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Myometrial contraction is a central feature of labor. Although a link between infection and preterm labor is widely accepted, surprisingly little is known about the mechanisms coupling infection-induced inflammation to myocyte contractile machinery. This study explores the myocyte response to pathogen-derived ligands in vitro. The pregnant human myometrial cell line PHM1-41 and primary cultured uterine myocytes responded to Toll-like receptor (TLR) ligands, including the bacterial wall component LPS, which at 100 ng/ml increased contraction of cells embedded within collagen gels over 72 h compared to PBS. LPS-treated myocytes secreted inflammatory mediators, including prostaglandin F2α, the cytokines TNF-α and IL-6, and a range of chemokines. The contractile response to LPS required TLR4 signaling and was independent of prostaglandin synthesis. Neutralizing TNF-α had no effect on LPS-mediated contraction; however, the Rho-associated protein kinase (ROCK) inhibitors Y-27632 (10 μM) and GSK-269962 (50 nM) both abrogated the contractile response. The finding of LPS-mediated contraction was supported by a 1.38 ± 0.072-fold (mean±se) increase in myosin light-chain phosphorylation 48 h post-treatment, assessed by in-cell Western blot analysis. Together, these data suggest that, in addition to modulating the local inflammatory environment, pathogen-derived ligands may directly promote myometrial contractility via Rho/ROCK signaling, thus contributing to preterm labor-mediated preterm birth.-Hutchinson, J. L., Rajagopal, S. P., Yuan, M., Norman, J. E. Lipopolysaccharide promotes contraction of uterine myocytes via activation of Rho/ROCK signaling pathways.