RasGRP1 Transduces Low-Grade TCR Signals which Are Critical for T Cell Development, Homeostasis, and Differentiation

AbstractTwo 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

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 KO and RasGRP1/3 DKO DN4 thymocytes show an increased frequency of γδ T cells, despite normal numbers and frequencies of mature thymic γδ T cells. A.

<p>CD3 by γδTCR profiles of bulk thymocytes from B6 (n = 7), 1KO (n = 3), 3KO (n = 6) and DKO (n = 9) thymi. <b>B.</b> Frequencies and numbers of mature CD3⁺γδTCR⁺ γδ T cells. <b>C.</b> left, frequencies of surface TCRβ⁺ DN4 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁻) cells from B6 (n = 8), 1KO (n = 6), 3KO (n = 9) and DKO (n = 12) thymi; right, frequencies of surface γδTCR⁺ DN4 cells. ***p<0.001.</p

RasGRP1 KO, RasGRP3 KO and RasGRP1/3 DKO thymocytes show intact survival.

<p>Percentages of DN3 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁺), DN4 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁻) and DP (CD4⁺CD8⁺Thy1.2⁺) showing active caspase 3.</p

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

Comparison of binding affinities for ingenol 3-angelate (I3A) and phorbol 12,13-dibutyrate (PDBu).

*<p>Value from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072331#pone.0072331-Kedei1" target="_blank">[9]</a>.</p>†<p>Values represent the mean ± SEM of four independent experiments.</p>‡<p>Value represents the mean ± SEM of three independent experiments.</p