MicroRNA-132 is enriched in developing axons, locally regulates Rasa1 mRNA, and promotes axon extension.

Developing axons can locally synthesize proteins, with roles in axon growth, guidance, and regeneration, but the mechanisms that regulate axonal mRNA translation are not well understood. MicroRNAs (miRNAs) are important regulators of translation but have still been little characterized in developing axons. Here we study mouse dorsal root ganglion (DRG) axons and show that their extension is impaired by conditional deficiency of the miRNA-processing enzyme Dicer in vitro and in vivo. A screen for axonal localization identifies a specific set of miRNAs preferentially enriched within the developing axon. High axonal expression and preferential localization were observed for miR-132, a miRNA previously known for roles in dendrites and dysregulation in major neurologic diseases. miR-132 knockdown reduced extension of cultured DRG axons, whereas overexpression increased extension. Mechanistically, miR-132 regulated the mRNA for the Ras GTPase activator Rasa1, a novel target in neuronal function. Moreover, miR-132 regulation of Rasa1 translation was seen in severed axons, demonstrating miRNA function locally within the axon. miR-132 expression in DRGs peaked in the period of maximum axon growth in vivo, consistent with its effect on axon growth, and suggesting a role as a developmental timer. Together, these findings identify miR-132 as a positive regulator of developing axon extension, acting through repression of Rasa1 mRNA, in a mechanism that operates locally within the axon.

Nonredundant functions for Ras GTPase-activating proteins in tissue homeostasis.

Inactivation of the small guanosine triphosphate-binding protein Ras during receptor signal transduction is mediated by Ras guanosine triphosphatase (GTPase)-activating proteins (RasGAPs). Ten different RasGAPs have been identified and have overlapping patterns of tissue distribution. However, genetic analyses are revealing critical nonredundant functions for each RasGAP in tissue homeostasis and as regulators of disease processes in mouse and man. Here, we discuss advances in understanding the role of RasGAPs in the maintenance of tissue integrity.

A role for p120 RasGAP in thymocyte positive selection and survival of naive T cells.

Activation of the Ras small GTP-binding protein is necessary for normal T cell development and function. However, it is unknown which Ras GTPase-activating proteins (RasGAPs) inactivate Ras in T cells. We used a T cell-specific RASA1-deficient mouse model to investigate the role of the p120 RasGAP (RASA1) in T cells. Death of CD4(+)CD8(+) double-positive thymocytes was increased in RASA1-deficient mice. Despite this finding, on an MHC class II-restricted TCR transgenic background, evidence was obtained for increased positive selection of thymocytes associated with augmented activation of the Ras-MAPK pathway. In the periphery, RASA1 was found to be dispensable as a regulator of Ras-MAPK activation and T cell functional responses induced by full agonist peptides. However, numbers of naive T cells were substantially reduced in RASA1-deficient mice. Loss of naive T cells in the absence of RASA1 could be attributed in part to impaired responsiveness to the IL-7 prosurvival cytokine. These findings reveal an important role for RASA1 as a regulator of double-positive survival and positive selection in the thymus as well as naive T cell survival in the periphery.

Innate immune function of the adherens junction protein p120-catenin in endothelial response to endotoxin.

Sepsis-induced acute lung injury is a common clinical disorder in critically ill patients that is associated with high mortality. In this study, we investigated the role of p120-catenin (p120), a constituent of endothelial adherens junctions, in regulating the innate immune function of lungs. In mice in which acute lung injury was induced by i.p. administration of LPS, we observed a rapid decrease in the expression of p120 in lungs. The p120 protein expression was correlated inversely with severity of inflammation. Suppression of p120 expression in lung endothelial cells in mice using small interfering RNA resulted in high sensitivity to endotoxin and greatly increased the mortality compared with controls. Knockdown of p120 also increased the expression of ICAM-1, neutrophil recruitment, production of cytokines TNF-α and IL-6, pulmonary transvascular protein permeability, and lung water content in response to LPS. We demonstrated that endothelial p120 modulates lung innate immune function by interfering with the association of TLR4 with its adaptor MyD88 to block TLR4 signaling and NF-κB activation in endothelial cells. In conclusion, these studies have uncovered a novel innate immune function of endothelial p120 in downregulating the lung inflammatory response to endotoxin through the suppression of TLR4 signaling.

p120Ras-GAP binds the DLC1 Rho-GAP tumor suppressor protein and inhibits its RhoA GTPase and growth-suppressing activities.

DLC1 (deleted in liver cancer 1), which encodes a Rho GTPase-activating protein (Rho-GAP), is a potent tumor suppressor gene that is frequently inactivated in several human cancers. DLC1 is a multidomain protein that has been shown previously to bind members of the tensin gene family. Here we show that p120Ras-GAP (Ras-GAP; also known as RASA1) interacts and extensively colocalizes with DLC1 in focal adhesions. The binding was mapped to the SH3 domain located in the N terminus of Ras-GAP and to the Rho-GAP catalytic domain located in the C terminus of the DLC1. In vitro analyses with purified proteins determined that the isolated Ras-GAP SH3 domain inhibits DLC1 Rho-GAP activity, suggesting that Ras-GAP is a negative regulator of DLC1 Rho-GAP activity. Consistent with this possibility, we found that ectopic overexpression of Ras-GAP in a Ras-GAP-insensitive tumor line impaired the growth-suppressing activity of DLC1 and increased RhoA activity in vivo. Our observations expand the complexity of proteins that regulate DLC1 function and define a novel mechanism of the cross talk between Ras and Rho GTPases.1R01CA129610

The p120 family of cell adhesion molecules.

p120 is the prototypic member of the p120 subfamily of armadillo-related proteins that includes p0071, delta-catenin/NPRAP, ARVCF and the more distantly related plakophilins 1-3. Like armadillo, beta-catenin and plakoglobin these proteins are involved in mediating cell-cell adhesion. Besides their junctional localization they also reveal a cytoplasmic and nuclear localization. Non-cadherin-associated, cytoplasmic p120 functions in Rho signaling and regulation of cytoskeletal organization and actin dynamics. The nuclear function remains largely unsolved. Some characteristics seem to be shared by the various members of the family but it seems unlikely that p120-related proteins have solely redundant functions and compete for interactions with identical binding partners. Stabilization of cadherins at the membrane seems a common function of p120, p0071, delta-catenin and ARVCF but it is not yet known if and how these proteins confer distinct properties to cellular junctions. Moreover, p0071, NPRAP and ARVCF have a C-terminal PDZ-binding motif that is lacking in p120 pointing to distinct roles of these proteins. PDZ domains are found in a series of proteins involved in establishing cell polarity in epithelial cells. Thus, p120 proteins may not only be master regulators of cadherin abundance and activity but play additional roles in regulating cell polarity. This review focuses on the putative roles of p120 proteins in cell polarity.

p120 Ras GTPase-activating protein associates with fibroblast growth factor receptors in Drosophila.

Btl (breathless) and Htl (heartless), the two FGFRs (fibroblast growth factor receptors) in Drosophila melanogaster, control cell migration and differentiation in the developing embryo. These receptors signal through the conserved Ras/mitogen-activated protein kinase pathway, but how they regulate Ras activity is not known. The present study shows that there is a direct interaction between p120 RasGAP (Ras GTPase-activating protein), a negative regulator of Ras, and activated FGFRs in Drosophila. The interaction is dependent on the SH2 (Src homology 2) domains of RasGAP, which have been shown to interact with a phosphotyrosine residue within the consensus sequence (phospho)YXXPXD. A potential binding site that matches this consensus is found in both Btl and Htl, located between the transmembrane and kinase domains of each receptor. A peptide corresponding to this region was capable of binding RasGAP only when the tyrosine residue was phosphorylated. This tyrosine residue appears to be conserved in human FGFR-1 and mediates the association with the adapter protein CrkII, but no association between dCrk (Drosophila homologue of CrkII) and the activated FGFRs was detected. RasGAP was a substrate of the activated FGFR kinase domain, and mutation of the tyrosine residue within the potential binding site on the receptor prevented tyrosine phosphorylation of RasGAP. RasGAP attenuated FGFR signalling in vivo and this ability was dependent on both its SH2 domains and its GAP activity. On the basis of these results, we propose that RasGAP is directly recruited into activated FGFRs in Drosophila and plays a role in regulating the strength of signalling through Ras and the mitogen-activated protein kinase pathway.

Tyrosine phosphorylation of p190 RhoGAP by Fyn regulates oligodendrocyte differentiation.

During development of the central nervous system, oligodendrocyte progenitor cells differentiate into mature myelinating cells. The molecular signals that promote this process, however, are not well defined. One molecule that has been implicated in oligodendrocyte differentiation is the Src family kinase Fyn. In order to probe the function of Fyn in this system, a yeast two hybrid screen was performed. Using Fyn as bait, p190 RhoGAP was isolated in the screen of an oligodendrocyte cDNA library. Coimmunoprecipitation and in vitro binding assays verified that p190 RhoGAP bound to the Fyn SH2 domain. Phosphorylation of p190 required active Fyn tyrosine kinase and was increased threefold upon differentiation of primary oligodendrocytes. Moreover, complex formation between p190 and p120 RasGAP occurred in differentiated oligodendrocytes. p190 RhoGAP activity is known to regulate the RhoGDP:RhoGTP ratio. Indeed, expression of dominant negative Rho in primary oligodendrocytes caused a hyperextension of processes. Conversely, constitutively activated Rho caused reduced process formation. These findings define a pathway in which Fyn activity regulates the phosphorylation of p190, leading to an increase in RhoGAP activity with a subsequent increase in RhoGDP, which in turn, regulates the morphological changes that accompany oligodendrocyte differentiation.

Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain.

Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, we circumvent the embryonic lethality of NF1 null mouse embryos by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background has ablated NF1 function in most differentiated neuronal populations. These mice have abnormal development of the cerebral cortex, which suggests that NF1 has an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism.