Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells.

Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H2O2) initiates an increase in Ca2+/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 (ERK1/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1/2 and CREB in C10 cells. Application of H2O2 increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H2O2 increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H2O2-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1/2 and CREB that mediates cell survival during oxidant stress.

Protective role for nitric oxide during the endoplasmic reticulum stress response in pancreatic beta-cells.

Higher requirements for disulfide bond formation in professional secretory cells may affect intracellular redox homeostasis, particularly during an endoplasmic reticulum (ER) stress response. To assess this hypothesis, we investigated the effects of the ER stress response on the major redox couple (GSH/GSSG), endogenous ROS production, expression of genes involved in ER oxidative protein folding, general antioxidant defense, and thiol metabolism by use of the well-validated MIN6 beta-cell as a model and mouse islets. The data revealed that glucose concentration-dependent decreases in the GSH/GSSG ratio were further decreased significantly by ER-derived oxidative stress induced by inhibiting ER-associated degradation with the specific proteasome inhibitor lactacystin (10 microM) in mouse islets. Notably, minimal cell death was observed during 12-h treatments. This was likely attributed to the upregulation of genes encoding the rate limiting enzyme for glutathione synthesis (gamma-glutamylcysteine ligase), as well as genes involved in antioxidant defense (glutathione peroxidase, peroxiredoxin-1) and ER protein folding (Grp78/BiP, PDI, Ero1). Gene expression and reporter assays with a NO synthase inhibitor (Nomega-nitro-L-arginine methyl ester, 1-10 mM) indicated that endogenous NO production was essential for the upregulation of several ER stress-responsive genes. Specifically, gel shift analyses demonstrate NO-independent binding of the transcription factor NF-E2-related factor to the antioxidant response element Gclc-ARE4 in MIN6 cells. However, endogenous NO production was necessary for activation of Gclc-ARE4-driven reporter gene expression. Together, these data reveal a distinct protective role for NO during the ER stress response, which helps to dissipate ROS and promote beta-cell survival.

Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 beta-cell line.

Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca2+ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca2+-regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca2+ in cAMP production. We report that depolarization with high external K+, tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca2+ and inhibited by nitrendipine, a Ca2+ channel blocker, or 2',5'-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca2+ and cAMP oscillations that were abolished in the absence of extracellular Ca2+ and blocked by 2',5'-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca2+ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca2+ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca2+ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca2+-dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.

Temporal changes of multiple redox couples from proliferation to growth arrest in IEC-6 intestinal epithelial cells.

Changes in intracellular redox couples and redox reactive molecules have been implicated in the regulation of a variety of cellular processes, including cell proliferation and growth arrest by contact inhibition. However, the magnitude, direction, and temporal relationship of redox changes to cellular responses are incompletely defined. The present work sought to characterize redox and metabolic changes associated with proliferative stages to contact inhibition of growth in rat IEC-6 intestinal epithelial cells. From the first day of culture until 1 day before confluence, an increase in GSH concentrations and a significant reduction in the redox potential of the GSSG/2GSH couple were observed. These changes were accompanied by a decrease in relative reactive oxygen species (ROS) and nitric oxide (NO) concentrations and oxidation of the redox potential of the NADP(+)/reduced NADP and NAD(+)/NADH couples. Postconfluent cells exhibited a significant decrease in GSH concentrations and a significant oxidation of the GSSG/2GSH couple. When cell proliferation decreased, relative ROS concentrations increased (P < 0.01), whereas NO concentrations remained unchanged, and the NAD(+)/NADH couple became more reduced. Together, these data indicate that the redox potential of distinct couples varies differentially in both magnitude and direction during successive stages of IEC-6 growth. This finding points out the difficulty of defining intracellular redox status at particular stages of cell growth by examining only one redox species. In addition, the data provide a numerical framework for future research of regulatory mechanisms governed by distinct intracellular redox couples.

Proteasome inhibition alters glucose-stimulated (pro)insulin secretion and turnover in pancreatic {beta}-cells.

Metabolic labeling studies were conducted in freshly isolated mouse islets and a beta-cell line (MIN6) to examine the effects of proteasome inhibition on glucose-stimulated (pro)insulin synthesis and secretion. Glucose-stimulated (pro)insulin synthesis, as determined by the incorporation of [(3)H]tyrosine, decreased significantly by 90% in islets and 71% in MIN6 cells pretreated with the proteasome inhibitor lactacystin (10 microM) for 2 h. To follow the fate of newly synthesized (pro)insulin, islets were pulse-labeled with [(3)H]tyrosine (40 microCi) for 20 min and chased +/- lactacystin (10 microM) for up to 4 h. The release of newly synthesized (pro)insulin ([(3)H]tyrosine-labeled) was similar between lactacystin-treated and control islets despite a 51% decrease (p <0.05) in total immunoreactive (pro)insulin secretion by lactacystin-treated islets. The specific radioactivity of [(3)H]tyrosine-labeled (pro)insulin in the extracellular medium of lactacystin-treated islets (0.52 +/- 0.16 cpm/microunits) was 2-fold greater relative to control islets (0.25 +/- 0.06 cpm/microunits). Induction of the unfolded protein response by lactacystin, as evidenced by the up-regulation of endoplasmic reticulum (ER) chaperones (GRP78/BiP, GRP94, protein disulfide isomerase) and induction of the stress-inducible transcription factor C/EBP-homologous protein/GADD153 (CHOP/GADD153), likely contributed to the release of newly synthesized (pro)insulin to relieve ER stress. The present data indicate proteasome inhibition did not prevent, but increased (p <0.05), the intracellular degradation of [(3)H]tyrosine-labeled (pro-)insulin from 8 to 24% in islets. Collectively, these data indicate beta-cells may balance glucose-stimulated (pro)insulin synthesis and secretion with the activity of the proteasome to regulate protein concentrations in the ER.