Endothelial Cell MLCK Isoform

EC MLCK is an ATP- and Ca2+/calmodulin-dependent enzyme essential for generation of centripetal cellular tension via its ability to enhance actomyosin motor activity [1]. EC MLCK appears to be enriched in microvascular endothelium compared to macrovascular cells, likely a reflection of the diversity of function within that tissue. Gene expression studies have noted that expression of the EC MLCK gene distinguishes micro- from macrovascular endothelium. Myosin light chain kinase (MLCK)-dependent stress fiber formation results in EC contraction and intercellular gap formation with decreased transmonolayer endothelial resistance (TER) and increased fluid and solute translocation [1]. Nonskeletal-muscle MLCK exists as high-molecular-weight (~210kDa) and low-molecular-weight (130-160kDa) isoforms that are derived from a single gene. Human ECs express only the high-molecular-weight form (EC MLCK) cloned by our group, with multiple splice variants also detected by RT-PCR. The two most predominantly expressed variants (EC MLCK 1 and 2) differ only by a single exon deletion (encoding AA# 436-505), which results in EC MLCK 2 containing 69 fewer amino acids than EC MLCK 1. This deleted region contains two sites for p60src-catalyzed tyrosine phosphorylation (Y464,

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Figure 2 Actin microfilaments distribution is altered by agents that affect vascular endothelial cell permeability. Human lung endothelial cells contain randomly distributed filamentous actin (red) in a stable monolayer (A). Edemagenic agents, such as thrombin, cause cytoskeletal rearrangement resulting in thick actin bundles "stress fibers" which span the cell. Increase intracellular tension and contraction results in formation of intercellular gaps (B, arrows). Platelet derived-phopholipids such as sphingosine 1-phophate are barrier protective and result in an increase in cortical actin at the cell periphery (C). (see color insert)

Figure 2 Actin microfilaments distribution is altered by agents that affect vascular endothelial cell permeability. Human lung endothelial cells contain randomly distributed filamentous actin (red) in a stable monolayer (A). Edemagenic agents, such as thrombin, cause cytoskeletal rearrangement resulting in thick actin bundles "stress fibers" which span the cell. Increase intracellular tension and contraction results in formation of intercellular gaps (B, arrows). Platelet derived-phopholipids such as sphingosine 1-phophate are barrier protective and result in an increase in cortical actin at the cell periphery (C). (see color insert)

Y471) that serve to produce differential regulation of EC MLCK splice-variant activity.

The EC MLCK isoform, like the endothelial cytoskeleton in general, is highly multifunctional and serves as an important effector in numerous endothelial processes. In multiple in vitro and in vivo models of EC permeability, increased MLCK activity produces increased MLC phosphorylation within newly formed stress fibers, intracellular tension, cell rounding, paracellular gap formation, and subsequent EC barrier disruption. Inhibition of EC MLCK attenuates or prevents vascular leak produced by ischemia reperfusion, neutrophils, TGFB, thrombin, and mechanical stress as well as neutrophil influx in response to LTB4 or FMLP. More recently, we have described increased MLC phosphoryla-tion in a cortical distribution during EC barrier enhancement [3], suggesting possible spatially-defined MLCK activation that can differentially regulate permeability. EC MLCK has been implicated in TNF-induced endothelial cell apoptosis, mechanotransduction, and calcium signaling.

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