Quantitation of mucin RNA by PCR reveals induction of both MUC2 and MUC5AC mRNA levels by retinoids.
The polydispersity of most human secretory mucin messages has made them difficult to detect specifically and quantitatively, impeding the evaluation of the relative expression of the various mucin genes and their role in normal and pathological conditions. For this reason, we developed competitive reverse transcriptase-polymerase chain reaction (PCR) methods to measure the airway mucins MUC2 and MUC5AC. Oligonucleotide pairs were designed that specifically detect MUC2 and MUC5AC, as demonstrated by the size and sequence of the PCR product and the expected tissue distribution. The mucin oligonucleotide primers were used to synthesize internal competitive standards, called MIMIC. Using this assay, the relative expression of these messages was analyzed in retinoid-replete or -deprived cultures of normal human tracheobronchial epithelial (NHTBE) cells. Retinoid deficiency induces squamous metaplasia in vivo and in vitro. Consistent with these observations and in contrast to a previous report, retinoid-deprived cultures produced at least an order of magnitude less MUC2 and MUC5AC message than retinoid-replete cultures. In summary, this paper describes methodology that can be applied to the specific and quantitative measurement of mucin messages and demonstrates that, in NHTBE cells, the level of MUC2 and MUC5AC mRNA is increased by retinoids.
Guzman
K
K
Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Gray
T E
TE
Yoon
J H
JH
Nettesheim
P
P
D002460:Cell Line; D004848:Epithelium / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D055271:Mucin 5AC; D055262:Mucin-2; D009077:Mucins / Q000032:analysis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis* / Q000187:drug effects; D012176:Retinoids / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D014132:Trachea / Q000378:metabolism*
C524625:MUC2 protein, human; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D055262:Mucin-2; D009077:Mucins; D012333:RNA, Messenger; D012176:Retinoids