JWA is required for the antiproliferative and pro-apoptotic effects of all-trans retinoic acid in Hela cells.

1. All-trans retinoic acid (ATRA) is known to inhibit cellular proliferation and induce differentiation and apoptosis. It usually activates gene expression by binding to a nuclear receptor that interacts with retinoic acid-response elements (RARE) and then activates the mitogen-activated protein kinase signal pathway. JWA, a newly identified ATRA-responsive gene, has recently been proposed as an important molecule for cellular differentiation induced by some chemicals, including ATRA. 2. To investigate the possible involvement of JWA in the inhibition of cellular proliferation and induction of apoptosis by ATRA, HeLa cells were stably transfected with sense or antisense JWA to establish cell lines that overexpressed or were deficient in JWA; ATRA (0.05-10 micromol/L) was used to induce cellular differentiation and apoptosis. 3. Western blot analysis revealed that ATRA caused increased expression of JWA in HeLa cells in a dose- and time-dependent manner, accompanied by activation of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. However, ERK1/2 phosphorylation induced by ATRA was inhibited in JWA-deficient HeLa cells. In JWA-overexpressing HeLa cells, ATRA showed more significant antiproliferative effects and induced more apoptosis. 4. The reporter gene assay showed that ATRA (5 mmol/L) enhanced the transcriptional activity of JWA by interacting with its promoter in the region from -194 to +107 bp (P < 0.01). Bioinformatic analysis indicated that the JWA promoter did not contain RARE, but did contain two CCAAT boxes in this fragment spanning -194 to +107 bp, which may be responsive to the ATRA-activated nuclear transcription factor CCAAT/enhancer binding proteins (C/EBP) or interacting proteins. Therefore, ATRA-inhibited cellular proliferation and -induced apoptosis in HeLa cells may be dependent on JWA transactivation via its C/EBP-binding motifs. 5. These data indicate that the inhibition of proliferation and the induction of apoptosis by ATRA are dependent on JWA expression in HeLa cells. The findings may represent a novel mechanism by which the effects of ATRA in regulating cellular proliferation and apoptosis are mediated, at least in part, by JWA expression.