Calcitriol Treatment Suppresses Contraction-Associated Gene Expression in Pregnant Mice Near Term

Preterm birth (PTB) is the leading cause of infant mortality during the first four weeks of life world-wide. This is due, in part, to our incomplete understanding of the mechanisms that mediate uterine quiescence during most of pregnancy and promote the transition to labor at term. Term and preterm labor are associated with increased levels of proinflammatory cytokines within maternal reproductive tissues where they activate inflammatory transcription factors (e.g. NF-κB) that enhance expression of genes encoding contraction-associated proteins (CAP) (i.e. connexin-43 (CX-43)), oxytocin receptor (OXTR)). By contrast, uterine quiescence is maintained throughout most of pregnancy by increased progesterone (P4) levels and enhanced progesterone receptor (PR) activity, which silence expression of proinflammatory mediators and CAP genes. Treatment of pregnant women at risk for preterm labor with progestins has negligible effects - underscoring the need for novel therapeutic targets. To identify such targets, our lab surveyed the myometrial transcriptome of timed-pregnant mice at 15.5-18.5 days post-coitum (dpc) and during labor at term (19.0 dpc) using RNA-sequencing. Interestingly, Cyp27b1 was one of the most highly downregulated transcripts at 18.5 dpc and in-labor, compared to 15.5 dpc. Cyp27b1 encodes 1α-hydroxylase, the key enzyme responsible for synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (calcitriol) which binds to the vitamin D receptor (VDR). Calcitriol/VDR have anti-inflammatory actions and are reported to mediate maternal tolerance to the hemi-allogeneic fetus. Interestingly, we previously observed that P4 treatment of timed-pregnant mice caused a significant increase in myometrial CYP27B1 mRNA levels, compared to controls. In the present study, we sought to assess effects of calcitriol treatment on CAP gene expression in timed-pregnant mice. To this end, pregnant mice were injected s.c. daily from 13.5-17.5 dpc with vehicle (n=3) of with 3 μg/kg of calcitriol (n=4). Mice were sacrificed and myometrial tissues were collected at 18.5 dpc. RT-qPCR revealed significantly reduced levels of CX43 (p<0.0001) and OXTR (p<0.05) in myometrium of calcitriol treated mice, compared to controls. Collectively, these data suggest that the decrease in Cyp27b1 expression, coupled with the decline in PR function near term may contribute to increased CAP gene expression leading to myometrial contractility and labor. Cyp27b1 may serve as a key P4/PR target gene that acts cooperatively to maintain myometrial quiescence via its anti-inflammatory actions. Thus, calcitriol may be a safe and effective treatment for the prevention of PTB.