Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death

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Abstract
In this study, we used comparative proteomics to identify proteins that were involved in the regulation of interdigital cell death. The protein profiles of embryonic day (E) 12.5 and 13.5 mouse hindlimb interdigital tissues were compared to identify proteins that were differentially expressed. The interdigital cells are irreversibly committed to programmed cell death (PCD) at E13.5, whereas they are developmentally plastic at E12.5. We established that protein disulfide isomerase (PDI) expression was up‐regulated at E13.5, while peroxiredoxin 1 (Prdx1) expression was down‐regulated at this time point. Semiquantitative reverse transcriptase‐polymerase chain reaction and Western blot analyses confirmed the data obtained from the two‐dimensional electrophoresis gels. Furthermore, we were able to up‐regulate PDI expression by manipulating the E12.5 interdigital tissues to die during culture, although this up‐regulation was not possible when cell survival was promoted. In addition, we could inhibit interdigital cell death and expression of proapoptotic genes (Bmp‐4 and Bambi) by treating interdigital tissues with PDI antibodies and bacitracin (a PDI enzyme inhibitor). These findings suggested that PDI was involved in the activation and maintenance of interdigital cell death. Conversely, we determined that Prdx1 expression was maintained when interdigital cultures were manipulated to survive but down‐regulated when the cultures were permitted to die. The result suggested that Prdx1 was involved in maintaining interdigital cell survival. However, we were unable to induce interdigital cell death by means of RNA interference‐mediated silencing of Prdx1 expression, indicating that Prdx1 down‐regulation is not sufficient for PCD to occur. Proteomic analysis of the Prdx1 knock‐down cells revealed that the level of NF‐kappaB inhibitor epsilon (IκBε) was dramatically reduced. Furthermore, we found an increase in NFκB activation and reactive oxygen species (ROS) levels in the cytoplasm as a result of Prdx1 knockdown. We also found that silencing Prdx1 made the interdigital cells more susceptible to ROS‐induced cell death. Taken together, our study identifies two new players in interdigital cell death and highlights that PCD is regulated by a delicate balance of proapoptotic and survival‐promoting activities. Developmental Dynamics 233:266–281, 2005.