of proges-terone in the uterine lumen of the rabbit. Wilh Roux Arch Dev Biol1981;190:127.

4. Bochskanl R, Thie M, Kichner C. Progesterone dependent uptake ofuteroglobin by rabbit endometrium. Histochemistry 1984;80:581.

5. Mukherjee AB, Laki K, Agrawal AK. Possible mechanism of successof an allotransplantation in nature mammalian pregnancy. Med Hy-pothesis 1980;6:1043.

6. Shiffmann E, Geetha V, Pencev D, Warabi H, Mato J, Hirata F. Adher-ence and regulation of leukotaxis. Agents Actions Suppl 1983;12:106.

7. Vasanthakumar G, Manjunath R, Mukherje AB, Warabi H, ShiffmannE. Inhibition of phagocyte chemotaxis by uteroglobin, an inhibitor ofblastocyst rejection. Biochem Pharmacol 1988;37:389.

8. Waite M. The phospholipases. Vol. 5. New York; Plenum;1987.9. Dennis EA. Diversity of group types, regulation and function of phos-

pholipase A2. J Biol Chem 1994;269:13057.10. Peri A, Dibin NH, Dhanireddy R, Mukherjee AB. Uteroglobin gene

expression in the rabbit uterus throughout gestation and in the fetallung. Relationship between uteroglobin and eicosanoid levels in the

developing fetal lung. J Clin Invest 1995;96(1):343.11. Singh G, Katial SL, Brown WE, Kennedy AL, Singh U, Wong-Chong

ML. Clara cell 10 kDa protein (CC10): comparison of structure andfunction to uteroglobin. Biochem Biophys Acta 1990;1039:348.

12. Baily A, Atger M, Atger P, Cerbón MA, Alizon M, Vu Hai MT, LogeatF, Milgrom E. The rabbit uteroglobin gene. Structure and interactionwith the progesterone receptor. J Biol Chem 1983;258(17):10384.

13. Suske G, Wenz M, Cato AC, Beato M. The uteroglobin gene region:hormonal regulation, repetitive elements and complete nucleotide se-quence of the gene. Nucleic Acids Res 1983;11(8):2257.

14. Miele L, Cordella-Miele E, Mukherjee AB. Uteroglobin: structure,molecular biology, and new perspectives on its function as a phospho-lipase A2 inhibitor. Endocr Rev 1987;8(4):474.

15. Misseyanni A, Klung J, Suske G, Beato M. Novel upstream elementsand the TATA-box region mediate preferential transcription from theuteroglobin promotor in endometrial cells. Nucleic Acids Res 1991;19(11):2849.

16. Klug J, Knapp S, Castro I, Beato M. Two distinct factors bind to therabbit uteroglobin TATA-box region and are required for efficienttranscription. Mol Cell Biol 1994;14(9):6208.

17. Klug J, Beato M. Binding of YYI to a site overlapping a weak TATAbox is essential for transcription from the uteroglobin promoter in en-dometrial cells. Mol Cell Biol 1996;16(11):6398.

18. Hagen G, Muller S, Beato M, Suske G. Cloning by recognition sitescreening of two novel GT box binding proteins: a family of Sp1 re-lated genes. Nucleic Acids Res 1992;20(21):5519.

19. Dennig J, Hagen G, Beato M, Suske G. Members of the Sp transcrip-tion factor family control transcription from the uteroglobin promoter.J Biol Chem 1995;270(21):12737.

20. Suske G, Lorenz W, Klug J, Gazdar AF, Beato M. Elements of the rab-bit uteroglobin promoter mediating its transcription in epithelial cellsfrom the endometrium and lung. Gene Expression 1992;2(4):339.

21. Pérez MM, García C, López de Haro MS, Nieto A. Interactions ofprogesterone-dependent endometrial nuclear factors with the promotorof the rabbit uteroglobin gene. Arch Biochem Biophys 1996;333(1):12.

22. Andrews NC, Faller DV. A rapid micropreparation technique for ex-traction of DNA-binding proteins from limiting numbers of mamma-lian cells. Nucleic Acids Res 1991;19(9):2499.

23. Arthur AT, Cowan BD, Daniel JC. Steroid binding to blastokinin. Fer-til Steril 1972;23:85.

24. Gutiérrez-Sagal R, Pérez-Palacios G, Langley E, Pasapera AM, CastroI, Cerbón MA. Endometrial expression of progesterone receptor anduteroglobin gene during early pregnancy in the rabbit. Mol Reprod De-velop 1993;34(3):244.

25. Loosfelt H, Fridlansky F, Savouret JF, Atger M, Milgrom E. Mecha-nism of action of progesterone in the rabbit endometrium. Induction ofuteroglobin and its messenger RNA. J Biol Chem 1981;256:3465.

26. Mukherjee AB, Ulane RE, Agrawal AK. Role of uteroglobin andtransglutamine in masking the antigenicity of implanting rabbit em-bryos. Am J Reprod Immunol Microbiol 1982;2:135.

27. Hagen G, Muller S, Beato M, Suske G. Sp1-mediated transcriptionalactivation is repressed by Sp3. EMBO J 1994;13(16):3843.

28. Scholz A, Truss M, Beato M. Hormone-induced recruitment of Sp1mediates estrogen activation of the rabbit uteroglobin gene in endome-trial epithelium. J Biol Chem 1998;273(8):4360.