Studies have shown that gamma-irradiation induces various biological responses, including oxidative stress and apoptosis, as well as cellular repair and immune system responses. However, most such studies have been performed using traditional two-dimensional cell culture systems, which are limited in their ability to faithfully represent in vivo conditions. A three-dimensional (3D) environment composed of properly interconnected and differentiated cells that allow communication and cooperation among cells via secreted molecules would be expected to more accurately reflect cellular responses. Here, we investigated y-irradiation induced changes in the secretome of 3D-cultured keratinocytes. An analysis of keratinocyte secretome profiles following fractionated-dose gamma-irradiation revealed changes in genes involved in cell adhesion, angiogenesis, and the immune system. Notably, peroxisome proliferator-activated receptor-alpha (PPAR alpha) was upregulated in response to fractionated-dose gamma-irradiation. This upregulation was associated with an increase in the transcription of known PPARa target genes in secretome, including angiopoietin-like protein 4, dermokine and kallikrein-related peptide 12, which were differentially regulated by fractionated-dose gamma-irradiation. Collectively, our data imply a mechanism linking gamma-irradiation and secretome changes, and suggest that these changes could play a significant role in the coordinated cellular responses to harmful ionizing radiation, such as those associated with radiation therapy. This extension of our understanding of gamma-irradiation-induced secretome changes has the potential to improve radiation therapy strategies. (C) 2016 Elsevier Inc. All rights reserved.