STAT3-Stathmin Interactions Control Microtubule Dynamics in Migrating T-cells

Abstract

T-cell migration is a complex highly coordinated process that involves cell adhesion to the high endothelial venules or to the extracellular matrix by surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. The mechanism(s) that regulates T-cell migration is of considerable relevance for understanding the pathogenesis of various diseases, such as chronic inflammatory diseases and cancer metastasis. This study was designed to identify potential involvement of STAT3, a latent transcription factor, in mediating integrin-induced T-cell migration. Using our previously characterized in vitro model for lymphocyte migration, we demonstrate that STAT3 is activated and translocated to the nucleus during the process of active motility of Hut78 T-lymphoma cells triggered via LFA-1. Blocking STAT3 signaling by multiple approaches inhibited LFA-1-induced T-cell locomotion via destabilization of microtubules and post-translational modification of tubulin. Here, we show that STAT3 physically interacts with stathmin to regulate microtubule dynamics in migrating T-cells. These observations strongly indicate that STAT3 is critically important for T-cell migration and associated signaling events.Efficient operation of the adaptive immune system requires migration of T-lymphocytes from the vascular compartment across tissue barriers and through the extracellular matrix. This process involves a series of integrin ligand-receptor interactions (1) that initially retards lymphocyte flow and ultimately leads to arrest and diapedesis across the endothelium (2, 3). T-cells utilize the integrin, lymphocyte function-associated antigen-1 (LFA-1), 3 when migrating in response to chemoattractants across the vasculature into lymph nodes or inflamed tissues (1, 4, 5). By engagement with ligands from the intercellular adhesion molecule group (ICAMs), in particular ICAM-1, LFA-1 also provides a strong adhesive force to promote and stabilize T-cell and antigen-presenting cell conjugate formation. We have demonstrated that LFA-1 transduces a variety of transmembrane signals in crawling T-cells involving protein kinase C activation and cytoskeletal rearrangement (4, 6 -9). However, the exact sequence of downstream integrin-mediated signaling events resulting in cytoskeletal rearrangements and cell locomotion is not fully understood.T-cell migration involves cross-talk between integrins and the cytoskeleton, coordinated changes in the cytoskeleton, and the controlled formation and dispersal of adhesion sites (10). Motile lymphocytes develop trailing extensions, which contain cytoskeletal and signaling elements (11). Microtubules (MTs) are essential components of the cytoskeleton and are important for many aspects of mammalian cell responses, including cell division, growth, migration, and signaling (12)(13)(14). Whereas the actin cytoskeleton provides the driving force at the cell front, the MT network assumes a regulatory function in coordinating rear retraction (15). MT retraction into the ...

The microtubule targeting agent PBOX-15 inhibits integrin-mediated cell adhesion and induces apoptosis in acute lymphoblastic leukaemia cells

“…Adhesion of cells on anti-integrin antibody or fibronectin coated surfaces was detected and subsequently quantified using a cell based high content analysis (HCA) system. The assay operates on the principle of fully automated fluorescent microscopy described previously (30)(31)(32) and allows multiplexing of key reporter parameters including cell-adhesion. Untreated or PBOX-15 pre-treated cells were added to fibronectin, anti-α4-, β1-, or β2-integrin antibody coated 24-well or 96-well plates (Nunc, Roskilide, Denmark) at a density of 2x10 5 or 1x10 4 cells/well respectively.…”

Analysis of dynamic tyrosine phosphoproteome in LFA‐1 triggered migrating t‐cells

“…Involvement of a number of serine/threonine kinases including protein kinase C and myosin light chain kinase in T-cell migratory processes has previously been established (Volkov et al, 1998(Volkov et al, , 2001Smith et al, 2003). Of note, other tyrosine kinase inhibitors including Herbimycin A (a broad-spectrum inhibitor of non-receptor tyrosine kinase), piceatannol (a Syk-selective tyrosine kinase inhibitor) and a dominant negative ZAP-70 tyrosine kinase have been shown to significantly reduce LFA-1 triggered T-cell migratory processes by our research group (Freeley et al, 2010;Verma et al, 2009;Kelleher et al, 1995) and others (Soede et al, 1998;Wang et al, 2009). Collectively these findings suggest that early tyrosine phosphorylation of proteins is an important event in T-cell migration.…”

“…Cross-linking of T-cell surface LFA-1 integrin receptor by mAb mimicking to a certain extent multivalent interactions with natural ligands has been successfully used as a model to study intracellular signalling processes (Volkov et al, 2001, Homasany et al, 2005Verma et al, 2009). Antibody-induced effects in this case are judged by morphological changes in the cells displaying migratory phenotypes and cytoskeletal rearrangement (Volkov et al, 2001, Verma et al, 2008.…”

“…has recently been shown to be essential for Jak-STAT signalling (Schoof et al, 2009), which is important for T-cell migration (Verma et al, 2009). The 14-3-3 protein identified in this study has been demonstrated to interact and regulate phosphatidylinositol 3-kinase (Bonnefoy-Berard et al, 1995), Tau (Chun et al, 2004;Yuan et al, 2004;Li and Paudel, 2007), 3BP2/SH3BP2 adaptor protein (Foucault et al, 2003) and PKC( ) (Robinson et al, 1994;Kim et al, 2005;Aitken, 2006;Meller et al, 1996), an enzyme crucial for cytoskeletal rearrangements in lymphocyte migration (Volkov et al, 1998(Volkov et al, , 2001).…”

“…The nodes added were restricted to proteins within our list and those which are directly interacting with each other. Some additional nodes such as cofilin1, STAT3 and stathmin previously demonstrated to be involved in LFA-1 signalling (Verma et al, 2009;Burkhardt et al, 2008) were subsequently added to further enrich the network (Fig. 8).…”