Abstract

Retinoic acids are morphogenic signaling molecules that are derived from vitamin A and involved in a variety of tissue functions. Two groups of their nuclear receptors have been identified: retinoic acid receptors (RARs) and retinoic acid X receptors (RXRs). All-trans retinoic acid is the high affinity ligand for RARs, and 9-cis retinoic acid also binds to RXRs with high affinity. In cells at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid via unknown mechanisms. It was previously shown that retinoic acids prevents activation-induced death of thymocytes. Here, we report that both all-trans and 9-cis retinoic acid induce apoptosis of mouse thymocytes and purified CD4⁺CD8⁺ cells in ex vivo cultures, with 9-cis retinoic acid being 50 times more effective. The induction of apoptosis by retinoic acids is mediated by RARγ because (a) the phenomenon can be reproduced only by RARγ-selective retinoic acid analogs, (b) the cell death induced by either retinoic acids or RARγ analogs can be inhibited by RARγ-specific antagonists, and (c) CD4⁺CD8⁺thymocytes express RARγ. In vivo administration of an RARγ analog resulted in thymus involution with the concomitant activation of the apoptosis-related endonuclease and induction of tissue transglutaminase. The RARγ pathway of apoptosis is RNA and protein synthesis dependent, affects the CD4⁺CD8⁺ double positive thymocytes, and can be inhibited by the addition of either Ca²⁺ chelators or protease inhibitors. Using various RAR- and RXR-specific analogs and antagonists, it was demonstrated that stimulation of RARα inhibits the RARγ-specific death pathway (which explains the lack of apoptosis stimulatory effects of all-trans retinoic acid at physiological concentrations) and that costimulation of the RXR receptors (in the case of 9-cis retinoic acid) can neutralize this inhibitory effect. It is suggested that formation of 9-cis retinoic acid may be a critical element in regulating both the positive selection and the “default cell death pathway” of thymocytes.