IL-4 induced MUC4 enhancement in respiratory epithelial cells in vitro is mediated through JAK-3 selective signaling.
BACKGROUND
Recent studies have identified MUC4 mucin as a ligand for activation of ErbB2, a receptor tyrosine kinase that modulates epithelial cell proliferation following epithelial damage in airways of asthmatics. In this study, we investigated the potential role of IL-4, one of the Th2 inflammatory cytokines persistent in asthmatic airways, in regulating MUC4 expression using a cell line NCI-H650.


METHODS
Real time PCR analysis was performed to determine concentration and time dependent effects of IL-4 upon MUC4 expression. Nuclear run on experiments were carried out to explore potential transcriptional modulation. Western blotting experiments using a monoclonal antibody specific to ASGP-2 domain of MUC4 were performed to analyze MUC4 glycoprotein levels in plasma membrane fractions. To analyze potential signal transduction cascades, IL-4 treated confluent cultures were co-incubated, separately with a pan-JAK inhibitor, a JAK-3 selective inhibitor or a MEK-1, 2 (MAPK) inhibitor at various concentrations before MUC4 transcript analysis. Corresponding transcription factor activation was tested by western blotting using a monoclonal p-STAT-6 antibody.


RESULTS
MUC4 levels increased in a concentration and time specific fashion reaching peak expression at 2.5 ng/ml and 8 h. Nuclear run on experiments revealed transcriptional enhancement. Corresponding increases in MUC4 glycoprotein levels were observed in plasma membrane fractions. Pan-JAK inhibitor revealed marked reduction in IL-4 stimulated MUC4 levels and JAK3 selective inhibitor down-regulated MUC4 mRNA expression in a concentration-dependent fashion. In accordance with the above observations, STAT-6 activation was detected within 5 minutes of IL-4 stimulus. No effect in MUC4 levels was observed on using a MAPK inhibitor.


CONCLUSIONS
These observations signify a potential role for IL-4 in MUC4 up-regulation in airway epithelia.
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA. gautam-damera@ouhsc.edu
Gautam
G
Damera
Baoyun
B
Xia
Goverdhan P
GP
Sachdev
D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D015847:Interleukin-4 / Q000494:pharmacology*; D053662:Interleukin-4 Receptor alpha Subunit; D053616:Janus Kinase 3; D055264:Mucin-4; D009077:Mucins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D019948:Receptors, Interleukin-4 / Q000235:genetics / Q000378:metabolism; D012137:Respiratory System / Q000187:drug effects / Q000378:metabolism; D050800:STAT6 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic
C504000:IL4R protein, human; D053662:Interleukin-4 Receptor alpha Subunit; C524658:MUC4 protein, human; D055264:Mucin-4; D009077:Mucins; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D019948:Receptors, Interleukin-4; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015847:Interleukin-4; D011505:Protein-Tyrosine Kinases; C507933:JAK3 protein, human; D053616:Janus Kinase 3