Abstract
The mouse skin model of carcinogenesis has been instrumental in our appreciation of the multistage nature of carcinogenesis. In this system, tumor promotion is a critical step in the generation of tumors and is usually achieved by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Although it is generally assumed that protein kinase C (PKC) is the sole receptor for TPA in this system, we sought to evaluate whether non-PKC pathways could also contribute to the effects of phorbol esters in skin. We documented expression of the high affinity non-PKC phorbol ester receptor and Ras activator RasGRP1 in mouse primary keratinocytes. Overexpression of RasGRP1 in keratinocytes increased the level of active GTP-loaded Ras. TPA treatment further elevated this Ras activation in a PKC-independent manner and induced the translocation and down-regulation of RasGRP1. Overexpression of RasGRP1 in keratinocytes also caused apoptosis. Finally, induction of keratinocyte differentiation by elevation of extracellular calcium suppressed expression of endogenous RasGRP1, whereas overexpression of RasGRP1 inhibited expression of the differentiation markers keratins 1 and 10 induced by high calcium in the medium. Taken together, our results demonstrate that RasGRP1 is an additional diacylglycerol/phorbol ester receptor in epidermal keratinocytes and suggest that activation of this novel receptor may contribute to some of the phorbol ester- and Ras-mediated effects in mouse epidermis.
Highlights
The mouse skin model of carcinogenesis has been instrumental in our appreciation of the multistage nature of carcinogenesis
To further examine the impact of RasGRP1 activation on differentiation, we looked at the effect of RasGRP1 overexpression on the differentiation induced by calcium in murine keratinocytes
We have shown that RasGRP1, a non-protein kinase C (PKC) phorbol ester receptor, is expressed in mouse epidermal keratinocytes and can mediate phorbol ester effects independently of PKC
Summary
Vol 278, No 52, Issue of December 26, pp. 52792–52801, 2003 Printed in U.S.A. RasGRP1 Represents a Novel Non-protein Kinase C Phorbol Ester Signaling Pathway in Mouse Epidermal Keratinocytes*. One of the experimental models that has been instrumental in investigating the stages of cancer formation is the induction of tumors in mouse skin In this model, a carcinogen initiates cells by introducing a mutation in a ras gene, and the initiated population expands under treatment with phorbol esters, non-genotoxic compounds with tumor-promoting activities. Calcium-induced differentiation suppresses the levels of RasGRP1 in a concentrationdependent manner, whereas overexpression of RasGRP1 in epidermal cells inhibits the expression of markers of differentiation and causes cell death, effects that may be mediated by activation of Ras. In summary, our findings indicate that RasGRP1 is an additional diacylglycerol/phorbol ester receptor in epidermal keratinocytes and suggest that activation of this novel receptor may contribute to some of the phorbol ester- and Ras-mediated effects in mouse skin
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