Biomedical subjects

J Bertoglio

Publications and source records attributed to J Bertoglio.

At least 19 recordsLinked to original sources

PubMed2001

Induction of the IL-13 receptor alpha2-chain by IL-4 and IL-13 in human keratinocytes: involvement of STAT6, ERK and p38 MAPK pathways.

IL-4 and IL-13 are related cytokines which induce both pro- and anti-inflammatory effects depending on the cell type they act upon and the nature of the receptors expressed. The type I receptor complex is composed of the IL-4Ralpha and gammac and only binds IL-4, whereas, in the type II receptor, IL-4Ralpha dimerizes with IL-13Ralpha1 upon either IL-4 or IL-13 binding. Another ligand binding chain potentially implicated in the IL-4/IL-13 receptor has been described, the IL-13Ralpha2, but the regulation of its expression and its role in IL-4/IL-13 transduction is poorly understood. In this study we report that IL-4 and IL-13 upregulate IL-13Ralpha2 at both the mRNA and protein levels in the keratinocyte cell line HaCaT. In these cells, IL-4 or IL-13 were shown to activate the Janus Kinases JAK1 and JAK2, the transcription factor STAT6, and the ERK and p38 mitogen-activated protein kinases. We show that IL-4 or IL-13-induced IL-13Ralpha2 mRNA expression was inhibited by the ERK inhibitor U0126, the JAK inhibitor AG490 and, to a lesser extent, the p38 MAPK inhibitor SB203580. Moreover, expression of a constitutive active mutant of STAT6 alone did not modify IL-13Ralpha2 mRNA expression, but potentiated the effects of IL-4 or IL-13 on IL-13Ralpha2 expression. The constitutive active mutants of MEK1 or MKK6 increased the level of expression of IL-13Ralpha2 mRNA even in absence of stimulation. Our findings demonstrate, for the first time, that IL-4 and IL-13 can induce IL-13Ralpha2 expression in keratinocytes, and that the ERK and p38 MAPK together with JAK2 and STAT6 play a critical role in this process.

Antineoplastic Agents↗

PubMed2001

Modulation of COX-2 expression by statins in human aortic smooth muscle cells. Involvement of geranylgeranylated proteins.

Cyclooxygenase (COX)-2 and COX-1 play an important role in prostacyclin production in vessels and participate in maintaining vascular homeostasis. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which is crucial in cholesterol biosynthesis. Recently, cholesterol-independent effects of statins have been described. In this study, we evaluated the effect of two inhibitors of HMG CoA reductase, mevastatin and lovastatin, on the production of prostacyclin and the expression of COX in human aortic smooth muscle cells. Treatment of cells with 25 microm mevastatin or lovastatin resulted in the induction of COX-2 and increase in prostacyclin production. Mevalonate, the direct metabolite of HMG CoA reductase, and geranylgeranyl-pyrophosphate reversed this effect. GGTI-286, a selective inhibitor of geranylgeranyltransferases, increased COX-2 expression and prostacyclin formation, thus indicating the involvement of geranylgeranylated proteins in the down-regulation of COX-2. Furthermore, Clostridium difficile toxin B, an inhibitor of the Rho GTP-binding protein family, the Rho selective inhibitor C3 transferase, and Y-27632, a selective inhibitor of the Rho-associated kinases, targets of Rho A, increased COX-2 expression whereas the activator of the Rho GTPase, the cytotoxic necrotizing factor 1, blocked interlukin-1alpha-dependent COX-2 induction. These results demonstrate that statins up-regulate COX-2 expression and subsequent prostacyclin formation in human aortic smooth muscle cells in part through inhibition of Rho.

Amides↗

PubMed2001

Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase.

The aim of this work was to investigate the coupling of human urotensin II (hU-II) to RhoA activation and regulation of RhoA-dependent functions. The use of the Rho-kinase inhibitor Y-27632 and the development of a membrane-permeant RhoA inhibitor (TAT-C3) allowed us to demonstrate that hU-II induced arterial smooth muscle contraction, actin stress fiber formation, and proliferation through the activation of the small GTPase RhoA and its downstream effector Rho-kinase.

ADP Ribose Transferases↗

PubMed2001

A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners.

p97/Gab2 is a recently characterized member of a large family of scaffold proteins that play essential roles in signal transduction. Gab2 becomes tyrosine-phosphorylated in response to a variety of growth factors and forms multimolecular complexes with SH2 domain-containing signaling molecules such as the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K), the tyrosine phosphatase SHP-2 and the adapter protein CrkL. To characterize the interactions between Gab2 and its SH2-containing binding partners, we designed a modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed in a regulated manner in yeast. Using this assay, we demonstrated that p97/Gab2 specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL. Interaction with p85-PI3K is mediated by tyrosine residues Y452, Y476 and Y584 of Gab2, while interaction with SHP-2 depends exclusively on tyrosine Y614. CrkL interaction is mediated by its SH2 domain recognizing Y266 and Y293, despite the latter being in a non-consensus (YTFK) environment.

Adaptor Proteins, Signal Transducing↗

PubMed2001

The interaction between Cdc42 and WASP is required for SDF-1-induced T-lymphocyte chemotaxis.

In studies aimed at further characterizing the cellular immunodeficiency of the Wiskott-Aldrich syndrome (WAS), we found that T lymphocytes from WAS patients display abnormal chemotaxis in response to the T-cell chemoattractant stromal cell-derived factor (SDF)-1. The Wiskott- Aldrich syndrome protein (WASP), together with the Rho family GTPase Cdc42, control stimulus-induced actin cytoskeleton rearrangements that are involved in cell motility. Because WASP is an effector of Cdc42, we further studied how Cdc42 and WASP are involved in SDF-1-induced chemotaxis of T lymphocytes. We provide here direct evidence that SDF-1 activates Cdc42. We then specifically investigated the role of the interaction between Cdc42 and WASP in SDF-1-responsive cells. This was achieved by abrogating this interaction with a recombinant polypeptide (TAT-CRIB), comprising the Cdc42/Rac interactive binding (CRIB) domain of WASP and a human immunodeficiency virus-TAT peptide that renders the fusion protein cell-permeant. This TAT-CRIB protein was shown to bind specifically to Cdc42-GTP and to inhibit the chemotactic response of a T-cell line to SDF-1. Altogether, these data demonstrate that Cdc42-WASP interaction is critical for SDF-1-induced chemotaxis of T cells.

Actins↗

PubMed2001

Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing.

Increased phosphorylation of myosin light chain (MLC) is necessary for the dynamic membrane blebbing that is observed at the onset of apoptosis. Here we identify ROCK I, an effector of the small GTPase Rho, as a new substrate for caspases. ROCK I is cleaved by caspase-3 at a conserved DETD1113/G sequence and its carboxy-terminal inhibitory domain is removed, resulting in deregulated and constitutive kinase activity. ROCK proteins are known to regulate MLC-phosphorylation, and apoptotic cells exhibit a gradual increase in levels of phosphorylated MLC concomitant with ROCK I cleavage. This phosphorylation, as well as membrane blebbing, is abrogated by inhibition of caspases or ROCK proteins, but both processes are independent of Rho activity. We also show that expression of active truncated ROCK I induces cell blebbing. Thus, activation of ROCK I by caspase-3 seems to be responsible for bleb formation in apoptotic cells.

Apoptosis↗

PubMed2001

Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not AP-1.

Cytokines and glucocorticoids (GCs) signaling pathways interfere with each other in the regulation of apoptosis and gene expression in the immune system. Interleukin-2 (IL-2), through the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways, activates STAT5 and activated protein-1 (AP-1) transcription factors, respectively, which are known to repress glucocorticoid receptor (GR) activity, at least in part, through protein-protein interactions. In this work, we have analyzed the mechanisms whereby IL-2 down-regulates the GC-induced transactivation of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5 binding site (-923/-914) was not required for IL-2-mediated inhibition but identified both glucocorticoid response elements (GREs) and the -104/+1 region as critical elements for this negative response. The DNA binding activities of transcription factors required for GC-mediated activation of the MMTV-LTR promoter and that bind to the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2 treatment. Overexpression of wild-type STAT5B enhanced the effect of IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B (Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1 activation had no effect in this system. Direct interaction between liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5 phosphorylation-independent manner. Overexpression of nuclear coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that the STAT5-repressive activity on the GC-dependent transcription may involve direct interaction of STAT5 with GR, is dependent on the promoter context and STAT5 activation level, and occurs independently of coactivators levels in T cells.

Animals↗

PubMed2001

Characterization of an IL-2 mimetic with therapeutic potential.

Human interleukin-2 (IL-2) interacts with two types of functional receptors (IL-2R alpha betagamma and IL-2R betagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. IL-2 is also used in different clinical trials aimed at improving the treatment of some cancers and the recovery of CD4 lymphocytes by HIV patients. The therapeutic index of IL-2 is limited by various side effects dominated by the vascular leak syndrome. We have shown that a chemically synthesised fragment of the IL-2 sequence can fold into a helical tetramer likely mimicking the quatemary structure of an hemopoietin. Indeed, peptide p1-30 (containing amino acids 1 to 30, including the sequence corresponding to the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T-cell lines expressing human IL-2R beta, whereas shorter versions of the peptide lack helical structure and are inactive. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8 low lymphocytes and natural killer cells, which constitutively express IL-2R beta. A significant IFN-gamma production is also detected following p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys) which is likely unable to induce vascular leak syndrome remains capable to generate LAK cells like the original p1-30 peptide. Altogether our data suggest that p1-30 has therapeutic potential.

CD8-Positive T-Lymphocytes↗

PubMed2000

The glucocorticoid receptor and STAT6 physically and functionally interact in T-lymphocytes.

In lymphocytes, glucocorticoids (GC)- and interleukin-4-signaling pathways are known to interact, as evidenced by inhibition of IL-4-mediated proliferation by dexamethasone or suppression of GC-induced apoptosis by IL-4. In this study, we characterized the molecular basis for this reciprocal interference. We report that, in murine CTLL-2 cells, IL-4 inhibits GC-induced MMTV (mouse mammary tumor virus) promoter transactivation, and that GC suppress IL-4-induced transactivation of a STAT6 (signal transducers and activators of transcription 6)-responsive promoter without affecting IL-4-stimulated STAT6 DNA-binding. Moreover, we evidenced a physical association between GC receptor and STAT6, which proved to be functionally relevant, since STAT6 overexpression increased the IL-4 inhibitory effect on GC-induced MMTV transactivation.

Animals↗

PubMed2000

Cdk2 associates with MAP kinase in vivo and its nuclear translocation is dependent on MAP kinase activation in IL-2-dependent Kit 225 T lymphocytes.

Cell proliferation is controlled by cdk2 which in association with cyclin E and A regulates G1/S transition and S phase progression. cdk2 activation is dependent on its localization in the nucleus where regulatory mediators are found. We report that activation of cdk2 is associated with the formation of cdk2/MAP Kinase complexes. cdk2 associates with both inactive and activated MAP Kinase. Prevention of MAP Kinase activation by the MEK inhibitor PD98059 inhibits both activation and nuclear localization of cdk2 and S phase entry. These findings indicate that the nuclear translocation of cdk2 is associated with the formation of molecular complexes containing active MAP Kinase and is dependent on MAP Kinase activation. Oncogene (2000) 19, 4184 - 4189

CDC2-CDC28 Kinases↗

PubMed2000

Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle.

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca(2+) sensitivity of contraction of smooth muscle via stimulation of myosin light chain phosphatase through unknown mechanisms (Wu, X., Somlyo, A. V., and Somlyo, A. P. (1996) Biochem. Biophys. Res. Commun. 220, 658-663). Myosin light chain phosphatase activity is controlled by the small GTPase RhoA and its target Rho kinase. Here we demonstrate cGMP effects mediated by cGK that inhibit RhoA-dependent Ca(2+) sensitization of contraction of blood vessels and actin cytoskeleton organization in cultured vascular myocytes. Ca(2+) sensitization and actin organization were inhibited by both 8-bromo-cGMP and sodium nitroprusside (SNP). SNP also caused translocation of activated RhoA from the membrane to the cytosol. SNP-induced actin disassembly was lost in vascular myocytes in culture after successive passages but was restored by transfection of cells with cGK I. Furthermore, cGK phosphorylated RhoA in vitro, and addition of cGK I inhibited RhoA-induced Ca(2+) sensitization in permeabilized smooth muscle. 8-Bromo-cGMP-induced actin disassembly was inhibited in vascular myocytes expressing RhoA(Ala-188), a mutant that could not be phosphorylated. Collectively, these results indicate that cGK phosphorylates and inhibits RhoA and suggest that the consequent inhibition of RhoA-induced Ca(2+) sensitization and actin cytoskeleton organization contributes to the vasodilator action of nitric oxide.

Actins↗

PubMed2000

IL-4 regulation of IL-6 production involves Rac/Cdc42- and p38 MAPK-dependent pathways in keratinocytes.

The stress-activated pathways leading to activation of p38 MAP kinase (p38 MAPK) and c-jun N-terminal kinases (JNK) have been shown to be activated by pro-inflammatory cytokines, physical and chemical stresses as well as a variety of hematopoietic growth factors. One exception is interleukin (IL)-4, which does not activate this pathway in hematopoietic cell. We report here that in A431, a keratinocytic cell line, IL-4 activates Rac and Cdc42 and their downstream effector p21-activated kinase (PAK). Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of PAK and leading to activation of p38 MAPK, since IL-4 stimulates tyrosine phosphorylation of p38 MAPK and increases its catalytic activity. As A431 cells are able to produce IL-6 in response to IL-4 stimulation, we assessed the involvement of p38 MAPK in IL-6 gene expression. A pyrimidazole compound, SB203580, a specific inhibitor of p38 MAPK, inhibits production and gene expression of IL-6. SB203580 reduced significantly the stability of IL-6 mRNA. Here we provide evidence that p38 MAPK is activated in response to IL-4 and is involved in IL-6 synthesis by stabilizing IL-6 mRNA.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2000

The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1-30 (containing amino acids 1-30, covering the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1-30 activates Tyk2, thus suggesting that IL-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma production is also detected after p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1-30 peptide. Altogether, our data suggest that p1-30 has therapeutic potential.

Amino Acid Sequence↗

PubMed2000

Association of STAT1, STAT3 and STAT5 proteins with the IL-2 receptor involves different subdomains of the IL-2 receptor beta chain.

Upon IL-2 stimulation of T lymphocytes, the IL-2 receptor (IL-2R) becomes phosphorylated on specific tyrosine residues which serve as docking sites for proteins containing SH2 or phosphotyrosine binding domains. To study the interaction of the IL-2Rbeta chain with Shc and STAT proteins, subdomains of the IL-2Rbeta chain were expressed as tyrosine-phosphorylated glutathione S-transferase fusion proteins and used to pull-down interacting proteins from Kit 225 cell lysates. These experiments provide direct biochemical evidence that binding to the IL-2R of the adaptor protein Shc requires phosphorylation of Tyr-338 in the IL-2Rbeta acidic subdomain. In addition, we report that STAT proteins that are activated by IL-2, i.e. STAT1, STAT3 and STAT5, indeed associate with the IL-2Rbeta chain. Both the A and B isoforms of STAT5 were found to associate with Tyr-510 of the IL-2Rbeta C-terminal region, depending on its phosphorylation. In contrast, STAT1 and STAT3 associated with the IL-2Rbeta chain through its acidic subdomain. These results indicate that the interaction between IL-2Rbeta and STAT1 or 3 does not require either phosphorylation of the receptor or even the presence of tyrosine residues of IL-2Rbeta. Thus, the IL-2R recruits STAT proteins through different modes of interaction.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Il-4 and IFN-gamma mRNA induction in human peripheral lymphocytes specific for beta-lactam antibiotics in immediate or delayed hypersensitivity reactions.

Beta-lactam antibiotics elicit CD4+ and CD8+ T-cell-mediated immune responses that play a central role in allergic reactions. However, the involvement of a type 1- (Th1 or Tc1) or a type 2-like (Th2 or Tc2) differentiation in drug allergy remains unclear. We investigated the expression of interleukin 4 (IL-4) and interferon gamma (IFN-gamma) mRNA by quantitative reverse transcription and polymerase chain reaction (RT-PCR) in patient-derived peripheral blood lymphocytes following specific in vitro stimulation. Samples were collected from a total of 19 patients who had developed immediate or delayed clinical manifestations of hypersensitivity to beta-lactam and from 11 control subjects. Peripheral blood mononuclear cells (PBMCs) were stimulated with either free antibiotics or antibiotic-human serum albumin (HSA) conjugates. Specific induction of IFN-gamma mRNA expression was observed in 11 of 11 allergic patients with immediate reactions, in 6 of 8 patients with delayed reactions, and in 4 of 11 control subjects. IL-4 mRNA expression was induced in 5 of 11 allergic individuals with immediate reactions but in none of the 8 patients with delayed responses and none of the 11 control subjects. IL-4 mRNA expression was only induced following activation with free drugs, while IFN-gamma mRNA expression was predominantly induced in CD4+ T cells following stimulation with HSA-conjugated drugs. Immediate-type hypersensitivity to beta-lactams was not associated with a pure type 2-like response when PBMCs were specifically stimulated in vitro: Some patients with well-documented history of beta-lactam-induced immediate allergic reaction showed a high IFN-gamma response. Contact dermatitis involved Tc1 and Th1 cells and other delayed hypersensitivity reactions to beta-lactams were restricted to Th1 responses.

Adolescent↗

PubMed1999

Binding of IL-4 to the IL-13Ralpha(1)/IL-4Ralpha receptor complex leads to STAT3 phosphorylation but not to its nuclear translocation.

Interleukin-4 (IL-4) is a pleiotropic cytokine, which acts on both hematopoietic and non-hematopoietic cells, through different types of receptor complexes. In this study, we report that in human B cells, IL-4 caused rapid phosphorylation of Janus kinase (JAK) 1 and JAK3 tyrosine kinases. In keratinocytes, the hematopoietic-specific receptor common gamma(c) chain is not expressed and the IL-13 receptor alpha(1) (IL-13Ralpha(1)) participates in IL-4 signal transduction. In keratinocytes, IL-4 induced JAK1 and JAK2 phosphorylation but, unlike in immune cells, IL-4 did not involve JAK3 activation for its signaling. In both cell types, IL-4 induced phosphorylation and DNA binding activation of the signal transducer and activator of transcription (STAT) 6 protein. Furthermore, IL-4 stimulation of keratinocytes also induced tyrosine phosphorylation of STAT3 which was found to bind to the phosphorylated IL-13Ralpha(1). STAT3 however did not significantly translocate to the nucleus, nor did it bind with high affinity to target DNA sequences.

Cell Line↗

PubMed1999

Involvement of small GTPases in Mycoplasma fermentans membrane lipoproteins-mediated activation of macrophages.

Mycoplasma fermentans lipoproteins (LAMPf) are capable of activating macrophages and inducing the secretion of proinflammatory cytokines. We have recently reported that mitogen-activated protein kinase (MAPK) pathways and NF-kappaB and activated protein 1 (AP-1) play a crucial role in the activation induced by this bacterial compound. To further elucidate the mechanisms by which LAMPf mediate the activation of macrophages, we assessed the effects of inhibiting small G proteins Rac, Cdc42, and Rho. The Rho-specific inhibitor C3 enzyme completely abolished the secretion of tumor necrosis factor alpha by macrophages stimulated with LAMPf and also inhibited the activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 kinase. In addition, we have shown that LAMPf stimulate Cdc42 and that inhibition of Cdc42 or Rac by dominant negative mutants abrogates LAMPf-mediated activation of JNK and transactivation of NF-kappaB and AP-1 in the murine macrophage cell line RAW 264.7. These results indicate that small G proteins Rho, Cdc42, and Rac are involved in the cascade of events leading to the macrophage activation by mycoplasma lipoproteins.

Animals↗

PubMed1999

Biochemical analysis of interleukin-2 receptor beta chain phosphorylation by p56(lck).

Tyrosine phosphorylation of multiple proteins, including the receptor itself, is an initial event in IL-2 signaling and leads to recruitment of SH2 or PTB domain-containing proteins to the receptor. In this study, we have used subdomains of the IL-2 receptor beta chain (IL-2Rbeta) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56(lck), one of the critical tyrosine kinases activated by IL-2. We report that recombinant p56(lck) phosphorylates in vitro tyrosine residues within the IL-2Rbeta chain but not those within the IL-2Rgamma chain. p56(lck) phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL-2Rbeta chain acidic subdomain. Interestingly, phosphorylation of Tyr-358 appears to require the presence of either Tyr-355 or Tyr-361. p56(lck) also phosphorylates very efficiently the two tyrosines present in the IL-2Rbeta chain C-terminal region, Tyr-392 and Tyr-510. We also investigated the association of p56(lck) with the IL-2Rbeta chain which was found to depend on a short stretch of the IL-2Rbeta chain acidic subdomain, and to be independent of the presence of its tyrosine residues.

Base Sequence↗