Michael T. Kirber

Regulation of ROS signal transduction by NADPH oxidase 4 localization

Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER). ER localization of Nox4 is critical for the regulation of protein tyrosine phosphatase (PTP) 1B, also an ER resident, through redox-mediated signaling. Nox4-mediated oxidation and inactivation of PTP1B in the ER serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking and specifically acts to terminate EGF signaling. Consistent with this notion, PTP1B oxidation could also be modulated by ER targeting of antioxidant enzymes but not their untargeted counterparts. These data indicate that the specificity of intracellular ROS-mediated signal transduction may be modulated by the localization of Nox isoforms within specific subcellular compartments.

Multifocal multiphoton microscopy (MMM) at a frame rate beyond 600 Hz

We introduce a multiphoton microscope for high-speed three-dimensional (3D) fluorescence imaging. The system combines parallel illumination by a multifocal multiphoton microscope (MMM) with parallel detection via a segmented high-sensitivity charge-couple device (CCD) camera. The instrument consists of a Ti-sapphire laser illuminating a microlens array that projects 36 foci onto the focal plane. The foci are scanned using a resonance scanner and imaged with a custom-made CCD camera. The MMM increases the imaging speed by parallelizing the illumination; the CCD camera can operate at a frame rate of 1428 Hz while maintaining a low read noise of 11 electrons per pixel by dividing its chip into 16 independent segments for parallelized readout. We image fluorescent specimens at a frame rate of 640 Hz. The calcium wave of fluo3 labeled cardiac myocytes is measured by imaging the spontaneous contraction of the cells in a 0.625 second sequence movie, consisting of 400 single images.

Impairment of PARK14-dependent Ca(2+) signalling is a novel determinant of Parkinson's disease.

The etiology of idiopathic Parkinsons disease (idPD) remains enigmatic despite recent successes in identification of genes (PARKs) that underlie familial PD. To find new keys to this incurable neurodegenerative disorder we focused on the poorly understood PARK14 disease locus (Pla2g6 gene) and the store-operated Ca2+ signalling pathway. Analysis of the cells from idPD patients reveals a significant deficiency in store-operated PLA2g6-dependent Ca2+ signalling, which we can mimic in a novel B6.Cg-Pla2g6ΔEx2-VB (PLA2g6 ex2KO) mouse model. Here we demonstrate that genetic or molecular impairment of PLA2g6-dependent Ca2+ signalling is a trigger for autophagic dysfunction, progressive loss of dopaminergic (DA) neurons in substantia nigra pars compacta and age-dependent L-DOPA-sensitive motor dysfunction. Discovery of this previously unknown sequence of pathological events, its association with idPD and our ability to mimic this pathology in a novel genetic mouse model opens new opportunities for finding a cure for this devastating neurodegenerative disease.

Caveolins sequester FA on the cytoplasmic leaflet of the plasma membrane, augment triglyceride formation, and protect cells from lipotoxicity

Ectopic expression of caveolin-1 in HEK293 cells enhances FA sequestration in membranes as measured by a pH-sensitive fluorescent dye (1). We hypothesized that sequestration of FA is due to the enrichment of caveolin in the cytosolic leaflet and its ability to facilitate the formation of lipid rafts to buffer high FA levels. Here we show that ec-topic expression of caveolin-3 also results in enhanced FA sequestration. To further discriminate the effect that caveolins have on transmembrane FA movement and distribution, we labeled the outer membrane leaflet with fluorescein-phosphatidylethanolamine (FPE), whose emission is quenched by the presence of FA anions. Real-time measurements made with FPE and control experiments with positively charged fatty amines support our hypothesis that caveolins promote localization of FA anions through interactions with basic amino acid residues (lysines and arginines) present at the C termini of caveolins-1 and -3.

Role of Molecular Determinants of Store-operated Ca2+ Entry (Orai1, Phospholipase A2 Group 6, and STIM1) in Focal Adhesion Formation and Cell Migration

Background: Store-operated Ca2+ entry is important for cell migration. Results: This study presents characterization of localization and roles of Orai1, STIM1, and PLA2g6 in adhesion dynamics during cell migration. Conclusion: Orai1 and PLA2g6 are involved in adhesion formation at the front, whereas STIM1 participates in both adhesion formation and disassembly. Significance: Results uncovered new parameters of Orai1, STIM1, and PLA2g6 involvement in cell migration. Store-operated Ca2+ entry and its major determinants are known to be important for cell migration, but the mechanism of their involvement in this complex process is unknown. This study presents a detailed characterization of distinct roles of Orai1, STIM1, and PLA2g6 in focal adhesion (FA) formation and migration. Using HEK293 cells, we discovered that although molecular knockdown of Orai1, STIM1, or PLA2g6 resulted in a similar reduction in migration velocity, there were profound differences in their effects on number, localization, and lifetime of FAs. Knockdown of STIM1 caused an increase in lifetime and number of FAs, their redistribution toward lamellae region, and an increase in cell tail length. In contrast, the number of FAs in Orai1- or PLA2g6-deficient cells was significantly reduced, and FAs accumulated closer to the leading edge. Assembly rate and Vinculin phosphorylation of FAs was similarly reduced in Orai1, PLA2g6, or STIM1-deficient cells. Although Orai1 and PLA2g6 accumulated and co-localized at the leading edge, STIM1 distribution was more complex. We found STIM1 protrusions in lamellipodia, which co-localized with FAs, whereas major accumulation could be seen in central and retracting parts of the cell. Interestingly, knockdown of Orai1 and PLA2g6 produced similar and non-additive effect on migration, whereas knockdown of STIM1 simultaneously with either Orai1 or PLA2g6 produced additional inhibition. Together these data suggest that although Orai1, PLA2g6, and STIM1 play major roles in formation of new FAs at the leading edge, STIM1 may also be involved in Orai1- and PLA2g6-independent disassembly of FAs in the back of cells.

Hypoxia influences CD40–CD40L mediated inflammation in endothelial and monocytic cells

The interaction between CD40 and its ligand (CD40L) has been implicated in the pathogenesis of atherosclerosis and is recognized as a central event in the development of immuno-inflammatory processes. Our previous studies have shown that the CD40-CD40L interaction modulates platelet, neutrophil, and endothelial reactive oxygen species (ROS) generation. Hypoxia, known to be associated with tissue ischemia and inflammation, also influences the ROS production and changes the cellular redox state. However, the effect of hypoxia on CD40-CD40L mediated vascular inflammation is unknown. We have investigated whether hypoxia influences CD40-CD40L mediated vascular inflammatory responses, ROS production, and cellular interactions. We found that hypoxia significantly enhances the inflammatory effect of CD40L in both endothelial and monocytic cells (THP1). CD40-CD40L interaction in the presence of hypoxia induces ROS production, the synthesis of an inflammatory adhesive protein intercellular adhesion molecule 1 (ICAM1) and activates stress response proteins (p38 MAP kinase and HSP27), indicating that CD40L mediates the induction of oxidative stress in these cells. Importantly, we found that the effects of CD40L can be transmitted between HUVECs and monocytic THP1 cells through intercellular CD40-CD40L interaction and these processes are augmented under hypoxia. Together, these data indicate that under hypoxic conditions the CD40-CD40L interaction significantly influences adhesion molecule expression, stress generation, actin polymerization, and monocytic adhesion to endothelial cells in addition to changes in signaling. In summary, we show that hypoxia can alter CD40-CD40L mediated endothelial-monocyte interaction, playing a significant role in vascular inflammation and cellular adhesion processes.

Proximal Events in 7,12-Dimethylbenz[a]anthracene-Induced, Stromal Cell-Dependent Bone Marrow B Cell Apoptosis: Stromal Cell–B Cell Communication and Apoptosis Signaling

Intercellular communication is an essential process in stimulating lymphocyte development and in activating and shaping an immune response. B cell development requires cell-to-cell contact with and cytokine production by bone marrow stromal cells. However, this intimate relationship also may be responsible for the transfer of death-inducing molecules to the B cells. 7,12-Dimethylbenz[a]anthracene (DMBA), a prototypical polycyclic aromatic hydrocarbon, activates caspase-3 in pro/pre-B cells in a bone marrow stromal cell-dependent manner, resulting in apoptosis. These studies were designed to examine the hypothesis that an intrinsic apoptotic pathway is activated by DMBA and that the ultimate death signal is a DMBA metabolite generated by the stromal cells and transferred to the B cells. Although a loss of mitochondrial membrane potential did not occur in the DMBA/stromal cell-induced pathway, cytochrome c release was stimulated in B cells. Caspase-9 was activated, and formation of the apoptosome was required to support apoptosis, as demonstrated by the suppression of death in Apaf-1fog mutant pro-B cells. Investigation of signaling upstream of the mitochondria demonstrated an essential role for p53. Furthermore, DMBA-3,4-dihydrodiol-1,2-epoxide, a DNA-reactive metabolite of DMBA, was sufficient to upregulate p53, induce caspase-9 cleavage, and initiate B cell apoptosis in the absence of stromal cells, suggesting that production of this metabolite by the stromal cells and transfer to the B cells are proximal events in triggering apoptosis. Indeed, we provide evidence that metabolite transfer from bone marrow stromal cells occurs through membrane exchange, which may represent a novel communication mechanism between developing B cells and stromal cells.