Yolanda Bayón

Signaling mechanisms involved in the activation of arachidonic acid metabolism in human astrocytoma cells by Tumor necrosis factor-α : Phosphorylation of cytosolic phospholipase A2 and transactivation of cyclooxygenase-2

Abstract : Tumor necrosis factor‐α (TNF‐α) is a cytokine that elicits cell responses by activating the mitogen‐activated protein kinase (MAP kinase) cascade and transcription factors such as nuclear factor‐kB (NF‐kB). As these elements play a central role in the mechanisms of signaling involved in the activation of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase‐2 (COX‐2), the effect of TNF‐α on arachidonate (AA) metabolism in 1321N1 astrocytoma cells was assayed. TNF‐α produced a phosphorylation of cPLA2, which was preceded by an activation of both c‐Jun N‐terminal kinase (JNK) and p38‐MAP kinase, and this was associated with the release of [3H]AA. In contrast, TNF‐α did not activate the extracellular signal‐regulated kinase (MAP kinase) p42, nor did it elicit a mitogenic response. Analysis of [3H]AA metabolites by reverse‐phase HPLC showed that all of the [3H]AA released during the first hour after TNF‐α addition eluted as authentic AA, whereas in samples obtained at 24 h after addition of TNF‐α, 25% of the [3H]AA had been converted into COX products as compared with only 9% in control cells. In keeping with these findings, TNF‐α produced an increase of COX‐2 expression, as judged from both RT‐PCR studies and immunoblot of COX‐2 protein, and a long‐lasting activation of NF‐kB. These data show that TNF‐α produces in astrocytoma cells an early activation of both p38‐MAP kinase and JNK, which is followed by the phosphorylation of cPLA2 and the release of AA. On the other hand, the activation of NF‐kB may explain the induction of the expression of COX‐2 and the delayed generation of prostanoids.

4-trifluoromethyl derivatives of salicylate, triflusal and its main metabolite 2-hydroxy-4-trifluoromethylbenzoic acid, are potent inhibitors of nuclear factor κB activation

The effect of two derivatives of salicylate, 2‐hydroxy‐4‐trifluoromethylbenzoic acid (HTB) and 2‐acetoxy‐4‐trifluoromethylbenzoic acid (triflusal), on the activation of NF‐κB elicited by tumour necrosis factor‐α (TNF‐α) on human umbilical vein endothelial cells (HUVEC) was tested. The expression of the mRNA of vascular cell adhesion molecule‐1 (VCAM‐1) was studied as an example of a gene the expression of which is regulated by NF‐κB. To extend these findings to other systems, the induction of nitric oxide synthase in rat adherent peritoneal macrophages was studied. Both HTB and triflusal were more potent than aspirin or salicylate as inhibitors of the nuclear translocation of NF‐κB. The calculation of the IC50 values showed ∼amp;2 mM for HTB, 4 mM for aspirin and >4 mM for salicylate. Comparison of the potency of these compounds on VCAM‐1 mRNA expression showed complete inhibition by both triflusal and HTB at a concentration of 4 mM whereas aspirin and salicylate produced only 36–43% inhibition at the same concentration. Inhibition of NF‐κB activation was also observed in rat peritoneal macrophages stimulated via their receptors for the Fc portion of the antibody molecule with IgG/ovalbumin immune complexes. This was accompanied by a dose‐dependent inhibition of nitrite production by the L‐arginine pathway via iNOS. IC50 values for this effect were 1.13±0.12 mM (triflusal), 1.84±0.34 (HTB), 6.08±1.53 mM (aspirin) and 9.16±1.9 mM (salicylate). These data indicate that the incorporation of a 4‐trifluoromethyl group to the salicylate molecule strongly enhances its inhibitory effect on NF‐κB activation, VCAM‐1 mRNA expression and iNOS induction, irrespective of the presence of the acetyl moiety involved in the inhibition of cyclo‐oxygenase.

KCTD5, a putative substrate adaptor for cullin3 ubiquitin ligases

Potassium channel tetramerization domain (KCTD) proteins contain a bric‐a‐brac, tramtrak and broad complex (BTB) domain that is most similar to the tetramerization domain (T1) of voltage‐gated potassium channels. Some BTB‐domain‐containing proteins have been shown recently to participate as substrate‐specific adaptors in multimeric cullin E3 ligase reactions by recruiting proteins for ubiquitination and subsequent degradation by the proteasome. Twenty‐two KCTD proteins have been found in the human genome, but their functions are largely unknown. In this study, we have characterized KCTD5, a new KCTD protein found in the cytosol of cultured cell lines. The expression of KCTD5 was upregulated post‐transcriptionally in peripheral blood lymphocytes stimulated through the T‐cell receptor. KCTD5 interacted specifically with cullin3, bound ubiquitinated proteins, and formed oligomers through its BTB domain. Analysis of the interaction with cullin3 showed that, in addition to the BTB domain, some amino acids in the N‐terminus of KCTD5 are required for binding to cullin3. These findings suggest that KCTD5 is a substrate‐specific adaptor for cullin3‐based E3 ligases.

Lipopolysaccharides of Brucella abortus and Brucella melitensis Induce Nitric Oxide Synthesis in Rat Peritoneal Macrophages

ABSTRACT Smooth lipopolysaccharide (S-LPS) and lipid A of Brucella abortus and Brucella melitensis induced the production of nitric oxide (NO) by rat adherent peritoneal cells, but they induced lower levels of production of NO than Escherichia coli LPS. The participation of the inducible isoform of NO synthase (iNOS) was confirmed by the finding of an increased expression of both iNOS mRNA and iNOS protein. These observations might help to explain (i) the acute outcome of Brucella infection in rodents, (ii) the low frequency of septic shock in human brucellosis, and (iii) the prolonged intracellular survival of Brucellain humans.

Coupling of C3bi to IgG inhibits the tyrosine phosphorylation signaling cascade downstream Syk and reduces cytokine induction in monocytes

The effect of coupling C3bi to immunoglobulin G (IgG) immune complexes (IC) on their ability to produce protein tyrosine phosphorylation and activation of the mitogen‐activated protein kinase (MAPK) and the Akt/protein kinase B (PKB) routes was assessedin human monocytes. Cross‐linking Fc receptors for IgG activated the protein tyrosine kinase Syk, phospholipases Cγ1 and Cγ2, the MAPK cascade, and the Akt/PKB route. Linkage of C3bi to the γ‐chain of IgG produced a decrease of the protein bands displaying tyrosine phosphorylation, whereas the MAPK cascades and the Akt/PKB route remained almost unaffected. Zymosan particles, which because of their β‐glucan content mimic the effect of fungi, produced a limited increase of tyrosine‐phosphorylated protein bands, whereas treatment of zymosan under conditions adequate for C3bi coating increased its ability to induce protein tyrosine phosphorylation. Noteworthy, this was also observed under conditions where other components of serum might be bound by zymosan particles, for instance, serum IgG, thereby suggesting their potential involvement in Syk activation. The induction of cytokines showed a changing pattern consistent with the changes observed in the signaling pathways. IC induced monocyte chemoattractant protein‐1 (MCP‐1)/CC chemokine ligand 2 (CCL2), interleukin (IL)‐1β, and eotaxin‐2/CCL24, which were not observed with C3bi‐coated IC. Zymosan induced the expression of tumor necrosis factor α (TNF‐α), TNF‐β, IL‐10, IL‐6, and MCP‐2/CCL8, whereas the cytokine signature of C3bi‐coated zymosan also included interferon‐inducible protein 10/CXC chemokine ligand 10, platelet‐derived growth factor‐BB, and I‐309/CCL1. Taken together, these findings indicate that C3bi targets the phagocytic cargo, and engagement or diversion of the Syk route determines the phagocyte response.

Characterization of New Substrates Targeted By Yersinia Tyrosine Phosphatase YopH

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.

Proline-serine-threonine phosphatase interacting protein 1 inhibition of T-cell receptor signaling depends on its SH3 domain

Proline‐serine‐threonine phosphatase interacting protein 1 (PSTPIP1) is an adaptor protein associated with the cytoskeleton that is mainly expressed in hematopoietic cells. Mutations in PSTPIP1 cause the rare autoinflammatory disease called pyogenic arthritis, pyoderma gangrenosum, and acne. We carried out this study to further our knowledge on PSTPIP1 function in T cells, particularly in relation to the phosphatase lymphoid phosphatase (LYP), which is involved in several autoimmune diseases. LYP–PSTPIP1 binding occurs through the C‐terminal homology domain of LYP and the F‐BAR domain of PSTPIP1. PSTPIP1 inhibits T‐cell activation upon T‐cell receptor (TCR) and CD28 engagement, regardless of CD2 costimulation. This function of PSTPIP1 depends on the presence of an intact SH3 domain rather than on the F‐BAR domain, indicating that ligands of the F‐BAR domain, such as the PEST phosphatases LYP and PTP‐PEST, are not critical for its negative regulatory role in TCR signaling. Additionally, PSTPIP1 mutations that cause the pyogenic arthritis, pyoderma gangrenosum and acne syndrome do not affect PSTPIP1 function in T‐cell activation through the TCR.

The Autoimmunity Risk Variant LYP-W620 Cooperates with CSK in the Regulation of TCR Signaling

The protein tyrosine phosphatase LYP, a key regulator of TCR signaling, presents a single nucleotide polymorphism, C1858T, associated with several autoimmune diseases such as type I diabetes, rheumatoid arthritis, and lupus. This polymorphism changes an R by a W in the P1 Pro rich motif of LYP, which binds to CSK SH3 domain, another negative regulator of TCR signaling. Based on the analysis of the mouse homologue, Pep, it was proposed that LYP and CSK bind constitutively to inhibit LCK and subsequently TCR signaling. The detailed study of LYP/CSK interaction, here presented, showed that LYP/CSK interaction was inducible upon TCR stimulation, and involved LYP P1 and P2 motifs, and CSK SH3 and SH2 domains. Abrogating LYP/CSK interaction did not preclude the regulation of TCR signaling by these proteins.

Immunoglobulin E-mediated anaphylaxis activates nuclear factor κB in rat small intestine

Abstract.Objective and Design: Since IgE-dependent reactions induce the inducible isoform of NO synthase, we postulated the involvement of the transcription factor NF-κB.¶Materials: 72 Wistar rats were divided into 6 groups and used to study the hemorrhagic necrosis of the small intestine elicited by anaphylaxis.¶Treatment: Passive anaphylaxis was produced by i.p. sensitization with IgE anti-dinitrophenyl monoclonal antibody and i.v. challenge with the cognate antigen.¶Methods: Competitive PCR was used to assay the expression of p50 subunit of NF-κB. κB-binding activity was assayed by electrophoretic mobility shift assay.¶Results: The PCR assay showed a time-dependent increase of mRNA coding for the p50 subunit of NF-κB, which was maximal 1 h after challenge (40 ± 3, versus 230 ± 32 f M, mean ± SEM) and decreased to prechallenge level at 4 h. κB-binding activity was also increased.¶Conclusions: IgE-mediated reactions trigger a pathway for nuclear signaling that seems to be related to the intermediate/late response of the allergic reaction.