Signalling with retinoids in the human lung: validation of new tools for the expression study of retinoid receptors

Abstract

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.

Conclusion: 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.

Figure 1

Schematic representation of the RARβ genomic context. Black arrows represent introns and black blocks are transcribed RARβ exons while grey blocks and arrows represent untranscribed spliced regions. All the promoters regions are grey circled while grey compact blocks indicate DR5 RAREs and grey rounds indicate CpG islands. P1, P1', P1", P2 and P3 indicate corresponding RAR-beta promoter regions. [GenBank:NM_000965] and [GenBank:NM_016152] are the Refseq IDs corresponding to the RARβ transcript isoforms 1 and 2 respectively. [GenBank:NM_000965] encodes the RARβ2 protein [Swiss-Prot:P10826-2] while [GenBank:NM_016152], resulting from the alternate splicing of [GenBank:NM_000965] at exon 3, encodes the RARβ4 protein [Swiss-Prot:P10826-3]. [GenBank:DA240288] and [GenBank:DC376623] are Expressed Sequence Tags. [GenBank:U52076] and [GenBank:DQ083391] are GenBank mRNAs. [GenBank:DC376623] and [GenBank:DQ083391] have specific first exons and share their other exons with [GenBank:NM_000965].

Figure 2

RARβ and RARβ2 relative expression ratios. RARβ x-fold ratios (R) were computed by Rest-RG^®, based on 2-log of absolute gene regulation, with their associated standard errors. In the different samples RARβ expressions are normalized to reference genes and NHBE expressions. The ratios significatively different from NHBE (p-values < 0.05) are indicated with asterisks.

Figure 3

Pan-RARβ antibody validation. MW: Molecular Weight. Panel A: Western-Blot of BEAS-2B and NHBE nuclear extracts. lane 1: BEAS-2B nuclear extracts. lane 2: NHBE nuclear extracts. Panel B: Immunoprecipitation of BEAS-2B nuclear extracts.