Toll-like receptor stimulation differentially regulates vasoactive intestinal peptide type 2 receptor in macrophages
Juan Luis Herrera
CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Seville, Spain
Search for more papers by this authorElena Gonzalez-Rey
CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Seville, Spain
Search for more papers by this authorRafael Fernandez-Montesinos
CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Seville, Spain
Search for more papers by this authorFrancisco J. Quintana
Center for Neurologic Diseases, Harvard Medical School, Boston, MA, USA
Search for more papers by this authorRafael Najmanovich
European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, UK
Search for more papers by this authorCorresponding Author
David Pozo
CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Seville, Spain
Correspondence to: David POZO, Ph.D., M.Sc., CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Américo Vespucio Ave, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.Tel.: +34-95-446 7841Fax: +34-95-446 1664E-mail: [email protected]Search for more papers by this authorJuan Luis Herrera
CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Seville, Spain
Search for more papers by this authorElena Gonzalez-Rey
CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Seville, Spain
Search for more papers by this authorRafael Fernandez-Montesinos
CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Seville, Spain
Search for more papers by this authorFrancisco J. Quintana
Center for Neurologic Diseases, Harvard Medical School, Boston, MA, USA
Search for more papers by this authorRafael Najmanovich
European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, UK
Search for more papers by this authorCorresponding Author
David Pozo
CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Seville, Spain
Correspondence to: David POZO, Ph.D., M.Sc., CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO-Junta de Andalucia), Américo Vespucio Ave, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.Tel.: +34-95-446 7841Fax: +34-95-446 1664E-mail: [email protected]Search for more papers by this authorAbstract
Vasoactive intestinal peptide (VIP) was originally isolated as a vasodilator intestinal peptide, then as a neuropeptide. In the immune system, VIP is described as an endogenous macrophage-deactivating factor. VIP exerts its immunological actions in a paracrine and/or autocrine manner, through specific receptors. However, very little is known about the molecular regulation of VIP type 2 receptor (VPAC2) in the immune system. We now report that different toll-like receptor (TLR) ligands selectively regulate the VPAC2 receptor gene and show a gene repression system controlled by key protein kinase signalling cascades in macrophages. VPAC2 gene expression is regulated by gram-positive (TLR2 ligands) and gram-negative bacteria wall constituents (TLR4 ligands). Moreover, VPAC2 is tightly regulated: TLR2- or TLR2/6- but not TLR2/1-mediated mechanisms are responsible for the induction of VPAC2. TLR stimulation by viral or bacterial nucleic acids did not modify the VPAC2 mRNA levels. Remarkably, imiquimod – a synthetic TLR7 ligand – led to a potent up-regulation of VPAC2 gene expression. TLR5 stimulation by flagellin present in gram-positive and gram-negative bacteria did not affect VPAC2 mRNA. The p38 mitogen-activated protein kinase (MAPK) activity accounted for the TLR4-mediated induction of VPAC2 gene expression. Surprisingly, our data strongly suggest for the first time a tightly repressed control of VPAC2 mRNA induction by elements downstream of MAPK kinase 1/2, PI3K/Akt, and particularly Jun-NH2-terminal kinase signalling pathways.
Supporting Information
Fig. S1 Effects of TLR ligands and protein kinase inhibitors on cytotoxicity. Raw 264.7 cells were previously seeded into 96-well plates to a final volume of 100 μl. After 24-hrs treatment with protein kinase inhibitors, LDH release was calculated according to the following equation: LDH release ∇ [LDH supernatant/(LDH supernatant + LDH cells)]*100. The proteasome inhibitor MG-132 (6 μM) was used as a positive control for cytotoxicity. Results are the mean ± S.D. of two independent experiments performed in duplicate.
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