Differentially expressed genes induced by TPA stimulation between the P2ry6+/+ and P2ry6-/- mice

We report that P2ry6-deficient mice exhibit marked resistance to 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage skin papilloma formation compared with wild-type mice. Consistent with these findings, epidermal hyperplasia in response to TPA stimulation was suppressed in the P2ry6 knockout or MRS2578 (P2RY6 antagonist)-treated mice. The dramatic decrease in hyperplasia and tumorigenesis due to P2ry6 disruption was associated with the suppression of TPA-induced keratinocyte proliferation and inflammatory reactions.We have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish differentially expressed genes induced by TPA stimulation between the P2ry6+/+ and P2ry6-/- mice. Notably, P2ry6 deletion prevented the TPA-induced increase in YAP nuclear accumulation and hence its downstream gene expression in an MST/LATS1-dependent manner. For the long-term two-stage carcinogenesis model, a single dose of 195 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) with very high purity (> 99.8%) (Sigma, St. Louis, MO,USA) in 200 μL of acetone was applied to the shaved dorsal skin of P2ry6+/+ (n=12) and P2ry6-/- (n=12) mice. A week later, 16 nmol of TPA (Sigma) with very high purity (> 99.5%) in 200 mL of acetone was applied twice a week to the shaved back skin until the 22nd week. The tumors on the backs of the mice were counted weekly after becoming visible. The incidence of mouse tumors and multiplicity of tumors were also calculated.We took the skin tumors of P2RY6 +/+ mice and P2RY6-/- mice, and checked the differential genes through RNA extraction and gene chip technology .