SUMMARY Phagocytosis and degradation of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) is fundamental to vision. Autophagy is also responsible for bulk degradation of cellular components but its role in POS degradation is not well understood. We report that the morning burst of RPE phagocytosis coincided with the enzymatic conversion of autophagy protein LC3 to its lipidated form. LC3 then associated with single membrane phagosomes containing engulfed POS in an Atg5 dependent manner that required Beclin1 but not the autophagy pre-initiation complex. The importance of this process was verified in mice with Atg5-deficient RPE cells that showed evidence of disrupted lysosomal processing. These mice also exhibited decreased photoreceptor responses to light stimuli and decreased chromophore levels that were restored with exogenous retinoid supplementation. These results establish that the interplay of phagocytosis and autophagy within the RPE are required for both POS degradation and the maintenance of retinoid levels to support vision.

The phosphoinositide 3-kinase (PI3K) catalytic subunit p110␦ is expressed in neutrophils and is thought to play a role in their accumulation at sites of inflammation by contributing to chemoattractantdirected migration. We report here that p110␦ is present in endothelial cells and participates in neutrophil trafficking by modulating the proadhesive state of these cells in response to tumor necrosis factor ␣ (TNF␣). Specifically, administration of the selective inhibitor of PI3K␦, IC87114, to animals reduced neutrophil tethering to and increased rolling velocities on cytokine-activated microvessels in a manner similar to that observed in mice deficient in p110␦. These results were confirmed in vitro as inhibition of this isoform in endothelium, but not neutrophils, diminished cell attachment in flow. A role for PI3K␦ in TNF␣-induced signaling is demonstrated by a reduction in Aktphosphorylation and phosphatidylinositol-dependent kinase 1 (PDK1) enzyme activity upon treatment of this cell type with IC87114. p110␦ expressed in neutrophils also contributes to trafficking as demonstrated by the impaired movement of these cells across inflamed venules in animals in which this catalytic subunit was blocked or genetically deleted, results corroborated in transwell migration assays. Thus, PI3K␦ may be a reasonable therapeutic target in specific inflammatory conditions as blockade of its activity reduces neutrophil influx into tissues by diminishing their attachment to and migration across vascular endothelium. (Blood.

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The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.

Phosphoinositide3-kinase gamma (PI3K␥) in neutrophils plays a critical role in the directed migration of these cells into inflamed tissues. In this study, we demonstrate the importance of the endothelial component of PI3K␥ activity relative to its leukocyte counterpart in supporting neutrophil interactions with the inflamed vessel wall. Despite the reconstitution of class-Ib PI3K function in neutrophils of p110␥ ؊/؊ mice, we observed a 45% reduction in accumulation of these cells in an acute lung injury model. Mechanistically, this appears to result from a perturbation in selectin-mediated adhesion as manifested by a 70% reduction in wild-type (WT) neutrophil attachment to and 17-fold increase in rolling velocities on p110␥ ؊/؊ microvessels in vivo in response to tumor necrosis factor alpha (TNF␣). This alteration in adhesion was further augmented by a deficiency in p110␦, suggesting that the activity of both catalytic subunits is required for efficient capture of neutrophils by cytokinestimulated endothelium. Interestingly, Eselectin-mediated adhesion in p110␥ ؊/؊ mice was impaired by more than 95%, but no defect in nuclear factor kappa B (NF-B)-induced gene expression was observed. These findings suggest a previously unrecognized partnership between class-I PI3Ks expressed in leukocytes and endothelium, the combination of which is required for the efficient trafficking of immunocompetent cells to sites of inflammation. IntroductionClass-I phosphoinositide 3-kinases (PI3Ks) play a pivotal role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells such as granulocytes and lymphocytes. [1][2][3][4][5] Structurally, they exist as heterodimeric complexes in which a catalytic p110 subunit (designated as ␣, ␤, ␥, or ␦) is in association with a particular regulatory subunit (designated p50, p55, p85, or p101). 1,6,7 Functionally, all class-I PI3Ks catalyze the phosphorylation of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) to form phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) in response to activation of either receptor tyrosine kinases (RTKs) or G-protein-coupled receptors (GPCRs), which ultimately regulates a diverse array of biologic functions. In regards to innate immunity, one major role of PI3Ks is to support chemoattractant-directed migration of neutrophils, macrophages, and specific populations of mast cells into sites of inflammation. [8][9][10][11] Mechanistically, activation of this intracellular signaling pathway is essential for reorganization of cytoskeleton and membrane structure in response to such agonists, events that result in cell polarity and pseudopodia extension. [12][13][14] In particular, it is believed that PI3K␥, a class-Ib PI3K, predominates in this process, as recruitment of neutrophils into inflamed tissues was reduced by more than 60% in p110␥-null mice as compared with wild-type (WT) controls. [9][10][11] These studies, however, did not take into account the contribution of other cell types required for neutrophil t...

Shiga toxin (Stx), cholera toxin (Ctx), and the plant toxin ricin are among several toxins that reach their intracellular destinations via a complex route. Following endocytosis, these toxins travel in a retrograde direction through the endosomal system to the trans-Golgi network, Golgi apparatus, and endoplasmic reticulum (ER). There the toxins are transported across the ER membrane to the cytosol, where they carry out their toxic effects. Transport via the ER from the cell surface to the cytosol is apparently unique to pathogenic toxins, raising the possibility that various stages in the transport pathway can be therapeutically targeted. We have applied a luciferase-based high-throughput screen to a chemical library of small-molecule compounds in order to identify inhibitors of Stx. We report two novel compounds that protect against Stx and ricin inhibition of protein synthesis, and we demonstrate that these compounds reversibly inhibit bacterial transport at various stages in the endocytic pathway. One compound (compound 75) inhibited transport at an early stage of Stx and Ctx transport and also provided protection against diphtheria toxin, which enters the cytosol from early endosomes. In contrast, compound 134 inhibited transport from recycling endosomes through the Golgi apparatus and protected only against toxins that access the ER. Small-molecule compounds such as these will provide insight into the mechanism of toxin transport and lead to the identification of compounds with therapeutic potential against toxins routed through the ER.

Class 1 phosphoinositide 3-kinases (PI3Ks), consisting of PI3K␣, ␤, ␥, and ␦, are a family of intracellular signaling molecules that play important roles in cellmediated immune responses. In thymocytes, however, their role is less clear, although PI3K␥ is postulated to partially contribute to pre-TCR-dependent differentiation. We now report that PI3K␦, in conjunction with PI3K␥, is required for thymocyte survival and ultimately for Tcell production. Surprisingly, genetic deletion of the p110␦ and p110␥ catalytic subunits resulted in a dramatic reduction in thymus size, cellularity, and lack of corticomedullary differentiation. Total thymocyte counts in these animals were 27-fold lower than in wild-type (WT) controls because of a diminished number of CD4 ؉ CD8 ؉ double-positive (DP) cells and were associated with T-cell depletion in blood and in secondary lymphoid organs. Moreover, this alteration in the DP population was intrinsic to thymocytes, because the reconstitution of p110␥␦ ؊/؊ animals with WT fetal liver cells restored the proportions of all thymocyte populations to those in WT controls. The observed defects were related to massive apoptosis in the DP population; TCRB expression, pre-TCR selection, and generation of DP cells appeared relatively unperturbed. Thus, class 1 PI3Ks work in concert to protect developing thymocytes from apoptosis. ( IntroductionThymocyte development relies on a series of intracellular signaling events that regulate cell differentiation, proliferation, and survival. This process can be followed based on the presence or absence of cell surface markers such as CD4, CD8, CD25, and CD44. [1][2][3] Early thymocyte progenitors lack CD4 and CD8 expression and are termed double-negative (DN) cells. The DN stage is subdivided into 4 categories. The DN1 stage is characterized by surface expression of CD44 (CD25 Ϫ CD44 ϩ ). Maturation of this earliest thymocyte subset then proceeds from the DN2 stage (CD25 ϩ CD44 ϩ ) to the DN3 stage (CD25 ϩ CD44 Ϫ ) and finally to the DN4 stage (CD25 Ϫ CD44 Ϫ ). The first regulatory checkpoint in thymocyte development, termed ␤-selection, occurs at the DN3 stage. This involves TCR␤ gene rearrangement and expression, which permits the subsequent formation of the pre-TCR complex. 4,5 Thymocytes unable to generate a functionally rearranged TCR␤ gene die by apoptosis. 6,7 Subsequently, signals provided by the pre-TCR and local microenvironment result in the proliferation and differentiation of DN thymocytes to the CD4 ϩ CD8 ϩ DP stage. A small subset of these cells ultimately bear a mature TCR␣␤ Ϫ CD3 complex and then further differentiate into CD4 ϩ or CD8 ϩ single-positive (SP) T cells.In addition to TCRB selection, thymocyte development is also shaped by the induction or inhibition of apoptosis. Although many different molecules can regulate this process, the proto-oncogene Bcl-2 appears to have a protective effect with regard to thymocyte survival. 8,9 This is supported by the observation that thymocytes in mice expressing a Bcl2 transgene are less pron...

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10 ؊12 to 10 ؊11 M) is sufficient for substantial increases in ERK activity.