Investigating integrin αMβ2 outside-in signaling in human monocytes
Date of Issue2014
School of Biological Sciences
Integrins are type I transmembrane and α/β heterodimeric adhesion receptors that mediate the interaction of cells and the extracellular matrix (ECM) or adjacent cells. They are expressed on most eukaryotic cells and involved in diverse biological functions, such as immune defense, cell growth and differentiation, hemostasis, bone resorption, tissue development, and neural functions. Although integrins do not have catalytic activities, they are bidirectional signaling receptors since their cytoplasmic tails are docking sites for many cytoplasmic molecules. Integrin αMβ2 is mainly expressed on cells of the myeloid lineage, natural killer (NK) cells and γδT cells. It plays crucial roles in cell adhesion, degranulation and phagocytosis. Integrin αMβ2 ligation-triggered intracellular signals are important for cell migration, cell growth and cell differentiation. This thesis describes the investigation of integrin αMβ2 outside-in signaling pathway. The results of this thesis contain three sections. In the first section (3.1), we provide data showing that protein kinase C (PKC)δ mediates integrin αMβ2 outside-in signaling in monocytes. Integrin αMβ2 clustering induces the translocation of PKCδ to the plasma membrane and PKCδ phosphorylation at Tyr311. PKCδ is activated by the src family kinases (SFKs), such as Hck and Lyn. We also show that the integrin β2 sequence Asn727-Ser734 is important in integrin αMβ2-induced PKCδ Tyr311 phosphorylation. We found that clustering of integrin αMβ2 induces down-regulation of forkhead box P1 (Foxp1), which is a transcription factor involved in monocyte differentiation. The expression of Foxp1 was reduced in monocytes following plating of cells on human lung microvascular endothelial (HMVE) cells. This was not observed in PKCδ-targeting small interfering RNA (siRNA)-treated monocytes. These results support a role for PKCδ in integrin αMβ2-induced Foxp1 regulation in monocytes. In the second section (3.2), we examined the activation of signaling conduits downstream of PKCδ that may lead into Foxp1 down-regulation. We found that mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinase (Erk) kinase (MEK)-Erk pathway is involved in integrin αMβ2-induced Foxp1 regulation. We also examined the molecules that are involved in the cross-talk between integrin αMβ2 signaling and lipopolysaccharide (LPS)-induced Toll-like receptor (TLR) signaling. These molecules include interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK1), PKCδ and MAPKs (such as Erk, c-Jun N-terminal kinase (JNK), and p38). We show that PKCδ interacts with IRAK1 and induces IRAK1T209 phosphorylation. These results suggest that the cross-talk between integrin αMβ2 signaling and LPS-induced signaling is involved in the interaction between PKCδ and IRAK1. In the third section (3.3), we show that the 4.1-Ezrin-Radixin-Moesin (FERM)-containing cytoplasmic protein kindlin3 is required for integrin αMβ2 outside-in signaling that modulates cell spreading. Reduced kindlin3 expression in K562 cells expressing wild type (WT) integrin αMβ2 or constitutively activated integrin αMβ2N329S abrogated the adhesion and spreading capacities of these cells on ligands inactive C3b (iC3b) and bovine serum albumin (BSA). We also show that kindlin3 is involved in the integrin αMβ2-spleen tyrosine kinase (Syk)-Vav1 signaling axis regulating the activities of Rac1 and Cdc42. These results provide further evidence that kindlin3 plays an important role in integrin outside-in signaling.
DRNTU::Science::Biological sciences::Molecular biology