Effect of 5‐lipoxygenase inhibitor MK591 on early molecular and signaling events induced by staphylococcal enterotoxin B in human peripheral blood mononuclear cells

Abstract

Staphylococcal enterotoxin B (SEB) is one of the many exotoxins produce by Staphylococcus aureus and is implicated in inducing diarrhea, vomiting, muscle numbness, possible involvement in autoimmune disorders and lethal shock [1]. The massive impact of T cell activation, proliferation, and cytokine production by CD4 + T cells via specific V b elements of T cell antigen receptor [2] has prompted a number of investigations to focus on the intricate signaling activities of SEB. Even though the molecular events of SEB-induced lethal shock in human peripheral blood mononuclear cell (PBMCs) are not very apparent, the actual response of lethal shock is expected to herald by changes in signaling pathways [3,4]. In mammalian cells, a variety of stimuli generate intracellular responses that converge on a limited number of components of multiple pathways [5]. Mitogen-activated protein kinase (MAPK) cascades together with arachidonic Staphylococcal enterotoxin B (SEB) has been the focus of a number of studies due to its ability to promote septic shock and a massive impact on the human immune system. Even though symptoms and pathology associated with SEB is well known, early molecular events that lead to lethality are still poorly understood. Our approach was to utilize SEB induced human peripheral blood mononuclear cells (PBMCs) as a prototype module to further investigate the complexity of signaling cascades that may ultimately lead to lethal shock. Our study revealed the activation of multiple divergent intracellular pathways within minutes of SEB induction including components that interconnect investigated pathways. A series of performed inhibi-tor studies identified a specific inhibitor of 5-LO (MK591), which has the ability to block JNK, MAPK, p38kinase and 5-LO signaling-cascades and drastically reducing the activity of pro-inflammatory cytokine TNF-a. Further evaluation of MK591 utilizing cell proliferation assays in PBMCs, human proximal tubule cells and in vivo studies (monkey) showed a decrease in cell proliferation. The inhibitory effect of MK591 was reconfirmed at a genetic level through the utilization of a set of SEB specific genes. Signaling activities, inhibitor studies, cellular analysis and gene expression analysis in unison illustrated the significance of pathway inter-connectors such as 5-LO as well as inhibiting such inter-connectors (using MK591) in SEB induced human PBMCs. Abbreviations 5-LO, 5-lipoxygenase; HIF, hypoxia-inducible factor; IL, interleukin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; p38kinase, p38-mitogen activated protein kinase; PBMC, peripheral blood mononuclear cell; REPTC, renal epithelial proximal tubular cell; SEB, staphylococcal enterotoxin B; TNF, tumor necrosis factor.

5-Lipoxygenase Activating Protein (FLAP) Dependent Leukotriene Biosynthesis Inhibition (MK591) Attenuates Lipid A Endotoxin-Induced Inflammation

“…They also demonstrated that inhibitors of the lipoxygenase pathway were able to block Staphylococcal enterotoxin B (SEB) induced ERK activation [25]. However, our studies show that MK591 pre-treatment enhanced and potentiated pERK activation.…”

Treatment of Epidermal Growth Factor (EGF) enhances Nuclear Maturation of Porcine Oocytes and Stimulates Expression of ER/Golgi Transport Proteins

“… Moreno et al (2002) had reported that treatment of brefeldin A, inhibitor of membrane trafficking, during in vitro maturation of mouse oocyte blocked nuclear maturation and this study indicated that membrane trafficking from ER to the Golgi apparatus is closely associated with oocyte maturation. As a one of transport protein, transport protein Sec61 subunit beta (Sec61β) is an important factor of the protein translocation of ER membrane and coatomer protein complex subunit gamma 2 (COPG2), a one of type-1 COP, is involved with retrograde transport of protein from Golgi apparatus to ER ( Mendis et al, 2008 ). These reports suggested that Sec61β and COPG2 are concerned with oocyte maturation through regulation of protein transport between Golgi apparatus and ER, however, roles of transport protein on oocyte maturation were not fully understood.…”

Structure-Based Discovery of Inhibitors of Microsomal Prostaglandin E2 Synthase−1, 5-Lipoxygenase and 5-Lipoxygenase-Activating Protein: Promising Hits for the Development of New Anti-inflammatory Agents

“…As preliminarily remarked, it should be put in evidence that compounds 4 and 7, inhibiting the two MAPEG family members, showed quite similar chemical features; on the contrary, the more encumbering ligand 20 seems to target no structurally related MAPEG enzymes. For our calculations, we used the 3D structure of FLAP in complex with the inhibitor 44 (MK-591, Chart 2) 52 solved by Ferguson et al 53 in 2007 [protein data bank (PDB) ID code 2Q7M]. Because of the lack of crystal structure information on 5-LO, we used a 15-LO 54 (PDB ID code 1LOX) enzyme, Taking into account the considerations reported above for the MGST-1 enzyme, also in the case of FLAP, the binding specificity was conferred by the H-bond with the Lys116.…”