Nuclear trafficking of proteins requires the cooperation between soluble transport components and nucleoporins. As such, classical nuclear import depends on the dimeric carrier importin-alpha/beta1, and CAS, a member of the importin-beta family, which exports importin-alpha to the cytoplasm. Here we analyzed the effect of oxidative stress elicited by diethyl maleate (DEM) on classical nuclear transport. Under conditions that do not induce death in the majority of cells, DEM has little effect on the nucleocytoplasmic concentration gradient of Ran, but interferes with the nuclear accumulation of several reporter proteins. Moreover, DEM treatment alters the distribution of soluble transport factors and several nucleoporins in growing cells. We identified nuclear retention of importin-alpha, CAS as well as nucleoporins Nup153 and Nup88 as a mechanism that contributes to the nuclear concentration of these proteins. Both nucleoporins, but not CAS, associate with importin-alpha in the nuclei of growing cells and in vitro. Importin-alpha generates high molecular mass complexes in the nucleus that contain Nup153 and Nup88, whereas CAS was not detected. The formation of high molecular mass complexes containing importin-alpha, Nup153 and Nup88 is increased upon oxidant treatment, suggesting that complex formation contributes to the anchoring of importin-alpha in nuclei. Taken together, our studies link oxidative stress to the proper localization of soluble transport factors and nucleoporins and to changes in the interactions between these proteins.

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) make profound contributions to both physiology and pathology. While it is widely believed that direct (PDGF-mediated) activation is the primary mode of activating PDGFRs, the discovery that they can also be activated indirectly begs the question of the relevance of the indirect mode of activating PDGFRs. In the context of a blinding eye disease, indirect activation of PDGFR␣ results in persistent signaling, which suppresses the level of p53 and thereby promotes viability of cells that drive pathogenesis. Under the same conditions, PDGFR␤ fails to undergo indirect activation. In this paper, we report that RasGAP (GTPase-activating protein of Ras) prevented indirect activation of PDGFR␤. RasGAP, which associates with PDGFR␤ but not PDGFR␣, reduced the level of mitochondrion-derived reactive oxygen species, which are required for enduring activation of PDGFRs. Furthermore, preventing PDGFR␤ from associating with RasGAP allowed it to signal enduringly and drive pathogenesis of a blinding eye disease. These results indicate a previously unappreciated role of RasGAP in antagonizing indirect activation of PDGFR␤, define the underlying mechanism, and raise the possibility that PDGFR␤-mediated diseases involve indirect activation of PDGFR␤.T he receptors for platelet-derived growth factor (PDGF) are essential for mouse development and are implicated in a variety of human diseases (1, 2). Furthermore, these observations are the basis for the consensus that, while there may be overlap in what the two PDGF receptors (PDGFRs), PDGFR␣ and PDGFR␤, are capable of, they also have nonredundant functions in physiology and pathology.Because the two PDGFRs engage nonidentical signaling events in acutely stimulated cultured cells (3), a plausible reason for the distinct phenotype of mice lacking pdgfra and/or pdgfrb (4, 5) relates to signaling. Characterization of mice that express chimeric receptors in which the cytoplasmic domains were interchanged indicated that PDGFR␤ was more capable than PDGFR␣. PDGFR␣/␤ chimeric mice had no phenotype, whereas PDGFR␤/␣ chimeric mice showed some of the defects seen in mice in which PDGFR␤ lacked a major portion of the cytoplasmic domain (6, 7). Thus, in the context of mouse embryogenesis, the two PDGFRs do not appear to trigger the same signaling events, and more specifically, PDGFR␤ does something that PDGFR␣ cannot.The disparity in signaling events between the two PDGFRs that is germane to this report involves RasGAP (GTPase-activating protein of Ras), which is recruited by PDGFR␤ but not PDGFR␣ (8-10). RasGAP promotes the inactivation of Ras (11-13). RasGAP is an SH2 domain-containing protein, and its association with PDGFR␤ is dependent on tyrosine phosphorylation of PDGFR␤ within a context that is preferred by the SH2 domains of RasGAP (14-19). PDGFR␣ does not interact with RasGAP because none of its phosphorylation sites are within such an amino acid motif (9,10,20).Consistent with the known function of RasGAP, PDGF stimu...

BackgroundSignaling through MEK→ERK1/2 and PI3 kinases is implicated in many aspects of cell physiology, including the survival of oxidant exposure. Oxidants play a role in numerous physiological and pathophysiological processes, many of which rely on transport in and out of the nucleus. However, how oxidative stress impacts nuclear trafficking is not well defined.Methodology/Principal FindingsTo better understand the effect of stress on nucleocytoplasmic trafficking, we exposed cells to the oxidant diethyl maleate. This treatment activated MEK→ERK1/2 as well as PI3 kinase→Akt cascades and triggered the inhibition of classical nuclear import. To define the molecular mechanisms that regulate nuclear transport, we examined whether MEK and PI3 kinase signaling affected the localization of key transport factors. Using recently developed tools for image acquisition and analysis, the subcellular distributions of importin-α, CAS, and nucleoporins Nup153 and Nup88 were quantified in different cellular compartments. These studies identified specific profiles for the localization of transport factors in the nucleus and cytoplasm, and at the nuclear envelope. Our results demonstrate that MEK and PI3 kinase signaling as well as oxidative stress control nuclear trafficking and the localization of transport components. Furthermore, stress not only induced changes in transport factor distribution, but also upregulated post-translational modification of transport factors. Our results are consistent with the idea that the phosphorylation of importin-α, CAS, Nup153, and Nup88, and the O-GlcNAc modification of Nup153 increase when cells are exposed to oxidant.Conclusions/SignificanceOur studies defined the complex regulation of classical nuclear import and identified key transport factors that are targeted by stress, MEK, and PI3 kinase signaling.

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PurposePrevious studies have shown that vitreous stimulates degradation of the tumor suppressor protein p53 and that knockdown of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Kα and -β) abrogates proliferation of p53-deficient cells. The purpose of this study was to determine whether vitreous stimulated expression of PI5P4Kα and -β and whether suppression of PI5P4Kα and -β would inhibit vitreous-induced cellular responses and experimental proliferative vitreoretinopathy (PVR).MethodsPI5P4Kα and -β encoded by PIP4K2A and 2B, respectively, in human ARPE-19 cells were knocked down by stably expressing short hairpin (sh)RNA directed at human PIP4K2A and -2B. In addition, we rescued expression of PI5P4Kα and -β by re-expressing mouse PIP4K2A and -2B in the PI5P4Kα and -β knocked-down ARPE-19 cells. Expression of PI5P4Kα and -β was determined by Western blot and immunofluorescence. The following cellular responses were monitored: cell proliferation, survival, migration, and contraction. Moreover, the cell potential of inducing PVR was examined in a rabbit model of PVR effected by intravitreal cell injection.ResultsWe found that vitreous enhanced expression of PI5P4Kα and -β in RPE cells and that knocking down PI5P4Kα and -β abrogated vitreous-stimulated cell proliferation, survival, migration, and contraction. Re-expression of mouse PIP4Kα and -β in the human PI5P4Kα and -β knocked-down cells recovered the loss of vitreous-induced cell contraction. Importantly, suppression of PI5P4Kα and -β abrogated the pathogenesis of PVR induced by intravitreal cell injection in rabbits. Moreover, we revealed that expression of PI5P4Kα and -β was abundant in epiretinal membranes from PVR grade C patients.ConclusionsThe findings from this study indicate that PI5P4Kα and -β could be novel therapeutic targets for the treatment of PVR.

ContextCortisol has been suggested as a risk factor for choroidal thickening, which may lead to retinal changes.ObjectiveTo compare choroidal thickness measurements using optical coherence tomography (OCT) in patients with endogenous active Cushing’s syndrome (CS) and to evaluate the occurrence of retinal abnormalities in the same group of patients.DesignCross-sectional study.SettingOutpatient clinic.PatientsEleven female patients with CS in hypercortisolism state as determined by the presence of at least two abnormal measurements from urinary cortisol 24 h, no suppression of cortisol with low dose dexamethasone suppression test, and nocturnal salivary cortisol levels and 12 healthy controls.MethodsChoroidal and retinal morphology was assessed using OCT.Main outcome measuresChoroidal thickness measurements and the presence of retinal changes.ResultsThe mean subfoveal choroidal thickness was 372.96 ± 73.14 µm in the patients with CS and 255.63 ± 50.70 µm in the control group (p < 0.001). One patient (9.09%) presented with central serous chorioretinopathy and one patient (9.09%) with pachychoroid pigment epitheliopathy.ConclusionChoroidal thickness is increased in the eyes of patients with active CS compared to healthy and matched control. Also, 18.18% of patients presented with macular changes, possibly secondary to choroidal thickening. While further studies are necessary to confirm our findings, excess corticosteroid levels seem to have a significant effect on the choroid and might be associated with secondary retinal diseases.

The involvement of gut microbiota in T-cell trafficking into tumor tissue of colorectal cancer (CRC) remains to be further elucidated. The current study aimed to evaluate the expression of major cytotoxic T-cell trafficking chemokines (CTTCs) and chemokine-associated microbiota profiles in both tumor and adjacent normal tissues during CRC progression. We analyzed the expression of chemokine C-X-C motif ligands 9, 10, and 11 (CXCL9, CXCL10, and CXCL11), and C-C motif ligand 5 (CCL5), characterized gut mucosa-associated microbiota (MAM), and investigated their correlations in CRC patients. Our results showed that the expression of CXCL9, CXCL10, and CXCL11 was significantly higher in tumor than in adjacent normal tissues in 136 CRC patients. Notably, the high expression of CXCL9 in tumor tissues was associated with enhanced CD8+ T-cell infiltration and improved survival. Moreover, the MAM in tumor tissues showed reduction of microbial diversity and increase of oral bacteria. Microbial network analysis identified differences in microbial composition and structure between tumor and adjacent normal tissues. In addition, stronger associations between oral bacteria and other gut microbes were observed. Furthermore, the correlation analysis between the defined MAM and individual CTTCs showed that the CTTCs’ correlated operational taxonomic units (OTUs) in tumor and adjacent normal tissues rarely overlap with each other. Notably, all the enriched OTUs were positively correlated with the CTTCs in either tumor or adjacent normal tissues. Our findings demonstrated stronger interactions between oral bacteria and gut microbes, and a shifted correlation pattern between MAM and major CTTCs in tumor tissues, underlining possible mechanisms of gut microbiota–host interaction in CRC.