Differential effects of bryostatin 1 and 12-O-tetradecanoylphorbol-13-acetate on the regulation and activation of RasGRP1 in mouse epidermal keratinocytes

Abstract

The antitumor agent bryostatin 1 and the tumorpromoting phorbol esters function as structural mimetics of the second lipid messenger diacylglycerol (DAG) by binding to the C1 domain of DAG receptors. However, bryostatin 1 and the phorbol esters often differ in their cellular actions. In mouse skin, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter, whereas bryostatin 1 lacks this activity and antagonizes the tumor-promoting effects of TPA. Although protein kinase C mediates many of the effects of DAG on skin, the exact mechanisms responsible for the biology of bryostatin 1 and TPA in the epidermis have not been elucidated. We recently reported that the novel DAG receptor RasGRP1 is expressed in mouse keratinocytes and mediates TPA-induced Ras activation. This finding prompted us to examine the regulation of RasGRP1 by bryostatin 1. We found that whereas TPA induced translocation of RasGRP1 to both the plasma and internal membranes of the keratinocytes, bryostatin 1 recruited RasGRP1 only to internal membranes and the nuclear envelope. In addition, TPA led to a concentration-dependent down-regulation of RasGRP1, whereas bryostatin 1 failed to induce full RasGRP1 downregulation. Interestingly, bryostatin 1 was less effective than TPA at activating Ras. The results presented here suggest the possibility that a differential modulation of RasGRP1 by bryostatin 1 compared with TPA could participate in the disparate responses of the epidermal cells to both DAG analogues. This result may have implications in the understanding of the antitumor effects of bryostatin 1 in the skin. [Mol Cancer Ther 2006;5(3):602 -10]

Transgenic Overexpression of RasGRP1 in Mouse Epidermis Results in Spontaneous Tumors of the Skin

“…Closer examination of the keratinocytes showed cytoplasmic and perinuclear localization of RasGRP1 in the cells (Fig. 3C), a localization that matched the subcellular distribution of RasGRP1 in keratinocytes in culture (8,10).…”

“…We have recently shown that the Ras exchange factor and phorbol ester receptor RasGRP1 is expressed in mouse primary keratinocytes, where it mediates Ras activation in response to the tumor-promoting phorbol ester TPA (8,10). To explore in more detail the role of RasGRP1 in the epidermis and in tumor promotion, we generated a transgenic mouse model in which overexpression of RasGRP1 was targeted to the basal layer of epidermal keratinocytes and the outer root sheath of hair follicles using the bovine K5 promoter (Fig.…”

RasGRP1 Overexpression in the Epidermis of Transgenic Mice Contributes to Tumor Progression during Multistage Skin Carcinogenesis

“…We have previously found that transient overexpression of RasGRP1 in primary keratinocytes in vitro causes Ras activation and that TPA further increases it (13,14). To test whether keratinocytes derived from K5.RasGRP1 mice were also more sensitive to the TPA-mediated activation of Ras than the wild-type cells, we measured the levels of active, GTPbound Ras using a pull-down assay in keratinocytes derived from both groups.…”

“…Levels of GTP-loaded Ras (Ras GTP ) were measured by using the GST-RBD domain of Raf-1 as a probe in an affinity precipitation or pull-down assay. Briefly, primary keratinocytes were first isolated from K5.RasGRP1 or wild-type mice as described elsewhere (14). Cells were serum starved overnight, treated with vehicle (Me 2 SO) or 1 Amol/L of TPA for 15 min, and harvested on ice in lysis buffer containing 25 mmol/L Tris-HCl (pH 7.5), 150 mmol/L of NaCl, …”

“…RasGRP1, for example, is expressed in T cells and at lower levels in B cells, neurons, mastocytes, and some kidney cells (8)(9)(10)(11)(12). Recently, we have also found RasGRP1 expression in epidermal keratinocytes, where it can mediate Ras activation in response to phorbol esters (13,14).…”