Steroid receptor coactivators 1 and 2 mediate fetal-to-maternal signaling that initiates parturition

Abstract

The precise mechanisms that lead to parturition are incompletely defined. Surfactant protein-A (SP-A), which is secreted by fetal lungs into amniotic fluid (AF) near term, likely provides a signal for parturition; however, SP-A-deficient mice have only a relatively modest delay (~12 hours) in parturition, suggesting additional factors. Here, we evaluated the contribution of steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2), which upregulate SP-A transcription, to the parturition process. As mice lacking both SRC-1 and SRC-2 die at birth due to respiratory distress, we crossed double-heterozygous males and females. Parturition was severely delayed (~38 hours) in heterozygous dams harboring SRC-1/-2-deficient embryos. These mothers exhibited decreased myometrial NF-κB activation, PGF2α, and expression of contraction-associated genes; impaired luteolysis; and elevated circulating progesterone. These manifestations also occurred in WT females bearing SRC-1/-2 double-deficient embryos, indicating that a fetal-specific defect delayed labor. SP-A, as well as the enzyme lysophosphatidylcholine acyltransferase-1 (LPCAT1), required for synthesis of surfactant dipalmitoylphosphatidylcholine, and the proinflammatory glycerophospholipid platelet-activating factor (PAF) were markedly reduced in SRC-1/-2-deficient fetal lungs near term. Injection of PAF or SP-A into AF at 17.5 days post coitum enhanced uterine NF-κB activation and contractile gene expression, promoted luteolysis, and rescued delayed parturition in SRC-1/-2-deficient embryo-bearing dams. These findings reveal that fetal lungs produce signals to initiate labor when mature and that SRC-1/-2-dependent production of SP-A and PAF is crucial for this process.

Figure 7. Rescue of parturition delay in SRC-1/SRC-2 dhet females bred to SRC-1/-2 dhet males is associated with increased myometrial PGF₂α synthesis and decreased ovarian P₄ production.

(A) Akr1b3 mRNA (n = 6 per group) and (B) protein (n = 3 for WT, PBS, and SP-A; n = 4 for PAF) in myometrium. The same immunoblot of β-actin was used for normalization of PGDH (D), which was probed on the same blot. (C) PGDH mRNA (n = 6 per group) and (D) protein (n = 3 for WT, PBS, and SP-A; n = 4 for PAF) in myometrium. (E) PGF₂α levels in myometrial homogenates for WT or SRC-1/-2 mice injected i.a. with PBS, SP-A, or PAF (n = 8 per group). In the box-and-whisker plot, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles. (F) StAR protein in ovaries of WT (n = 3) or SRC-1/-2 dhet females × SRC-1/-2 dhet males injected i.a. with PBS (n = 3), SP-A (n = 3), or PAF (n = 4). (G) Maternal serum P₄ levels, measured by ELISA, of 18.5 dpc WT females × WT males or SRC-1/-2 dhet females × SRC-1/-2 dhet males injected i.a. with PBS, SP-A, or PAF (n = 8 per group). (H) Proposed cooperative roles of SRC-1 and SRC-2 in the initiation of labor through regulation of SP-A and Lpcat1 gene expression. Data are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, compared with WT; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 compared with PBS injection (ANOVA).

Figure 6. Delayed parturition and decreased NF-κB activation and expression of Cx43 and Oxtr in SRC-1/-2 dhet females bred to SRC-1/-2 dhet males are rescued by i.a. injection of SP-A or PAF at 17.5 dpc.

(A) Gestation length of SRC-1/-2 dhet females × SRC-1/-2 dhet males injected i.a. at 17.5 dpc with PBS (n = 9), SP-A (n = 10), or PAF (n = 10). (B and C) Nuclear translocation of NF-κB subunits p65 and p50 in 18.5 dpc myometrial tissues of WT females × WT males (uninjected, n = 3) or SRC-1/-2 dhet females × SRC-1/-2 dhet males injected i.a. with PBS (n = 3), SP-A (n = 3), or PAF (n = 4) at 17.5 dpc. The same immunoblots of loading controls, histone H3, and GAPDH are shown for nuclear and cytoplasmic proteins, respectively, since p65 and p50 were probed on the same respective blots. (D) Cx43 mRNA levels (n = 6 per group) and (E) protein levels (n = 3 for WT, PBS, and SP-A; n = 4 for PAF) in myometrium. The same immunoblot of β-actin was used for normalization of OXTR (G), which was probed on the same blot. (F) Oxtr mRNA (n = 6 per group) and (G) protein (n = 3 for WT, PBS, and SP-A; n = 4 for PAF) in myometrium. Data are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with WT; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with PBS injection (ANOVA).

Figure 5. SRC-1 and SRC-2 enhance Lpcat1 promoter transactivation and expression by glucocorticoids in a GRE-dependent manner.

(A) Lpcat1:luciferase reporter constructs containing various amounts of 5′-flanking sequence of the mouse Lpcat1 gene; putative GREs, androgen response elements (ARE), and P₄ response element (PREs) are indicated. A549 cells cotransfected with these constructs and Renilla luciferase were treated with or without Dex (10^–7 M) for 24 hours; luciferase assays were performed (n = 4 for each construct). (B) Effects of mutation of GRE4 and GRE5 (Lpcat1_-2000MUT-Luc)and knockdown (KD) of SRC-1, SRC-2, or SRC-1 and SRC-2 on Dex-induced Lpcat1 promoter activity (n = 4, each construct). Data are mean ± SEM. (C–E) ChIP-qPCR of WT or SRC-1/-2 dKO (KO) fetal lung tissues using antibodies to (C) GR, (D) SRC-1, or (E) SRC-2 and specific primers for GRE1/2, GRE3, GRE4, or GRE5 within the Lpcat1 promoter (n = 4). (F) LPCAT1 mRNA levels in HFL type II cells treated with Dex with or without RU486 (n = 5). (G) Representative immunoblot of LPCAT1 protein in HFL type II cells treated with Dex with or without RU486. Five independent experiments were conducted. (H) LPCAT1 mRNA in HFL type II cells infected with recombinant lentiviruses expressing SRC-1 and/or SRC-2 shRNA and cultured with or without Dex (10^–7 M) (n = 4). NTC, silencer negative control siRNA. (I) Representative immunoblot of LPCAT1 protein in HFL type II cells infected with recombinant lentiviruses expressing SRC-1 or SRC-2 shRNA, cultured with or without Dex (n = 4). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ^##P < 0.01 compared with Dex treatment alone; ^###P < 0.001 compared with cells expressing WT Lpcat1_-2000-Luc and Silencer negative control siRNA (ANOVA).

Figure 4. LPCAT1 expression and PAF synthesis and secretion are reduced in lungs of SRC-1/-2 double-deficient fetuses.

(A) Lpcat1 mRNA in lungs of WT and SRC-1/-2 dhet (dhet) fetuses from 15.5 dpc to term (n = 5 mice per time point). (B) Representative immunoblot of LPCAT1 protein in lungs of WT or SRC-1/-2 dhet fetuses from 15.5 dpc to term (n = 2–3 per time point). (C) Lpcat1 mRNA in lungs of 18.5 dpc WT (n = 3), dhet (n = 14), dKO (n = 3), KO/het (n = 6), het/KO (n = 6), KO/WT (n = 3), het/WT (n = 13), and WT/het (n = 4) fetuses. (D) LPCAT1 protein in fetal lungs at 18.5 dpc. Representative immunoblot according to genotype, and densitometric scans of immunoblots of LPCAT1 normalized to β-actin for WT (n = 7), dhet (n = 8), KO/het (n = 8), and dKO (n = 4) fetuses are shown. (E) PAF in WT fetal lungs at 15.5 (n = 3), 16.5 (n = 3), 17.5 (n = 7), 18.5 (n = 5), and 19.5 (n = 3) dpc. (F) PAF in AF surrounding WT fetuses at 15.5 (n = 3), 16.5 (n = 3), 17.5 (n = 7), 18.5 (n = 6), and 19.5 (n = 4) dpc. (G) PAF in 18.5 dpc fetal lungs of WT (n = 5), SRC-1 KO (n = 4), SRC-2 KO (n = 3), dhet (n = 7), and dKO (n = 6). (H) PAF in AF surrounding 18.5 dpc WT (n = 6), SRC-1 KO (n = 4), SRC-2 KO (n = 4), dhet (n = 6), and dKO (n = 5) fetuses. (F and H) In the box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ^#P < 0.05, ^##P < 0.01 compared with WT at same dpc (ANOVA).

Figure 3. SRC-1/-2 dhet or WT females bred to dhet or 1-KO/2-het males, respectively, manifest decreased myometrial PGF₂α synthesis and delayed ovarian luteolysis.

(A) Akr1b3 mRNA and (B) protein in myometrium from WT females × WT males (n = 5), WT females × 1-KO/2-het males (n = 4), and dhet females × dhet males (n = 5). Immunoblots and densitometric scans normalized to β-actin are shown. (C) PGDH mRNA and (D) protein in myometrium of WT females × WT males (n = 5), WT females × 1-KO/2-het males (n = 4), and dhet females × dhet males (n = 5). Immunoblots and densitometric scans normalized to β-actin are shown. (E) PGF₂α levels in myometrial homogenates normalized to protein for WT females × WT males (n = 12), WT females × 1-KO/2-het males (n = 8) and dhet females × dhet males (n = 15). In the box-and-whisker plot, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles. (F) Maternal serum P₄, measured by ELISA for WT females × WT males (n = 12), WT females × 1-KO/2-het males (n = 8), and dhet females × dhet males (n = 14). (G) StAR protein in ovaries. Representative immunoblot of StAR, and densitometric scans of StAR immunoblots normalized to β-actin (n = 8 per group). (H) At 17 and 19 dpc, StAR immunostaining in corpus luteum of SRC-1/-2 dhet females × SRC-1/-2 dhet males (dhet) was more intense than that of WT. Three independent experiments conducted. Original magnification, ×200. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA).

Figure 2. NF-κB activation and contraction-associated protein gene expression are decreased in 18.5 dpc pregnant myometrium of SRC-1/-2 dhet females × SRC-1/-2 dhet males and WT females × 1-KO/2-het males, compared with WT females × WT males.

(A and B) Nuclear translocation of (A) NF-κB p65 and (B) p50 in myometrium. Representative immunoblots of p65 and p50 protein in nuclear (Nuc) or cytoplasmic (Cyto) fractions. The same immunoblots of histone H3 and GAPDH are shown for nuclear and cytoplasmic proteins, respectively, since p65 and p50 were probed on the same respective blots. Combined data from densitometric scans of nuclear immunoblots for p65 and p50 relative to cytoplasmic immunoblots, normalized to internal controls for WT females × WT males (n = 10), WT females × 1-KO/2-het males (n = 8), and dhet females × dhet males (n = 10). (C) Cx43 mRNA and (D) protein levels for WT females × WT males (n = 5), WT females × 1-KO/2-het males (n = 4), and dhet females × dhet males (n = 5). The same immunoblot of β-actin (D) was used for the normalization of AKR1B3 (Figure 3B) and PGDH (Figure 3D), which were probed on same blot. (E) Oxtr mRNA and (F) protein in myometrium from WT females × WT males (n = 5), WT females × 1-KO/2-het males (n = 4), and dhet females × dhet males (n = 5). Densitometric scans were normalized to β-actin. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA).

Figure 1. Delayed parturition in SRC-1/-2 dhet female × SRC-1/-2 dhet male and WT female × 1-KO/2-het male is associated with reduced SP-A expression in lungs of SRC-1/-2 double-deficient fetuses.

(A) Genotypes resulting from crosses of SRC-1/2 dhet females × SRC-1/2 dhet males and gestation length of pregnancies resulting from these double heterozygous crosses. (B) Gestation length of WT females × WT males (n = 13), SRC-1^–/– females × SRC-1^–/– males (n = 16), SRC-2^+/– females × SRC-2^+/– males (n = 10), SRC-1/-2 dhet females × SRC-1/-2 dhet males (n = 49), and WT females × 1-KO/2-het males (n = 25). (C) Gestation length of SRC-1/-2 dhet females × SRC-1/-2 dhet males is positively correlated with proportion of SRC-1 SRC-2 double-deficient pups. (D) SP-A mRNA in lungs of WT (n = 4), SRC-1/-2 dhet (dhet; n = 14), SRC-1/-2 dKO (dKO; n = 3), 1-KO/2-het (KO/het; n = 6), SRC-1 het/SRC-2 KO (het/KO; n = 6), SRC-1 KO/SRC-2 WT (KO/WT; n = 3), SRC-1 het/SRC-2 WT (het/WT; n = 13), and SRC-1 WT/SRC-2 het (WT/het; n = 4) fetuses at 18.5 dpc from matings of SRC-1/-2 dhet females × SRC-1/-2 dhet males. (E) SP-A protein in 18.5 dpc fetal lung from SRC-1/-2 dhet females × SRC-1/-2 dhet males. Representative immunoblot of SP-A protein according to genotype, and data from scans of SP-A immunoblots normalized to β-actin for WT (n = 6), SRC-1/-2 dhet (n = 6), and SRC-1/-2 dKO (n = 4) fetuses. (F) SP-A protein in AF surrounding WT and SRC-1/-2–deficient fetuses. In the representative immunoblot, the first 3 lanes were run on same gel but were noncontiguous with the remaining 5 lanes (divided by the black line). Combined data from scans of immunoblots of SP-A in AF surrounding WT (n = 5), dhet (n = 14), dKO (n = 3), KO/het (n = 6), het/KO (n = 6), KO/WT (n = 3), het/WT (n = 13), and WT/het (n = 4) fetuses. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA).