Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: Role of Rho‐dependent mechanisms

Abstract

Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr(696) and Thr(850) resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly.

Suppression of endotoxin-induced inflammation by taxol

“…Although, the relationship between microtubules and the contractile cytoskeleton is not fully explored, the disruption of either microfilaments or microtubule structure can cause endothelial cell contraction and barrier dysfunction via alterations in the actomyosin cytoskeleton. Microtubule depolymerisation, using either nocodazole or vinblastine, in pulmonary endothelial cells is associated with myosin light-chain phosphorylation, stress fibre formation, contraction and barrier dysfunction [2,4]. These effects are linked to activation of the small Rho guanosine triphosphatase and its effector Rho kinase, and can be attenuated by cell pre-treatment with taxol, which promotes microtubule assembly [2,4].…”

“…The capacity of inhibitors of microtubule polymerisation to increase transendothelial permeability to solutes in vitro has been reported [2]. Conversely, microtubule stabilisation by taxol attenuates solute permeability across the endothelial monolayer and paracellular gap formation induced by tumour necrosis factor (TNF)-a [3] and thrombin [4]. Furthermore, stabilisation of endothelial microtubules with taxol decreases leukocyte transmigration through endothelial monolayers, whereas disassembly of microtubules increases leukocyte migration [5].…”

GEF-H1 Couples Nocodazole-induced Microtubule Disassembly to Cell Contractility via RhoA

“…Although microtubules themselves do not generate contractile forces, it has been noticed that microtubule depolymerization induces the formation of actin stress fibers and cell contraction (Danowski, 1989;Brown et al, 1996;Verin et al, 2001;Birukova et al, 2004b). However, in spite of the apparent universality of this phenomenon, the molecular basis by which microtubule disassembly stimulates cell contractility is not clearly established.…”

“…One model suggests that microtubules function as rigid frames that resist actomyosin contraction in a cellular tensegrity array ). An alternative mechanism is that microtubules control cell contractility by enhancing myosin light chain (MLC) phosphorylation (Kolodney and Elson, 1995;Verin et al, 2001;Birukova et al, 2004b), rather than simply removing an opposing structural element. The phosphorylation of MLC at Ser19 regulates the interaction of myosin with actin to control cellular contractility.…”

Cytoskeletal Molecules Play a Major Role in Cancer Progression

“…Normal cell behavior regulated via the cytoskeletal filaments are altered in cancer. Several studies have demonstrated that alteration of cytoskeletal structures play a pivotal role in controlling cell behavior including in cancerous cells [1][2][3].…”