298 Regulation and Function of BMP-4 Expression in the Inflamed Gastric Mucosa

Abstract

BACKGROUND/AIMS: We previously reported that inhibition of Bone Morphogenetic Protein (BMP) signaling In Vivo, leads to enhancement of Helicobacter pylori (HP)-induced gastric inflammation suggesting that the BMPs can exert novel anti-inflammatory actions in the stomach. The aim of this study was to examine the function and the regulation of BMP-4 expression in gastric inflammation. METHODS: Inflammation was induced by infection of 3 month-old C57BL/6-BMP-4-β-gal/+ mice with HP. Animals were analyzed 2 months after inoculation. Expression of BMP-4 was assessed by X-Gal staining and QRT-PCR. Morphology of the gastric mucosa was examined in sections stained with H&E. Phosphorylation of Smad1-5-8 was determined by immunohistochemistry with anti-phospho-Smad1-5-8 antibodies (abs). Expression of INF-γ was measured by QRT-PCR. Gastric mesenchymal and mucus cells were isolated from the canine gastric mucosa and characterized by staining with anti-vimentin abs and the lectin GSII, respectively. Phosphorylation of STAT1 and ERK2 and expression of BMP-4 in the mesenchymal cells were assessed by western blots. IL-8 expression in the mucus cells was measured by QRT-PCR. INF-γ, IL-17-α, IL-12 and IL23 content in supernatants of cultures of mouse splenocytes and dendritic cells was measured by ELISA. RESULTS: Analysis of mucosal sections of C57BL/6-BMP-4-β-gal/+ mice stained with X-gal, demonstrated that BMP-4 is expressed in the mesenchymal layers of the gastric mucosa. HP induced inflammatory changes in the gastric mucosa and it stimulated the expression of both INF-γ and BMP-4. Phospho-Smad1-5-8 staining of the inflamed gastric mucosa indicated the presence of positively stained nuclei in gastric epithelial cells, but not in mesenchymal cells, or in cells of the inflammatory infiltrate. INF-γ induced the phosphorylation of both STAT-1 and ERK2 and it induced BMP-4 protein expression in the mesenchymal cells. Incubation of the cells with inhibitors of both STAT-1 and ERK2 phosphorylation abrogated the stimulatory effect of INF-γ. BMP-4 failed to affect LPS-induced release of INF-γ, IL-17-α, IL-12 and IL-23 in cultures of mouse splenocytes and dendritic cells. In contrast, BMP-4 potently inhibited both basal and TNF-α stimulated IL-8 gene expression in cultures of isolated mucus cells. CONCLUSIONS: Inflammatory stimuli induce the expression of BMP-4 both In Vivo and In Vitro, through mechanisms that appear to involve the activation of both ERK2 and STAT-1. BMP-4, in turn, inhibits the expression of inflammatory molecules in epithelial, but not in immune cells, suggesting that BMP-4 is a novel anti-inflammatoy peptide released by the mesenchyme to modulate the inflammatory response of the gastric epithelium.