Abstract
While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post–T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3′-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal–regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1–ERK1/2–Elk1 signaling required for optimal proliferation.
Highlights
T-cell lymphocytes are activated through the T-cell receptor (TCR) together with coreceptors such as CD28, which triggers cellular proliferation as a key step in promoting adaptive immunity [1]
Mechanistic data suggest that miR-15/16 can function as a tumor suppressor by directly targeting B-cell lymphoma 2 and affecting survival [17], but signaling molecules and pathways regulated by this miR pair in activated T cells may likely be different
By applying -omics and array approaches to evaluate signaling pathways regulated during T-cell activation, we established that mitogen-activated protein kinase kinase 1 (MEK1) and ERK1 are directly targeted by miR15a/16 and the MEK1–extracellular signal–regulated protein kinase 1/2 (ERK1/2)–Elk1 pathway as an important proproliferation pathway that is regulated by this miR cluster
Summary
Frank Urena1,2,‡ , Chi Ma1,‡, FuKun W. Khadka , Youping Deng, and Peter R. Hoffmann1,* From the 1Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA; 2Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA; 3Department of Anatomy, Physiology and Biochemistry, and 4Department of Quantitative Health Sciences, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA
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