SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles.

Primordial follicles, consisting of granulosa cell (GC)-enveloped oocytes are maintained in a state of developmental arrest until activated to grow. The mechanism that operates to maintain this arrested state in GCs is currently unknown. Here, we show the TGFß-activated transcription factor SMAD3 is expressed in primordial GC nuclei alongside the cell cycle proteins, cyclin D2 (CCND2) and P27. Using neonatal C57/Bl6 mouse ovaries densely populated with primordial follicles, CCND2 protein co-localised and was detected in complex with P27 by immunofluorescence and co-immunoprecipitation, respectively. In the same tissue, SMAD3 co-precipitated with DNA sequences upstream of Ccnd2 and Myc transcription start sites implicating both as direct SMAD3 targets. In older ovaries follicle growth was associated with nuclear exclusion of SMAD3 and reduced P27 and CCND2 in GCs, alongside elevated Myc expression. Brief (2 H) exposure of neonatal ovaries to TGFß1 (10 ng/ml) in vitro led to immediate dissociation of SMAD3 from the Ccnd2 and Myc promoters. This coincided with elevated Myc and phospho-S6, an indicator of mTOR signalling, followed by a small increase in mean primordial GC number after 48 H. These findings highlight a concentration-dependent role for TGFß signalling in the maintenance and activation of primordial follicles, through SMAD-dependent and independent signalling pathways, respectively.

Binding loci of RelA-containing nuclear factor-kappaB dimers in promoter regions of PHM1-31 myometrial smooth muscle cells.

Human parturition is associated with many pro-inflammatory mediators which are regulated by the nuclear factor-kappaB (NF-κB) family of transcription factors. In the present study, we employed a ChIP-on-chip approach to define genomic loci within chromatin of PHM1-31 myometrial cells that were occupied by RelA-containing NF-κB dimers in response to a TNF stimulation of 1 h. In TNF-stimulated PHM1-31 cells, anti-RelA serum enriched 13 300 chromatin regions; importantly, 11 110 regions were also enriched by anti-RelA antibodies in the absence of TNF. DNA sequences in these regions, from both unstimulated or TNF-stimulated PHM1-31 cultures, were associated with genic regions including IκBα, COX-2, IL6RN, Jun and KCNMB3. TNF-induced binding events at a consensus κB site numbered 1667; these were represented by 112 different instances of the consensus κB motif. Of the 1667 consensus κB motif occurrences, 770 (46.2%) were identified within intronic regions. In unstimulated PHM1-31 cells, anti-RelA-serum-enriched regions were associated with sequences corresponding to open reading frames of ion channel subunit genes including CACNB3 and KCNB1. Moreover, in unstimulated cells, the consensus κB site was identified 2116 times, being defined by 103 different sequence instances of this motif. Of these 2116 consensus κB motifs, 1089 (51.5%) were identified within intronic regions. Parallel expression array analyses in PHM1-31 cultures demonstrated that TNF stimulated a >2-fold induction in 51 genes and a fold repression of >1.5 in 18 others. We identified 14 anti-RelA-serum-enriched genomic regions that correlated with 17 TNF-inducible genes, such as COX2, Egr-1, Jun, IκBα and IL6, as well as five regions associated with TNF-mediated gene repression, including Col1A2.

The effect of trichostatin-A and tumor necrosis factor on expression of splice variants of the MaxiK and L-type channels in human myometrium.

The onset of human parturition is associated with up-regulation of pro-inflammatory cytokines including tumor necrosis factor (TNF) as well as changes in ion flux, principally Ca(2+) and K(+), across the myometrial myocytes membrane. Elevation of intra-cellular Ca(2+) from the sarcoplasmic reticulum opens L-type Ca(2+) channels (LTCCs); in turn this increased calcium level activates MaxiK channels leading to relaxation. While the nature of how this cross-talk is governed remains unclear, our previous work demonstrated that the pro-inflammatory cytokine, TNF, and the histone deacetylase inhibitor, Trichostatin-A (TSA), exerted opposing effects on the expression of the pro-quiescent Gαs gene in human myometrial cells. Consequently, in this study we demonstrate that the different channel splice variants for both MaxiK and LTCC are expressed in primary myometrial myocytes. MaxiK mRNA expression was sensitive to TSA stimulation, this causing repression of the M1, M3, and M4 splice variants. A small but not statistically significantly increase in MaxiK expression was also seen in response to TNF. In contrast to this, expression of LTCC splice variants was seen to be influenced by both TNF and TSA. TNF induced overall increase in total LTCC expression while TSA stimulated a dual effect: causing induction of LTCC exon 8 expression but repressing expression of other LTCC splice variants including that encoding exons 30, 31, 33, and 34, exons 30-34 and exons 40-43. The significance of these observations is discussed herein.

Regulation of GTP-binding protein (Gαs) expression in human myometrial cells: a role for tumor necrosis factor in modulating Gαs promoter acetylation by transcriptional complexes.

The onset of parturition is associated with a number of proinflammatory mediators that are themselves regulated by the nuclear factor κB (NF-κB) family of transcription factors. In this context, we previously reported that the RelA NF-κB subunit represses transcription and mRNA expression of the proquiescent Gαs gene in human myometrial cells following stimulation with the proinflammatory cytokine TNF. In the present study, we initially defined the functional consequence of this on myometrial contractility. Here we show that, contrary to our initial expectations, TNF did not induce myometrial contractility but did inhibit the relaxation produced by the histone deacetylase inhibitor trichostatin A, an effect that in turn was abolished by the NF-κB inhibitor N(4)-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine. This result suggested a role for TNF in regulating Gαs expression via activating NF-κB and modifying histone acetylation associated with the promoter region of the gene. In this context, we show that the -837 to -618 region of the endogenous Gαs promoter is occupied by cAMP-response element-binding protein (CREB), Egr-1, and Sp1 transcription factors and that CREB-binding protein (CBP) transcriptional complexes form within this region where they induce histone acetylation, resulting in increased Gαs expression. TNF, acting via NF-κB, did not change the levels of CREB, Sp1, or Egr-1 binding to the Gαs promoter, but it induced a significant reduction in the level of CBP. This was associated with increased levels of histone deacetylase-1 and surprisingly an increase in H4K8 acetylation. The latter is discussed herein.