Abstract
Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function.
Highlights
The acute respiratory distress syndrome (ARDS) is a highly morbid clinical entity marked pathophysiologically by the flooding of the alveolar space with protein rich fluid which leads to severe impairment of gas exchange and hypoxia (Rubenfeld and Herridge, 2007; Ware and Matthay, 2000)
In order to better characterize the putative site of CTTN binding within non-muscle myosin light chain kinase (nmMLCK), site directed mutagenesis was employed to generate a novel construct with key proline residues mutated to alanine
Wild-type and mutant flag-tagged constructs were transfected into HPAECs, stimulated with vehicle control or S1P × 10 minutes, immunoprecipitated with anti-flag antibodies, and CTTN levels in the isolated protein complexes were determined by western blotting
Summary
The acute respiratory distress syndrome (ARDS) is a highly morbid clinical entity marked pathophysiologically by the flooding of the alveolar space with protein rich fluid which leads to severe impairment of gas exchange and hypoxia (Rubenfeld and Herridge, 2007; Ware and Matthay, 2000). The pulmonary endothelium forms a thin semipermeable barrier between the vascular space and the interstitial and alveolar spaces allowing for effective gas exchange while preventing the excess leakage of fluid and protein (Dudek and Garcia, 2001). This barrier becomes severely compromised during ARDS. Pulmonary endothelial cell (EC) mechanics play a central role in the regulation of this barrier function as the formation of intercellular gaps and paracellular movement of fluid and solute are the primary mechanism producing vascular leak in pathologic conditions (Baldwin and Thurston, 2001; Garcia et al, 1995). S1P increases peripheral areas of dynamic actin rearrangement to produce membrane projections, such as lamellipodia, that decrease intercellular gaps and strengthen the EC barrier (Lee et al, 2006)
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