Induction of Snail family transcription factors on ultraviolet‐irradiated keratinocytes

Abstract

Zinc finger transcription factors of the Snail family play critical roles in embryonic development, in cancer progression and metastasis, and in normal physiologic processes. During development, expression of the Snail family members Slug and Snail is largely driven by growth factor binding to receptor tyrosine kinases and subsequent stimulation of downstream signaling cascades. Our initial studies showed that ultraviolet radiation (UVR) induced transient expression of Slug and Snail in cultured keratinocytes. Expression of Slug and Snail mRNA was stimulated by UVR in a dose‐dependent manner at physiological doses. Because growth factors have been shown to induce Snail family members through both MAPK and phosphatidylinositol 3‐kinase (PI3‐K) signaling pathways, we tested the contribution of these pathways to UVR induction of Slug and Snail in keratinocytes. In addition, because UVR is an environmental stressor, we also examined the role of the JNK stress response pathway in Snail family induction.Studies were performed using the 12F SCC human keratinocyte cell line, which was derived from a squamous cell carcinoma. Cells were allowed to reach confluence and serum‐starved for 48 hours. Two hours before UVR exposure, cells were pretreated with pharmacologic inhibitors (10 μM) of the MAPK pathway members MEK and p38, with an inhibitor of JNK activation, or with an inhibitor of the PI3‐K kinase pathway. Cells were exposed in PBS to 300 J/m2 UVR and harvested at 2 hours post‐exposure. Standard techniques were employed for RNA isolation, cDNA synthesis, and quantitative RT‐PCR. Levels of Slug and Snail mRNA were standardized to GAPDH levels and indexed to values in cells not exposed to UVR. Expression of Slug and Snail was significantly blocked by MAPK and PI‐3K pathways inhibitors. Inhibition of JNK activation had no effect on Slug induction.