Cyclophilin A is required for retinoic acid-induced neuronal differentiation in p19 cells.

Jun Song,Kazunari Yokoyama,Robert Chiu,John Rossi,Ying-Chun Lu

doi:10.1074/jbc.m311406200

Abstract

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me(2)SO-induced mesodermal differentiation. This difference suggests that CypA is specifically required for the RA-induced neuronal pathway. In addition to the loss of RA-induced RA receptor beta expression and retinoic acid response element (RARE)-binding activity, a dramatic reduction in RA-induced RARE-mediated luciferase activity in the CypA knockdown cell line suggests that CypA affects RARE-mediated regulation of gene expression. Silent mutation of target sequences confirms the specificity of RNA interference in p19 embryonal carcinoma cells. Collectively, our data reveal that a novel function of CypA is required in the processing of RA-induced neuronal differentiation in p19 embryonal carcinoma cells.

Highlights

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me2SO-induced mesodermal differentiation
We used siRNAs to block the expression of CypA in p19 embryonal carcinoma (EC) cells, which have been used as a model system to study neuronal differentiation
The activity of luciferase was not significantly enhanced by treatment with RA in the pcDNA3-CypA-wt-ransfected cells. pTK-Luc was transfected into cells as a control reporter (Fig. 5D). These results demonstrate that, p19 cells lacking CypA showed a loss of responsiveness to activation of RA receptors (RAR)-mediated gene expression through retinoic acid response element (RARE) binding, the restored expression of CypA recovered the activity

Summary

The abbreviations used are

CypA, cyclophilin A; siRNA, small interfering RNA; EC, embryonal carcinoma; RA, retinoic acid; RARE, RA response element; RAR, RA receptor; RNAi, RNA interference; EMSA, electrophoretic mobility shift assay; TK, thymidine kinase; Luc, luciferase. We demonstrate that the vector-based system produces siRNAs in p19 EC cells and results in specific and persistent knockdown of CypA in stable transfectants These CypA knockdown cell lines are unresponsive to retinoic acid (RA)induced neuronal differentiation. We confirm that the observed knockdown phenotype is the result of silencing of the intended target, by using a rescue plasmid to restore RA-induced differentiation potential as wild-type p19 EC cells. These results clearly indicate that CypA is essential during RA-induced neuronal differentiation of p19 EC cells

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION