Taking into account time for translation of mRNA, this increase is consistent with DNA microarray data that demonstrated a peak in expression of HB-EGF mRNA at 3 h after bacterial inoculation. Although the array analysis was conducted in a mouse OM model, it should be translatable to the rat ME, as they have nearly the same chronological inflammatory response following bacterial inoculation. Cleavage of transmembrane HB-EGF by metaloproteinases is required for activation, and the large influx of neutrophils that occurs within the first 24 h of bacterial inoculation would provide a rich source of MMP9. Considering that ME mucosal hyperplasia peaks around 2�C3d after inoculation, the result of the Western blot implies that HBEGF, which has a strong mitogenic effect and is highly PRIMA-1 expressed in early stages of infection, could play an important role in inducing mucosal hyperplasia. Finally, immunohistochemistry demonstrated that HB-EGF is produced by epithelial cells of the ME mucosa as well as by infiltrated cells. Similar results have been reported in inflammatory oral mucosa, and neutrophils have been shown to produce HB-EGF in response to GM-CSF. It has also been found that IL-1b, a major inflammatory cytokine for which we observed mRNA to be strongly upregulated early in OM, promotes release of sHB-EGF. Although no detailed mechanism regarding upregulation of HB-EGF in the ME following bacterial infection has been clarified, innate immune CP-547632 hydrochloride responses seem likely to play an important role in the induction of this growth factor in OM. The results of this study indicate significant involvement of HBEGF in the hyperplastic response of the ME mucosa during bacterial OM. HB-EGF was one of seven growth factors active on epithelial cells for which expression kinetics were consistent with a role in mucosal hyperplasia. It was also the only factor which stimulated the growth of normal or previously infected mucosa in vitro. Expression of HB-EGF protein was confirmed in vivo. These data are consistent with a dominant role for HB-EGF in regulating the growth of the ME mucosal epithelium during OM.