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BOR - Papers in Press, published online ahead of print February 27, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.065821
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BIOLOGY OF REPRODUCTION 78, 1029–1037 (2008)
DOI: 10.1095/biolreprod.107.065821
© 2008 by the Society for the Study of Reproduction, Inc.

Pregnancy; 

Delayed Parturition and Altered Myometrial Progesterone Receptor Isoform A Expression in Mice Null for Krüppel-Like Factor 91

Zhaoyang Zeng 4 5, Michael C. Velarde 3 4 5, Frank A. Simmen 5 6, and Rosalia C.M. Simmen 2 5 6

Department of Physiology and Biophysics,5 University of Arkansas for Medical Sciences, and Arkansas Children's Nutrition Center,6 Little Rock, Arkansas 72202

ABSTRACT

Preterm and delayed labor conditions are devastating health problems with currently unknown etiologies. We previously showed that the transcription factor Krüppel-like factor 9 (KLF9) influences the expression and/or transcriptional activity of receptors for estrogen and progesterone in endometrial cells in vivo and in vitro. Given that estrogen and progesterone differentially regulate uterine myometrial contractility during gestation, we hypothesized that lack of KLF9 could compromise myometrial function, leading to defects in parturition. To test this, we used mice null for Klf9 to evaluate gestation length, response to the progesterone receptor (PGR) antagonist RU486, expression levels of steroid receptor proteins, nuclear receptor coactivator and contractility-associated genes, and nuclear factor-kappaB (NF-kappaB) DNA binding activity in myometrium near term. Klf9 knockout (KO) mice exhibited delayed parturition by 1–2 days relative to wild-type (WT) counterparts, in the absence of fetal genotype contribution and differences in serum estrogen and progesterone levels. Knockout mice near term were refractory to the abortive action of RU486, and they displayed aberrant myometrial expression patterns of nuclear PGR-A and NF-kappaB p65/RELA relative to WT mice. Myometrial expression levels of nuclear estrogen receptor-alpha did not differ, whereas those for Oxtr and Crebbp mRNAs were lower, in KO versus WT mice. Results indicate that KLF9 contributes to the regulation of PGR-associated components in the myometrium necessary for timely onset of parturition in mice. The present study highlights the potential utility of Klf9 null mice to investigate the pathophysiology of parturition defects involving PGR signaling.

female reproductive tract, myometrium, parturition, progesterone, steroid hormone receptors

INTRODUCTION

The increasing incidence of preterm births, currently reported at 12% in the United States and up to 25% in developing countries [1, 2], is a significant global health problem, given that premature delivery is a major cause of neonatal morbidity and mortality. Although many preterm infants survive, they often suffer from extensive lifelong disabilities, including mental retardation, pulmonary and physical disorders, blindness, and hearing loss. Delayed onset of labor, albeit not as devastating as preterm births, represents 3%–5% of all complications during delivery and similarly results in neonatal morbidity and mortality [1]. Thus, understanding the molecular mechanisms underlying the timing of normal labor is critical for addressing the prevention, treatment, and management of these devastating health conditions.

Parturition is a complex physiological process regulated by numerous signals originating from the maternal and fetal units [3]. These signals likely are initiated at distinct stages of the parturition process and involve multiple pathways that must be fully integrated at the level of the myometrium. The "pregnancy" hormone progesterone (P) plays a major role in parturition events by maintaining uterine quiescence through suppression of genes that promote myometrial contractility [4, 5]. Thus, the withdrawal of P action promptly results in labor onset. For most mammalian species, this is achieved through a rapid decline in systemic P levels [6]. However, in humans and higher primates for whom P levels are maintained throughout pregnancy and during labor and delivery, changes in myometrial nuclear progesterone receptor (PGR) expression are implicated in "functional P withdrawal," leading to decreased myometrial P responsiveness [7, 8]. Induction of estrogen (E) signaling is also requisite for initiation of parturition by opposing P action, and thereby increasing the expression of genes that augment myometrial contractility [7, 9]. However, to what extent and at what level these steroid hormones counteract each other's activity to influence myometrial activation pathways leading to parturition, remain unclear. The lack of a systematic analysis of common or distinct gene targets of E and P in the late-term myometrium; the emergence of novel targets for regulating myometrial transformation and contraction, most of which have not been previously considered as E or P regulated; and differences in the spectrum of genes regulated in nonhuman (e.g., mouse) and human myometrium at term, despite conserved myometrial biochemical changes, may partly explain the current paucity in knowledge [10, 11].

Progesterone signals in target cells by binding to two PGR isoforms, PGR-A and PGR-B, which are transcribed from a single gene with distinct promoters and which differ only by the presence of a 165-amino acid sequence in the N-terminus of PGR-B [12, 13]. Progesterone receptor-A and PGR-B have discrete functions in reproduction [14]; whereas Pgr-A null mice, Pgrtm1Omc, exhibit severe uterine hyperplasia and ovarian abnormalities, leading to infertility, mice null for Pgr-B, Pgrtm2Omc, surprisingly, have no obvious uterine abnormalities but exhibit defective mammary gland development [15, 16]. The unique phenotypes of mice null for each PGR isoform have been attributed to the distinct gene regulatory activities of PGR-A and PGR-B, partly as a consequence of their divergent abilities to recruit coactivators and corepressors, which increase and decrease their transcriptional activities, respectively [17, 18]. Thus, the elucidation of the mechanisms underlying the specificity and selectivity of PGR activity has increasingly focused on the identification and regulation of nuclear coregulators that influence PGR-mediated transcription in P-target cells.

Our laboratory has identified Basic Transcription Element Binding Protein-1/Krüppel-like factor 9 (BTEB1/KLF9), a member of the Sp family of transcription factors [19, 20] as a PGR-interacting protein with a functional consequence in reproduction [21–24]. KLF9 enhances PGR-mediated transactivation in endometrial epithelial cells in vitro, in part by selective interactions with PGR-A and PGR-B isoforms under distinct physiological contexts [21, 22, 25]. Targeted deletion of Klf9 resulted in subfertility due to reduced embryo implantation, decreased uterine stromal PGR expression, and attenuated uterine sensitivity to P [23, 24]. More recently, we showed that KLF9 can negatively regulate estrogen receptor-{alpha} (ESR1) activity by promoting E-dependent ESR1 downregulation and decreasing ligand-activated ESR1 transactivation [26]. Thus, KLF9 supports PGR function by also opposing ESR1 signaling.

In light of the disparate regulation by KLF9 of ESR1 and PGR transactivities, and the requirement for withdrawal of P action coincident with activation of E signaling to initiate parturition, we considered the possibility of a regulatory role for KLF9 at parturition. In the present study, we used Klf9 null mice to investigate the consequence of Klf9 ablation on gestation length and on PGR and ESR1 signaling at late pregnancy. Our results demonstrate that KLF9 contributes to the initiation of parturition through a mechanism involving its regulation of the expression of PGR-A isoform and related signaling components in the myometrium, without affecting ESR1 expression.

MATERIALS AND METHODS

Animals and Myometrial Tissue Collection

All animal studies were conducted in accordance with the National Institutes of Health animal care guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Animals were allowed access to standard chow diet and water ad libitum and were housed in a pathogen-free barrier facility with a 12L:12D cycle. Heterozygous Klf9 mutants were intercrossed to generate wild-type (WT; +/+), heterozygous (HET; +/–), and null (KO; –/–) mice, which were propagated and genotyped as previously described [23, 24]. In timed pregnancy studies, age-matched WT and KO females (2–6 mo) were monitored for stage of estrous cycle by vaginal smears and were mated at proestrus of the second estrous cycle. The presence of a vaginal plug was considered as day postcoitum (dpc) 0.5. Wild-type and KO females were mated to KO and WT males, respectively, to generate pups of the same genotype (HET); this strategy eliminated the possible contribution of fetal genotype to parturition events. Uterine tissues were removed from mice at specified pregnancy days (n = 4–6 mice per genotype per gestation day), placed in ice, and opened longitudinally to retrieve the fetuses, which were counted and weighed. Myometrial tissues were isolated by careful dissection of all embryonic material and maternal decidua. For each animal, myometrial tissue from one uterine horn was divided into two pieces: one piece was homogenized in TriZol for subsequent RNA analyses, and the other piece was flash frozen and stored at –80°C for protein analyses. The myometrial tissue from the other horn was similarly split into two, with one part placed into fixative for paraffin embedding and the other part processed for X-Gal staining (below).

In studies to evaluate gestation length, time-mated WT and KO females were allowed to deliver, and the appearance of the first pup was considered as day of parturition. The number of born pups and their birth weights were determined within 8 h of birth. To induce parturition, the progesterone antagonist RU486 (mifepristone; Sigma-Aldrich, St. Louis, MO) was administered by subcutaneous injection (150 µg in 0.9% saline solution; Sigma-Aldrich) to pregnant mice at dpc 16 (WT), dpc 17 (KO), and dpc 18 (WT and KO; n = 4–6 per genotype per dpc), following a previously described protocol [27]. Parturition was monitored as indicated above.

X-Gal Staining

Isolated tissues were soaked in phosphate-buffered saline containing 20% sucrose, embedded in Tissue-Tek OCT medium (Fisher Scientific) and frozen in liquid nitrogen [23]. Sections (7 µm) were fixed in ice-cold 2% paraformaldehyde for 1 h at room temperature, incubated in 0.2% glutaraldehyde for 5 min, and stained with 5-bromo-4-chloro-3-indoyl-galactopyranoside (X-Gal; 1 mg/ml; Sigma-Aldrich) for 4 h at 37°C.

Serum E and P

Serum samples were processed from blood recovered by closed cardiac puncture as previously described [23] and stored at –20°C until assayed. Serum E and P levels were measured using Coat-A-Count Estradiol or Coat-A-Count Progesterone Diagnostic kits following the manufacturer's protocols (Diagnostic Products Corp., Los Angeles, CA). Assays were performed in duplicate per sample (n = 6–8 mice per genotype per dpc) in one run. Data are presented as least-squares mean ± SEM.

RNA Isolation and Quantitative RT-PCR

Total RNA from myometrial tissues was extracted using TriZol reagent, following the manufacturer's instructions (Invitrogen, Carlsbad, CA), and integrity of RNA was confirmed using the RNA6000 Nano LabChip kit with the Agilent 2100 bioanalyzer system (Agilent Biotechnologies, Palo Alto, CA). Complementary DNA was synthesized from 1 µg total RNA using random primers and a cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Polymerase chain reaction primers that span intron/exon junctions were designed using PrimerExpress software (Applied Biosystems). The forward and reverse primers and size of amplicons were: oxytocin receptor (Oxtr; 105 bp), 5'-GGC CGT GTT CCA GGT TCT C-3' and 5'-ATG CCC ACC ACC TGC AAG TA-3'; nuclear receptor coactivator 2 (Ncoa2; 121 bp), 5'-GGG CTG GGA AGA TCT GGT AAG-3' and 5'-ACT GAA CGC CAA CCC TTG TC-3'; CREB binding protein (Crebbp; 121 bp), 5'-CAA GCA AAT GGA GAG GTT CGA-3' and 5'-AAG ATG CAC AAT GGG CAA CTT-3'; gap junction membrane channel protein alpha 1 (Gja1; 102 bp), 5'-AAG GGA AGA AGC GAT CCT TAC C-3' and 5'-TGG TGA GGA GCA GCC ATT G-3'; and Rn18s (153 bp), 5'-TGC TCT TAG CTG AGT GTC CCG-3' and 5'-TCA TGG CCT CAG TTC CGA AA-3'. Standard curves were generated by serial dilution of a pool of total RNA isolated from myometrium. Quantitative RT-PCR (QPCR) was performed with the Light Cycler-based SYBR Green I detection system (Applied Biosystems) using an ABI Prism 7000 sequence detector (Applied Biosystems). Amplification was carried out as follows: preincubation at 50°C for 2 min, DNA polymerase activation at 95°C for 1 min, followed by 40 PCR cycles of 95°C for 15 sec, and 60°C for 1 min. For each sample, relative gene expression was calculated with Rn18s as internal reference. Results are expressed as fold change of mRNA compared with that for WT at dpc 18.5, which was arbitrarily assigned a value of 1, except when indicated.

Western Blot Analyses

Nuclear extracts from mouse myometrium and the human endometrial carcinoma cell line Ishikawa were prepared as previously described [26], and protein concentrations were measured using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL). Proteins were fractionated on SDS-10% polyacrylamide gels and transferred to nitrocellulose membranes. Blots were incubated with 5% dry milk in Tris-buffered saline containing 1% Tween-20 to block nonspecific binding and then with primary antibodies at 4°C overnight on a rocking platform. The antibodies used were: anti-rat KLF9 (1:1000; in-house) [21]; anti-human PGR (A0098; 1:500; Dako; sc-538; 1:500; Santa Cruz Biotechnology; Pgr1294; 1:250; Dako); anti-human ESR1 (sc-542; 1:500; Santa Cruz Biotechnology); and anti-human {alpha}-actinin (1:500; Sigma-Aldrich). An antibody to human lamin B1 (1:1000; Santa Cruz Biotechnology) was used as a control for protein loading and transfer. Membranes were washed with a series of buffers after incubation with primary antibody, and they were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology) for 1–2 h at room temperature. Immunoreactive proteins were visualized on X-ray film using an enhanced chemiluminescence detection system (ECL; Pierce Biotechnology). Signals were captured and quantified with the Bio-Rad molecular analyst detection system and Quantity One software. Blots were stripped with Restore Western blot stripping buffer (Pierce Biotechnology) for 1 h at room temperature prior to probing with a different antibody.

Nuclear Factor-{kappa}B p50/p65 Transcription Factor Assay

The nuclear factor-{kappa}B (NF-{kappa}B) p50/p65 DNA binding activity in myometrial samples isolated from WT and KO mice at late term was evaluated following the manufacturer's instructions (Chemicon International Inc., Temecula, CA). Briefly, nuclear extracts (100 µg protein) were incubated with a double-stranded biotinylated oligonucleotide consensus sequence (5'-GGGACTTTCC-3') that binds NF-{kappa}B p60/RELA and p50 proteins. After incubation, the mixtures were transferred to the streptavidin-coated plate, and unbound materials were washed with buffer. The bound p65/RELA and/or p50 proteins were detected with specific primary antibody to each and quantified by colorimetric assay (optical density at 450 nm) after incubation with horseradish peroxidase-secondary antibody. Positive and negative controls for the assay included cell extracts expressing NF-{kappa}B p65 and p50 proteins, a nonspecific double-stranded oligonucleotide, and a specific competitor double-stranded oligonucleotide.

Data Analysis

Comparisons of incidence of neonatal death and of gestation length in WT and KO mice were analyzed by chi-square and Fisher exact test, respectively. Comparisons of fetal weights and time of delivery after RU486 in WT and KO mice were analyzed by t-test. RNA and immunoblot analyses were presented as mean ± SEM. The values were subjected to analysis by Student t-test and one-way ANOVA, as indicated under each figure legend. Differences between means in one-way ANOVA were further analyzed by Tukey test. P < 0.05 was considered statistically significant.

RESULTS

KLF9 Is Expressed in Late Pregnancy Myometrium

We had previously shown that KLF9 is expressed in smooth muscle cells of the myometrium of early pregnant mice [23]. In those studies, X-Gal staining was used to evaluate KLF9 expression, since Klf9 KO mice were generated by knock-in of the LacZ gene into the open reading frame of Klf9 exon 1, Klf9tm1Yfk [28], and because in-house as well as commercially available anti-KLF9 antibodies, while useful for Western blot analysis, give high background by immunohistochemistry (R.C.M. Simmen, unpublished findings). Moreover, X-Gal staining was found to faithfully recapitulate the endogenous pattern of KLF9 expression in mouse tissues [23, 28]. X-Gal-stained cryosections of uterine tissues from Klf9 KO mice at dpc 18.5, dpc 19.5, and dpc 20.5 revealed robust and comparable Klf9 expression in myometrial circular and longitudinal muscle layers (Fig. 1A). A corresponding uterine section from WT mice at dpc 19.5 showed no staining, demonstrating the specificity of the staining procedure (Fig. 1A). To confirm KLF9 expression at the protein level, nuclear extracts prepared from myometrial tissues of WT mice at dpc17.5, dpc 18.5, and dpc 19.5 were evaluated by Western blot using anti-KLF9 antibody. Results indicate relatively high constitutive levels of KLF9 protein in myometrium near term (Fig. 1, B and C).


Figure 01
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  		FIG. 1. KLF9 gene and protein expression in mouse myometrium near term. A) Histological sections of X-Gal-stained uteri from WT at dpc 19.5 (parturition) and Klf9 null (KO) mice at dpc 18.5, dpc 19.5, and dpc 20.5 (parturition). Original magnification x20. Also shown are higher magnifications (x40) of a section of longitudinal smooth muscle layer (LM), circular smooth muscle layer (CM), and decidua (D) of X-Gal-stained uteri (boxed areas) from a KO mouse at dpc 19.5. Arrows indicate X-Gal-staining cells (blue). Note the absence of X-Gal stain in WT uterus. B) KLF9 protein levels (arrow, 32 kDa) in uterine myometria of WT mice as a function of day of pregnancy (n = 3 animals per dpc) by Western blot. Lamin B1 is 67 kDa. C) Abundance (percent adjusted volume) of KLF9 protein (assessed by densitometry of Western blot shown in B) relative to lamin B1 loading control. Data are mean ± SEM.

Delayed Parturition Is Associated with Loss of KLF9

Previously, we reported higher neonatal mortality (within 24 h of birth) for litters born to KO compared with WT dams [23]. To extend the analysis as a function of age of dams, we analyzed all birth records from homozygous matings (i.e., WT x WT and KO x KO) and segregated the data from dams of two age groups (≤6 mo and >6 mo, respectively). Consistent with a previous report [23], KO litters demonstrated higher neonatal mortality than corresponding age-matched WT litters (by 2.5-fold; P = 0.08) for 2- to 6-mo-old dams, and this difference was highly significant (P = 0.016) for older dams (Table 1). Our analysis also indicated that KO dams experienced greater problems in delivery, with 10.6% of 104 births showing complications (e.g., pups incompletely expelled from the uterus; delivery occurred in phases) as opposed to only 1% for WT dams (P = 0.014; R.C.M. Simmen, unpublished findings). The enhanced neonatal mortality with Klf9 ablation, the higher incidence of abnormal parturition in KO dams, and the presence of KLF9 in the myometrium at late pregnancy, together suggested a functional role for KLF9 in parturition events. To address this possibility in the absence of potential contribution(s) by the fetal/neonatal unit, WT and KO females between 2 and 8 mo of age were time mated with KO and WT males, respectively, to generate pups of the same genotype (i.e., HET), and gestation length was evaluated. Although WT females (82%) predominantly gave birth at dpc 19.5, KO females had delayed parturition, with a majority (70%) giving birth at dpc 20.5 or later (range: dpc 20.5–22.5; Table 2). The delayed births in KO dams were accompanied by increased incidences of neonatal death (19.5% for KO vs. 5.98% for WT; P < 0.05) and lower birth weights (1.265 ± 0.026 g for KO vs. 1.397 ± 0.024 g for WT; P < 0.001). Collectively, these results suggest that loss of KLF9 compromises timing of parturition and neonatal viability that can be directly attributed to maternal genotype, likely at the level of the uterus.


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  		TABLE 1. Incidence of neonatal death in WT and Klf9 KO dams.a


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  		TABLE 2. Gestation length in WT and Klf9 KO mice.

KLF9 Deficiency Does Not Affect Steroid Hormone Levels at Late Pregnancy

Consistent with the reported rapid decline in serum P levels near term [29], circulating levels of P dramatically decreased in pregnant WT mice (Fig. 2A). Knockout mice also showed a decline in P levels at late pregnancy, and there were no significant differences in these levels for WT and KO mice. Serum E levels remained unchanged between dpc 17.5 to dpc 19.5 for WT mice and did not differ between WT and KO (Fig. 2B).


Figure 02
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  		FIG. 2. Serum levels of progesterone (A) and estradiol (B) during late pregnancy in WT and Klf9 null mice. Values are presented as mean ± SEM (n = 6–8 mice per genotype per dpc). Bars with different letters differed at P < 0.05 by one-way ANOVA. No significant differences between genotypes were observed (Student t-test).

Loss of KLF9 Alters Response to PGR Antagonist RU486

To determine whether P-dependent PGR activity in the myometrium is compromised with Klf9 ablation, we used the well-characterized antagonist of PGR function RU486, which binds to PGR and impairs its gene regulatory activity [30]. Since RU486 is an abortive agent [31], we tested whether it can rescue parturition in KO mice. Wild-type and KO dams were injected with RU486 at dpc 16 (WT), dpc 17 (KO), and dpc 18 (WT, KO), and their times of delivery after RU486 were monitored. Consistent with a published report [27], RU486 induced parturition within 24 h of its administration in WT mice, and this response occurred when RU486 was injected at either dpc16 or dpc 18 (Table 3). Surprisingly, although KO mice at dpc 17 delivered 24 h after RU486 similarly to WT, KO mice at dpc 18 became completely refractory to this abortive agent and gave birth at their normal parturition day of ~dpc 20.5, which is ~2–3 days after RU486 injection (Table 3). These results suggest that absence of uterine KLF9 function 1 day prior to term can significantly alter uterine myometrial response to PGR-dependent parturition signal.


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  		TABLE 3. RU486-induced parturition in WT and Klf9 KO mice.

Loss of KLF9 Alters PGR-A Isoform Expression Levels in Term Myometrium

In humans, myometrial sensitivity to P at parturition is delicately controlled by the relative expression levels of the PGR isoforms PGR-B and PGR-A [9, 32, 33]. Given the loss of response of KO mice to RU486 at dpc 18, suggesting aberrant PGR signaling near term, we examined whether KLF9 influences myometrial sensitivity to parturition signals by regulating PGR expression. Since genomic responses of target cells to P are determined by the levels of nuclear (activated) PGR receptors, we evaluated PGR-B and PGR-A proteins in myometrial nuclear extracts by Western blot. Using an anti-PGR antibody (sc538) that was previously reported to detect PGR-B in late pregnant mouse uteri by another group [34], we found that nuclear extracts prepared from myometrium of WT mice showed a single immunoreactive band of ~100-kDa molecular weight that migrated at the same position as the protein designated PGR-B in that study [34]; however, we did not detect PGR-A in these myometrial extracts with this antibody (Fig. 3A). Progesterone receptor A of the correct molecular weight (~90 kDa) was detected in mouse myometrial extracts using anti-PGR antibody A0098 (Fig. 3A). When the blot was stripped and incubated with anti-{alpha}-actinin antibody, the ~100-kDa protein detected by the sc538 antibody co-migrated with {alpha}-actinin (Fig. 3A), consistent with a previous report [35]. Western blot analyses of the same myometrial samples upon incubation with another commercially available anti-PGR antibody (Pgr1294) that recognized PGR-B in human myometrial samples [33] detected both PGR isoforms in nuclear extracts prepared from human Ishikawa cells, but neither PGR isoform in the mouse samples (Fig. 3A).


Figure 03
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  		FIG. 3. Myometrial expression of PGR-A in WT and Klf9 null mice near term. A) Immunoblots of nuclear extracts from myometrium of WT mice (dpc 18.5) or from human Ishikawa endometrial carcinoma (I) cells. Blots were probed with different antibodies that were evaluated for detection of PGR-B (sc538; Pgr1294), PGR-A (A0098), and {alpha}-actinin (A2543). B) Myometrial levels of nuclear PGR-A (81 kDa) and lamin B1 (67 kDa) proteins of WT mice at dpc 17.5–19.5 (n = 3 mice per dpc) measured by Western blot. Abundance (percent adjusted volume, mean ± SEM) of PGR-A relative to lamin B1 loading control was determined by densitometry of Western blot. C) Abundance (percent adjusted volume, mean ± SEM) of nuclear PGR-A protein levels in myometria of WT and Klf9 null mice at dpc 17.5 (n = 4 per genotype), dpc 18.5 (n = 4 for WT; n = 5 for KO), and dpc 19.5 (n = 4 per genotype) relative to lamin B1 loading control, determined by densitometry of Western blots (not shown). Comparisons were between genotypes for each dpc.

Given the unreliability of available antibodies to recognize PGR-B protein in mouse myometrial tissues, only PGR-A protein levels were subsequently evaluated in the present study. Nuclear PGR-A protein levels increased dramatically between dpc 17.5 and dpc 19.5 in WT mice (Fig. 3B). Loss of KLF9 altered the expression pattern of PGR-A protein as a function of pregnancy day (Fig. 3C). Relative to lamin B1 (loading control), nuclear PGR-A levels were comparable at dpc 17.5 for WT and KO, tended to differ at dpc 18.5 (KO > WT; P = 0.08), and significantly differed at dpc 19.5 (KO < WT; P = 0.03) between genotypes.

Myometrial ESR1 Protein Levels Near Term Are Not Affected by Klf9 Null Mutation

Increased responsiveness of myometrium to E at late term, leading to the onset of labor may be due to increased myometrial expression of ESR1 [9]. To evaluate whether loss of KLF9 altered the pattern of myometrial ESR1 expression, nuclear extracts prepared from myometrial tissues of WT and KO mice at dpc 17.5, dpc 18.5, and dpc 19.5 were evaluated by Western blot using anti-ESR1 antibody. Nuclear ESR1 protein levels did not significantly change across late pregnancy for WT mice (Fig. 4A) and did not differ between WT and KO mice at each pregnancy day (Fig. 4B). Thus, in contrast to nuclear PGR-A, the levels of nuclear (activated) ESR1 were not altered by KLF9 in late pregnancy stage mouse myometrium.


Figure 04
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  		FIG. 4. Myometrial expression of ESR1 in WT and Klf9 null mice near term. A) Myometrial levels of nuclear ESR1 (arrow, 66 kDa) and lamin B1 (arrow, 67 kDa) proteins of WT mice at dpc 17.5–19.5 (n = 3 mice per dpc) measured by Western blot. Abundance (percent adjusted volume, mean ± SEM) of ESR1 relative to lamin B1 loading control was determined by densitometry of Western blot. B) Abundance (percent adjusted volume, mean ± SEM) of nuclear ESR1 protein levels in myometria of WT and Klf9 null mice at dpc 17.5 (n = 4 per genotype), dpc 18.5 (n = 4 for WT; n = 5 for KO), and dpc 19.5 (n = 4 per genotype), relative to lamin B1 loading control, determined by densitometry of Western blots (not shown). Comparisons were between genotypes for each dpc.

Selective Changes in Myometrial Expression of Parturition-Related Genes with Loss of KLF9

To further evaluate the contribution of KLF9 to parturition, we examined the transcript levels of nuclear receptor coactivators involved in P-dependent PGR activity near term (CREB binding protein, Crebbp; nuclear receptor coactivator 2, Ncoa2) [17, 18, 36] as well as of proteins involved in myometrial contractility (oxytocin receptor, Oxtr; gap junction membrane channel protein alpha 1, Gja1) in WT and KO mice at dpc 18.5 and dpc 19.5 [37–40]. In WT mice, Crebbp and Ncoa2 mRNA levels at dpc 19.5 did not differ from those of dpc 18.5 (Fig. 5, A and B). However, Oxtr transcript levels were higher (P < 0.05) at dpc 19.5 than at dpc 18.5, whereas those of Gja1 tended to differ (P = 0.094) between these pregnancy days (Fig. 5, C and D). An increase in myometrial expression of Oxtr and Gja1 from dpc 18.5 to dpc 19.5 was observed in KO mice. Relative to WT mice, KO mice at dpc 18.5 had lower Crebbp (P < 0.05) and Ncoa2 (P = 0.08) mRNA levels and comparable Oxtr and Gja1 transcript levels. At dpc 19.5, no differences for Crebpp, Ncoa2, and Gja1 were observed between WT and KO mice, whereas Oxtr levels were higher (P = 0.016) for WT than KO (Fig. 5). These results suggest that KLF9 may additionally influence onset of labor by directly or indirectly regulating the expression of a number of genes associated with parturition.


Figure 05
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  		FIG. 5. Transcript levels of A) Crebbp, B) Ncoa2, C) Oxtr, and D) Gja1 genes in myometria of WT and Klf9 null mice near term. The mRNA levels were quantified by QPCR and normalized to Rn18s levels (mean ± SEM; n = 4). Bars with different letters differed at P < 0.05 (ANOVA).

Loss of KLF9 Alters Pattern of NF-{kappa}B p65/RELA DNA Binding Activity

Previous studies have indicated that activation of NF-{kappa}B signaling accompanies the initiation of parturition [34, 41]. To determine whether delayed parturition in Klf9 null mice is due in part to altered NF-{kappa}B transcriptional activity, the ability of nuclear p65/RELA and p50 to bind the NF-{kappa}B binding motif of NF-{kappa}B-responsive genes was assessed in the myometrial tissues of WT and KO mice. DNA binding activity of p65/RELA, but not of p50 (data not shown), was detected in myometrial tissues of both genotypes (Fig. 6). However, whereas WT tissues showed increased p65/RELA DNA binding activity between dpc 17.5 and dpc 19.5 (P = 0.03), consistent with a previous report [41], those of KO mice did not exhibit a similar pattern of DNA binding activity (Fig. 6).


Figure 06
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  		FIG. 6. Myometrial levels of NF-{kappa}B p65/RELA DNA binding activity in WT and Klf9 null mice near term. Nuclear extracts (100 µg protein/assay) prepared from myometrium of WT and KO mice (n = 4–5 individual animals per dpc per genotype) were evaluated for binding activity to a NF-{kappa}B consensus sequence (see Materials and Methods). Binding activity (mean ± SEM) was calculated as the optical density at 450 nm (OD450) of enzyme product based on enzyme-linked immunosorbent assay relative to known standards. *P < 0.05 relative to dpc 17.5 WT values.

DISCUSSION

The present study tested the hypothesis that the Sp-related, PGR-interacting protein KLF9 contributes to the timing of parturition by mediating functional PGR signaling. Using mice null for Klf9 and corresponding WT controls, we showed that loss of KLF9 resulted in 1) delayed parturition; 2) aberrant response to the PGR antagonist RU486; 3) aberrant myometrial expression of PGR-A; 4) altered pattern of NF-{kappa}B p65/RELA DNA-binding activity; and 5) decreased myometrial expression of Oxtr and Crebbp, whose protein products are involved in ESR1 and PGR signaling. The distinct myometrial changes observed with Klf9 ablation were manifested in the absence of differences in steroid hormone profiles, nuclear ESR1 protein levels, and transcript levels for the contractility protein Gja1 and the nuclear receptor coactivator Ncoa2. Our findings that the delayed parturition in Klf9 null mice may be a consequence of altered myometrial PGR-A isoform expression and of NF-{kappa}B p65/RELA DNA-binding activity implicate these molecules in the timing of labor onset in this species, consistent with that noted in human and nonhuman primate parturition [7, 8, 33, 42]. Importantly, these results suggest the utility of the Klf9 null mouse in the study of mechanisms underlying P/PGR-regulated parturition. Given that the molecular events surrounding labor at term are highly orchestrated and that numerous overlapping mechanisms are considered to underlie myometrial contractility, the relatively modest delay in the timing of labor onset observed with Klf9 ablation (~1–2 days) is not surprising and, indeed, is consistent with the phenotypes of other mouse mutants where early or delayed parturition occurred by a similar time frame [38, 43].

The present studies indicate that KLF9 may participate in the regulation of myometrial PGR signaling, leading to the onset of parturition by several mechanisms. One mechanism relates to the ability of KLF9 to influence the expression levels of nuclear PGR-A, whose transcriptional activity in vitro has been shown to oppose that of PGR-B [44, 45]. Notably, while PGR-B is considered to mediate P-induced transcriptional activity, PGR-A can block PGR-B transactivation, and hence target cell responsiveness to P. Our inability to measure PGR-B protein levels in mouse myometrium with confidence, using currently available commercial antibodies, precluded the evaluation of whether an increased myometrial PGR-A:PGR-B ratio accompanies parturition in mice, which in the human myometrium has been linked to decreased myometrial sensitivity to P [33], and whether an alteration in this ratio underlies parturition defects with Klf9 null mutation. Nonetheless, our findings that loss of KLF9 interferes with PGR signaling by preventing the appropriate increase in PGR-A expression at term, in the absence of changes in serum E and P levels and independently of the contribution of the fetal genotype, suggest that the effects of KLF9 are predominantly, if not solely, at the level of the myometrium. A second potential mechanism may relate to KLF9's ability to selectively regulate nuclear coactivator Crebbp relative to Ncoa2 (this study), Ncoa1, and Ncoa3 (data not shown). The decreased levels of Crebbp with loss of KLF9 at dpc 18.5, leading to delayed parturition, appeared counter to an earlier report that a decline in Crebbp expression accompanied initiation of labor in both humans and mice [36]. However, we suggest that the limiting cellular levels of CREBBP as well as its ability to interact with a number of transcription factors, including PGR-A, PGR-B, and ESR1, could conceivably lead to competition among these receptors for formation of functional complexes, and hence alter the normal levels of CREBBP/PGR and CREBBP/ESR1 complexes found in myometria near or at term. Albeit speculative at the present time, we posit that a likely negative consequence of the latter is decreased ESR1 activation, consistent with the inability of an ESR1 target gene Oxtr [46, 47] to achieve the threshold levels comparable to those of WT, which is necessary for parturition to occur at dpc 19.5. However, it is also possible that KLF9 directly regulates Oxtr independently of ESR1 activation. Last, KLF9 may regulate NF-{kappa}B activation by affecting p65/RELA expression levels, resulting in aberrant pattern of p65 DNA binding activity in KO myometrial tissues. Since p65/RELA binding to an NF-{kappa}B response element within the human PGR promoter has been shown to increase PGR promoter activity [34], the decreased myometrial expression of PGR-A in KO versus WT mice at late term could be partly attributed to altered NF-{kappa}B signaling with loss of KLF9 expression. Studies to further explore these mechanisms are the subject of future investigations in our laboratory.

We used the response of WT and KO mice to the PGR antagonist RU486 as an indicator of defective PGR signaling near term. RU486, by interfering with P/PGR action, increases myometrial sensitivity to prostaglandins, leading to parturition [48]. The loss of myometrial sensitivity to RU486 at dpc 18 but not at dpc 17 in KO mice is consistent with the notion that aberrant PGR signaling near term impaired the ability of the myometrium to subsequently respond to or generate signals leading to parturition. Indeed, while no differences in levels of PGR-A isoform expression between WT and KO were noted at dpc 17, KO mice had numerically higher nuclear PGR-A protein and lower Crebbp transcript levels than corresponding WT mice at dpc 18.5. Nevertheless, because these observed changes were relatively modest, and P and E can trigger specific kinase signaling pathways, including those of MAPK and JNK [49, 50], which are known to influence myometrial contractility and onset of labor [51], it is possible that the altered myometrial response to RU486 with Klf9 KO is also due to defects in these pathways. A recent study [52] that detailed the ability of RU486 to alter MAPK, JNK, and P38 signaling pathways to modulate PGR-dependent gene transcription in the uterus, together with our previous findings that the JNK intracellular signaling pathway may interact with KLF9 for control of expression of the growth-associated Igfbp2 gene [53], support a role for KLF9 in RU486-mediated induction of labor.

Recent studies have suggested the potential importance of other PGR isoforms in the control of parturition [34, 42, 54, 55]. The expression of PGR-C, an N-terminally truncated form of PGR lacking the DNA binding domain [56], was reported in myometria of late pregnant mice and of women in labor [34]. Moreover, forced expression of PGR-C in human myometrial cells inhibited PGR-B transactivation [34]. A membrane-associated PGR, termed mPGR-{alpha}, which exhibits significant homology to G-protein-coupled receptors, has also been invoked in human pregnancy and labor, based on increased transcript levels in spontaneously laboring myometrium compared with nonpregnant and pregnant nonlaboring counterparts [54, 55]. We did not detect PGR-C in myometrial tissues of WT and KO mice at late pregnancy and term in the present study. Nuclear PGR-C was also not detected in human pregnancy myometrium in a recent study [33]. Thus, the importance of PGR-C and the membrane-associated PGR in parturition awaits further investigation.

In summary, the present findings demonstrate a supportive role for KLF9 in the timely onset of parturition through its regulation of PGR-A isoform expression, NF-{kappa}B p65/RELA DNA binding activity, and coregulator Crebbp expression, all of which are involved in PGR signaling. Given that KLF9 is also expressed by human myometrial cells of pregnancy (e.g., PHM1–31; our unpublished results) [57], we suggest that the Klf9 KO mouse may serve as a useful model to clarify the contribution of PGR activation pathways, distinct from that of the fetal component or the endocrine milieu, to human parturition. Since neonatal survival rate is dependent upon timely onset of parturition, with early and delayed labor similarly giving rise to morbidity and mortality, a complete understanding of key players in the regulation of the molecular processes leading to parturition will have significant consequences to human health.

ACKNOWLEDGMENTS

We thank Renea R. Eason and Dr. Yan Geng for technical assistance and members of our laboratories for discussion during the course of this work.

FOOTNOTES

1Supported by National Institutes of Health grant HD21961 and the Arkansas Children's Hospital Research Institute Intramural Grant Program. Back

Correspondence: 2Rosalia C.M. Simmen, University of Arkansas for Medical Sciences and Arkansas Children's Nutrition Center, 1212 Marshall St., Little Rock, AR 72202. FAX: 501 364 3161; e-mail: simmenrosalia{at}uams.edu

3Current address: Department of Obstetrics/Gynecology and Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143 Back

4These authors contributed equally to this work. Back

Received: 27 September 2007.

First decision: 10 October 2007.

Accepted: 19 February 2008.

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