Distinct sensitivity of neuroblastoma cells for retinoid receptor agonists: evidence for functional receptor heterodimers

Retinoic acid (RA) plays a major role in embryogenesis of the nervous system and has been reported to induce differentiation in neuroblastoma cell lines. To identify RA signaling pathways involved in such differentiation processes, two RA-sensitive neuroblastoma cell lines (LA-N-5 and SH-SY5Y) were extensively studied. Northern blot experiments determined that of the three RAR mRNAs, only RAR alpha was significantly expressed, with respectively weak or undetectable levels of RAR gamma and RAR beta. RXRs (alpha and beta) receptors were weakly expressed, Western blotting analysis confirmed the constitutive expression of RAR alpha and absence of RAR beta and weak levels of RXR alpha. Treatment with all-trans-RA up-regulated RAR alpha and induced a drastic increase of RAR beta (both at the RNA and protein level). To further characterize the function of RAR alpha, RAR beta and RXR alpha in NB cells, nuclear extracts from LA-N-5 cells were analysed by EMSA studies. Three specific retarded complexes were observed which were significantly decreased or shifted in the presence of monoclonal antibodies to RAR alpha, RAR beta and RXR alpha. RA treatment dramatically induced a DR5-binding RXR alpha-RAR beta heterodimer. Treatment with combinations of RAR alpha or RAR beta agonists with a RXR alpha agonist or with a RAR alpha agonist alone, induced neurite-outgrowth supporting the probability that both RXR alpha-RAR alpha or RXR alpha-RAR beta heterodimers are involved in RA-mediated differentiation of NB cells. The availability of novel synthetic RA-specific receptor ligands should provide the possibility of tissue specific therapeutic regimes.