Date of Award
Open Access Thesis
Medical Doctor (MD)
The goal of this project is to elucidate the signaling pathway involved in the T cell integrin lymphocyte function-associated antigen 1 (LFA-1) engagement-mediated, HuR nuclear-to-cytoplasmic translocation and subsequent mRNA stabilization. To examine the pathway transducing LFA-1 engagement-mediated mRNA stabilization, we employed quantitative, real time RT-PCR in the presence of a transcriptional inhibitor to examine the half-life of interferon gamma (IFN-γ) mRNA, a labile cytokine transcript. We used immunofluorescence to observe the nuclear-to-cytoplasmic tranlocation of HuR, an mRNA-binding and stabilizing protein. We also used Western blot analysis to analyze the activity levels of specific mitogen activated protein kinases (MAPKs) upon LFA-1 engagement. These techniques were performed using murine splenocyte-derived T cells from wild type as well as transgenic mice lacking one or both copies of the MAPK kinase MKK3 gene. They were also performed in the presence of MAPK pharmacologic inhibitors, RNA interference for HuR, and transfection vectors for constitutively active Rho-GTPases and MAPKs. Our data show that, in murine wild type T cells, LFA-1 engagement by its ligand, intercellular adhesion molecule-1 (ICAM-1), causes both HuR translocation and an increased half-life of IFN-γ mRNA transcripts. This mRNA stabilization is HuR-dependent: a decrease in HuR expression in T cells by RNA interference abrogates the LFA-1 induced IFN-γ mRNA stabilization. To examine the pathway responsible for LFA-1 engagement-mediated mRNA stabilization, we looked at MAPKs since T cell integrin engagement triggers mitogenesis. LFA-1 engagement activates both c-jun NH2 terminal protein kinase (JNK), and stress activated protein kinase (SAPK) (also known as the p38 MAPK). Pharmacologic inhibition of JNK, however, causes no decrease in HuR translocation or IFN-γ mRNA stabilization upon T cell integrin engagement, while pharmacologic inhibition of p38 MAPK inhibits both events. Interruption of the p38 MAPK pathway by using T cells lacking the p38 MAPK kinase MKK3 also causes a loss of HuR translocation and subsequent IFN-γ mRNA stabilization upon LFA-1 engagement. Activation of the p38 MAPK pathway alone via a constitutively active p38 MAPK kinase MKK6, however, was not sufficient to obtain IFN-γ mRNA stabilization. From these data, we conclude that T cells utilize LFA-1 engagement by ICAM-1 to modulate the stability of labile mRNA transcripts encoding proinflammatory cytokines. Modulation of the RNA binding molecule HuR is required for this mRNA stabilization. Furthermore, the MKK3/p38 MAPK pathway is necessary for LFA-1 engagement-mediated mRNA stabilization. Activation of the p38 MAPK pathway alone, however, is not sufficient to generate the stabilization of labile, proinflammatory cytokine transcripts.
DeGregorio, Scott David, "T Cell Integrin Dependent, MKK3/p38 Mapk-Mediated Stabilization of Labile Cytokine mRNAs" (2006). Yale Medicine Thesis Digital Library. 229.