Signalling with retinoids in the human lung: validation of new tools for the expression study of retinoid receptors.
BACKGROUND
Retinoid Receptors are involved in development and cell homeostasis. Alterations of their expressions have been observed in lung cancer. However, retinoid chemoprevention trials in populations at risk to develop such tumors have failed. Therefore, the pertinence of new clinical trials using second generation retinoid requires prior better understanding of retinoid signalling. This is our aim when validating extensively research tools, focused on Retinoic Acid Receptor beta, whose major role in lung cancer is documented.


METHODS
Biocomputing was used to assess the genomic organization of RAR beta. Its putative RAR-beta1' promoter features were investigated experimentally. Specific measures realized, with qRT-PCR Syber Green assays and a triplex of Taqman probes, were extensively validated to establish Retinoid Receptors mRNAs reference values for in vivo normal human bronchial cells, lung tumors and cell lines. Finally, a pan-RAR-beta antibody was generated and extensively validated by western-blot and immunoprecipitation.


RESULTS
No promoter-like activity was found for RAR-beta1'. RAR-beta2 mRNAs increase signs the normal differentiation of the human bronchial epithelium while a decrease is observed in most lung cancer cell lines. Accordingly, it is also, along with RXR beta, down-regulated in lung tumors. When using nuclear extracts of BEAS-2B and normal lung cells, only the RAR-beta2 long protein isoform was recognized by our antibody.


CONCLUSIONS
Rigorous samples processing and extensive biocomputing, were the key factors for this study. mRNA reference values and validated tools can now be used to advance researches on retinoid signalling in the lung.
Poulain
Stéphane
S
Faculté de Médecine, Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, INSERM U724, Centre de Ressources Biologiques, CHU de Nancy, 54505 Vandoeuvre-Lès-Nancy, France. Stephane.Poulain@nancy.inserm.fr
Lacomme
Stéphanie
S
Battaglia-Hsu
Shyue-Fang
SF
du Manoir
Stanislas
S
Brochin
Lydia
L
Vignaud
Jean-Michel
JM
Martinet
Nadine
N
D001483:Base Sequence; D015153:Blotting, Western; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D047468:Immunoprecipitation; D008168:Lung / Q000378:metabolism*; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000235:genetics* / Q000378:metabolism; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics*
D020033:Protein Isoforms; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta