Ehud Razin

Role of Platelet-Activating Factor in Ulcerative Colitis

Platelet-activating factor is released from inflammatory cells. It activates neutrophils, releases secondary messengers, and mediates mucosal ulceration and ischemia in the rat. We assessed its possible role in the pathogenesis of ulcerative colitis. Colonic biopsy specimens from patients with active ulcerative colitis and controls were incubated for 4 h in Tyrodes buffer in the presence or absence of 0.2 microM calcium ionophore (A23187) or 50 microliter of antihuman immunoglobulin E. Platelet-activating factor was determined in the tissue by aggregation assay after extraction with 80% ethanol and was confirmed by thin-layer chromatography and its inactivation by phospholipases. Platelet-activating factor was not detected in normal mucosa. Only A23187 and antihuman immunoglobulin E stimulated its activity: mean +/- SE, 43.2 +/- 8.6 and 33.0 +/- 6.1 pg/10 mg wet wt, respectively. In active ulcerative colitis basal platelet-activating factor activity was 8.9 +/- 3.5 pg/10 mg wet wt. A23187 and antihuman immunoglobulin E induced significantly higher stimulation of platelet-activating factor synthesis when compared with their effects on normal mucosa: 200 +/- 28 and 70 +/- 8.3 pg/10 mg wet wt, respectively. The enhanced stimulation induced by A23187 was dose-dependently inhibited by salazopyrine, 5-amino-salicylic acid, and prednisolone, but not by sulfapyridine. It is thus suggested that platelet-activating factor may be involved in the pathogenesis of the inflammatory response in ulcerative colitis and that its inhibition by steroids, 5-aminosalicylic acid, and salazopyrine may be an additional mechanism to explain their therapeutic effects.

The Function of Lysyl-tRNA Synthetase and Ap4A as Signaling Regulators of MITF Activity in FcϵRI-Activated Mast Cells

The involvement of microphthalmia transcription factor (MITF) in the function of mast cells, melanocytes, and osteoclasts has recently started to be investigated in depth. In a previous study, we found Hint to be associated with MITF in mast cells and showed that it suppresses MITFs transcriptional activity. Here, we have found that lysyl-tRNA synthetase (LysRS) is also associated with MITF and forms a multicomplex with MITF and Hint. We have also shown that Ap4A, an endogenous molecule consisting of two adenosine linked by four phosphate which is known to be synthesized by LysRS, is accumulated intracellularily above 700 microM in IgE-Ag-activated mast cells, binds to Hint, liberates MITF, and thus leads to the activation of MITF-dependent gene expression. This implies that LysRS plays a key role via Ap4A as an important signaling molecule in MITF transcriptional activity.

LysRS serves as a key signaling molecule in the immune response by regulating gene expression.

Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.

Structural Switch of Lysyl-tRNA Synthetase between Translation and Transcription

Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207 phosphorylation provokes a new conformer of LysRS that inactivates its translational function but activates its transcriptional function. The crystal structure of an MSC subcomplex established that LysRS is held in the MSC by binding to the N terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap(4)A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription.

Hierarchical Network between the Components of the Multi-tRNA Synthetase Complex IMPLICATIONS FOR COMPLEX FORMATION

The macromolecular tRNA synthetase complex consists of nine different enzymes and three non-enzymatic factors. This complex was recently shown to be a novel signalosome, since many of its components are involved in signaling pathways in addition to their catalytic roles in protein synthesis. The structural organization and dynamic relationships of the components of the complex are not well understood. Here we performed a systematic depletion analysis to determine the effects of structural intimacy and the turnover of the components. The results showed that the stability of some components depended on their neighbors. Lysyl-tRNA synthetase was most independent of other components for its stability whereas it was most required for the stability of other components. Arginyl- and methionyl-tRNA synthetases had the opposite characteristics. Thus, the systematic depletion of the components revealed the functional reason for the complex formation and the assembly pattern of these multi-functional enzymes and their associated factors.

Interplay between MITF, PIAS3, and STAT3 in Mast Cells and Melanocytes

ABSTRACT Microphthalmia transcription factor (MITF) and STAT3 are two transcription factors that play a major role in the regulation of growth and function in mast cells and melanocytes. In the present study, we explored the MITF-PIAS3-STAT3 network of interactions, how these interactions regulate gene expression, and how cytokine-mediated phosphorylation of MITF and STAT3 is involved in the in vivo interplay between these three proteins. In NIH 3T3 cells stimulated via gp130 receptor, transfected MITF was found to be phosphorylated at S409. Such phosphorylation of MITF leads to PIAS3 dissociation from MITF and its association with STAT3. Activation of mouse melanoma and mast cells through gp130 or c-Kit receptors induced the mobilization of PIAS3 from MITF to STAT3. In mast cells derived from MITF di/di mice, whose MITF lacks the Zip domain (PIAS3-binding domain), we found downregulation in mRNA levels of genes regulated by either MITF or STAT3. This regulatory mechanism is of considerable importance since it is likely to advance the deciphering of a role for MITF and STAT3 in mast cells and melanocytes.

Nonconventional Involvement of LysRS in the Molecular Mechanism of USF2 Transcriptional Activity in FcεRI-Activated Mast Cells

ABSTRACT Reports of the biological multifunctional activity of various aminoacyl tRNA synthetases have recently accumulated in the literature. The primary function of these critical enzymes is to charge various tRNAs with their appropriate amino acids, thus producing the building blocks of protein synthesis. We have previously shown that lysyl tRNA synthetase (LysRS) associates with microphthalmia transcription factor (MITF) and regulates its activity by synthesis of Ap4A in mast cells. Here, we show for the first time that LysRS associates with another transcription factor, USF2, which unlike MITF, is ubiquitously expressed in eukaryotic cells. Using mast cells, we have found that USF2 is negatively regulated by Hint and Ap4A acts as a positive regulator of USF2 by a molecular mechanism similar to that described for MITF. Since USF2 plays a significant role in a variety of cellular functions, our finding suggests that LysRS and Ap4A may be involved in general regulation of gene transcription.

Signal transduction in mast cells and basophils

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c-Fos as a Regulator of Degranulation and Cytokine Production in FcεRI-Activated Mast Cells

The AP-1 complex is composed of c-Jun and c-Fos and is a key component in the regulation of proinflammatory genes. Mast cells play a significant role in the initiation of many inflammatory responses, such as allergy and allergy-associated diseases. In the present work, we characterized the role of c-Fos in mast cell function by investigating IL-3-dependent cell proliferation, degranulation capability, and cytokine expression in c-Fos-deficient mice. In c-Fos-deficient mast cells, we found that FcεRI-mediated degranulation was significantly inhibited, which correlates with the reduced expression of SWAP-70, VAMP-7, and Synaptotagmin I genes, which are involved directly in the degranulation process. These findings show that c-Fos plays an important role in FcεRI-mediated regulation of mast cell function.

Signal transduction in the activation of mast cells and basophils

Abstract Mast cells are gaining recognition as participants in may inflammatory responses in addition to their well-documented role in anaphylaxis. However, the biochemical pathways underlying the ability of extracellular stimuli to activate intracellular events still require resolution. This report, from a recent workshop 1