FLCN regulates transferrin receptor 1 transport and iron homeostasis.

Birt-Hogg-Dubé (BHD) syndrome is a multiorgan disorder caused by inactivation of the folliculin (FLCN) protein. Previously, we identified FLCN as a binding protein of Rab11A, a key regulator of the endocytic recycling pathway. This finding implies that the abnormal localization of specific proteins whose transport requires the FLCN-Rab11A complex may contribute to BHD. Here, we used human kidney-derived HEK293 cells as a model, and we report that FLCN promotes the binding of Rab11A with transferrin receptor 1 (TfR1), which is required for iron uptake through continuous trafficking between the cell surface and the cytoplasm. Loss of FLCN attenuated the Rab11A-TfR1 interaction, resulting in delayed recycling transport of TfR1. This delay caused an iron deficiency condition that induced hypoxia-inducible factor (HIF) activity, which was reversed by iron supplementation. In a Drosophila model of BHD syndrome, we further demonstrated that the phenotype of BHD mutant larvae was substantially rescued by an iron-rich diet. These findings reveal a conserved function of FLCN in iron metabolism and may help to elucidate the mechanisms driving BHD syndrome.

Cytoprotective-selective activated protein C therapy for ischaemic stroke.

Despite years of research and efforts to translate stroke research to clinical therapy, ischaemic stroke remains a major cause of death, disability, and diminished quality of life. Primary and secondary preventive measures combined with improved quality of care have made significant progress. However, no novel drug for ischaemic stroke therapy has been approved in the past decade. Numerous studies have shown beneficial effects of activated protein C (APC) in rodent stroke models. In addition to its natural anticoagulant functions, APC conveys multiple direct cytoprotective effects on many different cell types that involve multiple receptors including protease activated receptor (PAR) 1, PAR3, and the endothelial protein C receptor (EPCR). Application of molecular engineered APC variants with altered selectivity profiles to rodent stroke models demonstrated that the beneficial effects of APC primarily require its cytoprotective activities but not its anticoagulant activities. Extensive basic, preclinical, and clinical research provided a compelling rationale based on strong evidence for translation of APC therapy that has led to the clinical development of the cytoprotective-selective APC variant, 3K3A-APC, for ischaemic stroke. Recent identification of non-canonical PAR1 and PAR3 activation by APC that give rise to novel tethered-ligands capable of inducing biased cytoprotective signalling as opposed to the canonical signalling provides a mechanistic explanation for how APC-mediated PAR activation can selectively induce cytoprotective signalling pathways. Collectively, these paradigm-shifting discoveries provide detailed insights into the receptor targets and the molecular mechanisms for neuroprotection by cytoprotective-selective 3K3A-APC, which is currently a biologic drug in clinical trials for ischaemic stroke.

The action of Semaphorin7A on thalamocortical axon branching.

Developing axons form extensive branches to make synaptic contacts with their target cells. Despite the important role of axon branching in neural circuit formation, its underlying molecular mechanism is still largely unknown. In this study, we investigated the involvement of Semaphorin7A (Sema7A) in thalamocortical (TC) axon branching. In situ hybridization demonstrated that sema7a was expressed specifically in layer 4, the TC recipient layer, when TC axons form extensive arbors. A similar protein expression pattern was observed by immunohistochemistry with an anti-Sema7A antibody. The effect of Sema7A on axon branching was investigated in dissociated cell cultures from embryonic rat thalamus. TC axon branching increased dramatically on Sema7A-coated dishes. We further studied the activity of Sema7A in vivo using loss- and gain-of-function analyses. The number of vesicular glutamate transporter 2-positive puncta was markedly reduced in the Sema7A-deficient cortex. In contrast, their number increased significantly when Sema7A was over-expressed in layer 4 cells by in utero electroporation. Taken together, these findings suggest that Sema7A acts as a positive regulator for TC axon branching and/or pre-synaptic puncta formation.

[Activated protein C, a protein at the crossroads between coagulation and inflammation]. / La protéine C activée: une protéine à l'interface de l'inflammation et de la coagulation.

Sepsis is defined as a systemic response to infection, characterized by an intense inflammatory response linked to coagulation activation and fibrinolysis inhibition, two processes which are intimately associated. In a field where mortality remains very high, administration of activated protein C, a physiological coagulation inhibitor with cytoprotective properties, has demonstrated its effectiveness and was able to reduce mortality. Protein C belongs to a system that involves plasma proteins and endothelial cell receptors. In addition to well documented effects on coagulation and fibrinolysis, activated protein C exhibits anti-inflammatory, anti-apoptotic but also anti-histone activities. Indeed, a recent study focusing on the cytoprotective effects of activated protein C showed that extracellular histones are released during severe sepsis and may participate in the pathophysiology of severe sepsis. These histones appear to be new targets of activated protein C.

Braking bad: blockade of inhibitory pathways improves interleukin-15 therapy.

Blockade of the CTLA-4 and PD-1 inhibitory pathways in T cells via the administration of neutralizing antibodies at the time of interleukin (IL)-15 therapy markedly enhanced the survival of tumor-bearing mice as compared with those receiving IL-15 alone or IL-15 in combination with just one of the antibodies.

Simultaneous blockade of multiple immune system inhibitory checkpoints enhances antitumor activity mediated by interleukin-15 in a murine metastatic colon carcinoma model.

PURPOSE: Interleukin 15 (IL-15) is a promising cytokine for immunotherapy of cancer due to its ability to stimulate the immunity of natural killer, B, and T cells. Its effectiveness, however, may be limited by inhibitory checkpoints and pathways that can attenuate immune responses. Finding strategies to abrogate these negative regulators and enhance the efficacy of IL-15 is a critical challenge. EXPERIMENTAL DESIGN: In a preclinical study, we evaluated IL-15 combined with antibodies to block the negative immune regulators cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) in a metastatic murine CT26 colon carcinoma model. RESULTS: IL-15 treatment resulted in a significant prolongation of survival in mice with metastatic tumor. Administration of IL-15, however, also increased expression of PD-1 on the surface of CD8(+) T cells including CD8(+)CD44(high) memory phenotype T cells. Moreover, IL-15 also increased the secretion of the immunosuppressive cytokine, IL-10. Combining IL-15 with anti-PD-L1 and anti-CTLA-4 (multiple immune checkpoint blockade) exhibited greater CTL killing and IFNγ secretion. Moreover, this combination resulted in a significant reduction in surface expression of PD-1 on CD8(+) T cells, a decrease in IL-10 secretion, and led to significantly longer survival of tumor-bearing animals compared with mice treated with IL-15 alone or combined singularly with anti-PD-L1 or anti-CTLA-4. CONCLUSIONS: Combining the immune stimulatory properties of IL-15 with the simultaneous removal of 2 critical immune system inhibitory checkpoints, we showed enhancement of immune responses leading to increased antitumor activity.

Reduced incidence and severity of experimental autoimmune arthritis in mice expressing catalytically inactive A disintegrin and metalloproteinase 8 (ADAM8).

A disintegrin and metalloproteinase 8 (ADAM8), a catalytically active member of the ADAMs family of enzymes, is expressed primarily on immune cells and thus probably involved in inflammatory responses. ADAM8 is also produced by chondrocytes, and recombinant ADAM8 can induce cartilage catabolism. We therefore decided to test the role of ADAM8 in autoimmune inflammatory arthritis using transgenic mice expressing catalytically inactive ADAM8. Transgenic DBA/1J mice expressing an inactivating point mutation in the ADAM8 gene to change Glu330 to Gln330 (ADAM8(EQ)) were generated to evaluate the proteolytic function of ADAM8 in an lipopolysaccharide-synchronized collagen-induced arthritis (LPS-CIA) model of autoimmune arthritis. The systemic inflammatory reaction to LPS was also evaluated in these mice. Expression profiling of paw joints from wild-type mice revealed that ADAM8 mRNA levels increased at the onset of clinical arthritis and correlated well with cellular macrophage markers. When subjected to LPS-CIA, ADAM8(EQ) mice demonstrated decreased incidence and severity of clinical arthritis compared to wild-type mice. Histological examination of paw joints from ADAM8(EQ) mice confirmed marked attenuation of synovial inflammation, cartilage degradation and bone resorption when compared to wild-type mice. However, transgenic mice and wild-type mice responded similarly to LPS-induced systemic inflammation with regard to mortality, organ weights, neutrophil sequestration and serum cytokine/chemokine production. We conclude that ADAM8 proteolytic activity plays a key role in the development of experimental arthritis and may thus be an attractive target for the treatment of arthritic disorders while minimizing risk of immunocompromise.

The impact of purinergic signaling on renal ischemia-reperfusion injury.

BACKGROUND: Adenosine provides renovascular protection in mouse models of ischemia-reperfusion injury (I/RI) through purinergic members of the G protein-coupled receptor family, such as the adenosine 2A receptor (A2AR). Ectonucleotidases CD39 and CD73 are integral vascular and immune nucleotidases that regulate extracellular adenosine signaling. Current investigation of CD39 and CD73 in renal I/RI has primarily focused on their respective roles in ischemic preconditioning. METHODS: In this study, we established a unilateral renal I/RI model and investigated the role of adenosine generation versus nucleotide removal in mediating protection in renal I/RI using mice deficient in CD39, CD73 or A2AR, thereby sequentially disrupting ectonucleotidase cascade and adenosinergic signaling. RESULTS: Compared with wild-type mice, Cd73 null mice showed reduced levels of serum creatinine and urea, apoptosis of renal cells, and histologic damage after I/RI. Deletion of CD39 was associated with severe renal injury. Administration of apyrase, a soluble form of CD39, decreased global apoptosis and I/RI induced renal injury in wild-type mice. Apyrase treatment also improved renal histology to some extent in A2AR null mice. CONCLUSION: The relative protective effect of CD73 deletion in renal I/RI may reflect an effect of AMP accumulation. Deletion of CD39 showed deleterious effects and administration of soluble CD39 exerted renal protection, which is partially mediated by A2AR. The protective effect conferred by apyrase suggests that supplementing CD39 NTPDase activity may be a useful therapeutic strategy in renal transplantation.

CD133 is a marker of bioenergetic stress in human glioma.

Mitochondria dysfunction and hypoxic microenvironment are hallmarks of cancer cell biology. Recently, many studies have focused on isolation of brain cancer stem cells using CD133 expression. In this study, we investigated whether CD133 expression is regulated by bioenergetic stresses affecting mitochondrial functions in human glioma cells. First, we determined that hypoxia induced a reversible up-regulation of CD133 expression. Second, mitochondrial dysfunction through pharmacological inhibition of the Electron Transport Chain (ETC) produced an up-regulation of CD133 expression that was inversely correlated with changes in mitochondrial membrane potential. Third, generation of stable glioma cells depleted of mitochondrial DNA showed significant and stable increases in CD133 expression. These glioma cells, termed rho(0) or rho(0), are characterized by an exaggerated, uncoupled glycolytic phenotype and by constitutive and stable up-regulation of CD133 through many cell passages. Moreover, these rho(0) cells display the ability to form "tumor spheroids" in serumless medium and are positive for CD133 and the neural progenitor cell marker, nestin. Under differentiating conditions, rho(0) cells expressed multi-lineage properties. Reversibility of CD133 expression was demonstrated by transfering parental mitochondria to rho(0) cells resulting in stable trans-mitochondrial "cybrid" clones. This study provides a novel mechanistic insight about the regulation of CD133 by environmental conditions (hypoxia) and mitochondrial dysfunction (genetic and chemical). Considering these new findings, the concept that CD133 is a marker of brain tumor stem cells may need to be revised.

[Molecular mechanisms of thalamocortical circuit formation].

Thalamocortical (TC) projection is one of the major neural circuitries in the brain. TC projection has characteristic aspects of cortical area and laminar specificities, and provides a suitable model system to investigate the developmental mechanisms of neural circuit formation in the mammalian brain including human beings. Recent studies with genetic, molecular and cellular biological approaches reveal area and lamina-specific gene expressions in the developing cortex, regulation mechanisms of TC axon growth and branching by these molecules, and activity-dependence of the mechanisms.

A critical review of the therapeutic potential of dibenzyl trisulphide isolated from Petiveria alliacea L (guinea hen weed, anamu).

The data compiled in the present review on dibenzyl trisulphide (DTS) isolated from Petiveria alliacea L (the guinea hen weed or anamu) revealed that the compound and its derivatives could be of tremendous pharmaceutical interest. The mode of action elucidated for DTS revealed that it is a mitogen activated protein extracellular regulated kinases 1 and 2 (MAPKinases erk1 and erk 2) signal transduction molecule. Dibenzyl trisulphide caused hyper-phosphorylation of growth factor induced MAPKinases (erk 1 and erk 2) phosphorylation, a process critical for the improvement of long term memory, and is implicated in neuronal growth. Dibenzyl trisulphide and its derivatives exhibited potent anti-proliferation/cytotoxic activity on a wide range of cancer cell lines. The cytotoxic activity of DTS was increased by 70-1000 fold when bound to albumin in vitro. Dibenzyl trisulphide seems to have a cytokine switching mechanism in which it down regulates cytokines from the Type I helper cells (Th -1 cell) pathway which contained several pro-inflammatory cytokines and up-regulates those on the Type 2 helper cells (Th-2) pathway. The trisulphide up-regulates some reticuloendothelial system parameters eg granulocyte counts and increased thymic and Peyer's patches masses via cell proliferation processes which are known to be regulated via the MAPKinase signal transduction pathway. When the zygotes ofAsternia pectinifera (Starfish) were exposed to DTS at concentration of 10 mM, a dose lethal to all cancer cells tested, it was observed that the sensitive process of protein biosynthesis was not affected Similarly, the proliferation of the HOFA human fibroblast, a noncancerous cell line, was not severely affected by DTS at 8.9 microM over seven days, a concentration also lethal to most cancer cell lines tested The implications of the findings will be highlighted in the present review.

The CD28/CTLA-4-B7 signaling pathway is involved in both allergic sensitization and tolerance induction to orally administered peanut proteins.

Dendritic cells are believed to play an essential role in regulating the balance between immunogenic and tolerogenic responses to mucosal Ags by controlling T cell differentiation and activation via costimulatory and coinhibitory signals. The CD28/CTLA-4-CD80/CD86 signaling pathway appears to be one of the most important regulators of T cell responses but its exact role in responses to orally administered proteins remains to be elucidated. In the present study, the involvement of the CD28/CTLA-4-CD80/CD86 costimulatory pathway in the induction of allergic sensitization and oral tolerance to peanut proteins was investigated. In both an established C3H/HeOuJ mouse model of peanut hypersensitivity and an oral tolerance model to peanut, CD28/CTLA-4-CD80/CD86 interactions were blocked using the fusion protein CTLA-4Ig. To examine the relative contribution of CD80- and CD86-mediated costimulation in these models, anti-CD80 and anti-CD86 blocking Abs were used. In the hypersensitivity model, CTLA-4Ig treatment prevented the development of peanut extract-induced cytokine responses, peanut extract-specific IgG1, IgG2a, and IgE production and peanut extract-induced challenge responses. Blocking of CD80 reduced, whereas anti-CD86 treatment completely inhibited, the induction of peanut extract-specific IgE. Normal tolerance induction to peanut extract was found following CTLA-4Ig, anti-CD86, or anti-CD80 plus anti-CD86 treatment, whereas blockade of CD80 impaired the induction of oral tolerance. We show that CD28/CTLA-4-CD80/CD86 signaling is essential for the development of allergic responses to peanut and that CD86 interaction is most important in inducing peanut extract-specific IgE responses. Additionally, our data suggest that CD80 but not CD86 interaction with CTLA-4 is crucial for the induction of low dose tolerance to peanut.

A critical review of the therapeutic potential of dibenzyl trisulphide isolated from Petiveria alliacea L (guinea hen weed, anamu) / Un estudio crítico del potencial terapéutico del trisulfuro de dibencilo aislado a partir de petiveria alliacea l (yerba de guinea, anamú)

The data compiled in the present review on dibenzyl trisulphide (DTS) isolated from Petiveria alliacea L (the guinea hen weed or anamu) revealed that the compound and its derivatives could be of tremendous pharmaceutical interest. The mode of action elucidated for DTS revealed that it is a mitogen activated protein extracellular regulated kinases 1 and 2 (MAPKinases erk1 and erk 2) signal transduction molecule. Dibenzyl trisulphide caused hyper-phosphorylation of growth factor induced MAPKinases (erk 1 and erk 2) phosphorylation, a process critical for the improvement of long term memory, and is implicated in neuronal growth. Dibenzyl trisulphide and its derivatives exhibited potent anti-proliferation/cytotoxic activity on a wide range of cancer cell lines. The cytotoxic activity of DTS was increased by 70-1000 fold when bound to albumin in vitro. Dibenzyl trisulphide seems to have a cytokine switching mechanism in which it down regulates cytokines from the Type I helper cells (Th -1 cell) pathway which contained several pro-inflammatory cytokines and up-regulates those on the Type 2 helper cells (Th-2) pathway. The trisulphide up-regulates some reticuloendothelial system parameters eg granulocyte counts and increased thymic and Peyer's patches masses via cell proliferation processes which are known to be regulated via the MAPKinase signal transduction pathway. When the zygotes ofAsternia pectinifera (Starfish) were exposed to DTS at concentration of 10 mM, a dose lethal to all cancer cells tested, it was observed that the sensitive process of protein biosynthesis was not affected Similarly, the proliferation of the HOFA human fibroblast, a noncancerous cell line, was not severely affected by DTS at 8.9 microM over seven days, a concentration also lethal to most cancer cell lines tested The implications of the findings will be highlighted in the present review.

Los datos compilados en el presente estudio sobre el trisulfuro de dibencilo (TSD) aislado a partir de Petiveria alliacea L (yerba de Guinea, ó anamú) revelaron que el compuesto y sus derivados podrían tener extraordinario interés farmacéutico. El modo de acción esclarecido en el TSD, reveló que se trata de una molécula de transducción de señales de proteínas kinasas 1 y 2 (MAP quinasas ERk 1 y 2) reguladas extracelularmente y activadas por mitógenos. El trisulfuro de dibencilo causó hiperfosforilación de la fosforilación de las quinasas MAP (Erk 1 y 2) inducidas mediante factor de crecimiento, un proceso crítico para el mejoramiento de la memoria a largo plazo, y que está implicado en el crecimiento neuronal. El trisulfuro de dibencilo y sus derivados mostraron una poderosa actividad citotóxica y antiproliferativa en una amplia gama de líneas celulares de cáncer. La actividad citotóxica del TSD se incrementaba de 70 á 1000 veces, cuando se vinculaba a la albúmin in vitro. El trisulfuro de dibencilo parece poseer un mecanismo conmutador citoquínico que regula por decremento las citoquinas provenientes de la vía de las células auxiliares de tipo 1 (células Th-1), que contiene varias citoquinas pro-inflamatorias y regula por incremento las de la vía de las células auxiliares de tipo 2 (Th-2). El trisulfuro regula por incremento los parámetros del sistema reticuloendotelial, p.ej. los conteos de granulocitos y el aumento tanto de las masas tímicas como de las masas de placas de Peyer, a través de los procesos de proliferación celular, de los cuales se sabe que son regulados mediante la vía de la transducción de señales de la quinasa MAP. Cuando los cigotos de Asternia pectinifera (estrella de mar) fueron expuestos al TSD a una concentración de 10 mM ­ una dosis letal para todas las células cancerosas sometidas a prueba ­ se observó que el proceso sensible de biosíntesis de la proteína no era afectado. De modo similar, la proliferación del fibroblasto humano HOFA ­ una línea celular no cancerosa ­ no fue afectada severamente por el TSD a 8.9 µM en siete días ­ una concentración letal para la mayoría de las líneas celulares cancerosas sometidas a prueba. Las implicaciones de los hallazgos se pondrán de relieve en el presente estudio

Novel treatments for autistic spectrum disorders.

In no area of developmental pediatric practice is there more controversy regarding the choice of treatment than related to children with autistic spectrum disorders (ASD). Complementary and alternative medical therapies (CAM) are often elected because they are perceived as treating the cause of symptoms rather than the symptoms themselves. CAM used for autism can be divided by proposed mechanism: immune modulation, gastrointestinal, supplements that affect neurotransmitter function, and nonbiologic intervention. Secretin as a therapy for autism is discussed as an example of how a clinical observation rapidly grew to a widespread treatment before well-designed studies demonstrated absence of effect. The plausibility for behavioral effect was not substantiated by clinical studies. CAM used for treatment of autism is examined in terms of rationale, evidence of efficacy, side effects, and additional commentary. Families and clinicians need access to well-designed clinical evidence to assist them in choice of therapies.

Role of CD39 (NTPDase-1) in thromboregulation, cerebroprotection, and cardioprotection.

Blood platelets maintain vascular integrity and promote primary and secondary hemostasis following interruption of vessel continuity. Biochemical or physical damage to coronary, carotid, or peripheral arteries promotes excessive platelet activation and recruitment culminating in vascular occlusion and tissue ischemia. Currently, inadequate therapeutic approaches to stroke and coronary artery disease (CAD) are a public health issue. Following our demonstration of neutrophil leukotriene production from arachidonate released from activated aspirin-treated platelets, we studied interactions among platelets and other blood cells. This led to concepts of transcellular metabolism and thromboregulation. Thrombosis has a proinflammatory component whereby biologically active substances are synthesized by different cell types that could not individually synthesize the metabolite(s). Endothelium controls platelet reactivity via at least three biochemical systems: autacoids leading to production of prostacyclin and nitric oxide (NO) and endothelial ecto-adenosine phosphatase (ADPase)/CD39/nucleoside triphosphate diphosphohydrolase (NTPDase-1). The autacoids are fluid phase reactants, not produced by tissues in the basal state, but are only synthesized intracellularly and released upon interactions of cells with an agonist. When released, they exert fleeting actions in the immediate milieu and are rapidly inactivated. CD39 is an integral component of the endothelial cell (EC) surface and is substrate activated. It maintains vascular fluidity in the complete absence of prostacyclin and NO, indicating that the latter are ancillary components of hemostasis. Therapeutic implications for the autacoids have not been compelling because of their transient and local action and limited potency. Conversely, CD39, acting solely on the platelet releasate, is efficacious in animal models. It metabolically neutralizes a prothrombotic releasate via deletion of ADP-the major recruiting agent responsible for formation of an occlusive thrombus. In addition, solCD39 reduced adenosine triphosphate (ATP)- and ischemia-induced norepinephrine release in the heart. This action can prevent fatal arrhythmia. Moreover, solCD39 ameliorated the sequelae of stroke in cd39 null mice. Thus, CD39 represents the next generation of cardioprotective and cerebroprotective molecules. This article focuses on our interpretations of recent data and their implications for therapeutics.

HAb18G/CD147 enhances the secretion of matrix metalloproteinases (MMP) via cGMP/NO-sensitive capacitative calcium entry (CCE) and accordingly attenuates adhesion ability of fibroblasts.

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca2+ mobilization to induce matrix metalloproteinases (MMP) production and attenuate adhesion ability of mouse fibroblast NIH/3T3 cells. HAb18G/CD147 cDNA was transfected into fibroblast 3T3 cells to obtain a cell line stably expressing HAb18G/CD147, t3T3, as demonstrated by immunofluorescence staining and flow cytometry assays. 8-Bromo-cGMP inhibited the thapsigargin-induced Ca2+ entry in 3T3 cells, whereas an inhibitor of protein kinase G, KT5823 (1 microM), led to an increase in Ca2+ entry. Expression of HAb18G/CD147 in t3T3 cells decreased the inhibitory response to cGMP. A similar effect on the Ca2+ entry was observed in 3T3 cells in response to an NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca2+ entry was also reduced in HAb18G/CD147-expressing t3T3 cells, indicating a role for HAb18G/CD 147 in NO/cGMP-regulated Ca2+ entry. Results of gelatin zymography assays showed that addition of extracellular Ca2+ induced MMP (MMP-2, MMP-9) release and activation in a dose-dependent manner, and expression of HAb18G/CD147 enhanced the secretion of MMP-2 and MMP-9 in 3T3 cells. 8-Bromo-cGMP and SNAP reduced the production of MMP in 3T3 cells but not in t3T3 with HAb18G/CD147 expression. RT-PCR experiments substantiated that the expression of MMP-2 and MMP-9 mRNA in HAb18G/CD 147-expressing t3T3 cell was significantly greater than that in 3T3 cells. Experiments investigating adhesion potentials demonstrated that HAb18G/CD147-expressing t3T3 cells pretreated with Ca2+ attached to Matrigel-coated culture plates significantly less efficiently than 3T3 cells. The proportion of attached cells could be increased by treatment with 8-bromo-cGMP and SNAP in 3T3 cells, but not in t3T3. These results suggest that HAb18G/CD147 attenuates adhesion potentials in fibroblasts by enhancing the secretion of MMP through NO/cGMP-sensitive capacitative Ca2+ entry.

Autocrine regulation of T cell motility by calreticulin-thrombospondin-1 interaction.

The mechanisms regulating T lymphocyte migration within the extracellular matrix are not understood. We show in this study that the thrombospondin-1 binding site of calreticulin, spanning aa 19-32, is a major triggering factor for T cell motility and migration within a three-dimensional collagen type 1 matrix, and that exogenous motogenic factors such as chemokines can stimulate migration via a calreticulin-thrombospondin-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous thrombospondin-1 elicited a motogenic signal to the T cells through the C-terminal domain of thrombospondin-1 and its cell surface receptor integrin-associated protein (CD47). Our data further revealed that thrombospondin-1 was expressed on the cell surface with a high turnover, and that PI3K and the Janus family of tyrosine kinases were required for T cell motility mediated through calreticulin, thrombospondin-1, and CD47. These results unveil an autocrine mechanism of calreticulin-thrombospondin-1-CD47 interaction for the control of T cell motility and migration within three-dimensional extracellular matrix substrata.

Recruitment of C-terminal Src kinase by the leukocyte inhibitory receptor CD85j.

The CD85j inhibitory receptor (also termed ILT2 or LIR-1) is a type-I transmembrane protein that belongs to the Ig superfamily and is expressed by different leukocyte lineages. The extracellular region of CD85j binds HLA class I molecules and its cytoplasmic domain displays four immunoreceptor tyrosine-based inhibition motifs (ITIM). Upon tyrosine phosphorylation CD85j recruits the SHP-1 tyrosine phosphatase, involved in negative signaling. In order to identify other molecules to which CD85j might interact with in a phosphotyrosine-dependent manner, a cDNA B-cell library was screened in a three-hybrid system in yeast using the CD85j cytoplasmic tail as bait in the presence of the Src-kinase c-fyn420, 531Y-F, 176R-Q mutant. In this system, the C-terminal Src kinase (Csk) was shown to interact with CD85j. Phosphorylation-dependent recruitment of Csk to the CD85j cytoplasmic tail was confirmed in CD85j-transfected mammalian cells by immunoprecipitation and Western blot analysis. Mutational analyses and phospho-peptide mapping suggested that the SH2 domain of Csk may preferentially bind to ITIM Y562 of CD85j; yet, mutation to phenylalanine of Y533, Y614, and Y644 also significantly reduced Csk recruitment by CD85j. Even though CD85j was detected in both anti-SHP1 and CSK immunoprecipitates, these two molecules did not co-precipitate together with CD85j. Our data support the possibility that Csk regulates the function of CD85j.

CEACAM1 enhances invasion and migration of melanocytic and melanoma cells.

Expression of the cell adhesion molecule CEACAM1 in melanomas is an independent factor for the risk of metastasis with a predictive value superior to that of tumor thickness. We have previously shown that CEACAM1 co-localizes at the tumor-stroma interface of invading melanoma masses with integrin beta(3) and that these two adhesion molecules interact via the CEACAM1 cytoplasmic domain. To address the functional consequences of CEACAM1 expression, we investigated invasion and migration of melanocytic and melanoma cells that stably express CEACAM1 using two different in vitro systems. Here, we demonstrate that CEACAM1 expression markedly enhances cell invasion and migration. The enhanced invasion and migration of CEACAM1-transfected cells was dependent on the presence of Tyr-488 within the full-length cytoplasmic CEACAM1 domain. Treatment with anti-CEACAM monoclonal antibodies blocked CEACAM1-enhanced cell invasion and cell migration in a dose-dependent manner. Furthermore, the enhanced invasion and migration of CEACAM1-transfected melanoma cells was blocked by integrin-antagonizing RGD peptides. Expression of integrin beta(3) induces the up-regulation of CEACAM1 in melanocytic MEL6 cells. These results strengthen the view that CEACAM1 and alpha(v)beta(3) integrin are functionally interconnected with respect to the invasive growth of melanomas.