Interleukin-6 is an essential determinant of on-time parturition in the mouse.

IL-6 abundance in amniotic fluid and uterine tissues increases in late gestation or with infection-associated preterm labor. A role in regulation of labor onset is suggested by observations that IL-6 increases expression of genes controlling prostaglandin synthesis and signaling in isolated uterine cells, but whether IL-6 is essential for normal parturition is unknown. To evaluate the physiological role of IL-6 in parturition in mice, we investigated the effect of Il6 null mutation on the timing of parturition and expression of genes associated with uterine activation. Il6 null mutant mice delivered 24 h later than wild-type mice, although circulating progesterone fell similarly in both genotypes during the prepartal period. Il6 null mutant mice were also refractory to low doses of lipopolysaccharide sufficient to induce preterm delivery in wild-type mice. The characteristic late-gestation elevation in uterine expression of Oxtr mRNA encoding oxytocin receptor, and peripartal increases in Ptgfr and Ptgs2 mRNAs regulating prostaglandin synthesis and signaling were delayed by 24 h in Il6 null mutant mice. Conversely, Ptger4 mRNA encoding the prostaglandin E receptor-4 was abnormally elevated in late-gestation in Il6 null mutant mice. Administration of recombinant IL-6 from d 11.5 postcoitum until term restored the normal timing of delivery and normalized Ptger4 mRNA expression in late gestation. We conclude that IL-6 has a key role in controlling the progression of events culminating in parturition and that it acts downstream of luteolysis in the uterus to regulate genes involved in the prostaglandin-mediated uterine activation cascade.

Inflammatory processes in preterm and term parturition.

A role for the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) is evident in term and preterm delivery, and this is independent of the presence of infection. All uterine tissues progress through a staged transformation near the end of pregnancy that leads from relative uterine quiescence and maintenance of pregnancy to the activation of the uterus that prepares it for the work of labour and production of stimulatory molecules that trigger the onset of labour and delivery. The uterus is activated by pro-inflammatory cytokines through stimulation of the expression and production of uterine activation proteins (UAPs). One of these actions is the stimulation of prostaglandin (PG) synthesis. Particularly important for labour is PGF(2alpha) and its receptor, PTGFR. In addition, pro-inflammatory cytokines are able to increase the synthesis of matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF) and the progesterone receptor C isoform, which leads to decreased tissue progesterone responsiveness. Some of these effects are replicated by PGF(2alpha), suggesting that it may act via its receptor to amplify the direct actions of cytokines. In turn, VEGF may enhance leukocyte recruitment to the uterus, and MMP-9 may promote activation of inactive pro-form cytokines. Pro-inflammatory cytokines also decrease the activity of 11beta-hydroxysteroid dehydrogenase, which likely increases intrauterine cortisol concentrations. In turn, cortisol may drive PG synthesis. Together these feed-forward mechanisms activate the uterus, trigger the production of uterine contractile stimulants and lead to labour and delivery.

Prostaglandin F2alpha and its receptor as activators of human decidua.

Prostaglandins produced by the intrauterine tissues of both mother and fetus (myometrium, decidua, placenta, chorion, and amnion) are involved with all of the physiologies of parturition (membrane rupture, cervical dilatation, myometrial contractility, placental separation, and uterine involution). For parturition to occur, however, the intrauterine tissues need to first be activated to prepare for the work of labor, then stimulated to initiate labor. Prostaglandins normally are considered to be stimulators of the physiologies of labor. This review presents evidence that one prostaglandin, PGF2alpha, and its receptor, FP, are also activators, especially of the decidua. Stimulated by cytokines, the decidual synthesis of PGF2alpha and the expression of FP lead to increased matrix metalloproteinase activity, further enhancement of cytokine activity, increased decidual oxytocin and oxytocin receptor expression, decreased progesterone responsiveness, and possibly, enhanced expression of vascular endothelial growth factor. These collective actions prepare the decidua for its role in parturition.

The interleukin 1beta-induced expression of human prostaglandin F2alpha receptor messenger RNA in human myometrial-derived ULTR cells requires the transcription factor, NFkappaB.

The molecular mechanisms that regulate the expression of genes involved in parturition are poorly understood. The mRNA expression of the prostaglandin F(2alpha) receptor (PTGFR), a uterine activating gene, is increased at labor and is required for uterine contractile activity in numerous animal models, although the signaling pathways responsible for this increased expression have not been identified. Proinflammatory cytokines have been proposed to regulate the expression of the uterine activating genes via activation of the nuclear transcription factor, NFkappaB, and initiate labor. However, it is uncertain whether uterine PTGFR is regulated this way. In this report, we demonstrate for the first time that treatment of immortalized human myometrial-derived ULTR cells with the proinflammatory cytokine IL1beta causes an increase in PTGFR mRNA levels. Furthermore, IL1beta treatment increased the nuclear levels of the RELA subunit of NFkappaB and increased binding of RELA to the NFkappaB DNA-binding site. Inhibition of NFkappaB activation with either the proteasome inhibitor MG132 or phenethyl caffeiate reduced PTGFR mRNA levels, which indicates that this transcription factor is important for basal transcription. Furthermore, this inhibition prevented IL1beta induction ofPTGFRmRNA, which confirms that NFkappaB is required for the IL1beta-induced increase inPTGFR. These results are consistent with the proposal that proinflammatory cytokines directly regulate uterine activation genes and that the transcription factor NFkappaB is involved in both basal and IL1beta-stimulated transcription of the PTGFR gene.

Cloning and characterization of the promoter region of the human prostaglandin F2alpha receptor gene.

The promoter region of the human prostaglandin F2alpha receptor (FP) gene was isolated, sequenced, and characterized. The 5'-flanking region has minimal homology to the bovine, mouse, and rat FP promoters with the exception of a 100-bp region. The human promoter similarly lacks a canonical TATA-box and a CAAT-box. Potential binding sites for SP-1, GATA-1, STAT-1, and AP-1 are present in the 5'-flanking region. One major transcription start site was identified using 5' RLM-RACE analysis and mapped to an adenine residue 262 nucleotides upstream from the initiator codon in exon 2. Transfection of HeLa cells with FP promoter-GFP deletion constructs indicates that the -2437/-1946 region contains repressor activity. DNase I footprinting analysis of this region identifies a footprint over the GATA-like site at -2400. This suggests repression of basal FP transcription may be mediated by a GATA binding site.

Cortisol infusion decreases renin, but not PGHS-2, EP2, or EP4 mRNA expression in the kidney of the fetal sheep at days 109-116.

Renal prostaglandins (PG), renin, and cortisol are necessary for normal kidney development and function during fetal life. We examined the effects of cortisol infusion before completion of nephrogenesis (d 109-116 gestation; 2.0-3.0 mg hydrocortisone succinate/24 h) on the renal mRNA expression of PGHS-2, the PGE(2) receptors, EP(2) and EP(4), and renin in fetal sheep. Cortisol infusion raised plasma cortisol levels to 42.8 +/- 6.0 nmol/L compared with saline infusion levels of 1.5 +/- 0.5 nmol/L (p < 0.001), but had no effect on fetal body weight, proportional kidney mass, or blood gases. Cortisol decreased significantly the relative expression of renin mRNA (saline: 0.93 +/- 0.06 units; cortisol: 0.32 +/- 0.03 units, p < 0.05), however it had no effect upon the expression of PGHS-2, EP(2), or EP(4) mRNA in fetal sheep kidney. Although there is substantial evidence that PGE(2) acting through either the EP(2) or EP(4) receptor stimulates renin synthesis in the adult kidney, our results have demonstrated that before the completion of nephrogenesis, cortisol down-regulation of renin mRNA expression is independent of any change in the expression of PGHS-2, EP(2), or EP(4) mRNA expression. During nephrogenesis, the insensitivity of PGHS-2, EP(2), and EP(4) expression to down-regulation by cortisol may permit continued PG regulation of renal development and urine formation.

Mouse placental prostaglandins are associated with uterine activation and the timing of birth.

We explored a potential mechanism linking placental prostaglandins (PGs) with a fall in plasma progesterone and increased expression of uterine activation proteins in the mouse. PG endoperoxide H synthase 2 (PGHS-2) mRNA expression increased in placenta in late gestation in association with an 8-fold increase in PGF(2alpha) concentration, reaching a peak on Gestational Day (GD) 18. This peak coincided with the final descent in plasma progesterone and birth on GD 19.3 +/- 0.2. Implantation of a progesterone-releasing pellet in intact pregnant dams on GD 16 delayed birth at term until GD 20.9 +/- 0.4 and inhibited the GD 18 increase in placental PGF(2alpha) levels in conjunction with a delayed fall in plasma progesterone that reached its lowest level 1 day after term birth. The mRNA levels of uterine activation proteins, connexin-43 (CX-43), oxytocin receptor, PGF(2alpha) receptor (FP), and PGHS-2, and the concentration of uterine PGF(2alpha) all increased at normal term birth. At progesterone-delayed term birth on GD 19.3, even though tissue PGF(2alpha) concentrations were at the same high levels observed at normal term birth, CX-43 and FP mRNA levels were lower than those at normal term birth, thereby possibly contributing to the delay of birth. These data are consistent with the hypotheses that fetal placental PGs affect the timing of birth by hastening luteolysis, that uterine activation initiates labor, and that birth may be delayed by blocking or decreasing the expression of two of the uterine activation proteins.

Placental restriction increases the expression of prostaglandin endoperoxide G/H synthase-2 and EP2 mRNA in the fetal sheep kidney during late gestation.

There is evidence that fetal growth restriction is associated with impaired nephrogenesis and reduced numbers of mature nephrons at birth. It has been proposed that such impairment of renal growth may contribute to increased blood pressure in later life. Although prostaglandins (PG) play a key role in kidney development, it is unknown whether a poor fetal substrate supply alters the synthesis or actions of PG within the fetal kidney. Using real-time reverse transcriptase PCR, we have measured the effect of chronic placental restriction (PR) on the renal expression of PG endoperoxide G/H synthase-2 (PGHS-2), PGE(2) receptors EP(2) and EP(4), and renin mRNA in the sheep fetus in late gestation. Restriction of placental growth reduced fetal body weight (PR: 3.2 +/- 0.2 kg, control: 4.8 +/- 0.2 kg) and total kidney weight (PR: 19.7 +/- 1.8 g, control: 25.1 +/- 1.3 g). Mean fetal arterial PO(2) was reduced by PR (PR: 15.03 +/- 0.67 mm Hg, control: 21.3 +/- 0.87 mm Hg). Renal PGHS-2 mRNA was increased in the PR group (PR: 2.26 +/- 0.38, control: 1.20 +/- 0.31) and was inversely related to mean fetal arterial PO(2) in the PR and control groups [PGHS-2: -0.17 (PO(2)) + 4.69, r(2) = 0.26]. PR also increased renal EP(2) (PR: 1.57 + 0.24, control: 0.82 + 0.13) but not EP(4) mRNA. Renin mRNA was directly related to renal EP(2) [renin = 0.37 (EP(2)) + 0.97, r(2) = 0.29] and EP(4), [renin = 0.75 (EP(4)) + 0.44, r(2) = 0.38] mRNA expression. Thus, the restriction of placental growth and associated chronic hypoxemia appear to increase the renal capacity to synthesize and respond to PG, which may play an important role in maintaining renin mRNA expression in the growth-restricted fetus.

Tyrphostins inhibit lipopolysaccharide induced preterm labor in mice.

OBJECTIVE: We tested whether lipopolysaccharide (LPS)-induced murine preterm birth was delayed by tyrphostins (inhibitors of tyrosine kinases). STUDY DESIGN: The tyrphostins, AG126 or AG1288, or the prostaglandin endoperoxide H synthase-2 (PGHS-2) inhibitor, NS-398, were administered 2 h before LPS, with LPS, or 2 h after LPS and their effect on gestational length was measured. RESULTS: Only tyrphostins and NS-398 administered 2 h before LPS successfully delayed preterm birth (p < 0.05). CONCLUSIONS: Both tyrphostins AG126 and AG1288 as well as NS-398 inhibited LPS induced preterm birth to the time of normal birth.