Genomic predictions for economically important traits in Brazilian Braford and Hereford beef cattle using true and imputed genotypes.

BACKGROUND: Genomic selection (GS) has played an important role in cattle breeding programs. However, genotyping prices are still a challenge for implementation of GS in beef cattle and there is still a lack of information about the use of low-density Single Nucleotide Polymorphisms (SNP) chip panels for genomic predictions in breeds such as Brazilian Braford and Hereford. Therefore, this study investigated the effect of using imputed genotypes in the accuracy of genomic predictions for twenty economically important traits in Brazilian Braford and Hereford beef cattle. Various scenarios composed by different percentages of animals with imputed genotypes and different sizes of the training population were compared. De-regressed EBVs (estimated breeding values) were used as pseudo-phenotypes in a Genomic Best Linear Unbiased Prediction (GBLUP) model using two different mimicked panels derived from the 50 K (8 K and 15 K SNP panels), which were subsequently imputed to the 50 K panel. In addition, genomic prediction accuracies generated from a 777 K SNP (imputed from the 50 K SNP) were presented as another alternate scenario. RESULTS: The accuracy of genomic breeding values averaged over the twenty traits ranged from 0.38 to 0.40 across the different scenarios. The average losses in expected genomic estimated breeding values (GEBV) accuracy (accuracy obtained from the inverse of the mixed model equations) relative to the true 50 K genotypes ranged from -0.0007 to -0.0012 and from -0.0002 to -0.0005 when using the 50 K imputed from the 8 K or 15 K, respectively. When using the imputed 777 K panel the average losses in expected GEBV accuracy was -0.0021. The average gain in expected EBVs accuracy by including genomic information when compared to simple BLUP was between 0.02 and 0.03 across scenarios and traits. CONCLUSIONS: The percentage of animals with imputed genotypes in the training population did not significantly influence the validation accuracy. However, the size of the training population played a major role in the accuracies of genomic predictions in this population. The losses in the expected accuracies of GEBV due to imputation of genotypes were lower when using the 50 K SNP chip panel imputed from the 15 K compared to the one imputed from the 8 K SNP chip panel.

Accuracy of genome-wide imputation in Braford and Hereford beef cattle.

BACKGROUND: Strategies for imputing genotypes from the Illumina-Bovine3K, Illumina-BovineLD (6K), BeefLD-GGP (8K), a non-commercial-15K and IndicusLD-GGP (20K) to either Illumina-BovineSNP50 (50K) or to Illumina-BovineHD (777K) SNP panel, as well as for imputing from 50K, GGP-IndicusHD (90iK) and GGP-BeefHD (90tK) to 777K were investigated. Imputation of low density (<50K) genotypes to 777K was carried out in either one or two steps. Imputation of ungenotyped parents (n = 37 sires) with four or more offspring to the 50K panel was also assessed. There were 2,946 Braford, 664 Hereford and 88 Nellore animals, from which 71, 59 and 88 were genotyped with the 777K panel, while all others had 50K genotypes. The reference population was comprised of 2,735 animals and 175 bulls for 50K and 777K, respectively. The low density panels were simulated by masking genotypes in the 50K or 777K panel for animals born in 2011. Analyses were performed using both Beagle and FImpute software. Genotype imputation accuracy was measured by concordance rate and allelic R(2) between true and imputed genotypes. RESULTS: The average concordance rate using FImpute was 0.943 and 0.921 averaged across all simulated low density panels to 50K or to 777K, respectively, in comparison with 0.927 and 0.895 using Beagle. The allelic R(2) was 0.912 and 0.866 for imputation to 50K or to 777K using FImpute, respectively, and 0.890 and 0.826 using Beagle. One and two steps imputation to 777K produced averaged concordance rates of 0.806 and 0.892 and allelic R(2) of 0.674 and 0.819, respectively. Imputation of low density panels to 50K, with the exception of 3K, had overall concordance rates greater than 0.940 and allelic R(2) greater than 0.919. Ungenotyped animals were imputed to 50K panel with an average concordance rate of 0.950 by FImpute. CONCLUSION: FImpute accuracy outperformed Beagle on both imputation to 50K and to 777K. Two-step outperformed one-step imputation for imputing to 777K. Ungenotyped animals that have four or more offspring can have their 50K genotypes accurately inferred using FImpute. All low density panels, except the 3K, can be used to impute to the 50K using FImpute or Beagle with high concordance rate and allelic R(2).

CX3CR1/CX3CL1 axis negatively controls glioma cell invasion and is modulated by transforming growth factor-ß1.

The chemokine CX3CL1 is constitutively expressed in the central nervous system by neurons and astrocytes controlling neuronal survival and neurotransmission. In this work, we analyzed the expression and function of the chemokine CX3CL1 and its receptor, CX3CR1, by human glioma cells. We show that both molecules are expressed on the tumor cell plasma membrane and that soluble CX3CL1 accumulates in the culture supernatants, indicating that the chemokine is constitutively released. We found that CX3CR1 is functional, as all the cell lines adhered to immobilized recombinant CX3CL1 and migrated in response to the soluble form of this chemokine. In addition, the blockade of endogenous CX3CL1 function by means of a neutralizing monoclonal antibody markedly delayed tumor cell aggregation and increased their invasiveness. We also show that CX3CL1 expression is potently modulated by the transforming growth factor-beta1 (TGF-beta1), a key regulator of glioma cell invasiveness. Indeed, both the treatment of glioma cells with recombinant TGF-beta1 and the inhibition of its endogenous expression by siRNA showed that TGF-beta1 decreases CX3CL1 mRNA and protein expression. Overall, our results indicate that endogenously expressed CX3CL1 negatively regulates glioma invasion likely by promoting tumor cell aggregation, and that TGF-beta1 inhibition of CX3CL1 expression might contribute to glioma cell invasive properties.

Lipid raft-dependent FcepsilonRI ubiquitination regulates receptor endocytosis through the action of ubiquitin binding adaptors.

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcepsilonRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcepsilonRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcepsilonRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway.

Although membrane phospholipid phosphatidylinositol-4,5bisphosphate (PIP2) plays a key role as signaling intermediate and coordinator of actin dynamics and vesicle trafficking, it remains completely unknown its involvement in the activation of cytolytic machinery. By live confocal imaging of primary human natural killer (NK) cells expressing the chimeric protein GFP-PH, we observed, during effector-target cell interaction, the consumption of a preexisting PIP2 pool, which is critically required for the activation of cytolytic machinery. We identified type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) alpha and gamma isoforms as the enzymes responsible for PIP2 synthesis in NK cells. By hRNA-driven gene silencing, we observed that both enzymes are required for the proper activation of NK cytotoxicity and for inositol-1,4,5-trisphosphate (IP3) generation on receptor stimulation. In an attempt to elucidate the specific step controlled by PI5KIs, we found that lytic granule secretion but not polarization resulted in impaired PI5KIalpha- and PI5KIgamma-silenced cells. Our findings delineate a novel mechanism implicating PI5KIalpha and PI5KIgamma isoforms in the synthesis of PIP2 pools critically required for IP3-dependent Ca(2+) response and lytic granule release.

The adaptor molecule CIN85 regulates Syk tyrosine kinase level by activating the ubiquitin-proteasome degradation pathway.

Triggering of mast cells and basophils by IgE and Ag initiates a cascade of biochemical events that lead to cell degranulation and the release of allergic mediators. Receptor aggregation also induces a series of biochemical events capable of limiting FcepsilonRI-triggered signals and functional responses. Relevant to this, we have recently demonstrated that Cbl-interacting 85-kDa protein (CIN85), a multiadaptor protein mainly involved in the process of endocytosis and vesicle trafficking, regulates the Ag-dependent endocytosis of the IgE receptor, with consequent impairment of FcepsilonRI-mediated cell degranulation. The purpose of this study was to further investigate whether CIN85 could alter the FcepsilonRI-mediated signaling by affecting the activity and/or expression of molecules directly implicated in signal propagation. We found that CIN85 overexpression inhibits the FcepsilonRI-induced tyrosine phosphorylation of phospholipase Cgamma, thus altering calcium mobilization. This functional defect is associated with a substantial decrease of Syk protein levels, which are restored by the use of selective proteasome inhibitors, and it is mainly due to the action of the ubiquitin ligase c-Cbl. Furthermore, coimmunoprecipitation experiments demonstrate that CIN85 overexpression limits the ability of Cbl to bind suppressor of TCR signaling 1 (Sts1), a negative regulator of Cbl functions, while CIN85 knockdown favors the formation of Cbl/Sts1 complexes. Altogether, our findings support a new role for CIN85 in regulating Syk protein levels in RBL-2H3 cells through the activation of the ubiquitin-proteasome pathway and provide a mechanism for this regulation involving c-Cbl ligase activity.

Inhibition of trail gene expression by cyclopentenonic prostaglandin 15-deoxy-delta12,14-prostaglandin J2 in T lymphocytes.

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a cyclopentenonic prostaglandin endowed with powerful anti-inflammatory activities, as shown in animal models of inflammatory/autoimmune diseases, where pharmacological administration of this prostanoid can ameliorate inflammation and local tissue damage via activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and/or covalent modifications of cellular proteins. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily expressed in most of the cells, including those of immune system such as T lymphocytes, in which it is up-regulated upon antigen-specific stimulation. This cytokine plays an important role in regulating various physiological and immunopathological processes, such as immunosurveillance of tumors and tissue destruction associated with different inflammatory and autoimmune diseases. Here, we demonstrate that 15d-PGJ(2) inhibits trail mRNA and protein expression by down-regulating the activity of its promoter in human T lymphocytes. Our data indicate that both the chemically reactive cyclopentenone moiety of 15d-PGJ(2) and the activation of PPARgamma may be involved in this repressive mechanism. We identified nuclear factor kappaB (NF-kappaB) as a direct target of the prostanoid. 15d-PGJ(2) significantly decreases the expression and/or DNA binding of c-rel, RelA, and p50 transcription factors to the NF-kappaB1 site of trail promoter. Moreover, 15d-PGJ(2)-mediated activation of the transcription factor heat shock factor-1 may contribute to inhibit trail promoter activity in transfected Jurkat T cells. These results suggest that modulation of TRAIL gene expression by 15d-PGJ(2) in T cells may provide a novel pharmacological tool to modify the onset and the progression of specific autoimmune and inflammatory disorders.

Antigen-activated human T lymphocytes express cell-surface NKG2D ligands via an ATM/ATR-dependent mechanism and become susceptible to autologous NK- cell lysis.

Recent evidence indicates that natural killer (NK) cells can negatively regulate T-cell responses, but the mechanisms behind this phenomenon as a consequence of NK-T-cell interactions are poorly understood. We studied the interaction between the NKG2D receptor and its ligands (NKG2DLs), and asked whether T cells expressed NKG2DLs in response to superantigen, alloantigen, or a specific antigenic peptide, and if this rendered them susceptible to NK lysis. As evaluated by FACS, the major histocompatibility complex (MHC) class I chain-related protein A (MICA) was the ligand expressed earlier on both CD4(+) and CD8(+) T cells in 90% of the donors tested, while UL16-binding protein-1 (ULBP)1, ULBP2, and ULBP3 were induced at later times in 55%-75% of the donors. By carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, we observed that NKG2DLs were expressed mainly on T cells that had gone through at least one division. Real-time reverse-transcription polymerase chain reaction confirmed the expression of all NKG2DLs, except ULBP4. In addition, T-cell activation stimulated phosphorylation of ataxia-telangiectasia mutated (ATM), a kinase required for NKG2DLs expression after DNA damage, and ATM/Rad3-related kinase (ATR) inhibitors blocked MICA induction on T cells with a mechanism involving NF-kappaB. Finally, we demonstrated that activated T cells became susceptible to autologous NK lysis via NKG2D/NKG2DLs interaction and granule exocytosis, suggesting that NK lysis of T lymphocytes via NKG2D may be an additional mechanism to limit T-cell responses.

15-deoxy-Delta12,14-prostaglandin J2 negatively regulates rankl gene expression in activated T lymphocytes: role of NF-kappaB and early growth response transcription factors.

Receptor activator of NF-kappaB ligand (RANKL) and its receptor RANK are cell surface proteins abundantly expressed in bone and lymphoid tissues, whose interaction triggers different signaling pathways leading to activation and differentiation of osteoclasts, pivotal actors of the normal bone remodeling cycle. Moreover, RANKL may act as an immunomodulator, representing an important dendritic cell survival factor produced by activated T cells. A large body of research has shown that not only does the RANKL/RANK system regulate the physiology of bone development but also plays an important pathological role in bone destruction mediated by inflammatory disorders or bone metastatic tumors. 15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a cyclopentenone-type PG endowed with anti-inflammatory properties and produced by different cells, including those of the immune system. Although 15d-PGJ(2) has been studied as a natural ligand of the peroxisome proliferator-activated receptor-gamma nuclear receptor, relevant peroxisome proliferator-activated receptor-gamma-independent actions mediated by this prostanoid have been described. In this study, we describe the effect of 15d-PGJ(2) on the expression of the rankl gene in T lymphocytes. We show that 15d-PGJ(2) inhibits rankl mRNA expression, protein, and rankl promoter activity by mechanisms mediated by its chemically reactive cyclopentenone moiety. Our data also indicate that 15d-PGJ(2) represses rankl activation by interfering with the expression and/or activity of the transcription factors NF-kappaB, early growth response-2, and early growth response-3, whose altered balancing and transactivation may contribute for the repression of this gene. These results place rankl as a novel molecular target for the different immunoregulatory activities mediated by 15d-PGJ(2). The physiological and pharmacological implications of these observations are discussed.

High-efficient lentiviral vector-mediated gene transfer into primary human NK cells.

OBJECTIVE: The long-term transfection of genes into primary natural killer (NK) cells without disrupting normal cellular functions has been proven to be difficult with currently available gene-transfer methods. In this study, we establish a lentiviral vector-based technique for improved gene transfer into human NK cells in vitro and we report on high-efficient transduction of freshly isolated as well as cultured primary NK cells. METHODS: Freshly isolated or primary cultured human NK cells, as well as the human NK cell line YTS, were transduced with replication-incompetent human immunodeficiency virus (HIV)-based lentiviral vector bearing a GFP reporter gene or a gene of interest under the control of the elongation factor 1alpha (EF1alpha) promoter. Transduction efficiencies were monitored by flow cytometry. RESULTS: A long-term transgene expression was detected in up to 98% of YTS NK cells, whereas in freshly isolated or primary cultured NK cells exposed to interleukin (IL)-2 plus IL-12 upon infection, efficiency was in the range of 50% to 90%. Moreover, in freshly isolated quiescent NK cells a transfection efficiency of 18% to 20% was achieved without stimulation. Notably, no major phenotypic and functional modifications were observed in transduced cells with respect to control cells: the expression levels of activating receptors, CD69-antigen induction as well as cytotoxic function were unaffected. CONCLUSION: Results of our study demonstrate that NK cells can be efficiently transduced by lentiviral vectors.

CIN85 regulates the ligand-dependent endocytosis of the IgE receptor: a new molecular mechanism to dampen mast cell function.

Ligation of the high-affinity receptor for IgE (Fc epsilonRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, Fc epsilonRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of Fc epsilonRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating Fc epsilonRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the Fc epsilonRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of Fc epsilonRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.

Arf6: a new player in FcgammaRIIIA lymphocyte-mediated cytotoxicity.

The activation of phosphoinositide metabolism represents a critical step in the signaling pathways leading to the activation of cytolytic machinery, but its regulation is partially understood. We report here that the stimulation of the low-affinity receptor for immunoglobulin G (IgG) (FcgammaRIIIA, CD16) on primary human natural killer (NK) cells induces a phosphatidylinositol 3-kinase (PI3K)-dependent activation of the small G protein Arf6. We first demonstrate a functional role for Arf6-dependent signals in the activation of the antibody-dependent cellular cytotoxicity (ADCC) attributable to the control of secretion of lytic granule content. We also show that Arf6 couples CD16 to the lipid-modifying enzymes phosphatidylinositol4phosphate 5-kinase type I alpha (PI5KIalpha) and phospholipase D (PLD) that are involved in the control of granule secretion; Arf6, but not Rho family small G proteins RhoA and Rac1, is required for receptor-induced PI5KIalpha membrane targeting as well as for PI5KIalpha and PLD activation. Our findings suggest that Arf6 plays a crucial role in the generation of a phosphatidylinositol4,5-bisphosphate (PIP2) plasma membrane pool required for cytolytic granule-mediated target cell killing.

Hyperthermia enhances CD95-ligand gene expression in T lymphocytes.

Hyperthermia represents an interesting therapeutic strategy for the treatment of tumors. Moreover, it is able to regulate several aspects of the immune response. Fas (APO-1/CD95) and its ligand (FasL) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T cells, the Fas-Fas-L system regulates activation-induced cell death, is implicated in diseases in which lymphocyte homeostasis is compromised, and plays an important role during cytotoxic and regulatory actions mediated by these cells. In this study we describe the effect of hyperthermia on activation of the fas-L gene in T lymphocytes. We show that hyperthermic treatment enhances Fas-L-mediated cytotoxicity, fas-L mRNA expression, and fas-L promoter activity in activated T cell lines. Our data indicate that hyperthermia enhances the transcriptional activity of AP-1 and NF-kappaB in activated T cells, and this correlates with an increased expression/nuclear translocation of these transcription factors. Moreover, we found that heat shock factor-1 is a transactivator of fas-L promoter in activated T cells, and the overexpression of a dominant negative form of heat shock factor-1 may attenuate the effect of hyperthermia on fas-L promoter activity. Furthermore, overexpression of dominant negative mutants of protein kinase Cepsilon (PKCepsilon) and PKCtheta; partially inhibited the promoter activation and, more importantly, could significantly reduce the enhancement mediated by hyperthermia, indicating that modulation of PKC activity may play an important role in this regulation. These results add novel information on the immunomodulatory action of heat, in particular in the context of its possible use as an adjuvant therapeutic strategy to consider for the treatment of cancer.

p38 MAPK activation controls the TLR3-mediated up-regulation of cytotoxicity and cytokine production in human NK cells.

Natural killer (NK) cells are a component of the innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. Although the synthetic double-stranded (ds) RNA polyinosinic-polycytidylic acid (poly I:C), a mimic of a common product of viral infections, is known to rapidly up-regulate their in vivo functions, NK cell ability to directly respond to dsRNA is still mostly unknown. Our results show that treatment with poly I:C significantly up-regulates both natural and CD16-mediated cytotoxicity of highly purified human NK cells. Poly I:C also induces the novel capability of producing CXCL10 chemokine in human NK cells and synergistically enhances interferon-gamma (IFN-gamma) production induced by either adaptive or innate cytokines. In accordance with the expression of Toll-like receptor-3 (TLR3) and of TRIF/TICAM-1 adaptor, poly I:C stimulation induces the activation of interferon regulatory factor-3 (IRF-3) transcription factor and of p38 mitogen-activated protein kinase (MAPK) in human NK cells. Finally, we demonstrate that p38 MAPK activity is required for the dsRNA-dependent enhancement of cytotoxicity and CXCL10 production. The occurrence of dsRNA-induced signaling and functional events closely correlates with the TLR3 mRNAprofile in different NK cell populations. Taken together, these data identify p38 as a central component of NK cell ability to directly respond to dsRNA pathogen-associated molecular pattern (PAMP).

Neonatal capsaicin treatment affects rat thymocyte proliferation and cell death by modulating substance P and neurokinin-1 receptor expression.

Herein we provide evidence that substance P (SP) and its neurokinin-1 receptor (NK-1R) expressed on thymocytes counteract thymus depletion induced by neonatal capsaicin (CPS) treatment by affecting thymocyte proliferation and apoptotic death. SP administration reversed the CPS-mediated inhibitory effects on the total thymocyte number and subset distribution, namely CD4+ and CD4- CD8- cells, through its interaction with NK-1R as shown by concomitant NK-1R (SR140333) antagonist administration. SP-induced enhancement of thymus cellularity parallels its ability of inhibiting the thymocyte apoptotic program. Indeed, exogenously administered SP completely nullified CPS-induced apoptosis, and SR140333 abrogated the SP-mediated protective effect. SP administration also stimulated concanavalin A (Con A)-induced thymocyte proliferation of CPS-treated rats, completely reversing the CPS-induced inhibition. The SP-mediated stimulation of Con A-induced thymocyte proliferation was NK-1R dependent as shown by concomitant administration of SP and SR140333 to CPS-treated rats. Our results also demonstrate that CPS treatment induces a marked decrease of thymocyte PPT-A mRNA level and endogenous SP content as evaluated by quantitative RT-PCR, in situ hybridization and cytofluorimetric analysis. By contrast, NK-1R mRNA levels were increased in thymocytes from CPS-treated rats. Exogenous SP administration augmented PPT-A, SP and NK-1R thymocyte expression in CPS-treated rats, and this enhancement was antagonized by SR140333 administration. Overall, our results strongly suggest that the immunomodulatory effects of neonatal CPS treatment on rat thymocyte functions are dependent on vanilloid-mediated regulation of SP and NK-1R functional expression by neuronal and immune cells.

The cyclopentenone-type prostaglandin 15-deoxy-delta 12,14-prostaglandin J2 inhibits CD95 ligand gene expression in T lymphocytes: interference with promoter activation via peroxisome proliferator-activated receptor-gamma-independent mechanisms.

15-Deoxy-delta(12,14)-PGJ(2) (15d-PGJ(2)) is a cyclopentenone-type PG endowed with anti-inflammatory properties and produced by different cells, including those of the immune system. 15d-PGJ(2) is a natural ligand of the peroxisome proliferator-activated receptor (PPAR)-gamma nuclear receptor, but relevant PPARgamma-independent actions mediated by this prostanoid have been described. Fas (APO-1/CD95) and its ligand (Fas-L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T cells, the Fas-Fas-L system regulates activation-induced cell death and has been implicated in diseases in which lymphocyte homeostasis is compromised. Moreover, several studies have described the pathogenic functions of Fas and Fas-L in vivo, particularly in the induction-progression of organ-specific autoimmune diseases. In this study we describe the effect of 15d-PGJ(2) on the activation of the fas-L gene in T lymphocytes. We show that 15d-PGJ(2) inhibits fas-L mRNA expression, activation-induced cell death, and fas-L promoter activity by mechanisms independent of PPARgamma and mediated by its chemically reactive cyclopentenone moiety. Our data indicate that 15d-PGJ(2) may repress fas-L activation by interfering with the expression and/or transcriptional activity of different transcription factors (early growth response types 3 and 1, NF-kappaB, AP-1, c-Myc, Nur77) whose altered balancing and transactivation may contribute for overall repression of this gene. In addition, the activation/expression of the heat shock response genes HSF-1 and HSP70 is not directly involved in the repression, and the electrophilic molecule cyclopentenone (2-cyclopenten-1-one) may reproduce the effects mediated by 15d-PGJ(2). These results suggest that modulation of Fas-L by 15d-PGJ(2) in T cells may represent an additional tool to consider for treatment of specific autoimmune and inflammatory disorders.

Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration.

Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of G(i) protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.

SH2-containing inositol phosphatase (SHIP-1) transiently translocates to raft domains and modulates CD16-mediated cytotoxicity in human NK cells.

Membrane recruitment of the SH2-containing 5' inositol phosphatase 1 (SHIP-1) is responsible for the inhibitory signals that modulate phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. Here we have investigated the molecular mechanisms underlying SHIP-1 activation and its role in CD16-mediated cytotoxicity. We initially demonstrated that a substantial fraction of SHIP-1-mediated 5' inositol phosphatase activity associates with CD16 zeta chain after receptor cross-linking. Moreover, CD16 stimulation on human primary natural killer (NK) cells induces the rapid and transient translocation of SHIP-1 in the lipid-enriched plasma membrane microdomains, termed rafts, where it associates with tyrosine-phosphorylated zeta chain and shc adaptor protein. As evaluated by confocal microscopy, CD16 engagement by reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly induces SHIP-1 redistribution toward the area of NK cell contact with target cells and its codistribution with aggregated rafts where CD16 receptor also colocalizes. The functional role of SHIP-1 in the modulation of CD16-induced cytotoxicity was explored in NK cells infected with recombinant vaccinia viruses encoding wild-type or catalytic domain-deleted mutant SHIP-1. We found a significant SHIP-1-mediated decrease of CD16-induced cytotoxicity that is strictly dependent on its catalytic activity. These data demonstrate that CD16 engagement on NK cells induces membrane targeting and activation of SHIP-1, which acts as negative regulator of ADCC function.

Activation of Syk tyrosine kinase is required for c-Cbl-mediated ubiquitination of Fcepsilon RI and Syk in RBL cells.

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells and basophils results in FcepsilonRI beta and gamma subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FcepsilonRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FcepsilonRI beta and gamma chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FcepsilonRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FcepsilonRI and activated Syk to the proteasome for degradation.

Candida albicans expresses a focal adhesion kinase-like protein that undergoes increased tyrosine phosphorylation upon yeast cell adhesion to vitronectin and the EA.hy 926 human endothelial cell line.

The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.