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Name of Direct Supervisor: Manuel Fresno, MD
Location: Universidad Autónoma de Madrid
Descriptive title of the research activity: The role of prostanoids in migration and inflammation.
Overall goals: The main goal is to study the regulation of leukocyte migration by different prostanoids and their influence on leukocyte response to chemokines and expression of chemokine receptors.
Rationale and Significance: Besides being a proinflammatory enzyme, COX-2 plays a very important role in proliferation, cell transformation and tumor migration and is the targe of the non-steroidal anti-inflammatory drugs (NSAIDS). Recent evidences indicate that migration of dendritic cells and other cell types in response to chemokines is blocked by NSAIDs and that PGE2 plays an obligatory role in this process. Moreover, we have also found that chemokines (SDF-1, Rantes, MIP1a, among others) induce COX-2 expression in macrophages. Transcriptional regulation of mouse COX-2 expression is mediated by different regulatory elements such as NF-B, NF-IL6 (C/EBP), CREB, AP-1 and NFAT, promoter sequence. We have found that some chemokines induce COX-2 via AP-1. On the other hand, COX-2 overexpression is able to induce NF-kB activation which is required for the expression of some chemokines. The role of G-protein coupled kinases (GRKs) in the desensitization and regulation of chemokine receptors has been described. However, the potential contribution of COX-2/prostaglandin pathways to the regulation of GRK levels has not been explored in detail.
Description of work: We will study the role of COX-2, PGs synthases and the PGs as well as NSAIDS in the function and migration of lymphocytes and monocytes. Specifically, we will address the role of COX-2, PG synthases (hematopoeitc PGDS and mPGES-1) and their metabolites in the expression of adhesion molecules and in the adhesive and migratory behaviour of lymphocytes and monocytes. For this, we can use wt or COX-2-/- leukocytes and alternatively cell lines or primary leukocytes in which some of those genes are silenced by RNAi. On the other hand, PGD is synthesized from PGH2 supplied by Cox-2 by PGD synthase, PGD can spontaneously transform in 15deoxy12,14 PGJ2 (PGJ2) which have anti-inflammatory properties. Hence, PGJ2 may have the opposite effect than PGE2 and reduce migration although this has not been proven yet. We will test PGJ2 on migration and silence PGDS expression in cell lines or primary leukocytes by siRNA. GRKs modulate migration by desensitizing chemokine receptors and GRK2 heterozygous knockout mice display an enhanced migration of leukocytes. In this context, we would be interested in addressing the potential contribution of the COX-2/prostaglandin pathway to the regulation of GRK levels in leukocytes. The molecular pathways by which chemokines signals to Cox-2 and PG synthases will be addressed in collaboration with Dr. Mayor. The effect of different PGs (and of altering Cox/PGDS/mPGES-1 expression) on GRK2, 5 and 6 protein and mRNA levels in lymphocytes, monocytes and endothelial cell models will be addressed by using the experimental approaches already established in Dr. Mayor’s laboratory.
Methodology: this work will make use of inducible expression vectors (variants of the Tet-on vector) to obtain a timely control of the expression of genes, such as PG synthases, whose downstream effects are both rapid and reversible. Detailed morphological analysis will be performed by scanning confocal as well as by deconvolution microscopy.
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