Mouse Platelet Ral GTPases Control P-Selectin Surface Expression, Regulating Platelet-Leukocyte Interaction.

OBJECTIVE: RalA and RalB GTPases are important regulators of cell growth, cancer metastasis, and granule secretion. The purpose of this study was to determine the role of Ral GTPases in platelets with the use of platelet-specific gene-knockout mouse models. APPROACH AND RESULTS: This study shows that platelets from double knockout mice, in which both GTPases have been deleted, show markedly diminished (≈85% reduction) P-selectin translocation to the surface membrane, suggesting a critical role in α-granule secretion. Surprisingly, however, there were only minor effects on stimulated release of soluble α- and δ-granule content, with no alteration in granule count, morphology, or content. In addition, their expression was not essential for platelet aggregation or thrombus formation. However, absence of surface P-selectin caused a marked reduction (≈70%) in platelet-leukocyte interactions in blood from RalAB double knockout mice, suggesting a role for platelet Rals in platelet-mediated inflammation. CONCLUSIONS: Platelet Ral GTPases primarily control P-selectin surface expression, in turn regulating platelet-leukocyte interaction. Ral GTPases could therefore be important novel targets for the selective control of platelet-mediated immune cell recruitment and inflammatory disease.

Evaluation and characterization of anti-RalA autoantibody as a potential serum biomarker in human prostate cancer.

Autoantibodies against intracellular tumor-associated antigens (TAAs) are commonly found in human cancers. In this study, we characterized the serum autoantibody response to the RalA, Ras-like GTPase, in patients with prostate cancer (PCa). The autoantibodies were detected by immunofluorescence assay in PCa cell lines, ELISA, and immunoblotting in 339 serum samples from patients with PCa and benign prostatic hyperplasia (BPH), and in normal human sera (NHS). The expression of RalA in prostate tumor tissues was evaluated by immunohistochemistry (IHC) in tumor microarrays. The autoantibody level to RalA (median) in NHS was significantly lower than in PCa (0.053 vs 0.138; P < 0.001) and BPH (0.053 vs 0.132; P < 0.005) groups. The circulating anti-RalA autoantibody could distinguish PCa patients from normal individuals with the area under the receiver operating characteristic (ROC) curve (AUC) performing at 0.861, with sensitivity of 52.9% and specificity of 91.0%. Elevation in serum immunoreactivity was observed in PCa patients after radical prostatectomy. The combined use of both anti-RalA autoantibody and PSA showed a significantly higher discriminatory ability compared with either of those markers alone. RalA protein expression was detected by IHC in 85.3% of tumor tissues from PCa patients, but without significant difference compared to BPH or normal control tissues. Together, our study shows the additional benefits of anti-RalA autoantibody as a potential serological biomarker for PCa, particularly in patients with normal PSA, and further demonstrate the utility of biomarker combinations in the immunodiagnosis of PCa.

Ras family of small GTPases in immunity and inflammation.

The Ras superfamily of small GTPases is a group of more than 150 small G proteins, all of which share some degree of homology to the founding member Ras. These small GTPases function as molecular switches within cells, impacting nearly all cellular processes. The Ras superfamily can be further divided into several smaller subfamilies, with those proteins that most closely resemble Ras belonging to the Ras subfamily. While heavily studied within the field of cancer biology, the Ras family of proteins also plays cardinal roles in immunity and inflammation. Here we review the roles of these molecular switches in regulating immune cell homeostasis and functions.

Activation of autophagy by inflammatory signals limits IL-1ß production by targeting ubiquitinated inflammasomes for destruction.

Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation, whereas inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1ß (IL-1ß) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered activation of the G protein RalB and autophagosome formation. The induction of autophagy did not depend on the adaptor ASC or capase-1 but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity, whereas stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.

Ral GTPases regulate cell-mediated cytotoxicity in NK cells.

NK cells are key components of the immune response to virally infected and tumor cells. Recognition of target cells initiates a series of events in NK cells that culminates in target destruction via directed secretion of lytic granules. Ral proteins are members of the Ras superfamily of small GTPases; they regulate vesicular trafficking and polarized granule secretion in several cell types. In this study, we address the role of Ral GTPases in cell-mediated cytotoxicity. Using a human NK cell line and human primary NK cells, we show that both Ral isoforms, RalA and RalB, are activated rapidly after target cell recognition. Furthermore, silencing of RalA and RalB impaired NK cell cytotoxicity. RalA regulated granule polarization toward the immunological synapse and the subsequent process of degranulation, whereas RalB regulated degranulation but not polarization of lytic granules. Analysis of the molecular mechanism indicated that Ral activation in NK cells leads to assembly of the exocyst, a protein complex involved in polarized secretion. This assembly is required for degranulation, as interference with expression of the exocyst component Sec5 led to reduced degranulation and impaired cytotoxicity in NK cells. Our results thus identify a role for Ral in cell-mediated cytotoxicity, implicating these GTPases in lymphocyte function.

CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions.

The chemokine CXCL12 is essential for the function of hematopoietic stem and progenitor cells. Here we report that secretion of functional CXCL12 from human bone marrow stromal cells (BMSCs) was a cell contact-dependent event mediated by connexin-43 (Cx43) and Cx45 gap junctions. Inhibition of connexin gap junctions impaired the secretion of CXCL12 and homing of leukocytes to mouse bone marrow. Purified human CD34(+) progenitor cells did not adhere to noncontacting BMSCs, which led to a much smaller pool of immature cells. Calcium conduction activated signaling by cAMP-protein kinase A (PKA) and induced CXCL12 secretion mediated by the GTPase RalA. Cx43 and Cx45 additionally controlled Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1. We suggest that BMSCs form a dynamic syncytium via connexin gap junctions that regulates CXC12 secretion and the homeostasis of hematopoietic stem cells.

Immunogenicity of Ra1A and its tissue-specific expression in hepatocellular carcinoma.

In order to understand the immunogenicity of a tumor-associated antigen (TAA), Ras family small GTP binding protein (Ra1A) in hepatocellular carcinoma (HCC), autoantibody responses to RalA were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and indirect immunofluorescence assay in sera from patients with HCC and sera from normal individuals. Immunohistochemistry (IHC) study with tissue array slides was also performed to analyze protein expression profiles of RalA in HCC and control tissues. This study demonstrated that RalA had a relative higher frequency of autoantibody response in HCC (20.1%) compared to liver cirrhosis (3.3%), chronic hepatitis (0%), and normal individuals sera (0%). RalA also showed a stepwise increased expression from normal liver tissues (26.7%), liver cirrhosis tissues (45.0%) to HCC tissues (63.3%). Sensitivity and specificity of anti-RalA antibody in detection of HCC was 20.1% and 99.3%, respectively. The data suggested that RalA might contribute to liver malignant transformation, and could be used as a potential tumor marker in HCC detection.

[Preparation and characterization of monoclonal antibodies against RalB].

OBJECTIVE: In order to explore the expression of RalB (ras related; GTP binding protein B) in mammal eucaryotic cell, we prepared and characterized monoclonal antibodies against RalB. METHODS: Hybridomas were generated by the fusion with Sp2/0 myelomas and spleen cells, which were from mice immunized with RalB recombinant proteins. The monoclonal antibodies against RalB were then used to identify the expression of RalB in mammal eucaryotic cell, including normal hepatic cell and hepatoma carcinoma cells, by Western blot and Immunohistochemistry. RESULTS: Two hybridoma cell lines, F001, F002, had been produced, each of which stably secrets antibodies against RalB. Subclass of IgG are both belonged to IgG1. Immunohistochemistry demonstrated that RalB was presented in plasma membrane of hepatoma tissue. Western-blot showed that RalB was expressed in all concerned cell. CONCLUSION: The monoclonal antibodies against RalB protein have been successfully prepared, which should provide useful reagent for further investigation into the biological function of RalB.