The microtubule-associated protein tau is phosphorylated by Syk Aberrant phosphorylation of tau protein on serine and threonine residues has been shown to be critical in neurodegenerative disorders called tauopathies . An increasing amount of data suggest that tyrosine phosphorylation of tau might play an equally important role in pathology , with at least three putative tyrosine kinases of tau identified to date . It was recently shown that the tyrosine kinase Syk could efficiently phosphorylate α-synuclein , the aggregated protein found in Parkinson 's disease and other synucleinopathies . We report herein that Syk is also a tau kinase , phosphorylating tau in vitro and in CHO cells when both proteins are expressed exogenously . In CHO cells , we have also demonstrated by co-immunoprecipitation that Syk binds to tau . Finally , by site-directed mutagenesis substituting the tyrosine residues of tau with phenylalanine , we established that tyrosine 18 was the primary residue in tau phosphorylated by Syk . The identification of Syk as a common tyrosine kinase of both tau and α-synuclein may be of potential significance in neurodegenerative disorders and also in neuronal physiology . These results bring another clue to the intriguing overlaps between tauopathies and synucleinopathies and provide new insights into the role of Syk in neuronal physiology . Many neurodegenerative disorders are characterised by intracellular inclusions of highly insoluble proteins , and a classification based upon the main protein component of these aggregates is widely used . The term `` synucleinopathies '' , for instance , refers to a group of disorders including Parkinson 's disease -LRB- PD -RRB- in which the synaptic protein α-synuclein -LRB- α-syn -RRB- forms neuronal or glial aggregates -LSB- 1 -RSB- . `` Tauopathies '' are another pathological entity in which the microtubule-associated protein tau self-assembles into filamentous inclusions called neurofibrillary tangles -LRB- NFT -RRB- -LSB- 2 -RSB- . The tauopathies include Alzheimer 's disease -LRB- AD -RRB- , progressive supranuclear palsy and corticobasal degeneration . Human brain tau consists of a family of six isoforms , generated by the alternative splicing of a single mRNA transcript . Tau proteins contain either three -LRB- tau 3R -RRB- or four -LRB- tau 4R -RRB- tubulin-binding domains depending on the splicing of exon 10 , and either one -LRB- tau 1N -RRB- two -LRB- tau 2N -RRB- or no -LRB- tau 0N -RRB- N-terminal inserts depending on the inclusion of exon 2 , exons 2 and 3 , or exclusion of both , respectively . Their apparent molecular weights range from 45 kDa for tau 0N3R isoform to 65 kDa for tau 2N4R . When aggregated in NFT , tau is abnormally hyperphosphorylated by a series of serine/threonine kinases , among which glycogen synthase kinase-3β , cyclin-dependent kinase 5 and casein kinase 1 appear to be the most relevant -LSB- 3,4 -RSB- . It has been hypothesized that this hyperphosphorylation contributes to neurodegeneration through the destabilisation of microtubules -LSB- 5 -RSB- . Recent data suggest that phosphorylation of tau also occurs on tyrosine residues and this could be of potential significance in pathological conditions -LSB- 6,7 -RSB- . Human tau protein possesses five tyrosine residues on positions 18 , 29 , 197 , 310 and 394 -LRB- numbered according to the longest 2N4R tau isoform -RRB- . Tau extracted from NFT of AD patients was found to be phosphorylated on tyrosine 18 -LRB- Y18 -RRB- by immunocytochemistry using a phosphospecific antibody -LSB- 6 -RSB- , and on tyrosine 394 -LRB- Y394 -RRB- by mass spectrometry -LSB- 8 -RSB- . Tyrosine 197 -LRB- Y197 -RRB- has been shown to be phosphorylated in tau aggregates from transgenic mice overexpressing mutant tau -LSB- 9 -RSB- . Recently , the dual specificity kinase tau-tubulin kinase 1 has been shown to phosphorylate tau in vitro on Y197 -LSB- 10 -RSB- . While Abl was recently described as a candidate tyrosine kinase for Y394 -LSB- 8 -RSB- , Fyn is considered to be the kinase for Y18 -LSB- 6 -RSB- . The tyrosine kinase Syk is known for its critical role in signalling through immune receptors in leucocytes -LSB- 11 -RSB- . Syk tyrosine kinase expression is however not confined to hematopoietic cells , and has been reported in a variety of tissues -LSB- 12 -RSB- . Syk expression has been confirmed in the central nervous system -LRB- CNS -RRB- by immunoblot analysis of mouse brain homogenates , and its localization in mouse neuronal cytoplasm affirmed by immunohistochemistry -LSB- 12 -RSB- . While its function in the CNS remains unclear , Syk was recently found to be a kinase for α-syn -LSB- 13 -RSB- . The direct interaction of Syk and α-syn was proven by confocal microscopy and a dual-hybrid system approach -LSB- 13 -RSB- . The intriguing overlap in the pathophysiology of synucleinopathies and tauopathies prompted us to assess whether tau protein was also a substrate for Syk tyrosine kinase -LSB- 14 -RSB- . We herein report that Syk can phosphorylate Tau on Y18 , and that the kinase and its substrate directly interact . We first investigated whether Syk can directly phosphorylate tau in vitro . Recombinant human tau 2N4R was incubated with or without recombinant Syk or Abl -LSB- 8 -RSB- . Tau was phosphorylated in vitro by Syk and by its previously recognized tyrosine kinase Abl as judged on Western blots probed with P-Tyr-100 anti-phosphotyrosine antibody -LRB- Cell Signaling -RRB- , whereas no phosphorylation was observed in experiments where tau was incubated without Syk -LRB- Fig. 1 -RRB- . In the presence of Syk , an additional band of approximately 70 kDa , migrating just above tau , was observed on the phosphotyrosine immunoblot . This band is likely to contain Syk since the kinase is known to autophosphorylate . Taken together , these results demonstrate that Syk can directly catalyze tau phosphorylation . To determine whether Syk can phosphorylate tau in cells , co-transfection experiments were performed in CHO cells using Syk expression vectors together with untagged or V5-tagged tau . Cells were lysed and tau was immunoprecipitated with either anti-V5 antibody -LRB- Invitrogen -RRB- or with anti-tau antibody -LRB- Tau-5 , BD Biosciences -RRB- . Western analysis was then performed on immunoprecipitated tau using the anti-phosphotyrosine -LRB- P-Tyr-100 , Cell Signaling -RRB- , anti-tau -LRB- Tau-5 -RRB- or anti-Syk -LRB- N-19 , Santa Cruz -RRB- antibodies as previously described -LSB- 8 -RSB- . In preliminary experiments , a V5-tagged tau 2N4R construct and wild-type Syk were transiently coexpressed in CHO cells . We have previously shown that V5-tagged tau 2N4R migrates at approximately 70 kDa on SDS-PAGE -LSB- 8 -RSB- . Because of the close molecular weights of Syk -LRB- 72 kDa -RRB- and the V5-tau construct , the two proteins co-migrated using conventional denaturing gel electrophoresis -LRB- data not shown -RRB- . To better resolve the two proteins , we used two complementary approaches . In a first set of experiments , we expressed V5-tagged tau 2N4R along with a Syk-GFP construct that migrates at 100 kDa . The Syk-GFP construct was proven to remain functionally active in previous studies -LSB- 15 -RSB- . In a second set of experiments , we coexpressed wild-type untagged Syk along with the shortest tau isoform -LRB- 0N3R -RRB- , which migrates at 45 kDa . Tau protein was phosphorylated on tyrosine when co-transfected with Syk-GFP construct or with its known tyrosine kinase Abl -LRB- Fig. 2A -RRB- . Additional positive controls included tau-transfected cells treated with the tyrosine-phosphatase inhibitor pervanadate -LRB- 100 μM for 30 min -RRB- . When Syk-GFP was co-transfected with tau , tau immunoprecipitates contained a ∼ 100-kDa tyrosine-phosphorylated protein , likely to contain Syk-GFP since this enzyme is tyrosine phosphorylated -LRB- Fig. 2A -RRB- . This was confirmed by blotting with Syk antibody after membrane stripping -LRB- Fig. 2A -RRB- . This co-immunoprecipitation of Syk with tau is indicative of Syk and its substrate being in the same protein complex . In order to exclude any effects of the V5 and GFP tags fused with tau and Syk , we wished to repeat this experiment using vectors expressing native proteins . The untagged and shortest isoform of tau -LRB- 0N3R -RRB- was phosphorylated on tyrosine when co-transfected with native 72 kDa Syk -LRB- Fig. 2B -RRB- , as compared with the transfection of tau alone . As in Fig. 2A , an additional 72-kDa band corresponding to phospho-Syk was visible on the phosphotyrosine blot when Syk was co-transfected with tau . The co-immunoprecipitation is therefore confirmed with native proteins . Taken together , these results demonstrate that Syk tyrosine kinase can both phosphorylate and co-immunoprecipitate with tau in cells , consistent with the direct phosphorylation of the substrate demonstrated in vitro -LRB- Fig. 1 -RRB- . The binding of Syk to both the shortest and longest isoform of tau suggests that it is independent of tau splicing . Tau has been shown to be tyrosine phosphorylated on residue 18 , 197 and 394 -LSB- 6,8 -- 10 -RSB- . To map the tyrosine residue -LRB- s -RRB- phosphorylated by Syk in tau , Syk was co-transfected into CHO cells along with wild-type or mutant tau constructs in which individual tyrosines were replaced by phenylalanine -LRB- Y18F , Y197F and Y394F -RRB- . These cDNA constructs have been described in detail previously -LSB- 8 -RSB- . The only such tyrosine mutation that resulted in a strong decrease in tau tyrosine phosphorylation was Y18F -LRB- Fig. 3 -RRB- . Tyrosine phosphorylation of the Y197F and Y394F constructs were not significantly different from the wild-type control -LRB- Fig. 3 -RRB- . This clearly demonstrates that Y18 is the major tyrosine phosphorylation site for Syk . In addition , the binding of Syk was independent of the tyrosine phosphorylation state of tau -LRB- Fig. 3 -RRB- , suggesting that the SH2 domains of Syk are not involved in its binding to tau . As Fyn and Syk phosphorylate the same tyrosine residue on tau , we decided to perform a time course of phosphorylation of tau by the two kinases under conditions designed to favour the measurement of stoichiometry . After 30 min , Syk had incorporated an average of 0.23 mol of phosphate/mol of tau and Fyn had incorporated an average of 0.25 mol of phosphate/mol of tau . These results show that Syk and Fyn phosphorylate tau with the same efficacy -LRB- Fig. 4 -RRB- . Our demonstration of binding of tau to Syk is consistent with tau being a specific substrate for Syk and for their involvement in a cell-signaling pathway in neurons . Interestingly , Syk plays an important role in signalling events of neurite induction and outgrowth in neuronal cell lines -LSB- 16 -RSB- . It is thus tempting to speculate that the phosphorylation of tau by Syk could be involved in neurite outgrowth . Our study also reinforces the role of Syk in neurodegenerative disorders . Remarkably , Y18 has been shown to be phosphorylated in NFT , suggesting that , like Fyn , Syk could be critical in the pathophysiology of AD -LSB- 6,7,17 -RSB- . Although early studies suggested a clear distinction between ` tauopathies ' and ` synucleinopathies ' , more recent studies demonstrate that there is often an overlap in the pathological findings in these disorders -LSB- 18 -- 20 -RSB- . Syk binds to and phosphorylates both tau and synuclein , suggesting that this kinase could be a link between synucleinopathies and tauopathies . In conclusion , our findings significantly contribute to the understanding of the signaling pathways involving tyrosine phosphorylation of tau in both physiological and pathological conditions . Further elucidation of the functional relationship between tau and the Syk in neurons could provide critical insights into the role of tau in cell signaling as well as the role of tau in neurodegenerative processes .

O O O B O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O B O O O O O O O O O O O O O B O O B O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O B O O O O O O O O O O O O O O O O B O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O B O O O B O O O O O B O O O O O O O B O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O B O O O O O B O O B O O O O O O O O O O O B O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O B O O O O B I O O B O O O O O O O O O O O B O O O O O O O B O O O O B O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O B O O O O O B O O O B O O O O O O O O O O O O O O B O B O O O B O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O B O O B O O O O O O O O O O B O O O O O O O O O B O O O O


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 -LRB- H2O2 -RRB- initiates an increase in Ca2 + / cAMP-response element binding protein -LRB- CREB -RRB- phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 -LRB- ERK1/2 -RRB- . Here , we investigated the role of crosstalk between protein kinase A -LRB- PKA -RRB- and epidermal growth factor receptor -LRB- EGFR -RRB- 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 . Introduction Lung epithelial cells , a target cell of inhaled oxidants , are subjected to a variety of environmental stresses , including oxidizing gases , particulates , and airborne microorganisms . Although the formation of oxidants is normally well regulated , excessive production may cause inflammation and cellular injury . Numerous studies suggest a role for oxidant stress in the pathogenesis of pulmonary diseases , including asthma , pulmonary fibrosis , and cancer -LSB- 1 -RSB- . Reactive oxygen species -LRB- ROS -RRB- are involved in multiple physiological processes through their capacity to regulate the expression of several growth factor receptors , protein kinases and phosphatases . In alveolar type II lung epithelial cells , hydrogen peroxide -LRB- H2O2 -RRB- induces tyrosine phosphorylation of the epidermal growth factor receptor -LRB- EGFR -RRB- -LSB- 2 -RSB- and leads to activation of protein kinase C -LRB- PKC -RRB- -LSB- 3 -RSB- and the mitogen activated protein kinases -LRB- MAPK -RRB- p38 , c-Jun amino-terminal kinase -LRB- JNK -RRB- , and extracellular signal-regulated kinase 1/2 -LRB- ERK1/2 -RRB- -LSB- 4 , 5 -RSB- . H2O2 also activates the expression of transcription factors , including Ca2 + / cyclic AMP-response element binding protein -LRB- CREB -RRB- -LSB- 6 -RSB- and members of the activator protein -LRB- AP-1 -RRB- family , c-jun and c-fos -LSB- 7 -RSB- , resulting in proliferation -LSB- 4 -RSB- and apoptosis -LSB- 6 , 8 -RSB- . CREB is a 43 kDa transcription factor belonging to the basic-leucine zipper -LRB- bZIP -RRB- family and is regulated by phosphorylation at serine residue 133 -LSB- 9 , 10 -RSB- . Phosphorylation of CREB initiates recruitment of co-factors to the Ca2 + / cAMP-response element -LRB- CRE -RRB- that are necessary for transcriptional activation such as CREB-binding protein -LRB- CBP300 -RRB- -LSB- 11 -RSB- . CREB activation is regulated by both Ca2 + and cAMP , which have been shown to regulate both ERK1/2 - and protein kinase A -LRB- PKA -RRB- - mediated CREB phosphorylation -LSB- 6 , 12 , 13 -RSB- . Many genes important for regulation of proliferation and apoptosis , including c-fos and bcl-2 , respectively , contain CREs in their promoter -LSB- 11 -RSB- . Our previous studies have shown that exposure of alveolar type II cells to bolus H2O2 or asbestos fibers results in CREB activation that is dependent on ERK1/2 activation -LSB- 6 , 14 -RSB- . Furthermore , we found that preventing CREB activation promoted cell survival and enhanced bcl-2 transcription , revealing a potential role for CREB in H2O2-mediated apoptosis -LSB- 6 -RSB- . Because of known crosstalk between PKA , ERK1/2 , and CREB in many cell types and the dose-dependent effects of H2O2 , the goal of this study was to determine their relative importance in oxidant-induced signaling . The effects of transient bolus H2O2 as well as a peroxide generating system were tested to represent a range of oxidant concentrations that lead to cell proliferation and/or apoptosis . The data support the hypothesis that PKA and EGFR are central regulators of oxidant -- induced ERK1/2 and CREB activation and demonstrate a link between PKA , EGFR , and CREB in cell survival following exposure to H2O2 in alveolar type II cells . Materials and methods Cell culture and treatments C10 cells , a contact-inhibited , non-transformed murine alveolar type II epithelial cell line -LSB- 15 -RSB- , were grown in CMRL 1066 medium supplemented with L-glutamine , penicillin/streptomycin , and 10 % fetal bovine serum -LRB- FBS -RRB- -LRB- GIBCO BRL , Rockville , MD -RRB- . Cells were grown to 90 % confluence , and then complete medium was replaced with CMRL 1066 medium supplemented with l-glutamine , penicillin/streptomycin , and 0.5 % FBS for 48 h before exposure to agents . H2O2 -LRB- Sigma , St. Louis , MO -RRB- was added to the medium at concentrations from 100 μM to 300 μM . Recombinant glucose oxidase -LRB- GO ; 5 mU/ml , 15 mU/ml , or 30 mU/ml -RRB- -LRB- Roche , Indianapolis , IN -RRB- was used to provide low level fluxes of H2O2 -LSB- 16 , 17 -RSB- . Forskolin and epidermal growth factor -LRB- EGF -RRB- -LRB- Sigma , St. Louis , MO -RRB- -LRB- 10 μM and 100 ng/ml , respectively -RRB- were used as positive controls for induction of phospho-CREB and phospho-EGFR , respectively . Tumor necrosis factor α -LRB- TNFα -RRB- -LRB- Calbiochem , LaJolla , CA -RRB- was used at 0.1 μg/ml to induce apoptosis . Control cultures received medium without agents and were treated identically . The ERK1/2 inhibitor , U0126 -LRB- 10 μM for 30 min prior to treatment -RRB- , the EGFR tyrosine kinase inhibitor , Tyrphostin AG1478 -LRB- 10 μM for 1 h prior to treatment -RRB- , and the antioxidant enzyme , catalase -LRB- 1000 U/ml 1 h prior to treatment -RRB- were obtained from Calbiochem . The PKA inhibitor , H89 -LRB- 10 μM for 1 h pre-treatment -RRB- was obtained from Biomol -LRB- Plymouth Meeting , PA -RRB- . N-acetyl-l-cysteine -LRB- NAC ; 10 mM 16 h prior to treatment -RRB- and DL-Buthionine - -LRB- S , R -RRB- - sulfoximine -LRB- BSO ; 10 μM 16 h prior to treatment -RRB- were purchased from Sigma -LRB- St. Louis , MO -RRB- . Western blot analysis After C10 cells were exposed to agents as described above , the cells were washed twice with cold PBS and collected in 4X sample buffer -LRB- 200 μM Tris , pH 6.8 , 4 % SDS , 4 mg/ml bromophenol blue , 0.04 % β-mercaptoethanol , 40 % glycerol , 2 μM pyronin-Y -RRB- . The amount of protein in each sample was determined using the RC/DC protein assay -LRB- Bio-Rad -RRB- . About 30 μg of protein was separated by a 10 % SDS-PAGE and transferred to nitrocellulose . Western blots were performed as described previously -LSB- 6 -RSB- using antibodies specific to total and phosphorylated CREB -LRB- 1:1000 ; rabbit polyclonal anti-CREB , Cell Signaling Technologies , Danvers , MA ; 1:500 ; rabbit polyclonal anti-phospho-CREB , Cell Signaling Technologies -RRB- , total and phosphorylated ERK1/2 -LRB- 1:1000 ; rabbit polyclonal anti-ERK1 / 2 , Cell Signaling Technologies ; 1:500 ; rabbit polyclonal anti-phospho-ERK1 / 2 , Cell Signaling Technologies -RRB- . Antibody binding was detected using horse radish peroxidase -LRB- HRP -RRB- - conjugated anti-rabbit secondary antibody -LRB- 1:5000 ; Jackson ImmunoResearch Laboratories , Inc. , West grove , PA -RRB- , followed by chemiluminescence -LRB- Kirkgaard and Perry Laboratories , Gaithersburg , MD -RRB- . QuantityOne -LRB- Bio-Rad , Hercules , CA -RRB- was used to quantify band density , and intensity of phospho-CREB bands were normalized to the intensity of the corresponding total CREB bands . Live cell imaging of cAMP and redox potential C10 cells grown on glass coverslips in 6-well tissue culture dishes were transiently transfected with 8 μg of a plasmid encoding a unimolecular fluoresecent resonance energy transfer -LRB- FRET -RRB- - based cAMP biosensor , Epac1-camps -LRB- provided by Dr. Martin Lohse , University of Würtzburg -RRB- -LSB- 18 , 19 -RSB- , or 1 μg of a plasmid encoding a redox-sensitive green fluorescent protein -LRB- GFP -RRB- , roGFP2 -LRB- a gift from Dr. James Remington , University of Oregon -RRB- -LSB- 20 -RSB- , using Lipofectamine 2000 -LRB- Invitrogen , Carlsbad , CA -RRB- , following the manufacturer 's instructions . The transfection efficiency was approximately 50 % . Imaging experiments were conducted after 48 -- 72 h of transfection , and the growth medium removed and replaced with Krebs-Ringer bicarbonate solution -LRB- KRB ; 119 mM NaCl , 4.7 mM KCl , 2.5 mM CaCl2 , 1 mM MgCl2 , 1 mM KH2PO4 , 25 mM NaHCO3 or 10 mM HEPES-NaOH -LRB- pH 7.40 -RRB- , and 2 mM glucose -RRB- . Coverslips were placed into a heated microperfusion chamber mounted on the specimen stage of an inverted fluorescence microscope -LRB- Nikon TE-2000U -RRB- equipped with a CARV spinning disk confocal system -LRB- Atto Bioscience Inc. , Rockville , MD -RRB- . Cells were continuously superfused with Krebs-Ringer bicarbonate solution -LRB- 2 -- 5 ml/min -RRB- at 37 °C . Dithiothreitol -LRB- DTT -RRB- -LRB- Calbiochem , LaJolla , CA -RRB- was used to calibrate the redox minimum . For imaging Epac1-camps FRET and roGFP2 , cells were visualized with a Nikon Super Fluor 40 × objective . MetaFluor/MetaMorph software was used for image acquisition and analysis -LRB- Universal Imaging -RRB- . The Epac1-camps EYFP excitation wavelength was 440 nm . Dual emission ratio imaging at 485 nm and 535 nm for Epac1-camps and dual excitation ratio imaging at 400 nm and 490 nm for roGFP2 were accomplished using a computer-controlled high speed filter wheel -LRB- Lambda 10-2 optical filter changer with rotation every 60 -- 80 ms , Sutter Instrument Co. , Novato , CA -RRB- . For roGFP2 , a 505DRLP dichroic mirror and an emission filter , 535DF25 , were used . Images -LRB- 50 -- 250 ms exposure -RRB- were captured every 10 s with a 16-bit Cascade 650 digital camera -LRB- Roper Instruments , Trenton , NJ -RRB- and background-corrected by manual selection of background regions -LSB- 19 -RSB- . The ratio values were normalized to the average baseline values measured 1-min prior to application H2O2 . Epac1-camps FRET decreases with increasing cAMP concentration , thus increased -LSB- cAMP -RSB- c is correlated with an increase in the relative emission ratio of ECFP/EYFP -LRB- 485/535 -RRB- -LSB- 18 -RSB- . Data were thus expressed as relative ratio 485/535 for Epac1-camps and fold increase in relative ratio 400/490 for roGFP2 . Immunofluorescence in C10 cells C10 cells were grown on glass coverslips for all experiments . After experimental exposures , immunofluorescence to detect the catalytic subunit of PKAα was performed as previously described -LSB- 6 , 21 -RSB- . Briefly , cells were washed with phosphate buffered saline -LRB- PBS -RRB- , fixed in 3.7 % formaldehyde , permeablized with − 20 °C methanol and incubated with blocking solution containing 2 % bovine serum albumin -LRB- BSA -RRB- in PBS . Cells were incubated with primary antibody -LRB- 1:100 ; rabbit polyclonal PKAα catalytic subunit antibody , Santa Cruz , Santa Cruz , CA -RRB- diluted in 2 % BSA plus 0.1 % Triton X-100 in PBS -LRB- BSA/PBS-T -RRB- overnight at 4 °C . Secondary antibody -LRB- 1:400 ; AlexaFlour 568 goat-anti-rabbit IgG , Molecular Probes -RRB- diluted in BSA/PBS-T was applied for 1 h at room temperature -LRB- RT -RRB- , and followed by incubation with nuclear counterstain , YOYO-1 iodide -LRB- Molecular Probes , Carlsbad , CA ; 1:10,000 -RRB- , 1 unit/ml RNase , and 0.1 % sodium azide in BSA/PBS-T for 30 min at RT. . Coverslips were mounted onto slides with AquaPolyMount -LRB- Polysciences , Inc. . Warrington , PA -RRB- . For each sample , confocal images were collected in fluorescence modes using a Bio-Rad MRC1024ES confocal scanning laser microscope -LRB- Bio-Rad , Hercules , CA -RRB- . For quantification , a nuclear mask was generated in Corel Photopaint using the YOYO-1 image template , and pixel intensities for PKAα were determined within the nuclear mask area as previously described -LSB- 22 -RSB- . Transient transfections with small interfering RNA -LRB- siRNA -RRB- The siCONTROL non-targeting siRNA # 2 and SMARTpool mouse CREB siRNA -LRB- 100 nM ; Dharmacon , Lafayette , CO -RRB- were transfected into C10 cells using Lipofectamine 2000 -LRB- Invitrogen , Carlsbad , CA -RRB- , following the manufacturer 's instructions . After 4 h in transfection media , FBS was added to 10 % and cells were incubated for 24 h. Cells were then transferred to reduced serum media -LRB- 0.5 % FBS -RRB- for 24 h prior to treatments . Real time quantitative PCR -LRB- RT qPCR -RRB- Total RNA was extracted from C10 cells using the RNeasy ™ PLUS protocol for total RNA isolation from animal cells -LRB- Qiagen , Valencia , CA -RRB- . cDNA was reverse transcribed from 500 ng total RNA using an Omniscript ™ Reverse Transcriptase RNase free DNase kit -LRB- Qiagen -RRB- , with an oligo dT primer , according to the manufacturer 's protocol . RT qPCR primers and probes for B-cell lymphoma-2 -LRB- bcl-2 -RRB- and c-fos were obtained as Assays-on-Demand ™ kits from Applied Biosystems -LRB- Foster City , CA -RRB- . PCR products were detected by TaqMan qPCR , as previously described -LSB- 23 -RSB- . Expression levels of target genes were determined using hypoxanthine-guanine phosphoribosyl transferase -LRB- hprt -RRB- as the internal standard . Samples were run in duplicate from 3 independent experiments and the comparative Ct -LRB- cycle threshold -RRB- method for relative quantity -LRB- RQ value -RRB- was used to calculate relative mRNA expression among samples . Detection and quantification of apoptosis Apoptosis was detected by measuring single-stranded DNA using Apostain ™ as previously described -LSB- 24 -RSB- . Briefly , cell monolayers grown on glass coverslips were treated with 30 mU/ml GO for 24 h , then fixed in methanol for 24 h at − 20 °C , boiled for 5 min in PBS containing 5 mM MgCl2 , and then immersed in ice-cold PBS for 10 min . Cells were blocked with 40 % FBS and then incubated with Apostain F7-26 -LRB- Alexis Biochemicals , San Diego , CA , 10 μg/ml -RRB- followed by HRP-conjugated secondary antibody -LRB- goat anti-mouse IgM ; Jackson Labortories , West Grove , PA , 1:400 -RRB- . To visualize secondary antibody binding , the peroxidase substrate DAB -LRB- Sigma -RRB- was used . Coverslips were mounted onto slides with AquaPolyMount -LRB- Polysciences , Inc. . Warrington , PA -RRB- for subsequent examination using bright field light microscopy . To determine the numbers of apoptotic cells and total cell numbers per field , 5 random fields were evaluated per experimental condition at x200 total magnification . Statistical analysis Statistical analyses were performed using non-normalized data , and pair-wise comparisons between treatment groups were achieved using Student 's t-test or Mann -- Whitney Rank Sum Test method for unequal variances . ANOVA -LRB- Holm -- Sidak method -RRB- was used for multiple comparisons . Differences were considered statistically significant at P < 0.05 . Results CREB is phosphorylated after exposure to H2O2 and glucose oxidase We have previously shown that H2O2 causes CREB phosphorylation in alveolar type II cells -LSB- 6 -RSB- . Here , we further show by Western blot that the CREB response to bolus addition of H2O2 -LRB- Fig. 1a -RRB- and H2O2 generated by glucose oxidase -LRB- GO -RRB- -LRB- Fig. 1b -RRB- is reduced when pretreated with the antioxidants NAC or catalase at effective concentrations documented previously in C10 cells -LSB- 25 -RSB- . Fig. 1Bolus and glucose oxidase generated H2O2 mediate CREB phosphorylation in an oxidant-dependent manner . -LRB- a -RRB- C10 cells , pre-incubated with 1,000 U/ml catalase -LRB- CAT -RRB- for 1 h , were treated with 10 μM forskolin or 100 , 200 , or 300 μM H2O2 for 10 min , and then analyzed by Western blot analysis for phospho-CREB -LRB- p-CREB -RRB- . -LRB- b -RRB- C10 cells , pre-incubated with 10 mM NAC for 16 h or 1,000 U/ml catalase -LRB- CAT -RRB- for 1 h , were treated with 10 μM forskolin or 5 , 15 , or 30 mU/ml glucose oxidase -LRB- GO -RRB- for 4 h , and then analyzed by Western blot analysis for phospho-CREB -LRB- p-CREB -RRB- . An antibody recognizing total CREB was used as a control for protein loading in A and B. Data are representative of 3 separate experiments H2O2-induced CREB phosphorylation is decreased after the reduction of PKA activity Because PKA is an important regulator of CREB -LSB- 9 -RSB- , we investigated its role in H2O2-induced CREB phosphorylation . Cells were examined after exposure to lower concentrations previously associated with expression of cyclin D -LRB- 100 μM -RRB- and higher concentrations known to induce apoptosis -LRB- 300 μM -RRB- -LSB- 6 , 26 -RSB- . Exposure to H2O2 led to CREB phosphorylation in a concentration-dependent manner that was significantly decreased after reduction of PKA activity using the specific inhibitor H89 -LRB- Fig. 2a , b -RRB- . The pattern of CREB phosphorylation in response to H2O2 closely paralleled that seen in response to activation of PKA by forskolin . H89 also reduced H2O2-mediated ERK1/2 activation , suggesting that PKA may regulate CREB activation directly or indirectly through ERK1/2-mediated CREB activation . Fig. 2H2O2-mediated CREB activation is inhibited by the PKA inhibitor , H89 . -LRB- a -RRB- C10 cells , pre-incubated with 10 μM H89 for 1 h , were treated with 10 μM forskolin or indicated concentrations of H2O2 for 10 min , and then analyzed by Western blot analysis for phospho-CREB -LRB- p-CREB -RRB- , total CREB , phospho-ERK1 / 2 -LRB- p-ERK1 / 2 -RRB- and total ERK1/2 . -LRB- b -RRB- Quantification of p-CREB band intensities from A corrected using the corresponding CREB band intensity and normalized to the untreated control . Data represent 4 separate experiments ; * P < 0.05 when compared with corresponding condition without H89 . -LRB- c -RRB- C10 cells , pre-incubated with 10 μM H89 for 1 h , were treated with 10 μM forskolin or indicated concentrations of glucose oxidase -LRB- GO -RRB- for 4 h , and then analyzed by Western blot for phospho-CREB -LRB- p-CREB -RRB- , total CREB , phospho-ERK1 / 2 -LRB- p-ERK1 / 2 -RRB- , and total ERK1/2 . -LRB- d -RRB- Quantification of p-CREB band intensities from C corrected using the corresponding CREB band intensity and normalized to the untreated control . Data represent mean ± SEM from 4 separate experiments ; * P < 0.05 when compared with corresponding condition without H89 Similar to results with bolus addition of H2O2 , H2O2 generated by GO stimulated CREB phosphorylation in a dose-dependent manner and the phosphorylation was significantly reduced by PKA inhibition at higher concentrations of GO -LRB- Fig. 2c , d -RRB- . However , unlike bolus addition , H2O2 generated by GO led to a dose-dependent increase in ERK1/2 phosphorylation that was marginally sensitive to inhibition of PKA activity , suggesting that the duration of the stimulus may cause differences in signaling patterns . H2O2 stimulates cAMP production in C10 cells Single cell imaging was utilized to determine whether H2O2 exerts a direct effect on upstream regulation of PKA through production of cAMP . Epac1-camps , a FRET-based cAMP biosensor , was expressed in C10 cells . Epac1-camps senses changes in cytoplasmic cAMP levels -LRB- -LSB- cAMP -RSB- c -RRB- through a cAMP-dependent conformational change resulting in reduced FRET between ECFP -LRB- 485 emission -RRB- and EYFP -LRB- 535 emission -RRB- -LSB- 18 , 19 -RSB- . Exposure to H2O2 caused a monophasic increase in -LSB- cAMP -RSB- c -LRB- Fig. 3a -RRB- that was comparable to the response elicited by direct activation of adenylyl cyclase with forskolin , but with a slower rise time -LRB- Fig. 3c -RRB- . H2O2 generated by GO stimulated a rise in -LSB- cAMP -RSB- c similar in magnitude to bolus addition of H2O2 , but with a longer lag-time presumably due to the slower generation of oxidant -LRB- Fig. 3b -RRB- . Fig. 3H2O2 stimulates an increase in cAMP levels . C10 cells were transiently transfected with the cAMP FRET indicator Epac1-camps and then exposed to 10 μM 250 μM H2O2 -LRB- a -RRB- , 15 mU/ml glucose oxidase -LRB- GO -RRB- -LRB- b -RRB- , or 10 μM forskolin -LRB- FSK -RRB- -LRB- c -RRB- for the times indicated . Data -LRB- means ± SEM -RRB- are expressed as the relative ratio -LRB- 485/535 -RRB- of the cAMP response from the live emission recording of a representative cell . Grey bars indicate time of addition and length of exposure . Results are representative of ≥ 9 , 5 , and 4 cells -LRB- A , B and C , respectively -RRB- Single cell imaging was also used to measure redox status and correlate cAMP levels with redox status . The redox sensitive GFP variant , roGFP2 , was expressed in C10 cells . Oxidation is detected by roGFP through a change in GFP excitation from 400 nm and 490 nm -LSB- 20 -RSB- . The time course of H2O2-mediated cAMP production correlated well with the level of intracellular oxidation measured by roGFP2 -LRB- Fig. 4a -RRB- . Upon removal of H2O2 , the redox state returned to baseline approximately 36 min after the initial exposure -LRB- Fig. 4b -RRB- . Fig. 4Redox status correlates with cAMP level in cells responding to H2O2 . C10 cells were transiently transfected with the redox indicator , roGFP2 and then exposed to 250 μM H2O2 for 10 min followed by 1 mM DTT -LRB- a -RRB- or 15 mU/ml glucose oxidase -LRB- GO -RRB- for 65 min , followed by 200 μM H2O2 , and then 10 mM DTT -LRB- b -RRB- . Data -LRB- means ± SEM -RRB- are expressed as the fold change in the excitation ratio at 400/490 nm normalized to the average base-line ratio values measured 1 min before stimulation . Results are averages of ≥ 23 and 11 cells -LRB- A and B , respectively -RRB- . Grey bars indicate time of addition and length of exposure , and WO indicates wash out with KREBs buffer EGFR tyrosine kinase activity is important for signaling from H2O2 to PKA and CREB It has been shown that the protein tyrosine activity of EGFR is important for EGF-mediated stimulation of adenylyl cyclase -LSB- 27 -RSB- . Thus to determine the contributions of EGFR in the observed H2O2-induced CREB phosphorylation , cells were evaluated with or without pretreatment with the specific EGFR tyrosine kinase inhibitor , AG1478 . Application of AG1478 significantly reduced CREB phosphorylation in response to bolus addition H2O2 and to H2O2 generated by higher concentrations of GO -LRB- Fig. 5 -RRB- , suggesting that EGFR activity is involved in H2O2-induced CREB phosphorylation . As expected , reduction of EGFR activity also inhibited H2O2-induced ERK phosphorylation -LRB- Fig. 5 -RRB- . Fig. 5Reduction of EGFR tyrosine kinase activity leads to the inhibition of both H2O2-induced CREB and ERK1/2 . C10 cells , pre-incubated with the EGFR tyrosine kinase inhibitor AG1478 -LRB- 10 μM -RRB- for 1 h , were treated with 10 μM forskolin or 100 ng/ml EGF for 5 min , the indicated concentrations of H2O2 for 10 min -LRB- a -RRB- or glucose oxidase -LRB- GO -RRB- for 4 h -LRB- c -RRB- , and then analyzed by Western blot analysis for phospho-CREB -LRB- p-CREB -RRB- , total CREB , phospho-ERK1 / 2 -LRB- p-ERK1 / 2 -RRB- , and total ERK1/2 . -LRB- b , d -RRB- Quantification of p-CREB band intensities from A and C respectively corrected using the corresponding CREB band intensity and normalized to the untreated control . Data represent mean ± SEM of 3 separate experiments ; * P < 0.05 when compared with corresponding condition without AG1478 Immunofluorescence evaluation of activated PKA translocation to the nucleus was used to more directly test the effects of H2O2 on PKA activation and to further evaluate a role for EGFR . Exposure to H2O2 led to an increase in the PKA free catalytic subunit that was significant in the nucleus at 300 μM and similar to the response to forskolin . Inhibition of EGFR tyrosine kinase activity with AG1478 prevented the H2O2-induced increases at both 200 μM and 300 μM -LRB- Fig. 6 -RRB- . Fig. 6Reduction of EGFR tyrosine kinase activity inhibits the H2O2-induced nuclear translocation of activated PKA . C10 cells , pre-incubated with 10 μM AG1478 for 1 h , were exposed to 10 μM forskolin and H2O2 for the indicated concentrations for 10 min . -LRB- a -RRB- The PKA free catalytic subunit -LRB- red -RRB- was detected by immunofluorescence and nuclei were stained with YOYO-1 -LRB- green -RRB- . -LRB- b -RRB- Quantification of PKA immunofluorescence pixel intensity within nuclei ; * P < 0.05 compared to control , # P < 0.05 compared to treatment , n = 3 experiments , 5 fields/experiment Direct activation of CREB by PKA is a minor pathway in EGF-induced CREB phosphorylation Because of the known crosstalk between EGFR , ERK1/2 , and PKA , we examined the role of PKA and ERK1/2 in forskolin - and EGF-induced CREB activation . Treatment with both forskolin and EGF resulted in a significant increase in CREB phosphorylation when compared to control cells -LRB- Fig. 7 -RRB- . Inhibition of PKA activity with H89 significantly decreased forskolin-induced CREB phosphorylation , but only blunted EGF-induced CREB phosphorylation . Inhibition of ERK1/2 phosphorylation with the MEK inhibitor , U0126 , also considerably decreased CREB phosphorylation . The inhibitory effects of H89 and U0126 were not additive towards either forskolin - or EGF-induced CREB phosphorylation , suggesting that direct activation of CREB through PKA may play a minor role in the activation of CREB . Furthermore , both forskolin and EGF induced ERK1/2 phosphorylation , but only forskolin-mediated CREB phosphorylation was inhibited by H89 , suggesting that PKA regulates CREB indirectly through the MAPK/ERK1/2 pathway . Fig. 7Crosstalk between PKA and ERK1/2 signaling is important in regulating CREB phosphorylation . -LRB- a -RRB- C10 cells , pre-incubated with 10 μM H89 , U0126 , or H89 and U0126 for 1 h , were treated with 10 μM forskolin for 10 min or 100 ng/ml EGF for 5 min , and then analyzed by Western blot analysis for phospho-CREB -LRB- p-CREB -RRB- , total CREB , phospho-ERK1 / 2 -LRB- p-ERK1 / 2 -RRB- , and total ERK1/2 . -LRB- b -RRB- Quantification of phospho-CREB in A corrected using CREB intensities and phospho-ERK/12 in A corrected using ERK1/2 intensities ; * P < 0.05 for DMSO treatment compared to AG1478 treatment ; # P < 0.05 for DMSO control compared to treatment . Data represent mean ± SEM of 3 separate experiments Knockdown of CREB using siRNA results in loss of oxidant-induced c-fos and bcl-2 transcription To more directly assess the role of CREB in oxidant-mediated responses , an siRNA approach was used to reduce levels of CREB within C10 cells . Transfection with siCREB resulted in greater than 80 % knockdown of both CREB mRNA and protein levels -LRB- Fig. 8a , b -RRB- . CRE-containing genes previously shown to be upregulated by H2O2 including bcl-2 and c-fos were found to be dose-dependently induced by GO . Induction was significantly reduced in cells transfected with siCREB -LRB- Fig. 8c , d -RRB- , suggesting that CREB is essential for the transcriptional response of these genes to H2O2 . Fig. 8Knockdown of CREB using siRNA results in loss of oxidant-induced c-fos and bcl-2 transcription . -LRB- a -RRB- C10 cells were transfected with 100 nM scrambled siRNA -LRB- siControl -RRB- or siCREB followed by Western blot analysis to detect CREB . β-actin was used as a loading control . -LRB- b -RRB- Cells were transfected as in A , followed by treatment with 5 or 15 mU/ml glucose oxidase -LRB- GO -RRB- for 6 h. Total RNA was extracted and cDNA was assessed by RT qPCR to quantify CREB , bcl-2 and c-fos mRNA levels . Shown are relative quantity -LRB- RQ -RRB- values normalized to the siControl sample . # P < 0.05 when compared to untreated siCon ; * P < 0.05 when compared to treatment siCon Knockdown of CREB significantly increases the number of cells undergoing apoptosis following exposure to glucose oxidase Recently , we have shown that reduction of CREB activity , by introducing a phosphorylation-null CREB construct , led to a decrease in apoptosis -LSB- 6 -RSB- . To resolve whether CREB is essential for H2O2-induced apoptosis , C10 cells transfected with scrambled siRNA -LRB- siControl -RRB- or siCREB were treated with 30 mU/ml GO for 8 h and apoptotic cells were identified by Apostain -LRB- Fig. 9 -RRB- . H2O2 generated from GO induced a significant increase in the percentage of cells undergoing apoptosis compared to the control in both siControl and siCREB cells . Cells expressing siCREB had significantly higher levels of apoptosis than the siControl cells in both the untreated condition and after GO exposure , supporting a role for CREB in the regulation of apoptosis induced by H2O2 . Interestingly , the apoptotic enhancing effect was not selective for H202-mediated apoptosis , as siCREB also enhanced the apoptotic effect of TNFα . Taken together , these results suggest that CREB plays an important role in the cellular response to oxidant stress including upregulation of survival genes and promoting cell survival . Fig. 9CREB is protective against oxidant-induced apoptosis . -LRB- a -RRB- C10 cells , transfected with siControl or siCREB , were exposed to 30 mU/ml glucose oxidase -LRB- GO -RRB- or 0.1 μg/ml tumor necrosis factor α -LRB- TNFα -RRB- for 8 h followed by identification of apoptotic cells using Apostain . -LRB- b -RRB- Quantification of % Apostain positive cells was determined by scoring the average of 5 fields/condition ; * P < 0.05 when compared to untreated siCon ; # P < 0.05 when compared to treatment siCon . Data represent mean ± SEM of 4 separate experiments . Bar = 100 μm Discussion Exposure to various environmental stresses , such as oxidative gases , metals , and particulates , initiates multiple physiological processes in alveolar type II cells . Oxidants have been implicated in the pathogenesis of lung cancer , pulmonary fibrosis , and asthma through their capacity to regulate an intricate network of protein kinases . Yet the relationship between oxidants and the pathogenesis of lung disease is unclear , primarily because of the lack of understanding of the mechanisms by which oxidants function in both normal physiological and disease states . In this study , we show for the first time that H2O2 exposure leads to an increase in cytoplasmic cAMP levels , followed by PKA-dependent CREB and ERK1/2 phosphorylation in alveolar type II cells . Furthermore , we show that EGFR activity is necessary for H2O2-stimulated nuclear accumulation of activated PKA and for PKA-mediated CREB phosphorylation . Together these data suggest that PKA is a central hub for interplay between EGFR , ERK1/2 , and CREB signaling pathways activated by oxidant stress . Several studies have shown crosstalk between PKA and ERK1/2 signaling pathways . This crosstalk appears to be cell type specific , since an increase in cAMP-dependent PKA activation stimulates ERK1/2 in some cells , but suppresses it in others -LSB- 28 -- 30 -RSB- . We have previously demonstrated that H2O2 exposure leads to ERK1/2-dependent CREB phosphorylation in C10 alveolar type II cells -LSB- 6 -RSB- . Studies here have dissected the underlying mechanisms to reveal that H2O2-induces cAMP formation and PKA activation that are important for both ERK1/2 and CREB phosphorylation . EGFR is overexpressed and activated in response to epithelial injury -LSB- 31 -RSB- . Its activation is believed to play an integral role in the cellular responses of lung epithelium to injury and oxidant stress , possibly by signal amplification through MAPK phosphorylation cascades . Oxidative modification of a reduced cysteine residue in the EGFR reversibly affects its activation -LSB- 32 , 33 -RSB- . Moreover , Goldkorn and colleagues have shown that in alveolar type II cells , H2O2 induces EGFR phosphorylation on tyrosine residues -LSB- 2 -RSB- . These findings suggest that even though there is no specific receptor for oxidants , their signaling may be efficiently transduced through interactions with the EGFR . In the present study , we show that EGFR activity is necessary for both activation of PKA nuclear translocation and subsequent CREB phosphorylation in response to H2O2 . Mitogenic signals transmitted through the EGFR have been shown to involve PKA-dependent signaling cascades . Tyrosine phosphorylation of the EGFR requires an increase in cAMP levels and PKA function in many , but not all cell types -LSB- 34 , 35 -RSB- . In addition , tyrosine kinase activity of EGFR is required for growth factor stimulation of adenylyl cyclase activity , which leads to the activation of PKA -LSB- 27 , 36 -RSB- . PKA activity has also been shown to promote downstream Raf-1 function and subsequent ERK activation -LSB- 37 -RSB- . Crosstalk has also been seen from EGFR to adenylyl cyclase activity and PKA in airway epithelial cells via beta adrenergic receptors -LSB- 38 , 39 -RSB- . Thus , communication between EGFR and PKA is potentially bi-directional . The H2O2-mediated generation of cAMP and the CREB activation response to H2O2 increased with oxidant concentration but , with minor exception , were not dependent on whether oxidant was added by bolus or generated by glucose/glucose oxidase . Although reduction of the CREB response by PKA or EGFR inhibition was only significant for the higher oxidant concentrations , a trend was observed with lower concentrations . While it is difficult to accurately predict the physiological significance of these oxidant levels , when put in context with previous findings , these observations suggest that CREB signaling through PKA and EGFR could participate in signaling that affects both oxidant-mediated proliferation and apoptosis . Previously we demonstrated that expression of phosphorylation-incompetent CREB paradoxically increases bcl-2 transcription and reduces apoptosis following bolus H2O2 exposure -LSB- 6 -RSB- . Findings here show that siRNA-mediated knockdown of CREB results in a loss of bcl-2 mRNA and an increase in apoptosis in cells responding to an H2O2 generating system . The difference is likely due to the method of CREB inhibition , in that the dominant-negative CREB may have altered chromatin interactions with other transcription factors or caused quenching of the response . Putting these results in context with the findings of others , we propose that CREB activation through PKA signaling serves as a key survival pathway in cells responding to oxidant stress . PKA-and CREB-mediated gene expression may thus be important in regulating oxidant-mediated apoptosis resulting from lung pathologies , and future experiments will explore the importance of these signaling pathways in promoting changes in gene expression following lung epithelial cell injury or oxidant exposure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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pulmonary epithelium
lung fibrosis
oxidative stress
fret biosensor