p38 signaling up-regulates BRAK expression in squamous cell carcinoma

Objectives: Stress signaling, for example, by ultraviolet (UV), TNF alpha or irradiation, is known to activate p38 and induce apoptosis of cells. BRAK, also known as CXCL14, is a non-ELR motif chemokine. It plays a key role not only in immune recognition processes but also acts as an anti-angiogenic factor. We recently found that BRAK expression was specifically down-regulated by epidermal growth factor treatment in HSC-3 tongue carcinoma cells and that over-expression of the gene in the cells showed anti-cancer activity (Ozawa et al., Biochem. Biophys. Res. Commun. 348: 406-412, 2006). Thus investigation of mechanisms of up-regulation of BRAK expression is very important for cancer prevention. Thus we investigated whether p38 signaling would stimulate BRAK gene expression or not.

Methods: HNSCC cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum. Nearly confluent cells were cultured overnight in serum-free DMEM with EGF (10 ng/ml). They were UV-irradiated (30J/m2) and then total RNA and proteins were extracted for analysis. We introduced si-RNA targeting p38 alpha, beta, gamma or delta into HSC-3 cells and subsequently extracted total RNA to determine the expression of BRAK. Protein levels were determined by western blotting by use of appropriate antibodies

Results: Irradiation with UV up-regulated BRAK mRNA expression accompanied by phosphorylation of p38. Furthermore, si-RNA targeting of p38 delta down-regulated BRAK expression.

Conclusions: Our results indicated that signaling by MAP kinase, especially p38 delta signaling plays a major role in the up-regulation of BRAK expression. Thus, activation of p38 delta by chemotherapy and/or irradiation therapy, leading to up-regulation BRAK may partially explain the effectiveness of such therapy.