()()i;}-7227/91/1292-0697$03.00/0 Endocrinology Copyright ^ 1991 by The Endocrine Society
Vol. 129, No. 2 Printed in U.S.A.
Regulation of the Uteroferrin Gene Promoter in Endometrial Cells: Interactions among Estrogen, Progesterone, and Prolactin* ALBERT E. FLISS, FRANK J. MICHEL, CHAO-LING CHEN, ANDREA HOFIG, FULLER W. BAZER, JANICE Y. CHOU, AND ROSALIA C. M. SIMMEN Animal (A.E.F., F.J.M., A.H., F. W.B., R.C.M.S.) and Large Animal Clinical (C.L.C.) Sciences Departments, University of Florida, Gainesville, Florida 32611; and the Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health (J. Y.C.), Bethesda, Maryland 20892
ABSTRACT. Expression of the gene for the porcine transplacental iron transport protein uteroferrin (UF) is largely restricted to the uterus, where it is differentially regulated by estrogen (E) and progesterone (P). To study the regulatory mechanisms subserving these effects, a 2-kilobase genomic fragment corresponding to —2005 to 48 nucleotides of the UF gene was ligated up-stream to the reporter gene chloramphenicol acetyltransferase (CAT). This construct (UF-CAT) was transiently transfected into rabbit endometrial (HRE-H9), mouse fibroblastic (AKR-2B), and human choriocarcinoma (JEG-3) cells. The basal gene promoter activity of UF-CAT was exhibited in H9 cells, but not in AKR-2B or JEG-3 cells. In contrast, a simian virus-40 early promoter (SV2) was functional in all three cell lines. The H9 cells were used to examine steroid regulation Of the UF gene promoter. The CAT expression in H9 cells primed with E and PRL, but not with E or PRL alone, was stimulated by P. In contrast, basal activity of SV2 in these cells was
unaffected by hormones, singly or in combination. To examine the basis for the E/PRL-dependent response to P, levels of P and E receptors in H9 cells were quantified. PRL and E plus PRL increased the number of high affinity sites for P, but had little effect on levels of high affinity sites for E in treated vs. untreated H9 cells. In vivo administration of PRL to cyclic gilts had no effect on levels of endometrial UF mRNA and secreted UF protein; however, E- plus PRL-treated gilts had higher {P < 0.05) levels of endometrial UF mRNA and luminal UF than PRL-treated gilts. These results demonstrate in vitro functional activity of the UF gene promoter and associated 5' flanking region and suggest that sequences within this region may mediate tissue-specific and steroid hormone-regulated expression of the UF gene. Moreover, interactions among E, PRL, and P modulate UF gene expression in vivo and in vitro. (Endocrinology 129: 697-704,1991)
E
The acid phosphatase of the human placenta has been characterized (4) and exhibits greater than 80% homology in amino acid and nucleotide sequences with UF (5). Bovine and human spleen (4, 6) and osteoclasts isolated from human (7) and rat (8) bones secrete an acid phosphatase whose activity can be inhibited by an antibody to porcine UF. Elevated levels of this enzyme are observed in certain pathological diseases, such as Gaucher's and Hodgkin's disease (9,10) and hairy cell leukemia (4, 9), and during increased bone turnover observed in metabolic bone diseases (8). The level of UF gene expression in porcine uterine endometrium varies during pregnancy (3). The UF gene is maximally expressed during mid- and late pregnancy (3), although a partial block in UF mRNA translation during late pregnancy has been postulated to account for the discrepancy in levels of UF mRNA and synthesized and secreted UF protein (3,11). Correlation between the serum progesterone (P) to estrogen (E) ratio (12) and levels of UF mRNA in endometrium are apparent up to midpregnancy (3), suggesting the importance of these
XPRESSION of porcine uteroferrin (UF), a transplacental iron transport protein exhibiting tartrate-resistant type 5 acid phosphatase activity, is largely restricted in vivo to glandular epithelium of uterine endometrium in pregnant and nonpregnant gilts (1-3). The function of UF in the uterus during the estrous cycle is not clear; however, during pregnancy, UF, by virtue of its ability to bind two atoms of iron per molecule, is proposed to act as a major iron carrier from the maternal uterus to the developing conceptus (embryo/fetus and associated membranes) (2). In other species, type 5 acid phosphatases are detected in tissues other than uterus. Received January 17, 1991. Address all correspondence and requests for reprints to: Dr. Rosalia C. M. Simmen, Animal Science Department, 125 Animal Science Building, 459 Shealy Drive, University of Florida, Gainesville, Florida 32611-0691. * This paper is published as Journal Series no. R-01615, University of Florida, Agricultural Experiment Station. This work was supported by NIH Grant HD-21961 (to R.C.M.S.). Portions of this work were presented at the Annual Meeting of the Society for the Study of Reproduction, July 1990 (Abstract 69).
697
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698
STEROID-REGULATED EXPRESSION OF UF GENE PROMOTER
hormones in regulating UF synthesis and secretion. Studies using immature and mature ovariectomized gilts subjected to steroid hormone administration indicated that while P increased and E decreased UF mRNA levels, both hormones stimulated UF protein secretion into the uterine lumen (13, 14). The isolation of the UF chromosomal gene has allowed tentative identification of the UF gene promoter region necessary for basal transcription (13). This region lacks characteristic TATA and CAAT boxes, but contains sequences exhibiting similarities to consensus sequences shown to mediate E (15) and P (16) regulation. However, the functional activities of these genetic elements have not been directly demonstrated. In this study we used a transient expression system and a hormone-responsive endometrial epithelial-like cell line to show that sequences up-stream of the transcription initiation site of the UF gene mediate tissue-specific and steroid hormoneregulated expression of UF. We also show that interactions among E, P, and PRL are relevant to the expression of the UF gene in vivo and in vitro.
Materials and Methods Materials Restriction and modifying enzymes were purchased from Promega Biotec (Madison, WI), Boehringer Mannheim (Indianapolis, IN), or Pharmacia (Piscataway, NJ) and were used according to recommended conditions. [3H]Estradiol and [3H] P were obtained from Amersham (Arlington Heights, IL). P and 17/3-estradiol were purchased from Sigma (St. Louis, MO). [a-32P]dNTPs and [a-35S]dATP were obtained from ICN (Irvine, CA). Porcine PRL was a gift from Dr. Douglas Bolt (USDA Animal Hormone Program). All other reagents used were of the highest grade available.
Endo«1991 Voll29«No2
Construction of CAT expression vector Standard cloning procedures, described by Maniatis et al. (18), were used to prepare the UF-CAT construct. The genomic clone pE9, subcloned into the EcoRl site of pGEM 4Z (Promega) and containing approximately 5 kilobases (kb) of sequences located up-stream of the structural sequences of the UF gene (13), was used as starting material. The region spanning from —2005 to 48 nucleotides of the UF gene was isolated from pE9 by digestion with Sacl and cloned into the unique Hindlll site of vector pSV0CAT, after addition of Hindlll linkers (Promega). The orientation of the resulting plasmids was determined by sequencing through the junctions using as primer, a 20-mer oligonucleotide corresponding to the most 5' sequence of the chloramphenicol acetyltransferase (CAT) reporter gene (19), which includes the ATG initiation codon. Plasmid pCSS27.1 was in the correct (5' to 3') orientation relative to the position of the start site of transcription of the CAT gene. The plasmid pSV2CAT containing the simian virus40 (SV40) virus early promoter linked to the CAT reporter gene was used as the positive control DNA in these studies. All plasmids were prepared by the alkaline lysis method, followed by CsCl-ethidium bromide gradient centrifugation, as described previously (18). The entire sequence of the UF DNA fragment cloned in the pSV0CAT vector (pCSS27.1) was determined by the Sanger dideoxy method (20), using synthetic oligonucleotides complementary to specific regions within the fragment as primers. The complete nucleotide sequence of this region will be presented in a separate publication (Fliss, A. E., and R. C. M. Simmen, manuscript in preparation). Cell culture and DNA transfections
The endometrial cell line (HRE-H9) used for transfection was immortalized from primary cultures of rabbit endometrial cells by transformation with an origin-defective SV40 temperature-sensitive (ts) A mutant (21). The growth characteristics and functional properties of this cell line have been described previously (22). The H9 cells were grown in a-Modified MiniAnimals mum Essential Medium supplemented with 10% fetal bovine serum (Sigma Chemical Co.), 5 Mg/ml insulin, streptomycin Gilts (primiparous female pigs) on day 6 of the estrous cycle (100 /xg/ml), and penicillin (100 IU/ml) and maintained in a were assigned randomly to one of the following treatments: 1) humidified atmosphere of 95% air-5% CO2 at 33 C (permissive saline twice daily at 0730 and 1930 h on days 6-11 and corn oil temperature). Approximately 1 x 105 cells were seeded/60-mm on day 11 (n = 3); 2) PRL in saline (1 mg/gilt) twice daily at plastic tissue culture dish and grown to 80-85% confluency 0730 and 1930 h on days 6-11 and corn oil on day 11 (n = 3); (~96 h after seeding). Cells were washed twice with Opti-MEM and 3) PRL in saline (1 mg/gilt) twice daily at 0730 and 1930 (Gibco, Grand Island, NY) before the addition of DNA. The h on days 6-11 and estradiol valerate in corn oil (E; 5 mg/gilt) culture medium was replaced with Opti-MEM, and the cells on day 11 (n = 3). The treatment regimen was demonstrated were transfected with supercoiled plasmid DNA using lipofectin previously to increase serum PRL in gilts (17), and subsequent reagent (Bethesda Research Laboratories, Gaithersburg, MD). treatment with E after PRL administration, as in no. 3 above, DNA (20 ng) and lipofectin (30 ng) were each diluted to 50 n\ was shown to increase the release of UF into the uterine lumen with sterile water, mixed gently in a polystyrene snap-cap tubes, (17). Gilts were hysterectomized 24 h after the last treatment and allowed to stand at room temperature for 15 min. The on day 12, following procedures outlined in Guidelines for Care resulting DNA-lipid mixture was added to each dish dropwise and Use of Experimental Animals. Hysterectomy was carried and with swirling. Cells were incubated for 24 h at 33 C. The out using thiamylal sodium and halothane to induce and mainmedium was removed and replaced with a-Modified Minimum tain anesthesia, respectively. Uterine horns were flushed with Essential Medium supplemented with 20% fetal bovine serum, PBS (0.01 M NaPO4, pH 7.4, containing 0.15 M NaCL; 30 ml/ and the cells were incubated for an additional 48 h at 33 C with horn), and flushings were collected for UF RIA, as previously or without the appropriate hormone treatment, as indicated in described (14). Endometrial tissues were dissected free from the text, before CAT assays. In studies where the effects of myometrium and stored at -70 C until use.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 07:51 For personal use only. No other uses without permission. . All rights reserved.
STEROID-REGULATED EXPRESSION OF UF GENE PROMOTER steroids were investigated, charcoal-stripped, phenol red-free medium and serum were used for plating cells. Neither medium nor serum had any detectable estrogens (
Vol. 129, No. 2 Printed in U.S.A.
Regulation of the Uteroferrin Gene Promoter in Endometrial Cells: Interactions among Estrogen, Progesterone, and Prolactin* ALBERT E. FLISS, FRANK J. MICHEL, CHAO-LING CHEN, ANDREA HOFIG, FULLER W. BAZER, JANICE Y. CHOU, AND ROSALIA C. M. SIMMEN Animal (A.E.F., F.J.M., A.H., F. W.B., R.C.M.S.) and Large Animal Clinical (C.L.C.) Sciences Departments, University of Florida, Gainesville, Florida 32611; and the Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health (J. Y.C.), Bethesda, Maryland 20892
ABSTRACT. Expression of the gene for the porcine transplacental iron transport protein uteroferrin (UF) is largely restricted to the uterus, where it is differentially regulated by estrogen (E) and progesterone (P). To study the regulatory mechanisms subserving these effects, a 2-kilobase genomic fragment corresponding to —2005 to 48 nucleotides of the UF gene was ligated up-stream to the reporter gene chloramphenicol acetyltransferase (CAT). This construct (UF-CAT) was transiently transfected into rabbit endometrial (HRE-H9), mouse fibroblastic (AKR-2B), and human choriocarcinoma (JEG-3) cells. The basal gene promoter activity of UF-CAT was exhibited in H9 cells, but not in AKR-2B or JEG-3 cells. In contrast, a simian virus-40 early promoter (SV2) was functional in all three cell lines. The H9 cells were used to examine steroid regulation Of the UF gene promoter. The CAT expression in H9 cells primed with E and PRL, but not with E or PRL alone, was stimulated by P. In contrast, basal activity of SV2 in these cells was
unaffected by hormones, singly or in combination. To examine the basis for the E/PRL-dependent response to P, levels of P and E receptors in H9 cells were quantified. PRL and E plus PRL increased the number of high affinity sites for P, but had little effect on levels of high affinity sites for E in treated vs. untreated H9 cells. In vivo administration of PRL to cyclic gilts had no effect on levels of endometrial UF mRNA and secreted UF protein; however, E- plus PRL-treated gilts had higher {P < 0.05) levels of endometrial UF mRNA and luminal UF than PRL-treated gilts. These results demonstrate in vitro functional activity of the UF gene promoter and associated 5' flanking region and suggest that sequences within this region may mediate tissue-specific and steroid hormone-regulated expression of the UF gene. Moreover, interactions among E, PRL, and P modulate UF gene expression in vivo and in vitro. (Endocrinology 129: 697-704,1991)
E
The acid phosphatase of the human placenta has been characterized (4) and exhibits greater than 80% homology in amino acid and nucleotide sequences with UF (5). Bovine and human spleen (4, 6) and osteoclasts isolated from human (7) and rat (8) bones secrete an acid phosphatase whose activity can be inhibited by an antibody to porcine UF. Elevated levels of this enzyme are observed in certain pathological diseases, such as Gaucher's and Hodgkin's disease (9,10) and hairy cell leukemia (4, 9), and during increased bone turnover observed in metabolic bone diseases (8). The level of UF gene expression in porcine uterine endometrium varies during pregnancy (3). The UF gene is maximally expressed during mid- and late pregnancy (3), although a partial block in UF mRNA translation during late pregnancy has been postulated to account for the discrepancy in levels of UF mRNA and synthesized and secreted UF protein (3,11). Correlation between the serum progesterone (P) to estrogen (E) ratio (12) and levels of UF mRNA in endometrium are apparent up to midpregnancy (3), suggesting the importance of these
XPRESSION of porcine uteroferrin (UF), a transplacental iron transport protein exhibiting tartrate-resistant type 5 acid phosphatase activity, is largely restricted in vivo to glandular epithelium of uterine endometrium in pregnant and nonpregnant gilts (1-3). The function of UF in the uterus during the estrous cycle is not clear; however, during pregnancy, UF, by virtue of its ability to bind two atoms of iron per molecule, is proposed to act as a major iron carrier from the maternal uterus to the developing conceptus (embryo/fetus and associated membranes) (2). In other species, type 5 acid phosphatases are detected in tissues other than uterus. Received January 17, 1991. Address all correspondence and requests for reprints to: Dr. Rosalia C. M. Simmen, Animal Science Department, 125 Animal Science Building, 459 Shealy Drive, University of Florida, Gainesville, Florida 32611-0691. * This paper is published as Journal Series no. R-01615, University of Florida, Agricultural Experiment Station. This work was supported by NIH Grant HD-21961 (to R.C.M.S.). Portions of this work were presented at the Annual Meeting of the Society for the Study of Reproduction, July 1990 (Abstract 69).
697
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 07:51 For personal use only. No other uses without permission. . All rights reserved.
698
STEROID-REGULATED EXPRESSION OF UF GENE PROMOTER
hormones in regulating UF synthesis and secretion. Studies using immature and mature ovariectomized gilts subjected to steroid hormone administration indicated that while P increased and E decreased UF mRNA levels, both hormones stimulated UF protein secretion into the uterine lumen (13, 14). The isolation of the UF chromosomal gene has allowed tentative identification of the UF gene promoter region necessary for basal transcription (13). This region lacks characteristic TATA and CAAT boxes, but contains sequences exhibiting similarities to consensus sequences shown to mediate E (15) and P (16) regulation. However, the functional activities of these genetic elements have not been directly demonstrated. In this study we used a transient expression system and a hormone-responsive endometrial epithelial-like cell line to show that sequences up-stream of the transcription initiation site of the UF gene mediate tissue-specific and steroid hormoneregulated expression of UF. We also show that interactions among E, P, and PRL are relevant to the expression of the UF gene in vivo and in vitro.
Materials and Methods Materials Restriction and modifying enzymes were purchased from Promega Biotec (Madison, WI), Boehringer Mannheim (Indianapolis, IN), or Pharmacia (Piscataway, NJ) and were used according to recommended conditions. [3H]Estradiol and [3H] P were obtained from Amersham (Arlington Heights, IL). P and 17/3-estradiol were purchased from Sigma (St. Louis, MO). [a-32P]dNTPs and [a-35S]dATP were obtained from ICN (Irvine, CA). Porcine PRL was a gift from Dr. Douglas Bolt (USDA Animal Hormone Program). All other reagents used were of the highest grade available.
Endo«1991 Voll29«No2
Construction of CAT expression vector Standard cloning procedures, described by Maniatis et al. (18), were used to prepare the UF-CAT construct. The genomic clone pE9, subcloned into the EcoRl site of pGEM 4Z (Promega) and containing approximately 5 kilobases (kb) of sequences located up-stream of the structural sequences of the UF gene (13), was used as starting material. The region spanning from —2005 to 48 nucleotides of the UF gene was isolated from pE9 by digestion with Sacl and cloned into the unique Hindlll site of vector pSV0CAT, after addition of Hindlll linkers (Promega). The orientation of the resulting plasmids was determined by sequencing through the junctions using as primer, a 20-mer oligonucleotide corresponding to the most 5' sequence of the chloramphenicol acetyltransferase (CAT) reporter gene (19), which includes the ATG initiation codon. Plasmid pCSS27.1 was in the correct (5' to 3') orientation relative to the position of the start site of transcription of the CAT gene. The plasmid pSV2CAT containing the simian virus40 (SV40) virus early promoter linked to the CAT reporter gene was used as the positive control DNA in these studies. All plasmids were prepared by the alkaline lysis method, followed by CsCl-ethidium bromide gradient centrifugation, as described previously (18). The entire sequence of the UF DNA fragment cloned in the pSV0CAT vector (pCSS27.1) was determined by the Sanger dideoxy method (20), using synthetic oligonucleotides complementary to specific regions within the fragment as primers. The complete nucleotide sequence of this region will be presented in a separate publication (Fliss, A. E., and R. C. M. Simmen, manuscript in preparation). Cell culture and DNA transfections
The endometrial cell line (HRE-H9) used for transfection was immortalized from primary cultures of rabbit endometrial cells by transformation with an origin-defective SV40 temperature-sensitive (ts) A mutant (21). The growth characteristics and functional properties of this cell line have been described previously (22). The H9 cells were grown in a-Modified MiniAnimals mum Essential Medium supplemented with 10% fetal bovine serum (Sigma Chemical Co.), 5 Mg/ml insulin, streptomycin Gilts (primiparous female pigs) on day 6 of the estrous cycle (100 /xg/ml), and penicillin (100 IU/ml) and maintained in a were assigned randomly to one of the following treatments: 1) humidified atmosphere of 95% air-5% CO2 at 33 C (permissive saline twice daily at 0730 and 1930 h on days 6-11 and corn oil temperature). Approximately 1 x 105 cells were seeded/60-mm on day 11 (n = 3); 2) PRL in saline (1 mg/gilt) twice daily at plastic tissue culture dish and grown to 80-85% confluency 0730 and 1930 h on days 6-11 and corn oil on day 11 (n = 3); (~96 h after seeding). Cells were washed twice with Opti-MEM and 3) PRL in saline (1 mg/gilt) twice daily at 0730 and 1930 (Gibco, Grand Island, NY) before the addition of DNA. The h on days 6-11 and estradiol valerate in corn oil (E; 5 mg/gilt) culture medium was replaced with Opti-MEM, and the cells on day 11 (n = 3). The treatment regimen was demonstrated were transfected with supercoiled plasmid DNA using lipofectin previously to increase serum PRL in gilts (17), and subsequent reagent (Bethesda Research Laboratories, Gaithersburg, MD). treatment with E after PRL administration, as in no. 3 above, DNA (20 ng) and lipofectin (30 ng) were each diluted to 50 n\ was shown to increase the release of UF into the uterine lumen with sterile water, mixed gently in a polystyrene snap-cap tubes, (17). Gilts were hysterectomized 24 h after the last treatment and allowed to stand at room temperature for 15 min. The on day 12, following procedures outlined in Guidelines for Care resulting DNA-lipid mixture was added to each dish dropwise and Use of Experimental Animals. Hysterectomy was carried and with swirling. Cells were incubated for 24 h at 33 C. The out using thiamylal sodium and halothane to induce and mainmedium was removed and replaced with a-Modified Minimum tain anesthesia, respectively. Uterine horns were flushed with Essential Medium supplemented with 20% fetal bovine serum, PBS (0.01 M NaPO4, pH 7.4, containing 0.15 M NaCL; 30 ml/ and the cells were incubated for an additional 48 h at 33 C with horn), and flushings were collected for UF RIA, as previously or without the appropriate hormone treatment, as indicated in described (14). Endometrial tissues were dissected free from the text, before CAT assays. In studies where the effects of myometrium and stored at -70 C until use.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 07:51 For personal use only. No other uses without permission. . All rights reserved.
STEROID-REGULATED EXPRESSION OF UF GENE PROMOTER steroids were investigated, charcoal-stripped, phenol red-free medium and serum were used for plating cells. Neither medium nor serum had any detectable estrogens (
