ANTISENSE OLIGONUCLEOTIDES TO CRABP I AND II ALTER THE EXPRESSION OF TGF-beta 3, RAR-beta, AND TENASCIN IN PRIMARY CULTURES OF EMBRYONIC PALATE CELLS

摘要

The cellular retinoic acid-binding proteins (CRABPs) are thought to modulate the responsiveness of cells to retinoic acid (RA). We have previously shown that primary cultures of murine embryonic palate mesenchymal (MEPM) cells express both CRABP-I andCRABP-II genes and that this expression is regulated by RA and transforming growth factor beta (TGF-P). These cells also express high levels of TGF-beta 3, which is also regulated by RA and TGF-beta. We have used an antisense strategy to investigate therole of the CRABPs in retinoid-induced gene expression. Subconfluent cultures of MEPM cells were treated for several days with phosphorothioale modified 18-meroligonucleotides antisense to CRABP-I or CRABP-II and then with all-fraras-retinoic acid at aconcentration of 3.3 mu M or 0.33 mu M for 5 or 22 h. Total RNA was then extracted and the expression of TGF-beta 3, retinoic acid receptor P (RAR-beta), and tenascin was assessed by northern blot analysis. Antisense oligonucleotides to CRABP-I partiallyinhibited the RA-induced TGF-beta 3, RAR-beta, and tenascin mRNA expression. The corresponding mis-sense oligonucleotides were without effect. Antisense oligonucleotides to CRABP-II also partially inhibited RA-induced expression of these genes. As withthe CRABP-I antisense, mis-sense oligonucleotides to CRABP-II had no effect. These data suggest that both CRABPs modulate the responsiveness of MEPM cells to retinoic acid. Inhibition of endogenous CRABP expression renders MEPM cells less responsive to RA with respect to induction of TGF-beta 3, RAR-beta, and tenascin gene expression. These results have important implications for our understanding of the role of the CRABPs in retinoid teratology.