Abstract
Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.
Highlights
Cellular stress response occurs when homeostasis is perturbed [1]
1234567890();,: RESULTS Multiple cellular stresses induce transcriptional induction of puma and noxa in zebrafish To first determine if pro-apoptotic mRNAs were upregulated in zebrafish following diverse cellular stresses, we analyzed the relative expression of puma, noxa, bax, and bid (Fig. S1 depicts zebrafish orthology analysis) in 24 hours post fertilization embryos exposed to either the genotoxic stress (30 Gy ionizing radiation, IR), ER stress (5 μM Thapsigargin, Thaps.), or oxidative stress (3.3 μM Phorbol 12-myristate 13-acetate, PMA)
Similar to IR, following Thaps. and PMA, both puma and noxa were significantly upregulated, bax and bid were not induced (Fig. 1B, C). Together these data have indicated that puma and noxa are strongly transcriptionally regulated by cellular stresses
Summary
Cellular stress response occurs when homeostasis is perturbed [1]. The apoptotic response to stress is often pathological and associated with human diseases [2,3,4,5,6,7,8]. DNA damage stress, unfolded protein stress, and oxidative stress responses have been linked to multiple human pathologies and can be distinguished by distinct proximal signaling components but can converge downstream on the p53 family of stress sensors and the apoptotic signaling network [9,10,11,12,13,14,15,16,17]. Deeper understanding of consequences of cellular stress and mediators of stress pathways in vivo will facilitate avenues to mediate disease pathogenesis
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