Myosin Light Chain Kinase MLCK

Actin and myosin are the major contractile components in the cytoskeleton. The cross-bridge movement between actin and myosin provides a mechanical basis for not only the maintenance of centripetal tension but also the development of contractile force in cells during physical or chemical stimulation. In vascular endothelial cells, the interaction between actin and myosin is mainly governed by the phos-phorylation status of the regulatory myosin light chain (MLC). Two major mechanisms control the activity of MLC. On one hand, myosin light-chain kinase directly phosphorylates MLC at Thr-18 and/or Ser-19, resulting in MLC activation and actin-myosin binding. On the other hand, myosin-associated protein phosphatase dephosphory-lates MLC and thus counteracts the MLCK effect, leading to cell relaxation. Certain types of agonists and cells can

Figure 1 A schematic diagram of microvascular endothelial barrier structure. The barrier is formed by a layer of endothelial cells that connect to each other (e.g., cell A to cell B) through the junctional adhesive molecule VE-cadherin, which binds to another VE-cadherin in the junction and connects to actin cytoskeleton via a family of catenins (apy, and p120). This endothelial lining is tethered to the extracellular matrix through the binding of transendothelial receptor integrins (composed of a and p subunits) and a family of cytoskeleton-linking proteins including focal adhesion kinase (FAK). The integrity of this barrier structure is maintained by VE-cadherin-mediated cell-cell adhesions and focal adhesion-supported cell-matrix attachment, whereas myosin light chain kinase (MLCK)-catalyzed myosin light chain (MLC) phosphorylation promotes cross-bridge movement between actin and myosin leading to cell contraction. A dynamic interaction among these structural elements controls the opening and closing of the paracellular pathways for fluid, proteins, and cells to move across the endothelium.

Endothelium Structure

Figure 1 A schematic diagram of microvascular endothelial barrier structure. The barrier is formed by a layer of endothelial cells that connect to each other (e.g., cell A to cell B) through the junctional adhesive molecule VE-cadherin, which binds to another VE-cadherin in the junction and connects to actin cytoskeleton via a family of catenins (apy, and p120). This endothelial lining is tethered to the extracellular matrix through the binding of transendothelial receptor integrins (composed of a and p subunits) and a family of cytoskeleton-linking proteins including focal adhesion kinase (FAK). The integrity of this barrier structure is maintained by VE-cadherin-mediated cell-cell adhesions and focal adhesion-supported cell-matrix attachment, whereas myosin light chain kinase (MLCK)-catalyzed myosin light chain (MLC) phosphorylation promotes cross-bridge movement between actin and myosin leading to cell contraction. A dynamic interaction among these structural elements controls the opening and closing of the paracellular pathways for fluid, proteins, and cells to move across the endothelium.

activate the contractile process by increasing MLCK activity through calcium/calmodulin signaling or by directly phosphorylating MLCK. Other agents are able to stimulate actomyosin contraction by inhibiting MLC phosphatase activity. In particular, a family of Rho-like small GTPases and their downstream effector, Rho kinase, have been implicated in the regulation of MLC phosphorylation through inhibition of MLC phosphatase.

The functional importance of the contractile mechanism in controlling microvascular permeability has been subject to extensive investigation. Recent studies provide strong evidence for a link between MLC phosphorylation and stress fiber formation or tension development in the endothelial cells. A causal role of MLCK in the endothelial hyperpermeability response to inflammatory mediators has been established through experiments in cultured endothe-lial monolayers as well as in intact isolated venules, where administration of pharmacological agents or transference of synthetic peptides that specifically block MLCK function can prevent the increase in permeability caused by thrombin, histamine, cytokines, oxygen radicals, and activated neutrophils. Furthermore, inhibition of MLCK or knockout of endothelial MLCK genes has proven to effectively attenuate microvascular leakage under clinically relevant con ditions such as severe burns and sepsis. These findings confirm that endothelial MLCK plays a critical role in mediating microvascular barrier dysfunction during inflammation and injury.

Protein Kinase C (PKC)

Protein kinase C represents a family of at least 10 serine/threonine kinases (a, pi, p2, g, 8, e, v, 9, m, X, and l). These isozymes show distinct expression patterns and are responsible for diverse cellular responses. Most of them can be activated by phorbol esters and diacylglycerol (DAG), as experimentally indicated by membrane translocation and increased phosphorylating activities. PKC activity is negatively regulated by serine/threonine protein phosphatases.

Functionally, despite its perhaps minor effect on the basal barrier property of the endothelium, PKC has been frequently referred to as a key mediator of microvascular hyperpermeability under stimulated conditions. Direct activation of PKC with DAG or phorbol esters increases the flux of fluid and macromolecules across the microvascular endothelium, whereas PKC inhibitors reduce the increases

VEGF Histamine

Myosin Light Chain

Figure 2 A model for the signal transduction of agonist-induced microvascular hyperpermeability. Histamine and vascular endothelial growth factor (VEGF) are used to represent permeability-increasing agonists. Histamine binding to its receptor (H) activates phos-pholipase C beta (PLCß) via a G protein (G), catalyzing the production from inositol bisphosphate (PIP2) to inositol trisphosphate (IP3) and diacylglycerol (DAG). DAG directly activates protein kinase C. In parallel, IP3 stimulates an internal release of Ca2+ followed by an influx of extracellular Ca2+, leading to upregulation of nitric oxide synthase (ecNOS) and nitric oxide (NO) production from its precursor larginine (l-Arg). Nitric oxide stimulates guanylate cyclase (GC) to produce cGMP, a potent activator of cGMP-dependent protein kinase (PKG). The elevated intracellular calcium also stimulates PKC and myosin light-chain kinase (MLCK); the latter phosphorylates myosin light chain (MLC-P). The hyperpermeability effect of VEGF is triggered by its tyrosine kinase receptor flkl/KDR and mediated through two major signaling pathways. On one hand, activated phospholipase C gamma (PLCg) elevates the intracellular calcium and subsequently activates the PKG, PKC, and MLCK cascades. On the other hand, KDR receptor occupancy results in the recruitment of several adaptor proteins, such as Grb2, Sos, and Src, which further produces the ras-raf-MEK-ERK cascade. In addition, the nonreceptor tyrosine kinases FAK and Src as well as the Rho-family of GTPases are also involved in the endothelial response to inflammatory stimulation. All of these protein kinases—PKG, PKC, MLCK, MAPK, and tyrosine kinases—may directly or indirectly target the endothelial barrier structure to cause an opening of the paracellular pathway for transendothelial flux of fluid and macromolecules. (see color insert)

Figure 2 A model for the signal transduction of agonist-induced microvascular hyperpermeability. Histamine and vascular endothelial growth factor (VEGF) are used to represent permeability-increasing agonists. Histamine binding to its receptor (H) activates phos-pholipase C beta (PLCß) via a G protein (G), catalyzing the production from inositol bisphosphate (PIP2) to inositol trisphosphate (IP3) and diacylglycerol (DAG). DAG directly activates protein kinase C. In parallel, IP3 stimulates an internal release of Ca2+ followed by an influx of extracellular Ca2+, leading to upregulation of nitric oxide synthase (ecNOS) and nitric oxide (NO) production from its precursor larginine (l-Arg). Nitric oxide stimulates guanylate cyclase (GC) to produce cGMP, a potent activator of cGMP-dependent protein kinase (PKG). The elevated intracellular calcium also stimulates PKC and myosin light-chain kinase (MLCK); the latter phosphorylates myosin light chain (MLC-P). The hyperpermeability effect of VEGF is triggered by its tyrosine kinase receptor flkl/KDR and mediated through two major signaling pathways. On one hand, activated phospholipase C gamma (PLCg) elevates the intracellular calcium and subsequently activates the PKG, PKC, and MLCK cascades. On the other hand, KDR receptor occupancy results in the recruitment of several adaptor proteins, such as Grb2, Sos, and Src, which further produces the ras-raf-MEK-ERK cascade. In addition, the nonreceptor tyrosine kinases FAK and Src as well as the Rho-family of GTPases are also involved in the endothelial response to inflammatory stimulation. All of these protein kinases—PKG, PKC, MLCK, MAPK, and tyrosine kinases—may directly or indirectly target the endothelial barrier structure to cause an opening of the paracellular pathway for transendothelial flux of fluid and macromolecules. (see color insert)

in endothelial permeability caused by thrombin, bradykinin, platelet activating factor, hydrogen peroxide, VEGF, or neutrophils. Importantly, clinical and experimental evidence is accumulating that PKC activation and subsequent microvascular barrier dysfunction may underlie the initiation and progress of circulatory disorders associated with diabetes, atherosclerosis, and ischemia-reperfusion injury.

Recent advances in molecular technologies enable direct targeting of particular PKC isoforms by selective inhibitors, synthetic peptides, or antisense oligonucleotides. Experiments employing these approaches reveal the relative importance of PKCa, PKCb, PKCS, and PKC|m (also known as PKD) in the regulation of microvascular permeability. Some studies demonstrate that endothelial hyperpermeabil-ity resulting from hyperglycemia, ischemia, and angiogene-sis or inflammatory stimulation is a PKCa-dependent event. In contrast, others suggest that PKCb overexpression in endothelial cells potentiates agonist-induced transendothe-lial flux of macromolecules, whereas pharmacological or antisense inhibitors of PKCß diminish the increase in endothelial permeability caused by phorbol esters or oxygen radicals. Oral administration of LY333531 or LY290181, selective pharmacological antagonists of PKCß, prevents microvascular leakage in the retina and kidney of diabetic animals. More recently, evidence is emerging that PKCS and PKD may be required for the microvascular permeability response to phorbol ester and diacylglycerol, typical exogenous and endogenous activators of PKC, respectively.

How PKC alters endothelial barrier function remains an interesting question. Both the cytoskeleton and cell-cell junctions are potential targets of this potent kinase. More specifically, it has been reported that PKC activates the endothelial contractile apparatus by inducing MLC phosphorylation and actin polymerization. Recent experiments suggest that the endothelial contractile response can be triggered by a PKC-dependent activation of the Rho pathway. Furthermore, disassembly of endothelial adherens junctions has been linked to PKC activation. In addition to its direct effect on the cytoskeletal and junctional structures, PKC may indirectly alter the endothelial barrier function through crosstalk with other intracellular signaling molecules. In this regard, the nitric oxide (NO) pathway has been increasingly recognized as a potential downstream target of PKC. An in vivo experiment in the hamster cheek pouch demonstrates that phorbol ester-induced macromolecular transport in the microcirculation is reduced when NO synthesis is blocked. This is further supported by evidence of enhanced nitric oxide synthase (NOS) phosphorylation and NO production in microvessels or endothelial cells exposed to PKC activators. A comparative analysis of the permeability response to PKC activators in isolated venules before and after NOS inhibition indicates that the barrier loosening effect of PKC is mediated, at least in part, through the endothelial production of NO.

Cytosolic calcium is an important cofactor required for the regulation of PKC function. Within this context, PKCa and PKCb are the predominant isozymes that are dependent on calcium for activation. Many permeability-increasing agonists are able to stimulate the release of calcium from intracellular stores followed by influx from extracellular space. This process, in addition to directly activating PKC, can upregulate the activity of other enzymes and second messengers in endothelial cells, such as MLCK and NO, which more likely serve as intracellular signals secondary to the PKC pathway in the transduction of endothelial permeability response. Currently, studies are ongoing to define the precise role of cytosolic calcium in the complex interactions among multiple protein kinases in microvascular endothelial cells upon inflammatory stimulation.

cAMP- and cGMP-Dependent Protein Kinases (PKA and PKG)

In general, an increase in the intracellular level of cAMP promotes endothelial barrier integrity, and the effect is most likely mediated through PKA. Studies have been carried out in cultured cells, perfused microvessels, and intact tissues in which application of PKA activators, such as b-adrenergic agonists or cAMP analogs, reduces endothelial permeability and prevents microvascular leakage during inflammation and ischemia-reperfusion injury. Recently, an endogenous PKA competitor, PKI, has been identified in vascular endothelial cells and is being used for selective inhibition of PKA. Infection of human dermal microvascular endothelial cells with adenovirus containing PKI gene results in overexpression of PKI and abrogates the cAMP-mediated protection against increased endothelial permeability.

The barrier protection effect of PKA may be related to its ability to stabilize endothelial cytoskeletal and adhesive structures. There are reports that PKA causes dephosphory-lation of MLC, dissociation of F-actin from myosin, stabilization of cytoskeletal filaments, and strengthening of cell-matrix adhesions. Moreover, PKA is well known for inhibiting leukocyte adhesion and platelet aggregation. This effect may play an indirect role in preventing the endothelial barrier from being damaged by activated leukocytes or platelets and their metabolites. Finally, it is possible that the PKA pathway functions through interactions with other signaling molecules such as PKG.

In contrast to the general consensus on the barrier-tightening effect of PKA, there is considerable controversy as to whether the cGMP-PKG cascade acts as a barrier protector or a permeability-increasing factor. Most in vitro experiments using cultured endothelial cells derived from large vessels or nonexchange microvessels show that an elevation of intracellular cGMP by NO donors or guanylate cyclase activators is associated with a decrease in endothe-lial permeability. On the other hand, in vivo observation and in vitro studies using microvascular endothelial cells report a PKG-dependent increase of microvascular permeability in response to a variety of agonists, including NO donors, his-tamine, bradykinin, tumor necrosis factor, platelet activating factor, and VEGF. These apparently contradictory findings may result from variations in cell types and experimental conditions. With regard to experimental models, cultured cells may not fully mimic the in vivo state of microcirculation. For example, it has been reported that PKG expression is dramatically reduced in cultured endothelial cells during serial passage. Whether the cells are exposed to a physiological range of shear stress and a normal calcium environment also affects the expression and activity of the kinase. Recent experiments in intact perfused microvessels show that selective inhibition of NOS, guanylate cyclase, or PKG suppresses venular hyperpermeability caused by shear stress, histamine-type agonists, and VEGF. It has been postulated that certain types of physical and chemical stimuli increase microvascular permeability by activating the NO-cGMP-PKG signaling cascade. Such a mechanism may account for the direct effect of the stimuli on microvascular endothelium where leukocytes and platelets are absent.

It seems that PKA and PKG form a pair of yin-and-yang-like functional antagonists in controlling microvascular permeability. Protein kinase A may play a dominant role in the maintenance of basal barrier property and counteract the effect of PKG in response to inflammation. The interaction between the two protein kinases can occur at several levels: competing for a common target at the cytoskeleton or junctions, counteracting to modulate leukocyte and platelet function, or feedback regulating through phosphodiesterases (PDE). Three forms of PDE, namely, cGMP-stimulated PDE II and IV and cGMP-inhibited PDE III, have been shown to affect PKA-associated alteration in endothelial permeability.

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