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Many recent studies indicate that dysregulation of autophagy is a common feature of many neurodegenerative diseases. The HIV-1-associated neurological disorder is an acquired cognitive and motor disease that includes a severe neurodegenerative dementia. We find that the neurodegeneration seen in the brain in HIV-1 infection is associated with an inhibition of neuronal autophagy, leading to neuronal demise. Neurons treated with supernatants from SIV-infected microglia develop a decrease in autophagy-inducing proteins, a decrease in neuronal autophagy vesicles, and an increase in sequestosome-1/p62. Examination of brains from HIV-infected individuals and SIV-infected monkeys reveals signs of autophagy dysregulation, associated, respectively, with dementia and encephalitis. Excitotoxic and inflammatory factors could inhibit neuronal autophagy, and stimulation of autophagy with rapamycin prevents such effects. Here we amplify on these findings, and propose that in the setting of HIV-infection, the decreased neuronal autophagy sensitizes cells to pro-apoptotic and other damaging mechanisms, leading to neuronal dysfunction and death. Hence, new therapeutic approaches aimed at boosting neuronal autophagy are conceivable to treat those suffering from the neurological complications of HIV.

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

In addition to their key role in protein biosynthesis, aminoacyl-tRNA synthetases have other biological functions that appeared during their long evolutionary development. In mammalian cells, specific members of this family of enzymes are also procytokines that, upon conversion, are active cytokines in pathways for angiogenesis, and thereby connect translation to control of blood vessel development. Here we describe an in vitro assay for tube formation by tumor endothelial cells on a matrigel substrate. In contrast to normal endothelial cells, tumor endothelial cells have strong angiogenic capabilities and the ability to form vessel-like tubes on a solid substrate. In particular, we found that a SV40-immortalized mouse lymphoid endothelial cell line was robust in this assay and yielded data that could be quantified with high precision. Consequently, this specific tube formation model provides an opportunity to discover and analyze potent agents that specifically affect angiogenesis. It has proven effective for studying the angiogenic functions of tRNA synthetase cytokines.

TLRs are an evolutionarily conserved family of cell membrane proteins believed to play a significant role in innate immunity and the response to tissue injury, including that induced by ischemia. TLR signaling pathways activate transcription factors that regulate expression of prosurvival proteins, as well as proinflammatory cytokines and chemokines through one of two proximal adapter proteins, MyD88 or Toll/IL-1R domain-containing adaptor-inducing IFN-β (Trif). Our study defines the constitutive protein expression of TLR2 in kidneys of humans and mice, and provides insight into the signaling mechanisms by which a deficiency of TLR2 protects from ischemic organ injury. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in TLR2, MyD88, Trif, and MyD88 × Trif. TLR2 protein was evident in many cell types in the kidney, including renal tubules of the outer stripe of the medulla, glomeruli, and in the renal vasculature. The pattern of protein expression was similar in humans and mice. The absence of TLR2, MyD88, and MyD88 × Trif conferred both physiologic and histologic protection against sublethal ischemia at 24 h. Interestingly, TLR2-deficient mice were better protected from ischemic renal injury than those deficient for the adapter protein MyD88, raising the intriguing possibility that TLR-2-dependent/MyD88-independent pathways also contribute to kidney injury. We conclude that TLR2 protein is constitutively expressed in the kidney and plays an important role in the pathogenesis of acute ischemic injury by signaling both MyD88-dependent and MyD88-independent pathways.

Growth of normal cells is anchorage dependent because signalling through multiple pathways including Erk, phosphatidylinositol-3-OH kinase (PI(3)K) and Rac requires integrin-mediated cell adhesion. Components of these pathways localize to low-density, cholesterol-rich domains in the plasma membrane named 'lipid rafts' or 'cholesterol-enriched membrane microdomains' (CEMM). We previously reported that integrin-mediated adhesion regulates CEMM transport such that cell detachment from the extracellular matrix triggers CEMM internalization and clearance from the plasma membrane. We now report that this internalization is mediated by dynamin-2 and caveolin-1. Internalization requires phosphorylation of caveolin-1 on Tyr 14. A shift in localization of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalization upon cell detachment, which mediates inhibition of Erk, PI(3)K and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1.

Pulmonary pathologies including adult respiratory distress syndrome are characterized by disruption of pulmonary integrity and edema compromising respiratory function. Sphingosine 1-phosphate (S1P) is a lipid mediator synthesized and/or stored in mast cells, platelets, and epithelial cells, with production up-regulated by the proinflammatory cytokines IL-1 and TNF. S1P administration via the airways but not via the vasculature induces lung leakage. Using receptor-null mice, we show that S1P, acting on S1P3receptor expressed on both type I and type II alveolar epithelial cells but not vascular endothelium, induces pulmonary edema by acute tight junction opening. WT but not S1P3-null mice showed disruption of pulmonary epithelial tight junctions and the appearance of paracellular gaps between epithelial cells by electron microscopy within 1 h of airways exposure to S1P. We further show by fluorescence microscopy that S1P induced rapid loss of ZO-1 reactivity, an essential component of the cytoplasmic plaque associated with tight junctions, as well as of the tetraspannin Claudin-18, an integral membrane organizer of tight junctions. S1P shows synergistic activity with the proinflammatory cytokine TNF, showing both pulmonary edema and mortality at subthreshold S1P doses. Specifically, preexposure of mice to subthreshold doses of TNF, which alone induced no lung edema, exacerbated S1P-induced edema and impaired survival. S1P, acting through S1P3, regulates epithelial integrity and acts additively with TNF in compromising respiratory barrier function. Because S1P3-null mice are resistant to S1P-induced pulmonary leakage, either alone or in the presence of TNF, S1P3antagonism may be useful in protecting epithelial integrity in pulmonary disease.

Shear stress is a fundamental determinant of vascular homeostasis, regulating vascular remodelling, cardiac development and atherogenesis, but the mechanisms of transduction are poorly understood. Previous work showed that the conversion of integrins to a high-affinity state mediates a subset of shear responses, including cell alignment and gene expression. Here we investigate the pathway upstream of integrin activation. PECAM-1 (which directly transmits mechanical force), vascular endothelial cell cadherin (which functions as an adaptor) and VEGFR2 (which activates phosphatidylinositol-3-OH kinase) comprise a mechanosensory complex. Together, these receptors are sufficient to confer responsiveness to flow in heterologous cells. In support of the relevance of this pathway in vivo, PECAM-1-knockout mice do not activate NF-kappaB and downstream inflammatory genes in regions of disturbed flow. Therefore, this mechanosensing pathway is required for the earliest-known events in atherogenesis.

Formation of a stable lamellipodium at the front of migrating cells requires localization of Rac activation to the leading edge. Restriction of alpha4 integrin phosphorylation to the leading edge limits the interaction of alpha4 with paxillin to the sides and rear of a migrating cell. The alpha4-paxillin complex inhibits stable lamellipodia, thus confining lamellipod formation to the cell anterior. Here we report that binding of paxillin to the alpha4 integrin subunit inhibits adhesion-dependent lamellipodium formation by blocking Rac activation. The paxillin LD4 domain mediates this reduction in Rac activity by recruiting an ADP-ribosylation factor GTPase-activating protein (Arf-GAP) that decreases Arf activity, thereby inhibiting Rac. Finally, the localized formation of the alpha4-paxillin-Arf-GAP complex mediates the polarization of Rac activity and promotes directional cell migration. These findings establish a mechanism for the spatial localization of Rac activity to enhance cell migration.

10 páginas, 9 figuras -- PAGS nros. 644-653, Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin αIIbβ3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. αIIbβ3 interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether αIIbβ3 might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKCβ co-immunoprecipitated with αIIbβ3 in response to the interaction of platelets with soluble or immobilized fibrinogen. PKCβ recruitment to αIIbβ3 was accompanied by a 9-fold increase in PKC activity in αIIbβ3 immunoprecipitates. RACK1, an intracellular adapter for activated PKCβ, also co-immunoprecipitated with αIIbβ3, but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKCβ recruitment to αIIbβ3 and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKCβ spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKCβI to associate with αIIbβ3 and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the β3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to β3. These studies demonstrate that the interaction of αIIbβ3 with activated PKCβ is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through αIIbβ3. Furthermore, the studies extend the concept of αIIbβ3 as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton., In addition to their roles in cell adhesion, integrins transmit signals in both directions across the plasma membrane to regulate cytoskeletal organization, motility, and other anchorage-dependent cellular responses (1). In platelets, for example, αIIbβ3 responds to “inside-out” signals with an increase in affinity for cognate ligands such as fibrinogen that bridge platelets to each other and mediate platelet adhesion to sites of vascular damage. In turn, ligand binding to αIIbβ3 triggers outside-in signals that promote cytoskeletal changes necessary for full platelet aggregation and spreading (2, 3). Bidirectional αIIbβ3 signaling is controlled, in part, by specific intracellular proteins that interact with the relatively short cytoplasmic tails of αIIb or β3. For example, binding of talin to β3 is a final common step in the cellular modulation of αIIbβ3 affinity (4), and binding of c-Src and Syk protein-tyrosine kinases to β3 is required for platelet spreading on fibrinogen (5, 6). Several other intracellular proteins, for example, CIB and β3-endonexin, can also bind to αIIb or β3 tails, respectively, and may influence αIIbβ3 functions (7-9). However, the full complement of intracellular proteins that are capable of interacting directly or indirectly with αIIbβ3 is unknown. The protein kinase C (PKC)1 subfamily of AGC serine/threonine kinases has been implicated in integrin function or dynamics in many cell types (10). In platelets, PKC is thought to regulate αIIbβ3 affinity, based on the stimulatory effects of phorbol esters, which bind to PKC C1 domains, and the blocking effects of broad spectrum PKC inhibitors (3). However, the lack of specificity of these compounds limits data interpretation and does not permit conclusions about the roles of specific PKC isoforms (11). PKCs have been categorized as classical (diacylglycerol- and Ca2+-regulated through C1 and C2 domains, respectively), novel (Ca2+-independent but diacylglycerol-regulated), or atypical (Ca2+- and diacylglycerol-independent) (12, 13). Platelets are reported to contain members of all three classes of PKC isozymes (14-21), as well as the related protein kinase D (22). Recently, experimental tools have become available to study specific PKC isoforms in cells, including overexpression, gene targeting and gene knock-down strategies, and molecular imaging (23-25). Some of these tools are potentially relevant to platelets., PKC function depends on the maturation of catalytic activity of the enzyme through phosphorylation and PKC binding to membranes or specific proteins. The latter interactions place PKC in proximity to substrates and relieve autoinhibitory restraints imposed by the binding of a pseudosubstrate sequence to the active site (12, 13, 26). One group of PKC targeting proteins has been termed RACK, which binds selectively to activated PKCs (27). The best characterized protein of this group is RACK1, a 36-kDa protein composed of seven WD40 repeats. RACK1 was originally identified based on its interaction with activated PKCβ and subsequently shown to interact with certain other PKC isoforms and with several other proteins, most notably integrin β cytoplasmic tails and c-Src (see Fig. 1A) (28-30)., Given the potential for the cytoplasmic tails of αIIbβ3 to serve as binding sites for signaling molecules and the apparent functional relationships between PKC and αIIbβ3, the present studies were carried out to determine whether specific PKCs associate with αIIbβ3 and if so to determine what the functional relevance of the association is. By using human and mouse platelets and a CHO cell model system, the results show that one particular PKC isoform, PKCβ, inducibly associates with αIIbβ3 in response to fibrinogen binding to cells. The PKCβ/αIIbβ3 interaction appears to be mediated by RACK1 and is required for cytoskeletal reorganization and platelet spreading on fibrinogen, but it is dispensable for the affinity modulation of αIIbβ3, This work was supported by National Institutes of Health Grants HL56595, HL57900, and AI38348. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in alphaIIbbeta3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these alphaIIbbeta3-tyrosine kinase interactions at nanometer proximity in living cells, we monitored bioluminescence resonance energy transfer between GFP and Renilla luciferase chimeras and bimolecular fluorescence complementation between YFP half-molecule chimeras. These techniques revealed that alphaIIbbeta3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of alphaIIbbeta3-c-Src, alphaIIbbeta3-Syk, and c-Src-Syk are observed in membrane ruffles and focal complexes, and the interactions involving Syk require Src activity. In contrast, FAK interacts with alphaIIbbeta3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin beta3 cytoplasmic tail. Thus, alphaIIbbeta3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of alphaIIbbeta3 signaling to the actin cytoskeleton.

Antagonists of alphavbeta3 and alphavbeta5 disrupt angiogenesis in response to bFGF and VEGF, respectively. Here, we show that these alphav integrins differentially contribute to sustained Ras-extracellular signal-related kinase (Ras-ERK) signaling in blood vessels, a requirement for endothelial cell survival and angiogenesis. Inhibition of FAK or alphavbeta5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin alphavbeta3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation. Furthermore, retroviral delivery of active Ras or c-Raf promoted ERK activity and angiogenesis, which anti-alphavbeta5 blocked upstream of Ras, whereas anti-alphavbeta3 blocked downstream of Ras, but upstream of c-Raf. The activation of c-Raf by bFGF/alphavbeta3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak. Thus, integrins alphavbeta3 and alphavbeta5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis.

Integrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from alpha4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient alpha4-mediated migration requires spatial control of alpha4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated alpha4 accumulated along the leading edge. Blocking alpha4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced alpha4-dependent migration and lamellipodial stability. alpha4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-alpha4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated alpha4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with alpha4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.

Hemodynamic shear stress is a fundamental determinant of vascular remodeling and atherogenesis. Changes in focal adhesions, cytoskeletal organization and gene expression are major responses of endothelial cells to shear stress. Here, we show that activation of the small GTPase Rac is essential for gene expression and for providing spatial information for shear stress-induced cell alignment. Fluorescence resonance energy transfer (FRET) localizes activated Rac1 in the direction of flow. This directional Rac1 activation is downstream of shear-induced new integrin binding to extracellular matrix. Additionally, Rac1 mediates flow-induced stimulation of nuclear factor kappaB (NF-kappaB) and the subsequent expression of intercellular cell adhesion molecule 1 (ICAM-1), an adhesion receptor involved in the recruitment of leukocytes to atherosclerotic plaque. These studies provide a unifying model linking three of the main responses to shear stress that mediate both normal adaptation to hemodynamic forces and inflammatory dysfunction of endothelial cells in atherosclerosis.

The Rho family GTPases Rac, Rho and Cdc42 are critical in regulating the actin-based cytoskeleton, cell migration, growth, survival and gene expression. These GTPases are activated by guanine nucleotide-exchange factors (GEFs). A biochemical search for Cdc42 activators led to the cloning of zizimin1, a new protein whose overexpression induces Cdc42 activation. Sequence comparison combined with mutational analysis identified a new domain, which we named CZH2, that mediates direct interaction with Cdc42. CZH2-containing proteins constitute a new superfamily that includes the so-called 'CDM' proteins that bind to and activate Rac. Together, the results suggest that CZH2 is a new GEF domain for the Rho family of proteins.

Multicellular animal development depends on integrins. These adhesion receptors link to the actin cytoskeleton, transmitting biochemical signals and force during cell migration and interactions with the extracellular matrix. Many integrin-cytoskeleton connections are formed by filamins and talin. The beta7 integrin tail binds strongly to filamin and supports less migration, fibronectin matrix assembly and focal adhesion formation than either the beta1D tail, which binds strongly to talin, or the beta1A tail, which binds modestly to both filamin and talin. To probe the role of filamin binding, we mapped the filamin-binding site of integrin tails and identified amino acid substitutions that led to selective loss of filamin binding to the beta7 tail and gain of filamin binding to the beta1A tail. These changes affected cell migration and membrane protrusions but not fibronectin matrix assembly or focal adhesion formation. Thus, tight filamin binding restricts integrin-dependent cell migration by inhibiting transient membrane protrusion and cell polarization.

The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum,75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.

Multiple steps in cancer metastasis, including tumorigenesis, tumor angiogenesis, invasion and colonization, can be successfully examined with well-established models. However, due to deficiency in experimental model systems that would accurately recapitulate tumor cell entry into the host vasculature, intravasation remains the least studied step in the metastatic cascade. Our laboratory has developed several independent avian and murine model systems to investigate the complex process of intravasation in a quantifiable and mechanistic manner. Chick embryo models of intravasation are based on the ability of a highly vascularized tissue, the chorioallantoic membrane (CAM), to sustain primary tumor development and spontaneous metastasis. Furthermore, the CAM serves as a repository of newly intravasated cells, which are quantified by human-specific Alu-qPCR. By employing different CAM models, the early events in spontaneous metastasis that ultimately lead to intravasation can be dissected and their individual input to overall intravasation can be quantified. These early processes include cell escape from the primary tumor, establishment of intratumoral and extratumoral angiogenic networks, invasion of adjacent stroma by escaped tumor cells, and migration of escaped tumor cells along tumor-associated blood vessels. In conjunction with confirmatory models of cancer metastasis in mice, we have identified several molecular systems that functionally contribute to the intravasation process. To investigate the specific mechanisms whereby tumor cells enter the angiogenic blood vessels, we have developed a new high-resolution confocal microscopy model based on acquisition and multiparameter analysis of primary microtumors imaged as entire units. This model was adapted to primary tumors developing within the mesoderm of the CAM as well as primary tumors developing in the dermis of mouse ear. In both avian and murine models, the volume of microtumors, the number of tumor cells per tumor, and the total volume of tumor-associated vasculature can be determined from 3D-reconstructed Z-stacks. Most importantly, the volume of intravascular tumor cells can be quantified by colocalizing tumor-vascular signals with a stringent threshold of a minimum volume of an individual tumor cell so that the cells that are localized within the vessels could be discriminated from the cells adherent to the abluminal vessel surface. In addition, high-resolution imaging of whole microtumors allows to visualize and quantify the tumor cells that appear as entering into the vasculature or in close association with the abluminal surface of the vessels. This approach allowed us for the fist time to visualize the intravasation events in numbers rendering statistical significance. By employing unique cell and protein markers in this novel system, the vascular cells interacting with the intravasating tumor cells can be identified and the candidate molecules involved in the close tumor-vascular interactions can be probed by treating the developing microtumors with specific reagents and small molecule inhibitors. By using these novel high-resolution models for intravasation, we have documented a majority of intravasation events within the primary tumor or in the nearest proximity to the primary tumor border. These data challenge the widely accepted concept that tumor cell escape, stromal invasion, and entry of escaped tumor cells into blood vessels adjacent to the primary tumor, are prerequisites of tumor cell intravasation. In contrast, our findings indicate that the bulk of cell intravasation events can take place within the primary tumor and prior to significant stromal invasion and migration of escaped tumor cells towards and along tumor-associated blood vessels. Our data suggest that tumor cell intravasation and metastatic spread can occur much earlier than commonly acknowledged by oncologists based on histological examination of resected tumors. This abstract is also presented as Poster A24. Citation Format: Elena I. Deryugina, William B. Kiosses, James P. Quigley. Quantitative and multiparameter analysis of tumor cell intravasation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR14.

Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.

The alpha4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses, possibly because alpha4 regulates cellular functions differently from other integrins through its cytoplasmic tail. We used novel mimics of the alpha4 tail to identify molecules that could account for alpha4-specific signalling. Here we report that the alpha4 tail, but not several other alpha-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with alpha4 integrins in Jurkat T cells at high stoichiometry, and joining the alpha4 tail to alphaIIb resulted in a complex of integrin alphaIIbbeta3 with paxillin. This association markedly enhanced the rates of alphaIIbbeta3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the alpha4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, alpha4beta1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the alpha4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.

We examined the role of Laminin-5 (Ln-5) an extracellular matrix component of breast gland basement membrane, in supporting migration of normal (HUMEC), immortalized (MCF-10A), and malignant breast epithelial cells that exhibit different degrees of metastatic potential (MDA-MB-435MDA-MB-231MCF-7). HUMEC, MCF-10A, and MCF-7 cells all adhered to purified Ln-5 through the alpha3beta1 integrin receptor in adhesion assays. However, HUMEC and MCF-10A cells remained statically adherent, while MCF-7 cells migrated on Ln-5 in Transwell and colloidal gold displacement assays. Anti-alpha3 integrin antibodies blocked migration of MCF-7 cells on Ln-5. MDA-MB-231 and MDA-MB-435 cells bound and migrated on Ln-5 through a beta1 integrin receptor that is insensitive to antibodies that block the function of alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphaV integrin subunits. Migration of all cell types tested was blocked by CM6, a monoclonal antibody directed to a cell adhesion site on the alpha3 chain of Ln-5. Thus, Ln-5 may play an important role in regulating adhesion and migration in normal and transformed breast epithelium. Our results indicate that the type of integrin utilized by breast cells to interact with Ln-5, as well as its functional state, may determine whether cells will be statically adherent or migratory on Ln-5.

The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and in-creased stress fibers and focal adhesions. The DN effect mapped to the most NH2-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged α-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.

Previous studies have shown that point mutations in the effector domain of Rac1 block specific downstream pathways such as PAK, JNK/SAPK kinases and membrane ruffling. Specifically, the F37A mutation, made in a constitutively activated Q61L background, activates PAK but fails to induce membrane ruffles. We now show that Q61L/F37A Rac not only fails to induce ruffling but potently blocks membrane ruffling induced by serum or PDGF. In the presence of serum, cells do extend filopodia, suggesting that this mutant only blocks a subset of the effectors that induce cytoskeletal reorganization. At later times, this rac mutant induces membrane blebbing, but not apoptosis. These results show that Q61L/F37A Rac, is constitutively activated with respect to PAK activation but functions as a dominant negative for another pathway, membrane ruffling. That an effector domain point mutant can simultaneously function as a dominant negative and dominant positive for different pathways implies that effects of these variants on cell functions must be interpreted with caution.

The immunocompetence and clinical accessibility of dermal tissue offers an appropriate and attractive target for vaccination. We previously demonstrated that pDNA injection into the skin in combination with surface electroporation (SEP), results in rapid and robust expression of the encoded antigen in the epidermis. Here, we demonstrate that intradermally EP-enhanced pDNA vaccination results in the rapid induction of a host humoral immune response. In the dermally relevant guinea pig model, we used high-resolution laser scanning confocal microscopy to observe direct dendritic cell (DC) transfections in the epidermis, to determine the migration kinetics of these cells from the epidermal layer into the dermis, and to follow them sequentially to the immediate draining lymph nodes. Furthermore, we delineate the relationship between the migration of directly transfected epidermal DCs and the generation of the host immune response. In summary, these data indicate that direct presentation of antigen to the immune system by DCs through SEP-based in vivo transfection in the epidermis, is related to the generation of a humoral immune response.

Pulmonary pathologies including adult respiratory distress syndrome are characterized by disruption of pulmonary integrity and edema compromising respiratory function. Sphingosine 1-phosphate (S1P) is a lipid mediator synthesized and/or stored in mast cells, platelets, and epithelial cells, with production up-regulated by the proinflammatory cytokines IL-1 and TNF. S1P administration via the airways but not via the vasculature induces lung leakage. Using receptor-null mice, we show that S1P, acting on S1P3 receptor expressed on both type I and type II alveolar epithelial cells but not vascular endothelium, induces pulmonary edema by acute tight junction opening. WT but not S1P3-null mice showed disruption of pulmonary epithelial tight junctions and the appearance of paracellular gaps between epithelial cells by electron microscopy within 1 h of airways exposure to S1P. We further show by fluorescence microscopy that S1P induced rapid loss of ZO-1 reactivity, an essential component of the cytoplasmic plaque associated with tight junctions, as well as of the tetraspannin Claudin-18, an integral membrane organizer of tight junctions. S1P shows synergistic activity with the proinflammatory cytokine TNF, showing both pulmonary edema and mortality at subthreshold S1P doses. Specifically, preexposure of mice to subthreshold doses of TNF, which alone induced no lung edema, exacerbated S1P-induced edema and impaired survival. S1P, acting through S1P3, regulates epithelial integrity and acts additively with TNF in compromising respiratory barrier function. Because S1P3-null mice are resistant to S1P-induced pulmonary leakage, either alone or in the presence of TNF, S1P3 antagonism may be useful in protecting epithelial integrity in pulmonary disease.

Toll-like receptors (TLRs) are pattern recognition receptors of innate immunity. TLRs initiate inflammatory pathways that may exacerbate chronic inflammatory diseases like atherosclerosis. En face laser scanning confocal microscopy (LSCM) of isolated aortic segments revealed the distribution of intimal TLR2 expression and the atheroprotective outcomes resulting from a TLR2 deficiency. TLR2 expression was restricted to endothelial cells in regions of disturbed blood flow, such as the lesser curvature region, in atherosclerosis-prone, low-density lipoprotein receptor-deficient (LDLr(-/-)) mice. Diet-induced hyperlipidemia in LDLr(-/-) mice increased this regional endothelial TLR2 expression. Bone marrow (BM) reconstitution of LDLr(-/-) and LDLr(-/-)TLR2(-/-) mice created chimeric mice with green fluorescent protein (GFP) expression in BM-derived cells (BMGFP(+)). Lesser curvature BMGFP(+) leukocyte accumulation, lipid accumulation, foam cell generation and endothelial cell injury were all increased by hyperlipidemia, whereas hyperlipidemic double mutant BMGFP(+)LDLr(-/-)TLR2(-/-) mice had reduced BMGFP(+) leukocyte accumulation, lipid accumulation, foam cells, and endothelial cell injury. This is the first report of in vivo site-specific expression of endothelial cell TLR2. Expression of this receptor on endothelial cells contributed to early atherosclerotic processes in lesion-prone areas of the mouse aorta.