We Pigs News for 05-06-2018

Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells

Guinea pig gastric mucosal cells in primary culture express Nox1 and spontaneously produce O2−. These cells are able to release O2− at much higher rates when exposed to Helicobacter pylori lipopolysaccharide from type I but not from less virulent type II strains, suggesting that Nox1 may be involved in the pathogenesis of H. pylori-associated diseases. Using a phosphoinositide 3-kinase inhibitor, LY-294002, and an adenoviral vector encoding constitutively active Rac1, we suggest that H. pylori LPS-stimulated O2− production in gastric mucosal cells is controlled by two distinct mechanisms: 1) transcriptional upregulation of Nox1 and NOXO1 and 2) activation of Rac1.Preparation of primary cultures of gastric mucosal cells under LPS-free conditions. 

On the other hand, the gp91phox and Nox4 mRNA were not detected in guinea pig gastric mucosal cells even after treatment with H. pylori LPS, as well as in the quiescent cells. Thus the O2− production induced by LPS appears to involve the enhanced expression of Nox1.Induction of NOXO1 mRNA expression by H. pylori LPS.We next investigated the expression of mRNA for Nox-activating proteins such as NOXO1 and NOXA1 in gastric mucosal cells untreated or treated with H. pylori LPS. 

For this purpose, we had cloned the guinea pig NOXO1 cDNA, the deduced amino acid sequence of which shows 72% identity with that of human NOXO1. Northern blot analysis performed with a probe derived from the cDNA revealed that H. pylori LPS stimulated the expression of NOXO1 mRNA, with a peak at 4 h, and a similar time course was observed in the upregulation of Nox1 mRNA. Lipid A of H. pylori, which is also capable of enhancing O2− generation by gastric mucosal cells, stimulated the expression of NOXO1 and Nox1 mRNA. 

The expression of these two mRNA was blocked by polymyxin B, an agent that interacts with the lipid A moiety of LPS and inhibits the H. pylori LPS- or lipid A-triggered elevation of O2− generation. In contrast to the NOXO1 mRNA, the p47phox mRNA does not seem to exist in quiescent guinea pig gastric mucosal cells or in cells stimulated with H. pylori LPS or its lipid A. Although we had previously obtained a very weak signal of a 47-kDa protein immunoreactive to an antibody against human p47phox in guinea pig gastric pit cells, the carefully performed Northern blot analysis and RT-PCR in the present study revealed that neither quiescent nor stimulated cells express p47phox mRNA.We also estimated the level of the mRNA for Nox activators, i.e., NOXA1 and p67phox, in gastric mucosal cells. 

This may be inconsistent with our previous finding that in guinea pig gastric mucosal cells, the amount of a 67-kDa protein that cross-reacted with an antibody against human p67phox increased in parallel with elevation of O2− generation after treatment with H. pylori LPS. To explore this inconsistency, we developed a novel polyclonal antibody against human recombinant p67phox that recognized the guinea pig p67phox with a molecular mass of 63 kDa, and the amount was not affected by H. pylori LPS. On the basis of these findings, we concluded that H. 

pylori LPS did not stimulate the induction of p67phox. The mRNA for p22phox, a possible partner of Nox1, was expressed in quiescent gastric mucosal cells, and the amount of the mRNA did not change even after the addition of H. pylori LPS. Taken together, gastric mucosal cells constitutively express the mRNA for NOXA1, p67phox, and p22phox, and the treatment with H. pylori LPS specifically induces the transcription of the Nox1 and NOXO1 genes, which is likely involved in the induction of O2− generation. 4B, Rac1 in guinea pig gastric mucosal cells stimulated by H. 

pylori LPS bound to GST-fused PAK2-RBD but not to GST alone. LY-294002 blocked the LPS-stimulated activation of Rac1 in association with the inhibition of O2− generation, suggesting the involvement of Rac1 activation, an event sensitive to LY-294002, in the LPS-induced O2− generation by Nox1.Restoration of LY-294002-inhibited O2− generation by expression of a constitutively active Rac1.To confirm the role of Rac in H. pylori LPS-stimulated O2− generation, we transduced an adenoviral vector encoding a constitutively active form of Rac1 to gastric mucosal cells and tested whether it affected the inhibition of O2− production using LY-294002. In the present study, we have shown that H. pylori LPS induced the transcription of the Nox1 and NOXO1 genes and activated the GTPase Rac1 in gastric mucosal cells, and also that the two events played crucial roles in O2− production induced by the bacterial component. 

Once guinea pig gastric mucosal cells are primed with H. pylori LPS, they continuously and spontaneously produce O2− for more than 24 h. To understand this unique feature of Nox1, studies to identify GEF and GAP as proteins that are involved in Nox1 activation in guinea pig gastric mucosal cells are underway. 

Keywords: [“cell”,”h”,”O2″]
Source: http://www.physiology.org/doi/full/10.1152/ajpcell.00319.2004

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