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Molecular Endocrinology, doi:10.1210/me.2003-0207
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Molecular Endocrinology 17 (11): 2340-2354
Copyright © 2003 by The Endocrine Society

Progesterone Regulation of the Mammalian Ortholog of Methylcitrate Dehydratase (Immune Response Gene 1) in the Uterine Epithelium during Implantation through the Protein Kinase C Pathway

Bo Chen, Damin Zhang and Jeffrey W. Pollard

Departments of Developmental and Molecular Biology and Obstetrics, Gynecology and Women’s Health, Center for the Study of Reproductive Biology and Women’s Health, Albert Einstein College of Medicine, New York, New York 10461

Address all correspondence and requests for reprints to: Jeffrey Pollard, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, New York, New York 10461. E-mail: pollard{at}aecom.yu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Implantation requires coordination between development of the blastocyst and the sex steroid hormone-regulated differentiation of the uterus. Under the influence of these hormones, the uterine luminal epithelium becomes receptive to attachment of the hatched blastocyst. In this study we sought to identify genes regulated by progesterone (P4) in the uterine epithelium. This resulted in the identification of one novel P4-regulated gene that had been previously found in lipopolysaccharide-stimulated macrophages and called immune response gene-1 (Irg1) and which is the mammalian ortholog of the bacterial gene encoding methylcitrate dehydratase. In adult mice Irg1 expression was limited to the uterine luminal epithelium where it is expressed only during pregnancy with a peak coinciding with implantation. Irg1 mRNA expression is regulated synergistically by P4 and estradiol (E2) but not by E2 alone. In macrophages Irg1 is induced by lipopolysaccharide through a protein kinase C (PKC)-regulated pathway. Now we demonstrate that the PKC pathway is induced in the uterine epithelium at implantation by the synergistic action of P4 and E2 and is responsible for the hormone induction of Irg1. These results suggest that the PKC pathway plays an important role in modulating steroid hormone responsiveness in the uterine luminal epithelium during the implantation window and that Irg1 will be an important marker of this window and may play an important role in implantation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE UTERINE PREPARATION for blastocyst implantation is carefully coordinated through the actions of the sex steroid hormones, 17ß-estradiol (E2) and progesterone (P4) (1). E2 secreted during estrus induces mating behavior, stimulates cell proliferation in the uterine epithelium (2, 3), and up-regulates uterine progesterone receptor (PR) expression (4). Copulation induces the formation and maintenance of the corpus luteum and the secretion of P4 through a neuroendocrine loop. P4 suppresses uterine epithelial cell proliferation (5) and induces its differentiation to become receptive to blastocyst attachment (6). Implantation in the mouse, however, requires that a nonreceptive uterus becomes receptive, a state that is achieved in response to a burst of E2 production known as nidatory estrogen, on the morning of d 4 of pregnancy (7, 8).

The switch from a nonreceptive to receptive epithelium suggests that P4 and E2 synergize to alter protein expression in these cells. Both these hormones act through their ligand-activated transcription factor receptors, which in turn are required for steroid hormone action during implantation, suggesting that alterations in gene expression control the preparation of the uterus for implantation (9, 10, 11). Consequently, several groups have identified P4-regulated genes expressed during this stage (12). These include the genes encoding growth factors (13, 14) such as leukemia inhibitory factor (LIF) (15, 16), amphiregulin (17), TGFß3 (18, 19), the homeobox proteins Hoxa-10 and Hoxa-11 (20), the peptide hormone calcitonin (21), and enzymes such as histidine decarboxylase (22), cyclooxygenase 2 (23), and cathepsins (24). Studies using targeted deletions or inhibitions of expression by antisense oligonucleotides have demonstrated that some of these genes are required for implantation to occur. For example, inhibition of calcitonin by antisense oligonucleotides injected into the uterus inhibited implantation (25). Ablation of Hoxa-10 resulted in a relative failure of decidualization (26) and of the IL-11 receptor in a truncated decidual response with the mesometrial decidual tissue not forming. Targeted ablation of the prostaglandin metabolizing enzyme, COX 2, was reported to block decidualization (23). However, subsequent studies failed to reproduce these results but showed instead a slight delay in decidualization but, subsequently, normal fertility (27). These proteins are expressed mostly in the stroma and therefore are unlikely to directly explain the uterine epithelial responsiveness to the blastocyst. However, LIF is expressed in the glandular epithelium just before implantation and is regulated by P4 together with nidatory E2 (16). Targeted deletions of LIF gene resulted in infertility (15). Interestingly, treatment of mice with LIF at the appropriate time restored implantation, and this LIF could completely substitute for nidatory estrogen in causing implantation (28). This suggests that E2 acts directly through LIF in the uterus. LIF is itself targeted to the LIF receptor expressed in the uterine luminal epithelium. LIF receptor is a member of the cytokine family of receptors that binds to gp130 and induces a signed transduction cascade that activates the transcription factor, signal transducer and activator of transcription 3a (29). This pathway has been demonstrated to be active in the luminal epithelium, and mice carrying C-terminal mutations in the gp130 common subunit are infertile (30).

Our previous studies have analyzed the mechanism of action of P4 inhibition of E2-induced cell proliferation in the mouse uterine epithelium (31). Because these hormones exert their effects in a cell type-specific manner these studies used purified luminal epithelial cell extracts for biochemical assays. Thus, in this study to analyze the action of P4 specifically in the uterine epithelium in its preparation for implantation, we isolated luminal epithelial cells, extracted RNA, and performed a differential PCR-based subtractive screen to identify genes responsive to estrogen in the presence or absence of P4 pretreatment. This revealed a novel P4-responsive gene that is an ortholog of the bacterial, methylcitrate dehydrogenase (PrpD), that had been originally identified previously in a macrophage cell line as a lipopolysaccharide (LPS)-responsive gene and named immune response gene-1 (Irg1) (32).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of Irg1 from the P4E2-Treated Uterine Epithelium
RNA isolated from the luminal epithelium 3 and 4 h after treatment of mice with either E2 or P4E2 was pooled as subjected to subtractive hybridization with P4E2 as the tester and E2 as the driver pool as described in Materials and Methods. The PCR products of the subtraction were subcloned into a TA vector, subjected to restriction endonuclease digestion, and sorted according to unique restriction maps and sequenced. A number of gene sequences were identified and their differential expression in the P4E2-treated group was confirmed by Northern blotting of uterine epithelial RNA samples isolated from E2 and P4E2-treated mice. Genes isolated that were expressed preferentially in the P4E2 group included cathepsin D, histidine decarboxylase, ß-galactosidase, Hoxa-10, and Irg 1 (data not shown). Only Irg 1 represented an unknown P4-regulated gene and, therefore, we decided to pursue this gene.

To obtain a full-length Irg1 cDNA corresponding to the 2.2-kb mRNA detected on the uterine RNA Northern blots, we performed 5'-RACE (rapid amplification of cDNA ends) and probed the {lambda}-ZAP cDNA mouse uterine epithelial P4E2 library with a 32P-labeled Irg 1 probe. Using RACE we obtained an additional 190 bp upstream of an ATG codon. The cDNA obtained from the library was approximately 2.1 kb. After sequencing of the cDNAs derived from these experiments and comparison with the public database of expressed sequence tags, the resultant nucleotide sequence was determined to be 2075 bp containing an open reading frame of 1467 bp, encoding 488 amino acids (Fig. 1AGoGoGoGoGo). This cDNA was consistent with the size detected on Northern blot. To confirm that the ATG is functional, this cDNA was subcloned into an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible PET-41a vector and expressed in BL21 bacterial cells. After (1.5 h) addition of IPTG, there was a strong band induced at approximately 80 kDa compared with the control group without IPTG induction (data not shown). After deducting the size of the glutathione-S-transferase tag, this strongly suggests Irg1 encodes a protein of approximately 50 kDa in size as predicted.



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  		Fig. 1. Irg1 Sequence and Amino Acid Alignment

A, Nucleotide and predicted amino acid sequence of Irg1. The nucleotide sequence also includes the 5'- and 3'-untranslated sequence derived from RACE and the cDNA libraries. B, Amino acid sequence alignment of mouse, rat, human, and Xenopus IRG1 protein with residue numbers of mouse Irg1 indicated above the sequence. The gray boxes highlight the identical residues among the four species. The secondary structure elements ({alpha}-helices are shown as solid lines, ß-sheet as checked boxes, coil regions as black boxes, and ß-turn as hatched arrows) were predicted using the PC Gene program. Consensus posttranslational modification sites among the mouse, rat, and human are indicated as follows: the putative glycosylation site is indicated as a black star, PKC phosphorylation sites as solid arrowheads, and the tyrosine phosphorylation site as an open arrowhead. C, The amino acid alignment between mouse Irg1 and PrpD from Escherichia coli. The identical amino acids are in gray, and the conservative amino acid changes are marked with an asterisk. The solid box shows the position of the alignment of PrpD with Irg1.

 


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  		Fig. 1A1. Continued

 


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  		Fig. 1A2. Continued

 


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  		Fig. 1B.
 


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  		Fig. 1C.
 
Amino acid sequence alignment using the Clustal method showed that the primary predicated protein sequence was well conserved between the mouse, rat, human, and Xenopus. The IRG1 protein between mouse and human shares more than 83% similarity, indicating it may play an important role across species (Fig. 1BGoGoGoGoGo). Sequence analysis predicted several putative posttranslational modification sites including a tyrosine phosphorylation site, phosphorylation site for protein kinase C (PKC), and glycosaminoglycan attachment site (Fig. 1BGoGoGoGoGo). A search of the GenBank DNA database for conserved domains revealed that more than 95% of the Irg1 sequence aligned to bacteria protein 2-methylcitrate dehydratase (Fig. 1CGoGoGoGoGo), which is responsible for converting 2-methylcitrate to 2-methylisocitrate in propionate catabolism (36).

Irg1 Is Expressed Strongly in the Uterus But Not Other Tissues
We used Northern blotting techniques to determine the tissue distribution of Irg1 expression. Total RNA from a variety of different tissues was isolated from d 4 pregnant mice. Irg1 transcripts were abundantly detected in the uterus, but there was no detectable signal in spleen, testis, brain, kidney, heart, liver, or lung and only a trace of expression in the ovary (Fig. 2Go).



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  		Fig. 2. Expression of Irg1 in Different Tissues

Autoradiograph of a Northern blot analysis of RNA isolated from different tissues as indicated, probed with a 32P-labeled Irg1 cDNA probe. The 18S rRNA in the bottom panel was used as RNA loading control.

 
Irg1 Is Expressed in the Uterine Epithelium during Implantation
Irg1 expression appeared to be restricted to the adult uterus. To examine this further, we isolated total uterine RNA at d 1–7 of pregnancy and used Northern blotting to determine Irg1 expression. The expression of Irg1 mRNA in the uterine samples was undetectable on d 1. It rose sharply on d 3 and increased further to reach a peak on d 4. By d 5, the concentration of Irg1 mRNA was dramatically reduced and was barely detectable on d 6 (Fig. 3AGo). The relative mRNA level of Irg1 was determined using densitometric scanning and normalized with the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression (Fig. 3BGo). The Irg1 signal on d 4 was approximately 2-fold higher than that on d 3, and we estimated an approximately 100-fold induction of Irg1 gene expression by comparing the normalized levels of Irg1 mRNA on d 1 and d 4 of pregnancy. By d 5, expression was reduced approximately 6-fold compared with d 4 and continued to decline thereafter (Fig. 3BGo).



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  		Fig. 3. Irg1 Expression Is Coincident with Implantation

Autoradiograph of a Northern blot of RNA isolated from mouse uteri from 1–7 d of pregnancy probed with a 32P-labeled Irg1 cDNA probe. Blots were reprobed with a GAPDH probe, and the signal was used as a RNA loading control. A, The ratio of Irg1 at different days of pregnancy relative to d 1 was determined by densitometric scanning using GAPDH to normalize RNA loading.

 
To investigate the cell type expression of Irg1 during pregnancy as well as to confirm our biochemical observations of its expression in the uterine epithelium under physiological conditions, we examined the site of Irg1 RNA expression in the uterus by in situ hybridization. Longitudinal and transverse sections of uteri isolated from mice at d 3–7 of pregnancy were hybridized with 32P-labeled antisense or sense Irg1 probes. In all of the sense control uterine sections from different time points, there was minimal signal with no specific localization (Fig. 4Go). In contrast, antisense probes detected expression uniformly along the luminal epithelium of the uterus on d 3 of pregnancy. This expression was dramatically elevated on d 4 (Fig. 4Go). At both these times, expression was largely restricted to the luminal epithelium with only a few glands in the superficial stroma showing positive. The level of Irg1 declined by d 5 in the uterine epithelium. On this day of pregnancy, the hybridization signal was no longer uniformly found along the uterine epithelium. Sporadic expression of Irg1 mRNA in the uterine epithelium immediately above the decidualized sites was maintained on d 6 of pregnancy, although epithelium between sites was negative for Irg1 expression (Fig. 4Go). By d 7 expression was very limited and barely above background (data not shown).



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  		Fig. 4. Localization of Irg1 Expression in the Mouse Uterus by in Situ Hybridization

In situ hybridization of mouse uterine sections prepared at d 3 (D), d 4 (F), d 5 (H), and d 6 (J) pregnant mice with a 35S-labeled antisense probe specific for Irg1 or a sense control probe (B) as described in Materials and Methods. The bottom right panel indicated that the residual luminal epithelium on the mesometrial side of the developing decidua retains Irg1 expression on d 6 whereas in the remaining epithelium the expression is largely lost (L). Morphology of the uterus is revealed in bright-field images (A, C, E, G, I, and K). Magnification, x40.

 
Irg1 Is Regulated by the Synergistic Action of P4 and E2
To determine the ovarian steroid hormone regulation of Irg1 gene expression, we reconstituted ovariectomized mice with physiological concentrations of E2 and P4 alone or in combination as described in Materials and Methods (groups a–d). In the initial experiments RNA was collected from animals 5 h after hormone administration and subjected to Northern blot analysis with the Irg1 cDNA probe. E2-treated samples had very low expression of Irg1, whereas samples treated with P4 showed an approximately 9-fold greater accumulation of Irg1 transcripts. Injection of both hormones to ovariectomized mice increased the level of Irg1 transcript by 50% compared with the samples administrated with P4 alone for 4 d (Fig. 5Go, A and B).



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  		Fig. 5. Irg1 Expression Is Regulated by P4 and Estrogen

A, Autoradiograph of a Northern blot analysis of total RNA isolated from the uterine epithelium of ovariectomized mice treated with E2 or P4 for 4 d alone, or E2 on the fourth day of the P4 treatment (P4E2) and killed 5 h after the last hormone treatment. The blots were hybridized with 32P-labeled Irg1 or GAPDH cDNA probes. B, The intensity of Irg1 expression was quantified by densitometric scanning and normalized with the GAPDH signal. Histograms show the relative level of expression (±SEM, n = 3) for each treatment group. Using Student’s t test, Irg1 expression in the uteri of mice treated with P4 and E2 or P4 alone is significantly higher than in the uteri of mice treated with E2 alone. Irg1 expression in the uteri of mice treated with P4 and E2 is also significantly higher than in the uteri of mice treated with P4 alone. C, Autoradiograph of a Northern blot probed as described in panel A of RNA isolated from the uterine epithelium of ovariectomized primed mice treated with progesterone for 3 d followed by the P4 plus E2 on the last day (P4E2), or a single injection of E2 (E2) on the last day and killed at the times indicated. The GAPDH expression level was used as a loading control. D, As in panel A except the RNA was isolated from uteri of mice that had been in the delayed implantation state treated with P4 or P4 E2 as indicated on the last day and 15 h before being killed. E, Histograms show the relative level of expression (±SEM, n = 3) for each treatment group. P value from Student’s t test was indicated above the bars. F, Autoradiogram of a Northern blot probed of RNA isolated from the uteri of d 4 pregnant mice heterozygous or homozygous for the LIF null mutation as indicated with a 32P-labeled Irg1 cDNA probe. Blots were reprobed with a GAPDH probe, and the signal was used as a RNA loading control. G (left), Autoradiograph of a Northern blot of RNA from either normally mated mice on d 4 of pregnancy (Prg) or pseudopregnant (Pseudo) mice probed with a 32P-labeled Irg1 cDNA probe. GAPDH signal was used as a RNA loading control, and the ratio of Irg1 signal to GAPDH is shown (right).

 
We next examined the time course of Irg1 mRNA induction after various hormonal treatments. Ovariectomized mice were injected with E2 alone or P4E2 after 3 d of P4 treatment. Mice were killed 4, 8, and 15 h later, and RNA was isolated from the uterine epithelium (Fig. 5CGo). Consistent with our previous results, uteri from mice treated only with E2 barely showed any Irg1 expression over this time course (not detectable at the autoradiographic exposure shown in Fig. 5AGo). However, strong expression of Irg1 transcripts was detected 4 h after E2 administration to the progestinized uterus. The level of Irg1 mRNA increased up to 8 h but was reduced by 15 h hormone treatment. Thus, P4 is required for E2 to induce maximal Irg1 expression.

To investigate this hormonal regulation further, we analyzed Irg1 expression in the delayed implantation model. In this experiment, mice were ovariectomized before the preimplantation estrogen surge on the morning of d 4, a treatment that results in blastocyst dormancy and inhibition of implantation. Daily P4 injection maintains this condition in a neutral phase, and embryos in these mice stay at the hatched blastocyst stage in the uterus (37). An estrogen injection to the P4-primed neutral uterus causes it to enter into the receptive phase and activates the dormant blastocyst in utero (38), resulting in implantation. In the current experiment, mice were treated daily with P4 after ovariectomy. On d 9 of gestation and 15 h after treatment with E2 in nidatory doses (Fig. 5DGo), the mice were killed and RNA was extracted from the uterine epithelium. Irg1 transcript levels were induced in these cells in response to P4E2 treatment when compared with those exposed only to P4 (Fig. 5EGo). Therefore, this experiment confirms that nidatory E2 together with P4 is required for maximal Irg1 mRNA induction during pregnancy.

Nidatory E2 has been shown to induce LIF, and the resultant transient burst of LIF on d 4 of implantation is required for the uterus to become receptive to blastocyst attachment (15, 39). Given the similarity of the hormonal control of Irg1 and LIF expression, we evaluated whether Irg1 mRNA expression was regulated by LIF using LIF-deficient mice. On d 4 of pregnancy, Irg1 expression was similar in LIF-/- mice when compared with the control heterozygous mice by Northern blotting (Fig. 5FGo) and in situ hybridization study (data not shown). Thus, Irg1 expression is not regulated by LIF.

Implantation is a mutually interactive process involving both the uterus and blastocyst (40). To determine whether Irg1 expression required the presence of a blastocyst, we took advantage of pseudopregnant mice, in which copulation between vasectomized males and fertile females results in normal P4 and E2 production in the absence of a fertilized embryo. Comparison of Irg1 expression in RNA isolated from the uterine epithelium of normal pregnant mice and pseudopregnant mice on d 4 revealed essentially the same level of expression (Fig. 5GGo). This indicates Irg1 induction is independent of the blastocyst and, therefore, of embryonic factors and entirely regulated by the ovarian sex steroid hormones.

Hormonal Regulation of Irg1 Expression Is via PKC
In macrophages, Irg1 mRNA expression is regulated by LPS acting through a PKC pathway (32). Therefore, we questioned whether there was a PKC involvement in its regulation in the uterus. First we determined whether PKC was active in the uterus over the implantation period in a pattern that would be consistent with Irg1 expression. Using anti-PKC class-specific antibodies we could detect PKC-{delta} and PKC-{eta} isoforms only in the uterus. Their concentration, however, did not significantly change through the first 6 d of pregnancy (Fig. 6AGo). However, using an antibody that detects the phosphorylated form of MARCKS (myristoylated alanine-rich protein kinase C substrate), a protein that is a major PKC substrate and which is widely distributed in various cell types and used as a marker of PKC activity (41), we found that uterine PKC activity changed during pregnancy. Phos-MARCKS was undetectable on d 1 and 2 of pregnancy but became evident on d 3 and reached a peak on d 4. Thereafter, it dropped to basal levels by d 6 (Fig. 6AGo). This pattern of expression is consistent with hormonal regulation by P4 and E2 with the peak activity being coincident with the uterine receptive period.



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  		Fig. 6. PKC Expression and Activity during Implantation

A, The cellular concentration of PKC-{delta}, PKC-{eta}, and phosphorylated (P) MARCKS was detected using relevant antibodies by Western blotting of cell extracts derived from the uterus from d 1–7 of pregnancy. Anti-GAPDH antibody was used to determine GAPDH concentrations as a control for protein loading. B, The cellular concentration of PKC-{delta}, PKC-{eta}, and phosphorylated MARCKS was detected by Western blotting on the uterine cell extracts from the uterine epithelium from mice treated with vehicle (OVX), E2, P4, or P4E2 after 4 continuous days of P4 injection as described in Materials and Methods.

 
To determine whether PKC activity is indeed steroid hormone regulated and also expressed in the uterine epithelium, we isolated uterine epithelial cell lysates from ovariectomized mice that had been treated with various P4 and E2 treatment regimens and probed them for PKC isoforms and for phospho-MARCKS. Both PKC-{delta} and PKC-{eta} are expressed in the epithelial cells but, as found in the total uterus, their levels were constant regardless of hormone treatment (Fig. 6BGo). In contrast, PKC activity changes in response to these hormones. In ovariectomized mice the phosphorylated form of MARCKS was at a low level. This level was increased by E2 and P4 treatment alone, with the former being slightly more potent (Fig. 6BGo). However, the highest level of phosphorylated MARCKS was found in cells after treatment of mice with the P4 and E2. This is consistent with the pattern of expression found during pregnancy.

Having established that PKC is activated during pregnancy in the uterus in a pattern similar to the induction of Irg1 expression and in the same cell type, we next determined whether Irg1 expression was dependent on PKC activity. To do this we injected the pan-PKC inhibitor (R031–8220) (42) into the uterine lumen of ovariectomized mice treated with P4 E2 immediately after the last hormone injection at doses consistent with the specific inhibition of PKC. In the same experiment, we also determined whether there was the involvement of protein kinase A (PKA) by the intraluminal injection of the PKA inhibitor, H89 (43). The efficacy of the PKC inhibitor (R031–8220) was examined using Western blots for phos-MARCKS of cellular uterine epithelial extracts derived from treated and untreated mice. Intraluminal injection of PKC inhibitor to the mouse uteri was performed immediately after administration with P4E2. Mice were killed 4 h after treatment. As assessed by the phosphorylated form of MARCKS, essentially all of PKC activity was blocked by the inhibitor (Fig. 7AGo). Having established that the PKC inhibitor is active in the uterus at this concentration and over this time, mice were killed 4 h after injection and RNA samples were collected from the luminal epithelium. As shown in Fig. 7BGo, Irg1 mRNA expression was inhibited by treatment with the PKC inhibitor, and the expression level was around 25% compared with the control untreated samples. In contrast, treatment with a PKA inhibitor did not significantly change the expression pattern of Irg1 mRNA (Fig. 7BGo).



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  		Fig. 7. PKC Regulates Irg1 Expression

A, Efficacy of PKC inhibitor in the mouse uterus. Epithelial protein extracts from the uteri of control mice and mice treated with P4E2 with and without the PKC inhibitor (R031-8220) as indicated for 4 h were isolated. Western blots were probed with anti-phospho-MARCKS antibody. The concentration of GAPDH protein was determined as a protein loading control. B (left), Autoradiograph of a Northern blot of RNA isolated from the uterine epithelium of ovariectomized mice treated with P4E2 in a regimen that allows implantation followed by an intraluminal injection of inhibitors for PKA (H89) or PKC (R031-8220) immediately after the last hormone injection and 4 h before killing. The Northern blots were probed with a 32P-labeled Irg1 or GAPDH cDNA probe. B (right), Histograms show the relative level of Irg1 mRNA expression level normalized to GAPDH mRNA for the various treatment groups as shown. Using Student’s t test, Irg1 expression in the uteri of mice treated with hormones (P4 and E2) and R031-8220 (P4E2+R031) is significantly lower than in the uteri of mice treated with hormones (P4E2) or hormones plus H89 (P4E2+H89). C, Autoradiograph of a Northern blot of RNA isolated from mice that had been in a delayed implantation state for 5 d and given a single injection of P4E2 sc and inhibitiors to PKA or PKC intraluminally, 15 h before killing the mice. The Northern blots were probed with a 32P-labeled cDNA to Irg1 and to GAPDH as a RNA loading control.

 
We also analyzed the effect of the PKC and PKA inhibitors in the delayed implantation model. As described previously, pregnant mice were ovariectomized on the early morning of d 4 and subsequently treated with P4 for the following 4 d. On d 9 of gestation and 15 h before killing the mice and 3 h after P4 plus estrogen treatment, PKC and PKA inhibitors were administrated by intraluminal injection. RNA samples isolated from the mouse uterus were monitored for Irg1 transcripts by Northern blot analysis (Fig. 7CGo). The level of Irg1 RNA in the delayed mice that had been treated with E2 and the PKC inhibitor was decreased dramatically when compared with the samples without inhibitor treatment. However, PKA inhibitor had no effect on Irg1 expression using this regimen (Fig. 7CGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Implantation requires synchronization between embryonic development and uterine differentiation. The uterine receptiveness is produced by the sequential and synergistic actions of ovarian steroid hormones. P4 is the predominant hormone, but a small increase in E2, known as the nidatory burst, is required by the uterus to enter a state in which the hatched blastocyst is allowed to implant (7, 8). This receptive period in the uterus begins on d 4 in the mouse and is immediately and compulsorily followed by a nonreceptive period when the blastocyst cannot implant (8). Removal of this nidatory E2 induces a delayed implantation state when the hatched blastocyst cannot implant although it remains viable (38). This state can be broken by subsequent injection of E2 provided that P4 levels have been maintained (38). Because the uterine epithelium is in direct contact with the blastocyst, these data have been construed to mean that the epithelium controls the implantation state of the embryo. In fact, the blastocyst can implant almost anywhere else except the uterus during delay (11).

There is still considerable controversy whether E2 and P4 act directly on the epithelial cells or through a paracrine mechanism (44, 45). In fact, at d 4 of pregnancy in the mouse uterus, PR can be barely detected in the uterine epithelium whereas it is abundantly expressed in the stroma (46). Paracrine mechanisms usually involve growth factors that act on transmembrane receptor kinases. These receptors trigger intracellular signaling pathways that often include the activation of PKC via inositol phospholipid hydrolysis. Using phosphorylated MARCKS as a marker of PKC activity, we showed that PKC activity, but not enzyme concentration, is regulated in the uterine epithelium during pregnancy. It shows a peak of activity at d 4 of pregnancy, a time that coincides with the receptive period for blastocyst implantation in the uterus. Others have shown that E2 and PKC pathways can interact. For example, in cell lines, PKC can increase the levels of and activate ER in a ligand-independent fashion (47, 48). In the rat ovary E2 induces PKC protein expression (49, 50). However, we did not find changes in PKC expression in the uterus with only the PKC-{delta} and PKC-{eta} isoforms being detected with the battery of antibodies used. Instead, we observed an increase in PKC activity that was regulated by E2 and P4 independently and synergistically in the luminal epithelium. To our knowledge, this is the first time that the ovarian hormone regulated alteration of PKC activity in the uterus during early pregnancy has been demonstrated, suggesting that this may play an important role during implantation.

In this study, we have identified a gene, Irg1, expressed predominantly in the luminal epithelium. This gene was originally found in a macrophage cell line that had been exposed to LPS (32). After identifying a full-length sequence with an open reading frame, analysis of its sequence showed that it is highly conserved in vertebrates and that it has high homology to the bacterial protein PrpD (36, 52). This enzyme is involved in the dehydration of (2S, 3S)-methylcitrate to 2-methyl-cisaconitate. The high homology of IRG1 protein sequences among mammals suggests it may have an indispensable role in metabolism. Possible functions could be odd-chain lipid degradation leading to the synthesis of propionyl-coenzyme A (CoA) that could be needed for the synthesis of other lipids required for implantation. Indeed, several enzymes that affect lipid metabolism are altered in the uterus during implantation. For example, lipoxygenase is an enzyme that converts arachidonic acid to leukotrieneA4, an essential fatty acid involved in inflammation and hypersensitivity reactions. Treatment of mouse uteri with lipoxygenase inhibitors block implantation (52). Similarly, cyclooxygenase 1 and 2, the rate-limiting enzymes in the synthesis of prostaglandins from common fatty acid, are expressed in the uterus. Cyclooxygenase-2 null mice have been described as being infertile due to implantation defects (23), although another group observed a much less severe phenotype that had no impact on fertility (27). IRG1 may also have a detoxification role because in humans, methylmalonik-CoA can be converted to the toxic propionyl-CoA via the B12-dependent methylmalonyl-CoA mutase and succinate decarboxylase. Therefore, the conversion of propionyl-CoA to pyruvate via the 2-methylcitric acid cycle could be a way to detoxify this molecule. The definitive role of Irg1 will be deduced from the gene targeting experiment designed specifically to disrupt its expression. These experiments are currently underway in our laboratory.

Our data have shown that Irg1 expression in the uterine epithelium is primarily regulated by P4 but its full physiological expression requires the synergistic interaction between P4 and E2. It also suggested that under physiological conditions, it is the nidatory estrogen secretion on d 4 that induced maximal Irg1 expression. Such conclusions are consistent with the expression pattern of Irg1 that shows a dramatic peak in the luminal epithelium at d 4 of pregnancy, a period of exposure to these two hormones. However, Irg1 is not regulated by LIF, suggesting that it is either on a parallel pathway or epistatic to LIF.

In macrophages, inhibitor studies indicated that Irg1 expression was regulated by LPS acting through the PKC pathway (32). Similarly, our inhibitor experiments suggest that the steroid hormone regulation of Irg1 expression in the uterus is mediated via PKC. This is consistent with the enhanced PKC activity in response to E2 and P4 in the uterine epithelial cells in a pattern similar to the accumulation of Irg1 mRNA in these cells. This could be via an effect by PKC on ER or PR activity, as has been observed in other systems, or through another mechanism such as a growth factor-mediated paracrine mechanism discussed above. Interestingly, Irg1 also has sites for PKC phosphorylation, suggesting that the protein’s functions could also be modulated by PKC in the luminal epithelium acting as another level of steroid hormone control. Taken together, our data show that Irg1 is expressed specifically in the luminal epithelium in response to the synergistic activity of E2 and P4 acting through the PKC pathway. These data suggest that both Irg1 and PKC may have important roles in implantation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animal and Treatments
All animal experiments were conduced under NIH guidelines for the care and use of experimental animals. Virgin female CD1 mice, obtained from Charles River Laboratories, Inc. (Wilmington, MA), were maintained on 12-h light,12-h dark cycles. Natural pregnancies were followed after detection of vaginal plug, which was designated as d 1 of pregnancy. To induce and maintain delayed implantation, mice were ovariectomized on the morning of d 4 of pregnancy, and injected daily with P4 from d 5–8. On d 9, mice were separated into two groups and given: 1) 1 mg of P4 or 2) 1 mg of P4 with one injection of 10 ng of E2 to induce uterine receptivity.

Hormone replacement experiments were performed as described (31). Briefly, mice were ovariectomized at 10–12 wk of age. After resting for 2–3 wk, they were primed for 2 d with 100 ng of E2 6 d before the experiment. Groups of two to five mice were killed by cervical dislocation at different time points after one of the following treatments: 1) no treatment (control); 2) one injection of 50 ng of E2; 3) 4 d of 1 mg of P4 (P4); or 4) 4 d 1 mg of P4 with one injection of 50 ng of E2 at the same time as the last P4 injection (P4E2). Day 4 pregnant uteri from the LIF null mutant and heterozygote control mice were provided frozen in dry ice as a kind gift of Dr. Colin Stewart (National Cancer Institute, Frederick, MD). All hormones were given sc in peanut oil. Steroid hormones were purchased from Sigma Chemical Co. (St. Louis, MO).

Preparation of Epithelial Cell Protein or RNA Extracts
After hormone treatment, uteri were removed, split longitudinally, and vortexed with Teflon beads (Small Parts, Inc., Miami, FL) in extraction buffer for 1 min as described (33). For protein analysis the extraction buffer contained 10 mM HEPES-KOH (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 2.5 mM EGTA, 10 mM ß-glycerophosphate, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM NaF, 0.1 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg of aprotinin per milliliter, 10 µg of leupeptin per milliliter, 10 µg of pepstatin A per milliliter. The beads were washed in a buffer containing 90 mM HEPES-KOH (pH 7.5), 0.2 M NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.2% Tween 20, 10% glycerol, 10 mM ß-glycerophosphate, 1 mM DTT, 1 mM NaF, 0.1 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg of aprotinin per milliliter, 10 µg of leupeptin per milliliter, and 10 µg of pepstatin A per milliliter. Lysates were sonicated and clarified by centrifugation, and for each experiment, equal amounts of protein, measured by Bradford assay (34) (Bio-Rad Laboratories, Hercules, CA), were used.

For RNA extraction from the epithelium, the extraction buffer contained 10 mM HEPES-KOH (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 2.5 mM EGTA, 10 mM ß-glycerophosphate, and 10% glycerol. To the 2 ml of extraction buffer 6 ml 6 M guanidinium thiocyanate with 37.5 mM sodium citrate and 0.75% Sarkosyl (vol/vol) was added and homogenized, followed by addition of 6 ml of phenol, 1.2 ml of chloroform, and 0.6 ml of sodium acetate (pH 4.0) with shaking. The mixture was kept on ice for 15 min and centrifuged at 12,000 x g for 20 min. For each sample the aqueous phase was removed to a fresh tube and precipitated with isopropyl alcohol. The RNA pellet was harvested by centrifugation at 8,000 x g for 15 min followed by a wash with 75% ethanol and another centrifugation to reharvest the RNA pellet. RNA was isolated from the total LIF -/- and +/- uteri using the guanidium isothyocyanate method as described previously (35).

Isolation of P4E2-Responsive Genes
RNA samples from the uterine epithelium were purified from DNA contamination by treatment with DNase I (Roche Clinical Laboratories, Indianapolis, IN) and converted into cDNA with reverse transcriptase. cDNA obtained from the mouse uterine epithelial cells treated with P4E2 was used as tester (cDNA pool containing the specific expressed gene) and cDNA from the mouse uterine epithelial cells treated with E2 was used as driver (reference sample for the tester cDNA pool) and subjected to the differential display reaction using a subtractive PCR kit (CLONTECH, Palo Alto, CA). The PCR products were subcloned into a TA plasmid vector (Invitrogen, San Diego, CA), and the resultant cDNA clones were subjected to restriction endonuclease digestion analysis with AluI, EcoRI, and HindIII (Roche Clinical Laboratories). Clones with unique restriction endonuclease digestion patterns were sequenced and compared with the appropriate DNA sequence databases.

Northern Blot Analysis
Total RNA (15 µg) was separated using a formaldehyde agarose gel electrophoresis and transferred to nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). After transfer, RNA was UV cross-linked to the membrane as described previously (35). Blots were prehybridized in Rapid-hyb buffer (Amersham Bioscience) for 1 h at 65 C. Hybridization was carried out for 5–24 h in the Rapid-Hyb buffer with 32P-labeled cDNA fragment probes at the concentration of 106 cpm/ml. The membranes were washed twice in 2x standard saline citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) washing solution at room temperature for 30 min, followed by another two washes in 0.5% SSC with 0.1% SDS at 65 C. The membranes were exposed to x-ray film or to Phospho-Screens. The intensity of the positive bands was determined by densitometry. To adjust for variations in RNA loading, the resultant signals were normalized either by the signal of GAPDH or 28S rRNA intensity.

5'-RACE Experiment and cDNA Library Construction
5'-RACE experiments were performed using SMART-RACE kit (CLONTECH). Briefly, total RNA from the mouse uterine epithelium treated with P4E2 was reverse transcribed with the Smart II oligo from the kit, followed by PCR amplification with the specific PCR primer for Irg1 (Irg1–3: 5'-TCGGTGGGAGCCTGAAGTCTGGTC-3'). The parameters for the PCR were: 94 C for 3 min, followed by 25 cycles of denaturing at 94 C for 30 sec, annealing at 65 C for 30 sec, and extension at 72 C for 2 min. The PCR product was cloned into TA plasmid vector (Invitrogen) for sequence analysis.

To prepare a representative progestinized uterine epithelial cell cDNA library in the Hybri-ZAP l vector, 1 mg RNA extracted from the uterine epithelium of 200 mice 3 and 4 h after the last treatment with P4E2 (group 4 in the animals section) was provided to Stratagene for their customer library construction. The titer of the primary library produced was 3.5 x 106 pfu, and the average insert size was 2.5 kb.

In Situ Hybridization
Uteri were collected from mice from d 1–7 of pregnancy and frozen in optimal cutting compound (Tissue-Tek) in liquid N2. In situ hybridization was performed by postfixing 10–15 µm uterine cryosections in 0.1 M sodium phosphate-buffered 4% paraformaldehyde, pH 7.4, for 30 min. They were rinsed in PBS for 1 min, and in 2x SSC for 1 min, followed by acetylation with 0.5% acetic anhydride in 0.1 M triethanolamine, pH 8.0, for 10 min, rinsed again in 2x SSC and then in PBS, and finally dehydrated in a graded series of ethanol washes. The slides were prehybridized in 2x SSC and 50% formamide at 50 C for 2 h and hybridized using hybridization buffer containing 2 x 104 cpm/ml antisense or sense RNA probes (hybridization buffer; 0.75 M NaCl, 50% formamide, 1x Denhardt’s solution, 10% dextran sulfate, 30 mM DTT, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 µg/ml salmon sperm DNA, and 0.5 mg/ml yeast tRNA) at 50 C for 16 h. Slides were washed twice in 2x SSC for 2 min; in 2x SSC, 50% formamide, and 0.1% ß-mercaptoethanol (BME) at 50 C for 1 h; in 20 mg/ml RNase A at 37 C for 30 min; in 0.5 M NaCl and 10 mM Tris-HCl, pH 8.0; in 2x SSC, 50% formamide, and 0.1% BME at 58 C for 30 min; and in 0.1x SSC and 0.1% BME at 63 C for 30 min before final dehydration. The sections were exposed to x-ray film for 4 or 5 d to obtain autoradiograms and then dipped in photographic emulsion and exposed for 6–8 wk. After development, sections were counterstained with cresyl violet.

Analysis of PKC and PKA in the Uterus During Implantation
To study the role of PKC and PKA during implantation, uterine protein lysates prepared from mice at d 1–7 of pregnancy were boiled in gel sample buffer containing SDS and separated by electrophoresis, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), and blotted with the appropriate antibodies (31). The following rabbit antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): {alpha}-PKC {delta} (sc-213), {alpha}-PKC {eta} (sc-215). Antibody against phosphor-MARCKS was acquired from Cell Signaling Technology (Beverly, MA). To study the hormonal regulation of PKC, ovariectomized mice that had been treated with various hormone regimens described above were anesthetized with avertin and the uterus exposed through a dorsal incision. Antagonists of PKC (R031-8220) and PKA (H89) (kind gifts of Dr. C. Rubin, Albert Einstein College of Medicine, New York, NY) were injected intraluminally in PBS. An equal volume of PBS was used in the control mice. Mice were killed at different times afterward and analyzed for Irg1 mRNA expression or PKC activity as described in Results.


   ACKNOWLEDGMENTS
 
We thank Jim Lee for his technical assistance in the mouse facility, Drs. Hui Feng and Min Ren and C. Rubin for the PKC and PKA inhibitors and advice on the concentration to use, Dr. A. Niklaus for helpful discussions, and Dr. C. Stewart (NCI, Frederick, MD) for providing uteri from LIF-deficient and LIF-positive pregnant mice.


   FOOTNOTES
 
E-mail addresses for B.C. and D.Z.: bochen{at}aecom yu.edu and ZDAM{at}novonordisk.com, respectively.

This work was supported by NIH Grants RO1 CA 89617 (to J.W.P.) and the Cancer Center core grant P30-CA13330. J.W.P. is the Betty and Sheldon E. Feinberg senior faculty scholar in cancer research.

Abbreviations: BME, ß-Mercaptoethanol; CoA, coenzyme A; DDT, dithiothreitol; E2, 17ß-estradiol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IPTG, isopropyl-ß-D-thiogalactopyranoside; Irg1, immune response gene 1; LIF, leukemia-inhibitory factor; LPS, lipopolysaccharide; MARCKS, myristoylated alanine-rich PKC substrate; P4, progesterone; PKA, protein kinase A; PKC, protein kinase C; PR, progesterone receptor; PrpD, methylcitrate dehydratase; RACE, rapid amplification of cDNA ends; SDS, sodium dodecyl sulfate; SSC, standard saline citrate.

Received for publication June 2, 2003. Accepted for publication July 18, 2003.


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