Understanding how NF-kappaB Regulates Expression and Repression of the Galphas GTP-binding Protein in the Myometrium

Scientists need to understand which molecules are used to ensure the uterus is programmed to stay relaxed throughout pregnancy and how they are modified when labour is triggered.

One important protein, called G-alpha-s, is found on individual muscle cells called myocytes. G-alpha-s serves as a molecular relay between the signals outside the myocyte and the chain of molecules within it that stop it contracting during pregnancy.

Importantly, the level of G-alpha-s in the muscle of the uterus, the myometrium, as a whole is very high during pregnancy. In myometrium from women in active labour, however, the number of G-alpha-s molecules is greatly reduced suggesting that the myocytes of the myometrium have the ability to shut down G-alpha-s production at term.

We wish to know what causes this rise and fall in G-alpha-s synthesis. We also know that the bodily inflammatory response can shut down G-alpha-s production. Consequently, we would like to examine whether plant-derived anti-inflammatory chemicals can inhibit this shut-down of G-alpha-s, which in turn may then prevent premature uterine contractions. We will test this by seeing if these anti-inflammatory chemicals can stop small pieces of human myometrium from contracting when inflammatory molecules are present.

The GTP-binding protein, Galphas, is a key regulator of myometrial quiescence, being highly expressed during quiescence and then repressed in labouring human myometrium. Moreover, both normal and preterm human labour is associated with increased expression of pro-inflammatory compounds, many of which activate the nuclear factor kappaB (NF-kappaB) which in turn stimulates gene expression. We recently reported that NF-kappaB associates with protein kinase A (PKA) in myometrial homogenates suggesting cross-talk between Galphas/cAMP/PKA signalling and NF-kappaB occurs within the myometrium (Chapman et al. 2004 JCEM 89; 5683). Significantly, we also showed that NF-kappaB could repress myometrial Galphas expression (Chapman et al. 2005 Endocrinology 146: 4994). Consequently, this proposal will extend these observations by: 1) Identifying those regions of the Galphas promoter that facilitate NF-kappaB-mediated repression. 2) Demonstrating that the anti-inflammatory compound, curcumin (an NF-kappaB inhibitor) and the histone deacetylase inhibitor, trichostatin-A (TSA), can prevent inflammation-induced repression of Galphas expression. 3) Using the chromatin immunoprecipitation assay to determine which DNA-binding proteins are required for NF-kappaB-mediated Galphas repression. 4) Providing evidence that curcumin and TSA can also antagonise inflammation-induced myometrial contractility in isolated strips of human myometrium.