ABSTRACT
Slit proteins and their receptors, the Roundabout (Robo) family, are known to have a pivotal role in the vascular system. Slit2/Robo1 regulates the migration of human umbilical vein endothelial cells (HUVECs) and tumor-associated endothelial cells. Robo4, the endothelial-specific Robo, is also considered to be involved in vascular cell migration. However, the Slit/Robo signaling pathway is still unclear. Using a Boyden chamber assay, we found that Slit2 induces the migration of HUVECs under a Robo4 knockdown condition. This effect disappeared in Robo1 knockdown cells. The co-existence of the N-terminal extracellular portion of Robo1 blocked the Slit2-evoked migration of HUVECs, while that of Robo4 caused no effect. These results show that the Slit2 signal is transduced through Robo1, while the negative regulation of Robo4 is an intracellular event. Targeted proteomics using an anti-Robo1 monoclonal antibody identified CdGAP, an adhesion-localized Rac1-and Cdc42-specific GTPase activating protein, as a candidate for Slit2/Robo1 signaling. Robo1 and CdGAP were co-immunoprecipitated from CHO cells co-transfected with Robo1 and CdGAP genes. These results suggest that Slit2/Robo1 binding exerts an effect on cell migration, which is negatively regulated by Robo4, and Robo1 may function by interacting with CdGAP in HUVECs.
Subject(s)
Cell Movement/physiology , Endothelial Cells/physiology , GTPase-Activating Proteins/metabolism , Nerve Tissue Proteins/metabolism , Phosphoproteins/metabolism , Receptors, Cell Surface/metabolism , Receptors, Immunologic/metabolism , Cell Line , Endothelial Cells/cytology , Humans , Intercellular Signaling Peptides and Proteins , Signal Transduction/physiology , Roundabout ProteinsABSTRACT
We present an alternative method for evaluating cardiac fat tissue-dual gradient-echo in-phase and opposed-phase magnetic resonance imaging (IPOP-MRI) with electrocardiographic (ECG) gating. Conventional IPOP-MRI can be used to evaluate small amounts of fat and is widely used for abdominal imaging, but cardiac motion artifacts make its use difficult for cardiac imaging. Using ECG gating prior to IPOP-MRI, we evaluated lipomatous metaplasia after myocardial infarction. The areas of lipomatous metaplasia measured by IPOP-MRI with ECG gating correlated well with those areas on black-blood T(1)-weighted imaging (r=0.82, P<0.0001, mean bias-0.29 cm(2), limit of agreement+/-2.06 cm(2)).
Subject(s)
Electrocardiography/methods , Lipomatosis/pathology , Magnetic Resonance Imaging/methods , Myocardial Infarction/pathology , Adipose Tissue/pathology , Aged , Female , Humans , Lipomatosis/complications , Male , Myocardial Infarction/complications , Myocardium/pathologyABSTRACT
ROBO1 is a receptor mediating Slit-induced repulsive action on axon guidance and differentially expressed in human cancers. Although ROBO1 ectodomain has been detected, the cleavage site had not been determined. In this study we identified the precise cleavage site of ROBO1. We also report multi-step proteolysis of ROBO1 by metalloproteinases and gamma-secretase, producing two carboxy-terminal fragments, ROBO1-CTF1 at 129-kDa and ROBO1-CTF2 at 118-kDa. We have further demonstrated nuclear accumulation of ROBO1, which is abolished by either a metalloproteinase inhibitor TAPI-1 or a gamma-secretase inhibitor L-685,458. ROBO1 may function beyond the receptor through stepwise cleavages and translocation to the nucleus.
