Michael Glogauer

The role of actin-binding protein 280 in integrin-dependent mechanoprotection.

To survive in a mechanically active environment, cells must adapt to variations of applied membrane tension. A collagen-coated magnetic bead model was used to apply forces directly to the actin cytoskeleton through integrin receptors. We demonstrate here that by a calcium-dependent mechanism, human fibroblasts reinforce locally their connection with extracellular adhesion sites by inducing actin assembly and by recruiting actin-binding protein 280 (ABP-280) into cortical adhesion complexes. ABP-280 was phosphorylated on serine residues as a result of force application. This phosphorylation and the force-induced actin reorganization were largely abrogated by inhibitors of protein kinase C. In a human melanoma cell line that does not express ABP-280, actin accumulation could not be induced by force, whereas in stable transfectants expressing ABP-280, force-induced actin accumulation was similar to human fibroblasts. Cortical actin assembly played a role in regulating the activity of stretch-activated, calcium-permeable channels (SAC) since sustained force application desensitized SAC to subsequent force applications, and the decrease in stretch sensitivity was reversed after treatment with cytochalasin D. ABP-280-deficient cells showed a >90% increase in cell death compared with ABP-280+ve cells after force application. We conclude that ABP-280 plays an important role in mechanoprotection by reinforcing the membrane cortex and desensitizing SACs.

CHEMOTACTIC SIGNALING PATHWAYS IN NEUTROPHILS: FROM RECEPTOR TO ACTIN ASSEMBLY

In this review, we present an overview of the signaling elements between neutrophil chemotactic receptors and the actin cytoskeleton that drives cell motility. From receptor-ligand interactions, activation of heterotrimeric G-proteins, their downstream effectors PLC and PI-3 kinase, the activation of small GTPases of the Rho family, and their regulation of particular cytoskeletal regulatory proteins, we describe pathways specific to the chemotaxing neutrophil and elements documented to be important for neutrophil function.

Rac1 and Rac2 differentially regulate actin free barbed end formation downstream of the fMLP receptor

Actin assembly at the leading edge of migrating cells depends on the availability of high-affinity free barbed ends (FBE) that drive actin filament elongation and subsequent membrane protrusion. We investigated the specific mechanisms through which the Rac1 and Rac2 small guanosine triphosphatases (GTPases) generate free barbed ends in neutrophils. Using neutrophils lacking either Rac1 or Rac2 and a neutrophil permeabilization model that maintains receptor signaling to the actin cytoskeleton, we assessed the mechanisms through which these two small GTPases mediate FBE generation downstream of the formyl-methionyl-leucyl-phenylalanine receptor. We demonstrate here that uncapping of existing barbed ends is mediated through Rac1, whereas cofilin- and ARP2/3-mediated FBE generation are regulated through Rac2. This unique combination of experimental tools has allowed us to identify the relative roles of uncapping (15%), cofilin severing (10%), and ARP2/3 de novo nucleation (75%) in FBE generation and the respective roles played by Rac1 and Rac2 in mediating actin dynamics.

The N. gonorrhoeae Type IV Pilus Stimulates Mechanosensitive Pathways and Cytoprotection through a pilT-Dependent Mechanism

The Neisseria gonorrhoeae type IV pilus is a retractile appendage that can generate forces near 100 pN. We tested the hypothesis that type IV pilus retraction influences epithelial cell gene expression by exerting tension on the host membrane. Wild-type and retraction-defective bacteria altered the expression of an identical set of epithelial cell genes during attachment. Interestingly, pilus retraction, per se, did not regulate novel gene expression but, rather, enhanced the expression of a subset of the infection-regulated genes. This is accomplished through mitogen-activated protein kinase activation and at least one other undefined stress-activated pathway. These results can be reproduced by applying artificial force on the epithelial membrane, using a magnet and magnetic beads. Importantly, this retraction-mediated signaling increases the ability of the cell to withstand apoptotic signals triggered by infection. We conclude that pilus retraction stimulates mechanosensitive pathways that enhance the expression of stress-responsive genes and activate cytoprotective signaling. A model for the role of pilus retraction in influencing host cell survival is presented.

Modulation of reactive oxygen species by Rac1 or catalase prevents asbestos-induced pulmonary fibrosis

The release of reactive oxygen species (ROS) and cytokines by alveolar macrophages has been demonstrated in asbestos-induced pulmonary fibrosis, but the mechanism linking alveolar macrophages to the pathogenesis is not known. The GTPase Rac1 is a second messenger that plays an important role in host defense. In this study, we demonstrate that Rac1 null mice are protected from asbestos-induced pulmonary fibrosis, as determined by histological and biochemical analysis. We hypothesized that Rac1 induced pulmonary fibrosis via generation of ROS. Asbestos increased TNF-alpha and ROS in a Rac1-dependent manner. TNF-alpha was elevated only 1 day after exposure, whereas ROS generation progressively increased in bronchoalveolar lavage cells obtained from wild-type (WT) mice. To determine whether ROS generation contributed to pulmonary fibrosis, we overexpressed catalase in WT monocytes and observed a decrease in ROS generation in vitro. More importantly, administration of catalase to WT mice attenuated the development of fibrosis in vivo. For the first time, these results demonstrate that Rac1 plays a crucial role in asbestos-induced pulmonary fibrosis. Moreover, it suggests that a simple intervention may be useful to prevent progression of the disease.

The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils.

In inflammatory diseases, circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not prevent neutrophil recruitment effectively. The Slit family of secreted proteins and their transmembrane receptor, Robo, repel axonal migration during CNS development. Emerging evidence shows that by inhibiting the activation of Rho‐family GTPases, Slit2/Robo also inhibit migration of other cell types toward a variety of chemotactic factors in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow and detected Robo‐1 expression. Using video‐microscopic live cell tracking, we found that Slit2 selectively impaired directional migration but not random movement of neutrophils toward fMLP. Slit2 also inhibited neutrophil migration toward other chemoattractants, namely C5a and IL‐8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant‐induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2 but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and MIP‐2. In all instances, Slit2 reduced neutrophil recruitment effectively (P<0.01). Collectively, these data demonstrate that Slit2 potently inhibits chemotaxis but not random motion of circulating neutrophils and point to Slit2 as a potential new therapeutic for preventing localized inflammation.

Neural crest cell-specific deletion of Rac1 results in defective cell-matrix interactions and severe craniofacial and cardiovascular malformations.

The small GTP-binding protein Rac1, a member of the Rho family of small GTPases, has been implicated in regulation of many cellular processes including adhesion, migration and cytokinesis. These functions have largely been attributed to its ability to reorganize cytoskeleton. While the function of Rac1 is relatively well known in vitro, its role in vivo has been poorly understood. It has previously been shown that in neural crest cells (NCCs) Rac1 is required in a stage-specific manner to acquire responsiveness to mitogenic EGF signals. Here we demonstrate that mouse embryos lacking Rac1 in neural crest cells (Rac1/Wnt1-Cre) showed abnormal craniofacial development including regional ectodermal detachment associated with mesenchymal acellularity culminating in cleft face at E12. Rac1/Wnt1-Cre mutants also displayed inappropriate remodelling of pharyngeal arch arteries and defective outflow tract septation resulting in the formation of a common arterial trunk (persistent truncus arteriosus or PTA). The mesenchyme around the aortic sac also developed acellular regions, and the distal aortic sac became grossly dysmorphic, forming a pair of bilateral, highly dilated arterial structures connecting to the dorsal aortas. Smooth muscle cells lacking Rac1 failed to differentiate appropriately, and subpopulations of post-migratory NCCs demonstrated aberrant cell death and attenuated proliferation. These novel data demonstrate that while Rac1 is not required for normal NCC migration in vivo, it plays a critical cell-autonomous role in post-migratory NCCs during craniofacial and cardiac development by regulating the integrity of the craniofacial and pharyngeal mesenchyme.

Nitric oxide enhances osteoclastogenesis possibly by mediating cell fusion

Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. Here, we investigate how nitric oxide (NO) affects osteoclastogenesis. Time lapse photomicrography, using the fluorescent NO indicator dye, 4,5-diaminofluorescein diacetate, revealed an intense NO signal in pre-osteoclasts preceding cell fusion. Osteoclastogenesis in RAW264.7 cells increased when exposed to the NO synthase inhibitor, L-NMMA (0.25 microM), for the initial 48 h. In contrast, pre-osteoclast fusion decreased when RAW264.7 cells were exposed to L-NMMA from 48 to 96 h. Both NO synthase inhibitors, L-NMMA and L-NAME, decreased osteoclast formation during this time period. The inhibitory effect of L-NMMA on osteoclast formation was abolished with increasing concentrations (25-200 ng/ml) of sRANKL suggesting signaling cross talk. NO donors increased osteoclast formation in a dose-dependent manner, with greatest stimulation at 15 microM NOC-12 (2.3 fold) and 5 microM NOC-18 (2.4 fold). Measuring nitrite (NO end product) daily from culture media of RAW264.7 cells undergoing osteoclastogenesis revealed that an increase in NO production coincided with the fusion of pre-osteoclasts (day 4). Inhibiting fusion by plating cells on polystyrene dishes pre-coated with poly-(L-lysine) decreased both osteoclast formation and NO production. To address if NO mediates fusion through the actin cytoskeleton, actin free barbed ends were measured. 0.25 microM L-NMMA decreased, while 15 microM NOC-12 and 5 microM NOC-18 increased actin free barbed ends. We hypothesize that while NO initially negatively regulates pre-osteoclast differentiation; it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling.

Blockade of TLR2 Inhibits Porphyromonas gingivalis Suppression of Mineralized Matrix Formation by Human Dental Pulp Stem Cells

INTRODUCTIONnHuman dental pulp stem/progenitor cells (hDPSC) can differentiate into odontoblast-like cells and express dentin sialophosphoprotein (DSPP) and osteocalcin (OCN); thus, they may be used to regenerate dentin. However, residual bacterial components in the root canal may suppress this activity.nnnPURPOSEnThis study investigated the effect of a Porphyromonas gingivalis component on the expression of DSPP and OCN by stimulated hDPSCs and the influence of blockade of TLR2-mediated P. gingivalis host recognition.nnnMETHODSnStimulated hDPSCs were exposed to varying concentrations of P. gingivalis lipopolysaccharide (LPS), and the expression of DSPP and OCN was measured. Similar groups of stimulated hDPSCs were exposed to TLR2 blocking agents before exposure to LPS.nnnRESULTSnhDPSCs exposed to 5, 10, and 20 μg/mL LPS exhibited a dose-dependent reduction in the expression of DSPP (3.19 ± 0.18, 2.60 ± 0.49, and 1.15 ± 0.29, respectively) and OCN (3.51 ± 1.18, 2.60 ± 0.67 and 1.66 ± 0.89, respectively). The expression of DSPP and OCN after exposure to 20 μg/mL of LPS was significantly lower than measured for unexposed stimulated cells (analysis of variance and post hoc Tukey test, P < .05). The blockade of TLR2 using an extra- and intracellular agent affected DSPP (4.67 ± 0.97 and 5.29 ± 1.66, respectively) and OCN (5.25 ± 1.69 and 5.82 ± 2.38, respectively) expression at levels comparable to stimulated cells unexposed to 20 μg/mL LPS (6.32 ± 2.47 and 4.70 ± 1.60 for DSPP and OCN, respectively).nnnCONCLUSIONSnThe suppressing effect of P. gingivalis on mineralized matrix formation by hDPSCs is confirmed, and this suppression can be moderated by TLR2 blockade.

Targeting the isoprenoid pathway to abrogate progression of pulmonary fibrosis

Fibrotic remodeling in lung injury is a major cause of morbidity. The mechanism that mediates the ongoing fibrosis is unclear, and there is no available treatment to abate the aberrant repair. Reactive oxygen species (ROS) have a critical role in inducing fibrosis by modulating extracellular matrix deposition. Specifically, mitochondrial hydrogen peroxide (H2O2) production by alveolar macrophages is directly linked to pulmonary fibrosis as inhibition of mitochondrial H2O2 attenuates the fibrotic response in mice. Prior studies indicate that the small GTP-binding protein, Rac1, directly mediates H2O2 generation in the mitochondrial intermembrane space. Geranylgeranylation of the C-terminal cysteine residue (Cys(189)) is required for Rac1 activation and mitochondrial import. We hypothesized that impairment of geranylgeranylation would limit mitochondrial oxidative stress and, thus, abrogate progression of pulmonary fibrosis. By targeting the isoprenoid pathway with a novel agent, digeranyl bisphosphonate (DGBP), which impairs geranylgeranylation, we demonstrate that Rac1 mitochondrial import, mitochondrial oxidative stress, and progression of the fibrotic response to lung injury are significantly attenuated. These observations reveal that targeting the isoprenoid pathway to alter Rac1 geranylgeranylation halts the progression of pulmonary fibrosis after lung injury.