The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). 1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that 1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.

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Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by functionblocking autoantibodies in autoimmunity.

Epidermal growth factor receptor (EGFR) exaggerated (oncogenic) function is currently targeted in cancer treatment with drugs that block receptor ligand binding or tyrosine kinase activity. Because endocytic trafficking is a crucial regulator of EGFR function, its pharmacological perturbation might provide a new anti-tumoral strategy. Inhibition of phosphatidic acid (PA) phosphohydrolase (PAP) activity has been shown to trigger PA signaling towards type 4 phosphodiesterase (PDE4) activation and protein kinase A inhibition, leading to internalization of empty/inactive EGFR. Here, we used propranolol, its L-and D-isomers and desipramine as PAP inhibitors to further explore the effects of PAP inhibition on EGFR endocytic trafficking and its consequences on EGFR-dependent cancer cell line models. PAP inhibition not only made EGFR inaccessible to stimuli but also prolonged the signaling lifetime of ligand-activated EGFR in recycling endosomes. Strikingly, such endocytic perturbations applied in acute/intermittent PAP inhibitor treatments selectively impaired cell proliferation/ viability sustained by an exaggerated EGFR function. Phospholipase D inhibition with FIPI (5-fluoro-2-indolyl des-chlorohalopemide) and PDE4 inhibition with rolipram abrogated both the anti-tumoral and endocytic effects of PAP inhibition. Prolonged treatments with a low concentration of PAP inhibitors, although without detectable endocytic effects, still counteracted cell proliferation, induced apoptosis and decreased anchorageindependent growth of cells bearing EGFR oncogenic influences. Overall, our results show that PAP inhibitors can counteract EGFR oncogenic traits, including receptor overexpression or activating mutations resistant to current tyrosine kinase inhibitors, perturbing EGFR endocytic trafficking and perhaps other as yet unknown processes, depending on treatment conditions. This puts PAP activity forward as a new suitable target against EGFR-driven malignancy.Abbreviations DAG, diacylglycerol; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, EGF receptor; GFP, green fluorescent protein; LPP, lipid phosphate phosphatase; PA, phosphatidic acid; PAP, phosphatidic acid phosphohydrolase; PARP, poly ADP ribose polymerase; PDE4, type 4 phosphodiesterases; PKA, protein kinase A; PLD, phospholipase D; TfR, transferrin receptor.