Adrenomedullin signals through NF‐κB in epithelial cells

Adrenomedullin is a peptide found in a variety of cells and tissues and involved in a multitude of biological processes. Recently, adrenomedullin has been identified as a host defense peptide and as such it plays a role in the inflammatory response. The transcription factor NF-kappaB is a major regulator of genes involved in the inflammatory response and the aim of this study was to determine whether NF-kappaB played a role in the inflammatory process triggered by adrenomedullin. Skin epithelial cells (HaCaTs) were used as our model in vitro. Western blot analysis from adrenomedullin-stimulated HaCaT cells revealed a rapid degradation of NF-kappaB inhibitor alpha and beta followed by the translocation of free NF-kappaB to the nucleus, where it was detected by Texas Red immunostaining after incubation with adrenomedullin for 15 min. Electromobility shift assay showed that NF-kappaB present in the nucleus was active, since it bound to a probe containing an NF-kappaB binding site. Supershift assays indicated that p50 and p65, members of the NF-kappaB family, were both part of the NF-kappaB dimmers involved in adrenomedullin cell signaling. HaCaTs secreted interleukin-6 in response to AM, which was significantly attenuated by the NF-kappaB inhibitor SN-50. Taken together, the data lend support for an immunoregulatory role for AM.