Most glandular tissues comprise polarized epithelial cells organized around a single central lumen. Although there is active research investigating the molecular networks involved in the regulation of lumenogenesis, little is known about the extracellular factors that influence lumen formation and maintenance. Using a three-dimensional culture system of epithelial endometrial cells, we have revealed a new role for pro-inflammatory cytokines such as TNF alpha and IL1 alpha in the formation and, more importantly, maintenance of a single central lumen. We also studied the mechanism by which glucocorticoids repress TNF alpha and IL1 alpha expression. Interestingly, regulation of pro-inflammatory cytokine expression and subsequent lumen formation is mediated by estrogen receptor alpha (ER alpha) but not by the glucocorticoid receptor. Finally, we investigated the signaling pathways involved in the regulation of lumen formation by pro-inflammatory cytokines. Our results demonstrate that activation of the ERK/MAPK signaling pathway, but not the PI3K/Akt signaling pathway, is important for the formation and maintenance of a single central lumen. In summary, our results suggest a novel role for ER alpha-regulated pro-inflammatory cytokine expression in lumen formation and maintenance.Supported by grants from Fondo de Investigaciones Sanitarias (FIS) [grant numbers FISPI10/00604, FISPI070304, FISPI070276]; Ministerio de Ciencia e Innovacion [grant numbers SAF2002-10529-E, SAF2004-05250]; National Cancer Institute [grant number CA90400]; Marato de TV3 2005-47 (Fundacio la Marato de TV3), RD06/0020/1034 and RD06/0020/0013(Red Tematica de Investigacion Cooperativa en Cancer), 2009SGR794 (Grups Consolidats de Recerca) and 2004XT00090 (Xarxa Tematica); AECC, Catalunya contra el cancer and programa de intensificacion de la investigacion; and Instituto Carlos III. N.E. holds a fellowship from Fundacion Alicia Cuello de Merigo and a predoctoral fellowship from FIS [grant number FI08/0012]. Deposited in PMC for release after 12 months.