Kristine Debolt

Membrane depolarization is the trigger for PI3K/Akt activation and leads to the generation of ROS

Loss of fluid shear stress (ischemia) to the lung endothelium causes endothelial plasma membrane depolarization via ATP-sensitive K(+) (K(ATP)) channel closure, initiating a signaling cascade that leads to NADPH oxidase (NOX2) activation and ROS production. Since wortmannin treatment significantly reduces ROS production with ischemia, we investigated the role of phosphoinositide 3-kinase (PI3K) in shear-associated signaling. Pulmonary microvascular endothelial cells in perfused lungs subjected to abrupt stop of flow showed membrane depolarization and ROS generation. Stop of flow in flow-adapted mouse pulmonary microvascular endothelial cells in vitro resulted in the activation of PI3K and Akt as well as ROS generation. ROS generation in the lungs in situ was almost abolished by the PI3K inhibitor wortmannin and the PKC inhibitor H7. The combination of the two (wortmannin and H7) did not have a greater effect. Activation of NOX2 was greatly diminished by wortmannin, knockout of Akt1, or dominant negative PI3K, whereas membrane depolarization was unaffected. Ischemia-induced Akt activation (phosphorylation) was not observed with K(ATP) channel-null cells, which showed minimal changes in membrane potential with ischemia. Activation of Akt was similar to wild-type cells in NOX2-null cells, which do not generate ROS with ischemia. Cromakalim, a K(ATP) channel agonist, prevented both membrane depolarization and Akt phosphorylation with ischemia. Thus, Akt1 phosphorylation follows cell membrane depolarization and precedes the activation of NOX2. These results indicate that PI3K/Akt and PKC serve as mediators between endothelial cell membrane depolarization and NOX2 assembly.

Phenotypic control of gap junctional communication by cultured alveolar epithelial cells

We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed approximately 3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed ∼3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.

Ascorbic acid regulates multiple metabolic activities of cartilage cells.

Bones grow in length because of the activities of cartilage cells in the epiphyseal growth plate. We have examined selected events that occur in the growth cartilage by the use of cultured epiphyseal cells; we have also evaluated the influence of ascorbate on these activities. Our studies indicate that 1) ascorbate induces the expression of a unique collagen isoform, type X collagen; 2) ascorbate stimulates alkaline phosphatase activity of maturing chondrocytes; and 3) ascorbate regulates the energy status of the maturing chondrocyte. We have found that in the presence of ascorbate there is a change in oxidative activity. Thus, lactate formation is inhibited, there is an increase in the adenylate energy charge ratio, and there is an elevation in the activity of isocitrate dehydrogenase. The results of these studies point to multiple effects of vitamin C on chondrocyte maturation involving changes in protein synthesis and energy metabolism.

PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium

We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however, a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATP channel, caveolae (caveolin-1), and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule-1 (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1(-/-) lungs compared with lungs from wild-type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1(-/-) PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in WT mice was significantly lowered in PECAM-1(-/-) mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker VEGF compared with untreated (sham) and PECAM-1(-/-) lungs. Thus PECAM-1 (and caveolae) are parts of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal.

Rab38 targets to lamellar bodies and normalizes their sizes in lung alveolar type II epithelial cells.

Rab38 is a rat Hermansky-Pudlak syndrome gene that plays an important role in surfactant homeostasis in alveolar type II (ATII) pneumocytes. We examined Rab38 function in regulating lamellar body (LB) morphology in ATII cells. Quantitative electron microscopy revealed that LBs in ATII cells were ∼77% larger in Rab38-null fawn-hooded hypertension (FHH) than control Sprague-Dawley (SD) rats. Rab38 protein expression was restricted in lung epithelial cells but was not found in primary endothelial cells. In SD ATII cells, Rab38 protein level gradually declined during 5 days in culture. Importantly, endogenous Rab38 was present in LB fractions purified from SD rat lungs, and transiently expressed enhanced green fluorescent protein (EGFP)-tagged Rab38 labeled only the limiting membranes of a subpopulation (∼30%) of LBs in cultured ATII cells. This selective targeting was abolished by point mutations to EGFP-Rab38 and was not shared by Rab7 and Rab4b, which also function in the ATII cells. Using confocal microscopy, we established a method for quantitative evaluation of the enlarged LB phenotype temporally preserved in cultured FHH ATII cells. A direct causal relationship was established when the enlarged LB phenotype was reserved and then rescued by transiently reexpressed EGFP-Rab38 in cultured FHH ATII cells. This rescuing effect was associated with dynamic EGFP-Rab38 targeting to and on LB limiting membranes. We conclude that Rab38 plays an indispensible role in maintaining LB morphology and surfactant homeostasis in ATII pneumocytes.

Functional Interaction of Glutathione S-transferase pi and Peroxiredoxin 6 in Intact Cells

Peroxiredoxin 6 (Prdx6) is a 1-Cys member of the peroxiredoxin superfamily that plays an important role in antioxidant defense. Glutathionylation of recombinant Prdx6 mediated by π glutathione S-transferase (GST) is required for reduction of the oxidized Cys and completion of the peroxidatic catalytic cycle in vitro. This study investigated the requirement for πGST in intact cells. Transfection with a plasmid construct expressing πGST into MCF7, a cell line that lacks endogenous πGST, significantly increased phospholipid peroxidase activity as measured in cell lysates and protected intact cells against a peroxidative stress. siRNA knockdown indicated that this increased peroxidase activity was Prdx6 dependent. Interaction between πGST and Prdx6, evaluated by the Duolink Proximity Ligation Assay, was minimal under basal conditions but increased dramatically following treatment of cells with the oxidant, tert-butyl hydroperoxide. Interaction was abolished by mutation of C47, the active site for Prdx6 peroxidase activity. Depletion of cellular GSH by treatment of cells with buthionine sulfoximine had no effect on the interaction of Prdx6 and πGST. These data are consistent with the hypothesis that oxidation of the catalytic cysteine in Prdx6 is required for its interaction with πGST and that the interaction plays an important role in regenerating the peroxidase activity of Prdx6.

Pentose phosphate shunt metabolism by cells of the chick growth cartilage

We have measured the activity of the pentose shunt pathway in the chick growth cartilage. Measurement of D-[1-14C] glucose and D-[6-14C] glucose metabolism by chondrocytes indicated that pentose phosphate shunt activity was low. However, when the cells were stimulated with phenazine methosulfate (PMS) and t-butyl hydroperoxide, a significant elevation in shunt activity was observed. This activity was further increased by dithiothreitol. Enzymatic and substrate requirements of the shunt pathway were examined and related to morphology of the tissue. It was found that as chondrocytes mature, there is increased glucose-6-phosphate dehydrogenase activity, and decreased quantities of glucose-6-phosphate and NADPH. While these investigations indicated that shunt activity was maximum in hypertrophic cartilage, the results of cytochemical studies suggested that the activity was greatest in those cells that were most removed from the O2 supply. Experiments were performed to examine O2 requirements of chondrocytes in relationship to the pentose phosphate shunt. First, using a phosphorescence quenching technique, total O2 uptake by these cells was found to be constant over a large part of the physiological range of O2 tensions. Over the same range, when stimulated by PMS, O2 uptake by CN- treated cells was increased. In the 1-5 microM O2 range, non-mitochondrial O2 consumption decreased more slowly than total respiration. Finally, the observation that NADPH directly stimulated chondrocyte O2 consumption suggest that cartilage cells may be able to form O2 metabolites.