Understanding the basis of CYP26 mediated regulation of lens regeneration using ex vivo eye cultures and 4-oxo-RA

PURPOSE Xenopus has the remarkable ability to regenerate a lens from the basal cornea epithelial cells in response to signals from the retina. Previous work demonstrated that the Retinoic Acid (RA) metabolizing enzyme CYP26 is expressed in the cornea, and that its activity is required for lens regeneration. Gaps remain in our knowledge as to whether CYP26 is needed only to attenuate RA signaling via RA elimination, or whether it also acts to generate retinoid metabolites, such as 4-oxo-RA, to act as signaling ligands. Other key questions are why CYP26 antagonism, but not exogenous retinoids, can reduce cell division in the cornea, and when during regeneration CYP26 is important. MATERIALS AND METHODS Ex vivo cultures supplemented with RA, 4-oxo-RA, or the CYP26 inhibitor Liarozole were used to assay the effects of these compounds on lens regeneration. Similarly, corneas were explanted, cultured in the presence of these compounds, and assayed for mitotic changes by counting anti-Histone H3 positive nuclei. qPCRs validated responsiveness to these compounds. RESULTS Ex vivo cultures showed that when the media was supplemented with the RA metabolite 4-oxo-RA in addition to Liarozole, lens regeneration was still inhibited. 4-oxo-RA also does not rescue the loss of cell division in the cornea that is observed upon CYP26 antagonism. Liarozole inhibited regeneration when added 12 hours after lentectomy, but not when added 48 hours after. CONCLUSIONS These data show that the necessity of CYP26 is not explained as a generator of 4-oxo-RA for regeneration. Moreover, Liarozole-induced mitotic reduction is not explained by 4-oxo-RA deficiency. These results support a model of RA-independent mitotic regulation by CYP26, though other retinoid metabolites may be active. Finally, CYP26 activity is only needed between 12 and 48 hours post-surgery, showing that its action is required only during the earliest stages of lens regeneration. Financial interests The authors declare no competing financial interests.