The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (ECs). In the late embryonic and early postnatal period, these cells respond to inducing factors found in the brain environment by adopting a set of defined characteristics, including high-electrical-resistance tight junctions. Although the factors have not been identified definitively, a great deal of information about brain ECs has been obtained, especially recently. This review concentrates on a cell biological analysis of the BBB, with an emphasis on regulation of the specialized intercellular junctions. The development of these junctions seems to depend on two primary processes: the appearance of high levels of the tight junction protein occludin and intracellular signaling processes that control the state of phosphorylation of junctional proteins. Recent studies have revealed that the BBB can be modulated in an ongoing way to respond to environmental stimuli.

In epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability.

Native Pasteurella multocida toxin (PMT) is shown to be an extremely potent mitogen for Swiss 3T3 fibroblasts. Half-maximal stimulation of DNA synthesis was obtained at concentrations of 1 and 2 pM for recombinant PMT (rPMT) and PMT, respectively. The degree of rPMT-induced DNA synthesis was comparable to that elicited by 10% fetal bovine serum and, moreover, was observed in the complete absence of other factors. Cell proliferation was also enhanced by rPMT. The toxin was also a potent mitogen for BALB/c and NIH 3T3 cells, 3T6 cells, and tertiary mouse embryo or human fibroblasts. The mitogenic activity of rPMT was heat-labile. A polyclonal antiserum to PMT inhibited DNA synthesis when added early, but not late, during treatment of the Swiss 3T3 cells with rPMT. A similar time-dependent action of methylamine was also observed. Furthermore, transient exposure of the cells to rPMT at 37C, but not at 4°C, resulted in a stimulation of DNA synthesis. Thus, toxin action may require cell entry and processing via an acidic compartment. The toxin, at mitogenic concentrations, caused a large increase in the production of inositol phosphates. In contrast, rPMT did not increase the intracellular concentration of cyclic AMP in Swiss 3T3 cells. The basis of rPMT action may afford a unique insight into molecular signaling events involved in the control of cell proliferation.

The G2019S leucine rich repeat kinase 2 (LRRK2) mutation is the most common genetic cause of Parkinson's disease (PD), clinically and pathologically indistinguishable from idiopathic PD. Mitochondrial abnormalities are a common feature in PD pathogenesis and we have investigated the impact of G2019S mutant LRRK2 expression on mitochondrial bioenergetics. LRRK2 protein expression was detected in fibroblasts and lymphoblasts at levels higher than those observed in the mouse brain. The presence of G2019S LRRK2 mutation did not influence LRRK2 expression in fibroblasts. However, the expression of the G2019S LRRK2 mutation in both fibroblast and neuroblastoma cells was associated with mitochondrial uncoupling. This was characterized by decreased mitochondrial membrane potential and increased oxygen utilization under basal and oligomycin-inhibited conditions. This resulted in a decrease in cellular ATP levels consistent with compromised cellular function. This uncoupling of mitochondrial oxidative phosphorylation was associated with a cell-specific increase in uncoupling protein (UCP) 2 and 4 expression. Restoration of mitochondrial membrane potential by the UCP inhibitor genipin confirmed the role of UCPs in this mechanism. The G2019S LRRK2-induced mitochondrial uncoupling and UCP4 mRNA up-regulation were LRRK2 kinase-dependent, whereas endogenous LRRK2 levels were required for constitutive UCP expression. We propose that normal mitochondrial function was deregulated by the expression of G2019S LRRK2 in a kinase-dependent mechanism that is a modification of the normal LRRK2 function, and this leads to the vulnerability of selected neuronal populations in PD.

Abstract. Cadherins and catenins play an important role in cell-cell adhesion. Two of the catenins, [3 and ~/, are members of a group of proteins that contains a repeating amino acid motif originally described for the Drosophila segment polarity gene armadillo. Another member of this group is a 120-kD protein termed p120, originally identified as a substrate of the tyrosine kinase pp60 ~r¢. In this paper, we show that endothelial and epithelial cells express p120 and pl00, a 100-kD, p120-related protein. Peptide sequencing of pl00 establishes it as highly related to p120. p120 and pl00 both appear associated with the cadherin/catenin complex, but independent p120/catenin and pl00/catenin complexes can be isolated. This association is shown by coimmunoprecipitation of cadherins and catenins with an anti-p120/ pl00 antibody, and of pl20/pl00 with cadherin or catenin antibodies. Immunocytochemical analysis with a p120-specific antibody reveals junctional colocalization of p120 and 13-catenin in epithelial cells. Catenins and p120/p100 also colocalize in endothelial and epithelial cells in culture and in tissue sections. The cellular content of p120/p100 and 13-catenin is similar in MDCK cells, but only ~20% of the p120/p100 pool associates with the cadherin/catenin complex. Our data provide further evidence for interactions among the different arm proteins and suggest that p120/p100 may participate in regulating the function of cadherins and, thereby, other processes influenced by cell-cell adhesion.

During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.

Brain capillary endothelial cells

Death-associated protein kinase (DAP kinase) is a proapoptotic, calcium/calmodulin-dependent serine/threonine kinase. Here, we report that DAP kinase phosphorylates the regulatory light chain of myosin II (MLC) both in vitro and in vivo, and that this phosphorylation occurs preferentially at residue Ser19. In quiescent fibroblasts, DAP kinase stabilizes stress fibers through phosphorylation of MLC, but it is dispensable for the formation of peripheral microfilament bundles. This cytoskeletal effect of DAP kinase occurs before the onset of apoptosis and does not require an intact death domain. In addition, DAP kinase is required for serum-induced stress-fiber formation, which is associated with the upregulation of its catalytic activity. Despite being both sufficient and necessary for the assembly or maintenance of stress fibers, DAP kinase is incapable of stimulating the formation of focal adhesions in quiescent cells. Moreover, it promotes the disassembly of focal adhesions but not stress fibers in cells receiving serum factors. Together, our results identify a novel and unique function of DAP kinase in the uncoupling of stress fibers and focal adhesions. Such uncoupling would lead to a perturbation of the balance between contractile and adhesion forces and subsequent cell detachment, which might contribute to its pro-apoptotic activity.