Chemokine signaling via PTX-insensitive G proteins : activation of transcription factors and chemotaxis

Abstract

Chemokine receptors are members of the seven-transmembrane-domain G protein-coupled receptors. Previous studies have shown that CC chemokine receptors, such as CCR1 and CCR2b, could utilize Gα14 and Gα16 to activate phospholipase Cβ (PLCβ) and induce extracellular signal-regulated kinase (ERK) phosphorylation via Gα14 in cotransfection systems. The aim of this research is to investigate whether CCR1-mediated Gα14 or Gα16 activation can modulate the activities of other mitogen-activated protein kinases (MAPKs), signal transducer and activator of transcription 3 (STAT3), inhibitor κB kinase (IKK) and nuclear factor κB (NF-κB) in cotransfected HEK293 cells, and chemotaxis of monocytic THP-1 cells expressing CCR1, CCR2b, Gα14 and Gα16. The phosphorylations of c-Jun N-terminal kinase (JNK), p38 MAPK, STAT3, IKK and NF-κB was identified by Western blotting analysis while cell migration was determined using transwell chemotaxis plate. It was found that both CCR1 and CCR2b-mediated chemotaxis could not be completely blocked by pertussis toxin (PTX) pretreatment in THP-1 cells, suggesting the involvement of PTX-insensitive G proteins. CCR1 and CCR2b agonists were less efficacious in mediating PTX-insensitive chemotaxis of THP-1 cells transfected with small interfering RNA (siRNA) against Gα16, showing the participation of G16 in CCR1 and CCR2b agonist-induced cell migration. The CCR1 agonist also activated chemotaxis of PMA-pretreated Jurkat T cells and butyric acid-pretreated HL-60 leukemic cells. The cell movement was resistant to PTX pretreatment in both cells, demonstrating the involvement of PTX-insensitive G proteins. In addition, it was illustrated that CCR1 could activate JNK, p38 MAPK, IKK, NF-κB and STAT3 at both Tyr705 and Ser727 via G14 and G16 in cotransfected HEK293 cells. The CCR1 agonist-enhanced phosphorylations of IKK and STAT3 at Tyr705 through G14 and G16 was dependent on Raf-1, mitogen-activated protein kinase kinase 1/2, PLCβ, protein kinase C, calmodulin, calmodulin-dependent kinase II and c-Src. The restrictive expression of G14 and G16 may facilitate chemokines to induce local expression of cytokine or chemokine genes in specific organs or tissues.

Date of Award	2007
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology