The endothelium provides a semi-selective barrier between the vasculature and many tissues. When this barrier function is disrupted in the lung, as occurs in acute lung injury syndromes such as ARDS and sepsis, vascular permeability increases and life-threatening pulmonary edema often occurs. Despite its relevance to these morbid clinical syndromes, the cellular regulation of endothelial barrier function is poorly understood. Cell culture models of inflammatory-induced endothelial cell (EC) barrier dysfunction have demonstrated the critical importance of a chain of events which includes cytoskeletal rearrangement, cellular contractility, and paracellular gap formation leading to enhanced fluid and protein transport. Myosin light chain (MLCK) plays a central role in initiating this series of events by phosphorylating myosin light chains (MLC) and thereby increasing their interaction with the actin cytoskeleton to produce cell contraction. Endothelial cells express only a recently cloned high MW isoform of MLCK whose regulation is unclear. This EC MLCK isoform increases its activity when tyrosine phosphorylated by p60src, and under these conditions EC MLCK is found in stable association with the actin-binding protein, cortactin. Although cortactin is associated with many aspects of cytoskeletal rearrangement, its function is still unclear. This background suggests the following hypothesis: cortactin is an important participant in EC cytoskeletal rearrangement and barrier dysfunction through interaction with EC MLCK.
In Specific Aim number 1, the interaction of MLCK and cortactin will be characterized using deletion constructs of both proteins to identify structural sites of interaction using in vitro binding assays and transient transfections of cultured EC. The effects of MLCK-cortactin interaction and cortactin overexpression on physiologic aspects of EC cytoskeletal rearrangement such as barrier function and migration will be studied.
Specific Aim number 2 will address the role of cortactin phosphorylation (Tyr and Ser/Thr) on interaction with MLCK and EC barrier function by using additional constructs.
| Li, Yansong; Uruno, Takehito; Haudenschild, Christian et al. (2004) Interaction of cortactin and Arp2/3 complex is required for sphingosine-1-phosphate-induced endothelial cell remodeling. Exp Cell Res 298:107-21 |
| Dudek, Steven M; Birukov, Konstantin G; Zhan, Xi et al. (2002) Novel interaction of cortactin with endothelial cell myosin light chain kinase. Biochem Biophys Res Commun 298:511-9 |
| Dudek, S M; Garcia, J G (2001) Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 91:1487-500 |
