educed ability to repress the expression of mucin genes. We inves tigated the derepression of the transcription of MUC5B and MUC5AC genes, as well as the biosynthesis and secre tion these mucins in lung epithelial cells after treatment with PCN, by qRT PCR, western blotting, ELISA and im munofluorescence. Previously, we have shown that PCN significantly induced sellckchem MUC5B expression Inhibitors,Modulators,Libraries in human pri mary bronchial epithelial cells and in 16HBE cells cultured at the air liquid interface. In the presence of 12. 5 ug ml of PCN, qRT PCR analyses revealed that the expression of MUC5AC and MUC5B genes were increased significantly by 11 and 21 fold, respectively. Densitometry analyses of western blots indicate that the expression of MUC5AC and MUC5B pro teins increased by 4 and 5 fold, respectively.
Inhibitors,Modulators,Libraries These results were confirmed by ELISA analyses, which showed dose dependent induction of both MUC5AC and MUC5B mucins by PCN in both NCI H292 and 16HBE cells. It is also apparent that MUC5B was expressed in higher concentrations both in the presence and absence of PCN, but the level of induction by PCN was similar between the two mucins. Immunofluorescence staining indicated that, simi lar to MUC5B, PCN induced the expression of MUC5AC in NHBE and 16HBE cells cultured at the air liquid interface to similar Inhibitors,Modulators,Libraries extent as the positive control IL 13. PCN deficient PA mutant is attenuated in its ability to induce the goblet cell hyperplasia and metaplasia in mouse airways We have previously shown that chronic exposure to PCN induces GCHM and mucus hypersecretion.
However, no studies thus far have comparatively exam ined the induction of GCHM and mucus secretion by wild type PA versus PCN deficient mutant. C57BL6 mice were repeatedly challenged with 1 �� 106 of wild type PA PAO1 or the isogenic PCN deficient phzS mutant on Day 1, Inhibitors,Modulators,Libraries 3, 5 and 7. All eight mice challenged with the wild type PAO1 developed robust GCHM and mucus hypersecretion as indicated by PAS stained mucins. In contrast, only one out of eight mice infected with the phzS mutant showed low levels of isolated mucin expressing goblet cells. IHC analyses indicate that the expression of MUC5AC and MUC5B mucin were sig nificantly higher in PAO1 infected airways when com pared to the phzS infected airways. These results concur with the results from in vitro studies in NCI H292 and 16HBE cells, and ex vivo studies using NHBE cells, which indicate that PCN is a strong inducer of GCHM and mucus hypersecretion in airways.
GSH alleviates the RNS mediated FOXA2 modification and degradation Next, we examined whether the antioxidant GSH could attenuate the toxicity of PCN generated ROS RNS. We postulated that GSH could relieve the suppression and reduce nitrosylation of FOXA2 in the NCI H292 cells. As shown in Figure 7A, PCN reduced the expression of FOXA2 by 43%. However, GSH restored AV-951 selleck bio the expression of FOXA2 in a concentration dependent manner. At concentrations of 1 mM, GSH increased the expression of FOXA2 in PCN exposed NCI