Abstract 315: RasGRP1 is a critical link to Ras activation by phorbol esters in mouse epidermal keratinocytes

Abstract

Abstract RasGRP1 is a member of the guanine nucleotide exchange factor family for Ras that binds with high affinity to ultrapotent diacylglycerol analogs like the phorbol esters. Recent work from our lab has demonstrated expression of RasGRP1 in epidermal keratinocytes and a role of this exchange factor in skin carcinogenesis. Further, the studies suggested that RasGRP1 participates in the action of tumor promoting phorbol esters like 12 - tetradecanoylphorbol - O - 13 - acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in epidermal carcinogenesis, we sought to discern if RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout mouse model (KO) to examine the response of primary keratinocytes to TPA. In contrast to the response of wild type (Wt) keratinocytes, which showed a rapid and sustained stimulation of Ras up to 30 minutes in response to TPA (Ras-GTP/Total Ras arbitrary units, 0 min TPA= 0.14 ± 0.06, 30 min TPA= 0.47 ± 0.06; n=5, **p<0.01), Ras-GTP levels in the KO cells were barely detected after TPA treatment (0 min TPA= 0.05 ± 0.02, 30 min TPA= 0.04 ± 0.02; n=7, ns). The lack of response was rescued by enforced expression of RasGRP1 in the KO keratinocytes, suggesting that the effect on Ras was specific to RasGRP1. To further confirm a RasGRP1-specific effect and rule out any indirect developmental effects that arise form early loss of RasGRP1 in the mouse mutant, we used an RNAi approach to silence RasGRP1 in Wt keratinocytes. This approach induced more than 90% depletion of RasGRP1 from the cells and led to a significant reduction in the ability of TPA to stimulate Ras, similar to the one observed in the KO keratinocytes. Analysis of specific Ras isoforms demonstrated that both H- and N-Ras depended on RasGRP1 for Ras activation by TPA, while activation of K-Ras could not be detected under our experimental conditions. Interestingly, while RasGRP1 was dispensable for ERK phosphorylation in response to TPA, phosphorylation of JNK was reduced in the KO keratinocytes. Notably, phosphorylation levels of the JNK2 isoform were significantly lower in the KO cells exposed to TPA compared to the levels observed in the Wt controls (p-JNK2/Total JNK2 arbitrary units, 30 min TPA, Wt= 0.15 ± 0.03, KO=0.05 ± 0.01; n=6-7, **p<0.01). Moreover, enforced expression of RasGRP1 was able to rescue the reduction in phopho-JNK2 levels observed in the RasGRP1 KO cells. Taken together, our data identify RasGRP1 as a critical molecule in the activation of Ras by TPA in primary mouse keratinocytes, and suggest JNK2 as one of the relevant downstream targets. These findings raise questions about the events taking place during TPA-induced tumor promotion in the epidermis -so far primarily attributed to PKC modulation-, and support further investigation on the role of RasGRP1 in keratinocyte transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 315.