Induction of apoptosis by retinoids and retinoic acid receptor gamma-selective compounds in mouse thymocytes through a novel apoptosis pathway

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 RAR gamma because (a) the phenomenon can be reproduced only by RAR gamma-selective retinoic acid analogs, (b) the cell death induced by either retinoic acids or RAR gamma analogs can be inhibited by RAR gamma-specific antagonists, and (c) CD4(+)CD8(+) thymocytes express RAR gamma. In vivo administration of an RAR gamma analog resulted in thymus involution with the concomitant activation of the apoptosis-related endonuclease and induction of tissue transglutaminase. The RAR gamma 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 RAR alpha inhibits the RAR gamma-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 g-cis retinoic acid may be a critical element in regulating both the positive selection and the ''default cell death pathway'' of thymocytes.