TBX2, a Novel Regulator of Labour

Abstract

Background and Objectives: Therapeutic interventions targeting molecular factors involved in the transition from uterine quiescence to overt labour are not substantially reducing the rate of spontaneous preterm labour. The identification of novel rational therapeutic targets are essential to prevent the most common cause of neonatal mortality. Based on our previous work showing that Tbx2 (T-Box transcription factor 2) is a putative upstream regulator preceding progesterone withdrawal in mouse myometrium, we now investigate the role of TBX2 in human myometrium. Materials and Methods: RNA microarray analysis of (A) preterm human myometrium samples and (B) myometrial cells overexpressing TBX2 in vitro, combined with subsequent analysis of the two publicly available datasets of (C) Chan et al. and (D) Sharp et al. The effect of TBX2 overexpression on cytokines/chemokines secreted to the myometrium cell culture medium were determined by Luminex assay. Results: Analysis shows that overexpression of TBX2 in myometrial cells results in downregulation of TNFα- and interferon signalling. This downregulation is consistent with the decreased expression of cytokines and chemokines of which a subset has been previously associated with the inflammatory pathways relevant for human labour. In contrast, CXCL5 (C-X-C motif chemokine ligand 5), CCL21 and IL-6 (Interleukin 6), previously reported in relation to parturition, do not seem to be under TBX2 control. The combined bioinformatical analysis of the four mRNA datasets identifies a subset of upstream regulators common to both preterm and term labour under control of TBX2. Surprisingly, TBX2 mRNA levels are increased in preterm contractile myometrium. Conclusions: We identified a subset of upstream regulators common to both preterm and term labour that are activated in labour and repressed by TBX2. The increased TBX2 mRNA expression in myometrium collected during a preterm caesarean section while in spontaneous preterm labour compared to tissue harvested during iatrogenic preterm delivery does not fit the bioinformatical model. We can only explain this by speculating that the in vivo activity of TBX2 in human myometrium depends not only on the TBX2 expression levels but also on levels of the accessory proteins necessary for TBX2 activity.

Keywords: TBX2; TERT-HM cells; delivery; inflammation; myometrium; premature delivery; progesterone withdrawal; proinflammatory cytokines and chemokines; uterine smooth muscle cells.

Figure 1

Heatmap of Differentially expressed genes in hTERT-HM cells overexpressing TBX2.

Figure 2

String network analysis of TBX2 induced differentially expressed genes.

Figure 3

Cytokines and chemokines regulated by TBX2 overexpression. In bold: cytokines/chemokines involved in the initiation in parturition based on PubMed query.

Figure 4

TBX2 mRNA expression in human preterm myometrial samples. (a): TBX2 mRNA expression measured by Illumina microarray profiling (* t-test p value < 0.05). (b): TBX2 mRNA expression measured by quantitative polymerase chain reaction (qPCR) (* t-test p value < 0.05). Dots indicate individual data points. The width of the density curves corresponds to the frequency of the observations. The box and whisker plot in the center with a median represents the interquartile range and the 95% confidence intervals. PNL: Preterm no labour; PSL: Preterm spontaneous labour.

Figure 5

TBX2 mRNA expression in human preterm myometrial samples. (a): TBX2 expression in human myometrium by RNA-Sequencing (extracted from Chan et al., 2014). (b): TBX2 expression in human myometrium by microarray profiling (extracted from Sharp et al., 2016). A blue asterisk marks outliers. Dots indicate individual data points. The width of the density curves in the violin plot corresponds to the frequency of the observations. The box & whisker plot in the center with a median represents the interquartile range and the 95% confidence intervals. TNL: Term no labour; TSL: Term spontaneous labour. * t-test p value < 0.05.

Figure 6

Upstream regulators governing preterm and term labour.

Figure 7

The Z-scores of the top 50 upstream regulators. The top 50 upstream regulators identified in study (A–C) are represented as a network plot. The four central nodes (A–D) and their corresponding edges (the coloured lines) represented in this figure correspond to four independent datasets. Dataset A: hTERT-HM cells overexpressing TBX2 (light blue lines, source current study).Dataset B: Preterm Labour in human myometrium (pink lines, source current study).Dataset C: Term labour in human myometrium (light green lines, source Chan et al., 2014). Dataset D: Term labour in human myometrium (purple lines, source Sharp et al., 2016).