Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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PUMA Regulates Radiation Induced Fibrosis by Activating Epithelial to Mesenchymal Transition through Snail1 Expression
Nicholas Brownstone, BA, William Hambley, BA, Karan Mehta, BS, Nakul Rao, MD, Chin Park, MD, Rohini Kadle, BS, Anna Zhou, BA, Camille Kim, BS, Abigail Sartor, BA, Piul Rabbani, PhD, Pierre Saadeh, MD, Daniel Ceradini, MD.
NYU Langone Medical Center, New York, NY, USA.

Purpose: p53 upregulated mediator of apoptosis (PUMA) has gained attention as a critical factor in fibrosis and we have previously demonstrated the role of PUMA in radiation-induced fibrosis through a SMAD3 independent pathway. Recently, Snail1 has been identified as a central mediator of epithelial-to-mesenchymal transition (EMT), which promotes fibrosis after injury. Here, we examine the impact of radiation on the expression of Snail1 and determine the interactions of Snail1 and PUMA in radiation injury.
Methods: We examined 3T3 fibroblasts exposed to a single dose of radiation (15 Gy) following treatment with small interfering RNA (siRNA) targeting PUMA and non-sense siRNA by quantitative RT-PCR for expression of Snail1, E-Cadherin and PUMA. Additionally, cytoplasmic and nuclear extracts from C57BL6 dorsal mouse skin were analyzed for Snail1 expression by western blotting. In vivo, C57BL6 mice were irradiated (45 Gy) and their skin was subsequently analyzed for Snail1 expression.
Results: Irradiation increased gene expression of Snail1 and E-cadherin in vitro (relative fold change of 1.27 and 1.81 respectively, p<0.05) as compared to a control. Irradiation also increased gene expression of Snail1 in vivo (relative fold change of 3.6, p<0.05). Puma silencing caused a downregulation in PUMA (relative fold change of 0.72, p<0.05), but an upregulation in E-cadherin (relative fold change of 6.96, p<0.05) suggesting that PUMA inhibition prevents EMT. Irradiation after PUMA silencing in vivo caused a downregulation in the gene expression level of Snail1 (relative fold change of 0.78, p<0.05). Additionally, western blotting of C57BL6 dorsal mouse skin demonstrated that Snail1 was mainly localized in the nucleus as opposed to the cytoplasm, which correlates with activation and maintenance of EMT.
Conclusion: Irradiation increases Snail1 both in vivo and in vitro, suggesting that activation of EMT is a major contributor to radiation injury. Inhibition of PUMA signaling prevents EMT by decreasing Snail1 expression and downstream target E-Cadherin. These data characterize a novel SMAD3 independent molecular pathway for radiation fibrosis, therefore identifying potential new targets for therapeutic intervention.


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