Studies of the zinc finger transcription factors Slug and Snail have largely focused on their important role in modulating epithelial-mesenchymal transition during embryonic development and tumor progression. However, these transcription factors also appear to regulate local inflammation. In previous studies, we showed that Slug knockout mice were resistant to sunburn, an acute cutaneous inflammatory response, compared to wild type mice. In the present studies, we used Slug knockout, chimeric Slug knockout/wild type, conditional Slug knockout, and Slug transgenic mice to study the role of Slug in ultraviolet radiation (UVR)-induced cutaneous inflammation. We demonstrated, using immunohistochemistry, in vivo imaging, and neutrophil migration studies, that it was Slug expression in the epidermis rather than in neutrophils that modulated the UVR response. Microarray analysis indicated that this modulation was effected by the production of epidermal cytokines, including CCL3, CCL4, oncostatin, and interleukin-1β, that recruited neutrophils to UVR-exposed skin. The pattern of pro-inflammatory gene regulation by Slug suggested a central role for NF-κB in mediating gene induction. Immunohistochemical analysis of UVR-induced NF-κB activation demonstrated reduced activation in Slug knockout and enhanced activation in Slug transgenic epidermis. Slug thus appears to modulate NF-κB activation in response to UVR.
Overall design: Shaved Slug knockout and wild type mice were exposed to ~3 MED of UVR or left untreated. 24 hours later, mice were sacrificed and skin samples were frozen. Epidermal RNA was isolated from frozen skin samples using Trizol. Several replicates of each exposure condition were performed. RNA isolation was completed as recommended by the TRIzol supplier and RNA quality was assessed using Agilent’s RNA 6000 Nano kit. No RNA was used if the RIN value was less than 7.5. A total of 1000 ng of RNA was used to synthesize cDNA using the High Capacity cDNA RT Kit from Applied Biosystems. SABiosciences PAMM-3803Z RT-PCR array was used to quantify mRNA expression. Equal amounts of cDNA were added to each well, cDNA was mixed with iTAQ SYBR Green Supermix with ROX from Bio-Rad Laboratories, and each plate was run using the Applied Biosystems Prism 7900 HT qPCR protocol as follows: one cycle at 95° C for 10 minutes; 40 cycles at 95° C for 0.15 seconds and at 60° C for 60 seconds; and a final cycle of 15 seconds each at 95° C, 60° C, and 95° C. Analyses of each plate was performed using the SDS 2.3 program from Applied Biosciences, using the same threshold and baseline for all samples.
Less...