RasGRPs are targets of the anti-cancer agent ingenol-3-angelate.

Ingenol-3-angelate (I3A) is a non-tumor promoting phorbol ester-like compound identified in the sap of Euphoria peplus. Similar to tumor promoting phorbol esters, I3A is a diacylglycerol (DAG) analogue that binds with high affinity to the C1 domains of PKCs, recruits PKCs to cellular membranes and promotes enzyme activation. Numerous anti-cancer activities have been attributed to I3A and ascribed to I3A's effects on PKCs. We show here that I3A also binds to and activates members of the RasGRP family of Ras activators leading to robust elevation of Ras-GTP and engagement of the Raf-Mek-Erk kinase cascade. In response to I3A, recombinant proteins consisting of GFP fused separately to full-length RasGRP1 and RasGRP3 were rapidly recruited to cell membranes, consistent with direct binding of the compound to RasGRP's C1 domain. In the case of RasGRP3, IA3 treatment led to positive regulatory phosphorylation on T133 and activation of the candidate regulatory kinase PKCδ. I3A treatment of select B non-Hodgkin's lymphoma cell lines resulted in quantitative and qualitative changes in Bcl-2 family member proteins and induction of apoptosis, as previously demonstrated with the DAG analogue bryostatin 1 and its synthetic analogue pico. Our results offer further insights into the anticancer properties of I3A, support the idea that RasGRPs represent potential cancer therapeutic targets along with PKC, and expand the known range of ligands for RasGRP regulation.

RasGRP1, but not RasGRP3, is required for efficient thymic ß-selection and ERK activation downstream of CXCR4.

T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4(-)CD8(-) 'double negative' (DN) thymocytes, pass through a checkpoint termed "ß-selection" before maturing into CD4(+)CD8(+) 'double positive' (DP) thymocytes. ß-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR) undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive ß-selection. Additionally, it has long been known that ERK is activated during ß-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the ß-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRß chain. As a result of impaired ß-selection, the pool of TCRß(+) DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of ß-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the ß-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

Bryostatin analogue-induced apoptosis in mantle cell lymphoma cell lines.

The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally been attributed to its action on protein kinase C. However, we previously documented apoptosis in a B non-Hodgkin lymphoma cell line involving diacylglycerol analogue stimulation of Ras guanyl-releasing protein, a Ras activator, and Bim, a proapoptotic Bcl-2 family protein. To further explore the role of Bim, we examined several Bim-deficient B non-Hodgkin lymphoma cells for their responses to pico, a synthetic bryostatin-1-like compound. The Bim(-) mantle cell lymphoma cell lines Jeko-1, Mino, Sp53, UPN1, and Z138 and the Bim(+) cell line Rec-1, as well as the Burkitt lymphoma cells lines BL2 (Bim(-)) and Daudi (Bim(+)), were examined for their response to pico using assays for proliferation and apoptosis as well as biochemical methods for Ras guanyl-releasing proteins and Bcl-2 family members. With the exception of UPN1, mantle cell lymphoma cell lines underwent pico-induced apoptosis, as did BL2. In some cases, hallmarks of apoptosis were substantially diminished in the presence of mitogen-activated protein kinase kinase inhibitors. Pico treatment generally led to increased expression of proapoptotic Bik, although the absolute levels of Bik varied considerably between cell lines. A pico-resistant variant of Z138 exhibited decreased Bik induction compared to parental Z138 cells. Pico also generally decreased expression of anti-apoptotic Bcl-XL and Mcl1. Although, these changes in Bcl-2 family members seem unlikely to fully account for the differential behavior of the cell lines, our demonstration of a potent apoptotic process in most cell lines derived from mantle cell lymphoma encourages a re-examination of diacylglycerol analogues in the treatment of this subset of B non-Hodgkin lymphoma cases.

Familial recurrence of cerebral palsy with multiple risk factors.

The recurrence of cerebral palsy in the same family is uncommon. We, however, report on two families with two or more affected siblings. In both families, numerous potential risk factors were identified including environmental, obstetric, and possible maternal effects. We hypothesize that multiple risk factors may lead to the increased risk of recurrence of cerebral palsy in families. Intrinsic and maternal risk factors should be investigated in all cases of cerebral palsy to properly counsel families on the risk of recurrence. Recent studies of genetic polymorphisms associated with cerebral palsy are considered with reference to our observations in these two families.

RasGRP1 is essential for ras activation by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in epidermal keratinocytes.

RasGRP1 is a guanine nucleotide exchange factor for Ras that binds with high affinity to diacylglycerol analogs like the phorbol esters. Recently, we demonstrated a role for RasGRP1 in skin carcinogenesis and suggested its participation in the action of tumor-promoting phorbol esters like 12-O-tetradecanoylphorbol-13-acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in carcinogenesis, we sought to discern whether RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout (KO) mouse model to examine the response of keratinocytes to TPA. In contrast to the effect seen in wild type keratinocytes, Ras(GTP) levels were barely detected in RasGRP1 KO cells even after 60 min of exposure to phorbol esters. The lack of response was rescued by enforced expression of RasGRP1. Furthermore, small hairpin RNA-induced silencing of RasGRP1 abrogated the effect of TPA on Ras. Analysis of Ras isoforms showed that both H-Ras and N-Ras depended on RasGRP1 for activation by TPA, whereas activation of K-Ras could not be detected. Although RasGRP1 was dispensable for ERK activation in response to TPA, JNK activation was reduced in the KO keratinocytes. Notably, TPA-induced phosphorylation of JNK2, but not JNK1, was reduced by RasGRP1 depletion. These 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. Given the role of TPA as a skin tumor promoter, our findings provide additional support for a role for RasGRP1 in skin carcinogenesis.

RasGRP1 transgenic mice develop cutaneous squamous cell carcinomas in response to skin wounding: potential role of granulocyte colony-stimulating factor.

Models of epidermal carcinogenesis have demonstrated that Ras is a critical molecule involved in tumor initiation and progression. Previously, we have shown that RasGRP1 increases the susceptibility of mice to skin tumorigenesis when overexpressed in the epidermis by a transgenic approach, related to its ability to activate Ras. Moreover, RasGRP1 transgenic mice develop spontaneous papillomas and cutaneous squamous cell carcinomas, some of which appear to originate in sites of injury, suggesting that RasGRP1 may be responding to signals generated during the wound-healing process. In this study, we examined the response of the RasGRP1 transgenic animals to full-thickness incision wounding of the skin, and demonstrated that they respond by developing tumors along the wounded site. The tumors did not present mutations in the H-ras gene, but Rasgrp1 transgene dosage correlated with tumor susceptibility and size. Analysis of serum cytokines showed increased levels of granulocyte colony-stimulating factor in transgenic animals after wounding. Furthermore, in vitro experiments with primary keratinocytes showed that granulocyte colony-stimulating factor stimulated Ras activation, although RasGRP1 was dispensable for this effect. Since granulocyte colony-stimulating factor has been recently associated with proliferation of skin cancer cells, our results may help in the elucidation of pathways that activate Ras in the epidermis during tumorigenesis in the absence of oncogenic ras mutations.

A proapoptotic signaling pathway involving RasGRP, Erk, and Bim in B cells.

OBJECTIVE: Bryostatin-1 and related diacylglycerol (DAG) analogues activate RasGRPs in lymphocytes, thereby activating Ras and mimicking some aspects of immune receptor signaling. To define the role of RasGRPs in lymphocyte apoptosis and to identify potential therapeutic uses for DAG analogues in lymphocyte disorders, we characterized the response of lymphoma-derived cell lines to DAG analogues. MATERIALS AND METHODS: Human lymphoma-derived B cell lines and mouse primary B cells were treated with bryostatin-1 or its synthetic analogue "pico." Ras signaling partners and Bcl-2 family members were studied with biochemical assays. Cellular responses were monitored using growth and apoptosis assays. RESULTS: Stimulation of B cells with DAG analogues results in activation of protein kinase C/RasGRP-Ras-Raf-Mek-Erk signaling and phosphorylation of the proapoptotic BH3-only protein Bim. In vitro, Bim is phosphorylated by Erk on sites previously associated with increased apoptotic activity. In Toledo B cells derived from a non-Hodgkin's lymphoma (B-NHL), DAG analogue stimulation leads to extensive apoptosis. Apoptosis can be suppressed by either downregulation of Bim or overexpression of Bcl-2. It is associated with the formation of Bak-Bax complexes and increased mitochondrial membrane permeability. Toledo B-NHL cell apoptosis shows a striking dependence on sustained signaling. CONCLUSION: In B cells, Erk activation leads directly to phosphorylation of Bim on sites associated with activation of Bim. In Toledo B-NHL cells, the dependence of apoptosis on sustained signaling suggests that Bcl-2 family members could interpret signal duration, an important determinant of B cell receptor-mediated negative selection. Certain cases of B-NHL might respond to DAG analogue treatment by the mechanism outlined here.

The role of RasGRPs in regulation of lymphocyte proliferation.

RasGRP1 links TCR signaling to Ras in T cells, while both RasGRP1 and RasGRP3 link BCR signaling to Ras in B cells. T cells deficient in RasGRP1 have defective proliferative responses as do B cells deficient in both RasGRP1 and RasGRP3, confirming the importance of Ras activation in lymphocyte proliferation. While aged Rasgrp1-/- mice develop late-onset autoimmunity characterized by splenomegaly and the presence of anti-nuclear antibodies (ANA), the additional loss of RasGRP3 expression inhibits this phenotype. We show here that the autoimmunity in Rasgrp1-/- mice is T cell dependent. Compared to wildtype, Rasgrp1-/- T cells induce greater in vitro B cell proliferation that is due, at least in part, to increased production of interleukin-4 (IL-4). Rasgrp1 Rasgrp3 double mutant B cells are less responsive to this T cell stimulation. The reduced double mutant B cell proliferative response was paralleled by decreased induction of cyclin D2 upon stimulation with IL-4 and anti-IgM. Taken together these results suggest that double mutant mice fail to generate autoimmunity due to their decreased B cell cyclin D2 accumulation, and thus proliferation, in response to the elevated levels of IL-4 produced by mutant T cells.

RasGRP1 and RasGRP3 regulate B cell proliferation by facilitating B cell receptor-Ras signaling.

The RasGRPs are a family of Ras activators that possess diacylglycerol-binding C1 domains. In T cells, RasGRP1 links TCR signaling to Ras. B cells coexpress RasGRP1 and RasGRP3. Using Rasgrp1 and Rasgrp3 single and double null mutant mice, we analyzed the role of these proteins in signaling to Ras and Erk in B cells. RasGRP1 and RasGRP3 both contribute to BCR-induced Ras activation, although RasGRP3 alone is responsible for maintaining basal Ras-GTP levels in unstimulated cells. Surprisingly, RasGRP-mediated Ras activation is not essential for B cell development because this process occurs normally in double-mutant mice. However, RasGRP-deficient mice do exhibit humoral defects. Loss of RasGRP3 led to isotype-specific deficiencies in Ab induction in immunized young mice. As reported previously, older Rasgrp1-/- mice develop splenomegaly and antinuclear Abs as a result of a T cell defect. We find that such mice have elevated serum Ig levels of several isotypes. In contrast, Rasgrp3-/- mice exhibit hypogammaglobulinemia and show no signs of splenomegaly or autoimmunity. Double-mutant mice exhibit intermediate serum Ab titers, albeit higher than wild-type mice. Remarkably, double-mutant mice exhibit no signs of autoimmunity or splenomegaly. B cell proliferation induced by BCR ligation with or without IL-4 was found to be RasGRP1- and RasGRP3-dependent. However, the RasGRPs are not required for B cell proliferation per se, because LPS-induced proliferation is unaffected in double-mutant mice.

Synthetic bryostatin analogues activate the RasGRP1 signaling pathway.

The functional properties of four diacylglycerol (DAG) analogues were compared using cell-signaling assays based on the protein RasGRP1, a DAG-regulated Ras activator. Compounds 1 and 2, synthetic analogues of bryostatin 1, were compared to authentic bryostatin 1 and phorbol 12-myristate-13-acetate (PMA). The two "bryologues" were able to activate RasGRP1 signaling rapidly in cultured cells and isolated mouse thymocytes. They elicited expression of the T cell activation marker CD69 in human T cells. DAG analogues promptly recruited RasGRP1 to cell membranes, but they did not induce RasGRP1 proteolysis. Bryostatin 1 and compounds 1 and 2 appeared to be less potent than PMA at inducing aggregation of mouse thymocytes, a PKC-dependent, RasGRP1-independent response. In addition to sharing potential anticancer properties with bryostatin 1, compounds 1 and 2 might be clinically useful as modulators of the immune system.

Autoimmunity as the consequence of a spontaneous mutation in Rasgrp1.

A mouse strain was identified with a recessive genetic lesion, which spontaneously developed a lymphoproliferative autoimmune syndrome exhibiting features of systemic lupus erythematosus. Positional mapping of the disease-associated locus revealed a lesion in Rasgrp1 that prevented the translation of the RasGRP1 protein. T cells from these mice failed to activate Ras or proliferate vigorously following antigen encounter and showed defects in positive selection. Peripheral RasGRP1lag T cells spontaneously adopted a memory phenotype and were able to transfer disease to lymphopenic recipient mice. CD4+ T cells accumulated in the lymphoid tissues of older RasGRP1lag mice and were resistant to activation-induced cell death. RasGRP1lag B cells were functionally normal, but activated B cells were detected in older mice, as were autoantibodies directed against self-antigens. Our findings indicate that Ras signaling pathways are required to maintain T cell tolerance and to prevent autoimmune disease.

RasGRP1 transduces low-grade TCR signals which are critical for T cell development, homeostasis, and differentiation.

Two important Ras-guanyl nucleotide exchange factors, Sos and RasGRP1, control Ras activation in thymocytes. However, the relative contribution of these two exchange factors to Ras/ERK activation and their resulting impact on positive and negative selection is unclear. We have produced two lines of RasGRP1(-/-) TCR transgenic mice to determine the effect of RasGRP1 in T cell development under conditions of defined TCR signaling. Our results demonstrate that RasGRP1 is crucial for thymocytes expressing weakly selecting TCRs whereas those that express stronger selecting TCRs are more effective at utilizing RasGRP1-independent mechanisms for ERK activation and positive selection. Analysis of RasGRP1(-/-) peripheral T cells also revealed hitherto unidentified functions of RasGRP1 in regulating T cell homeostasis and sustaining antigen-induced developmental programming.