Dataset of KCNQ1, KCNN4, KATP channel expression and dexamethasone modulation of protein kinase signaling in airway epithelial cells

Dexamethasone produces anti-secretory responses in airway epithelium through the inhibition of basolateral membrane K+ channels [1–3]. We have used the human bronchial epithelial cell line 16HBE14o− to investigate the effects of dexamethasone on the expression of K+ channels and regulatory protein kinases. The data demonstrate the expression of three distinct K+ channel types – KCNQ1:KCNE3, KCNN4 and KATP which are differentially regulated by protein kinase A and protein kinase C. The data also provide evidence for rapid non-genomic actions of dexamethasone on PKC and PKA phosphorylation and their association with the various K+ channel sub-types. Biotinylation experiments provide data on the effects of dexamethasone on membrane expression of the K+ channels. Antibody co-immunoprecipitation, rtPCR and western blotting data are given for the non-genomic dexamethasone transcription-cell signaling pathway involving Gi-protein coupled receptor, PKC, adenylyl cyclase Type IV, cAMP, PKA and ERK1/2 activation.


Data
We have previously described a rapid non-genomic anti-secretory effect of corticosteroids on airway Clsecretion via inhibition of basolateral membrane K þ channels [1e3]. Here we present the original data underpinning these findings.
2. Experimental design, materials, and methods 2.1. Expression of the KCNN4 protein in 16HBE14o À airway epithelial cells Western blot analysis was performed to determine expression of the KCNN4 channel in 16HBE14o À cells. Primers specific for KCNN4 (GenBank accessionNo. AF000972) were designed using the Primer 3 software (Table 1). A PCR product of the expected size was generated from complimentary DNA extracted from 16HBE14o À cells (Fig. 1). The T84 cell line was used as a positive control. As illustrated in Fig. 2, an anti-KCNN4 antibody recognized prominent bands with the molecular masses of 46 kDa.

Membrane expression of KCNN4 in 16HBE14o À cells
Biotinylation experiments were performed to investigate the effect of dexamethasone on the expression and membrane localization of KCNN4 channels (Fig. 3). The presence of KCNN4 at the basolateral membrane was confirmed by cell surface biotinylation. However, no change in surface expression of KCNN4 following dexamethasone (15mins, 1 nM) or vehicle (15mins, methanol, 0.001% v/v) treatment was observed. Table 1 First-strand cDNA was subjected to PCR amplification using primers designed with the Primer 3 PCR primer design program (Whitehead Institute for Biomedical Research).

Protein
Primer Sequence 5 0 / 3 0 Product size bp KCNN4 (F) GCCGTGCGTGCAGGATTTAGG 403 KCNN4 (R) GCCCGGCACCACGTCACCATA GAPDH (F) CATTGGGGGTAGGAACACGGA 373 GAPDH(R) GCCAAAAGGGTCATCATCTCCG Fig. 1. Semi-quantitative RT-PCR analysis of KCNN4 expression in 16HBE14o ¡ cells. Total RNA was isolated from 16HBE14o À cells using RNA easy kit (Qiagen). cDNA was reverse transcribed using poly dT primers and ImProm™ Reverse Transcriptase System (Promega, USA) and 1 ml of this reaction was directly amplified using GoTaq® Green Master Mix. (Promega, USA) using specific primers for human KCNN4 isoform and synthesised by MWG Biotech (Germany). The PCR reaction produced DNA fragments at the expected length for KCNN4 in T84 and 16HBE14o À cells. GAPDH (cDNA and GAPDH primer pairs) was used as a control and neg (negative control, primers pairs without cDNA). Total protein (100 mg/lane) was transferred to nitrocellulose membrane after fractionating by SDS-PAGE and blotted with anti-KCNN4. Bands at 46 kDa corresponding to KCNN4 were detected. b-actin was used as a control to estimate protein loading.
Values represent mean ± SEM, n ¼ 3; n.s. denotes values were not significant between T84 and 16HBE14o À samples. Statistical analysis was performed using the Student's paired t-test.  Dexamethasone (1nM) or vehicle (methanol, 0.001% v/v) was added to 16HBE14o À cells for 15 mins. Barchart summary for the effect of dexamethasone on cell surface biotinylation in 16HBE14o À cells. Values are mean ± SEM, n ¼ 3, n.s. denotes values are not significant. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison tests.

Expression of KCNQ1 channels in 16HBE14o À airway epithelial cells
Semi-quantitative RT-PCR was carried out to determine expression of KCNQ1. Different primer pairs, designed to specifically amplify human KCNQ1 was used to generate PCR products from cDNAs of 16HBE14o À cells (Table 2). Human KCNQ1 (hKCNQ1) cloned into pTLN6, was kindly provided by Prof. Thomas Jentsch (Leibniz-Institute for Molecular Pharmacology, Berlin, Germany), and was used as a positive control for KCNQ1 expression. Agarose gels showing RT-PCR products amplified from 16HBE14o À cDNA with PCR primer pairs for hKCNQ1. Fig. 4 shows that the hKCNQ1 product was detected in 16HBE14o À cells. Western blot analysis was performed to determine expression of the KCNQ1 channel and its regulatory subunit KCNE3 in 16HBE14o À cells (Fig. 5). As illustrated, anti-KCNQ1 and anti-KCNE3 antibodies recognized prominent bands with the molecular masses of 37 kDa and 27 kDa respectively. The T84 cell line was used as a positive control as these cells are known to express KCNQ1 protein. These data confirm that the cAMP-dependent KCNQ1 channels and their KCNE3 regulatory subunit are expressed in 16HBE14o À cells.

Expression of K ATP channels in 16HBE14o À epithelial cells
The molecular identity of the lung K ATP channel subunits was investigated in the 16HBE14o À cells. Different primer pairs, designed to specifically amplify human Kir 6.1, Kir 6.2, SUR 1 and SUR 2A (Table 3) were used to generate PCR products from cDNAs of 16HBE14o À cells. The Kir 6.1 and Kir 6.2 primer pairs amplified bp products, respectively (Fig. 6). In addition, the SUR 1 primer pairs Table 4 List of primers for PKC isoforms and GAPDH.

Protein
Primer Sequence 5 0 / 3 0 Product size bp . Semi-quantitative RT-PCR analysis of PKC isoforms in human bronchial epithelial cells. Total RNA was isolated from 16HBE14o À cells using RNA easy kit (Qiagen). cDNA was reverse transcribed using poly dT primers and ImProm™ Reverse Transcriptase System (Promega, USA) and 1 ml of this reaction was directly amplified using GoTaq® Green Master Mix. (Promega, USA) using specific primers for human PKC isoforms and PKD (Table 3) and synthesised by MWG Biotech (Germany). The PCR reaction produced DNA fragments at the expected length for PKCa, PKCd, PKCε and PKCm (PKD1). GAPDH (þ) (cDNA and GAPDH primer pairs) was used as a control. Image representative of three independent experiments. amplified a 340 bp product. No product could be detected with primers for SUR2A. These results suggest that the K ATP channels in 16HBE14o À cells could be formed from Kir 6.1, Kir 6.2 and SUR2A subunits. The sulfonylurea receptors SUR 2A and subunits Kir 6.1 and Kir 6.2 were found to be expressed in the 16HBE14o À cell line. Values are given as reflective PKC expression in 16HBE14o À cell lysates compared to MCF-7. Values are displayed as mean ± SEM (n ¼ 3). ** Denotes p < 0.001, * denotes p < 0.01, n.s. denotes not significant (p > 0.05) between PKC isoform in MCF-7 and 16HBE14o À . Statistical analysis was performed using the Students paired t-test.

Cell surface expression of KCNQ1
Biotinylation experiments were performed to investigate the effect of dexamethasone on the localization of the KCNQ1 channel. The presence of KCNQ1 at the membrane was confirmed by cell surface biotinylation (Fig. 7). There was no change in surface expression of KCNQ1 following dexamethasone (1 nM) or vehicle (0.001% methanol) treatment indicating that dexamethasone does not change the cellular localization or expression of the KCNQ1 channel.

Effects of dexamethasone on expression and activation of PKC isoforms in human bronchial epithelial cells
Candidate protein kinase C isoforms of the cPKC and the nPKC groups were investigated in mediating the rapid anti-secretory responses to dexamethasone. Primers specific for PKCa, PKCd, PKCε and . PKCa is rapidly activated in response to dexamethasone in 16HBE14o ¡ cells. Representative Western blot analysis of phospho-PKCa in cellular extracts of 16HBE14o À . The activation of PKCa by dexamethasone was monitored using antibodies specific to the phosphorylated form of PKCa (Ser 657). b-actin (42 kDa) was used as an internal control to estimate protein loading. The graph represents densitometric analysis at specific time points of dexamethasone treatment. Values are given as fold changes in PKCa activation of 16HBE14o À cell lysates. Values are displayed as mean ± SEM (n ¼ 4). ** Denotes significance: p < 0.001, * denotes p < 0.01 between control and treated values. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison tests.

Table 5
Summary of the fold increase in PKCa activity induced by dexamethasone.

Expression of PKC isoforms in human bronchial epithelial cells
The results obtained from RT-PCR analysis were confirmed by western blotting. Western blots were performed on three independently derived cell lysates to establish PKC isoform expression. As a positive control lysates from MCF-7 breast cancer cell line was used. Western blot analysis revealed expression of these selected isoforms in 16HBE14o À cells. An equivalent amount of protein (50 mg) was loaded in each track and equal loading of samples was confirmed by probing the same blot with b-actin monoclonal antibody.
Immunoblots using antibodies for individual isoforms of PKC were performed: PKCa (  Representative Western blot analysis of phospho-PKCd in cellular extracts of 16HBE14o À . Using antibodies specific to the phosphorylated form of PKCd (Ser 643), the activation by dexamethasone was monitored by Western blot analysis. b-actin (42 kDa) was used as an internal control to estimate protein loading. The graph represents densitometric analysis at specific time points of dexamethasone treatment. Bombesin (BOM) was used as a positive control for PKCd activity. Values are given as fold changes in PKCd activation of 16HBE14o À cell lysates. Values are displayed as mean ± SEM (n ¼ 4); n.s. denotes no significance between control and treated values. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison tests. revealed the expression of the classical isoform PKCa (80 kDa), the novel isoforms PKCd (78 kDa) and PKCε (95 kDa) and also expression of PKD (115 kDa). PKCa and PKD1 were expressed in equal quantities in 16HBE14o À cells compared to MCF-7 cells (positive control). PKCd and PKCε were significantly (**p < 0.001, *p < 0.01) respectively, less expressed in 16HBE14o À cells compared to MCF-7 control. This reflected non-uniform expression of PKC isoform levels (PKCa > PKD1 > PKCε > PKCd levels of expression).

Effect of dexamethasone on PKCa, PKCd and PKCε activity in 16HBE14o À cells
The levels of PKC isoform activation can be observed through changes in phosphorylation states at key amino acid residues. For PKCa activation Ser657 autophosphorylation in the hydrophobic C-terminal is required for catalytic activation and stabilisation of the protein upon translocation to the plasma membrane. PKCd activity was assessed using an antibody to PKCd phosphorylated at Ser643. PKCd autophosphorylates at Ser643 in the turn motif after initial phosphorylation at Thr505 by PDK-1  Table 7. Bombesin (BOM) was used as a positive control for PKCε activity. Values are displayed as mean ± SEM (n ¼ 3); n.s. denotes no significance between control and treated values. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison tests.  (2), Ser729 in the C-terminal hydrophobic region and (3) autophosphorylation at Thr710. The level of phosphorylation at these residues can be used as a measure of catalytic activity of the kinase isoforms. By employing antibodies specific to the phosphorylated form of PKCs, activation was monitored in response to dexamethasone by Western blot analysis. PKCa activation was assessed by probing with a specific antibody to phosphorylation at Ser 657. Dexamethasone treatment produced a biphasic activation of PKCa at 2 and 10 mins (Fig. 10, Table 5). PKCd activity was measured using a specific antibody to phosphorylation at Ser643. Dexamethasone treatment had no effect on PKCd phosphorylation levels (Fig. 11, Table 6). PKCε activity was measured Table 8 List of primers for human adenylyl cyclase isoforms AC1-9 and GAPDH.
. Semi-quantitative RT-PCR analysis of adenylyl cyclase isoforms AC1-9 in human bronchial epithelial cells. Total RNA was isolated from 16HBE14o À cells using RNA easy kit (Qiagen). cDNA was reverse transcribed using poly dT primers and ImProm™ Reverse Transcriptase System (Promega, USA) and 1 ml of this reaction was directly amplified using GoTaq® Green Master Mix.
(Promega, USA) using specific primers for human AC isoforms (Xu, D, Isaaca, C (2001)) ( Table 3)  using a specific antibody to phosphorylation at Ser 729. Dexamethasone treatment had no effect on PKCε phosphorylation levels (Fig. 12, Table 6). Bombesin was used as a positive control for PKC activation. Stripping the blot and reprobing with b-actin showed equal loading of all lanes. These results show that dexamethasone selectively activates classical PKCa and does not rapidly activate the novel PKC isoforms: PKCd and PKCε.
2.9. Expression of cAMP-adenylyl cyclase -PKA signaling pathway in 16HBE14o À cells Adenylyl cyclase (AC) expression was determined in 16HBE14o À cells. Because of the unavailability of satisfactory antibodies for most of the AC isoforms, the expression of AC isoforms was investigated by RT-PCR (Table 8). As shown in Fig. 13, the AC isoforms AC3, AC4, AC6 and AC7 were found to be expressed in 16HBE14o À cells.

Expression of PKA regulatory and catalytic subunits in human bronchial epithelial cells
Since AC isoforms are expressed in 16HBE14o À cells, it was of interest to investigate the expression levels of the catalytic and regulatory subunits of PKA in 16HBE14o À cells. The PKA isoform I (PKA I ) the soluble cytosolic isoform, was investigated in cellular extracts in this study, as distinct from isoform II which is membrane bound. Total untreated cellular lysates of 16HBE14o À cells were prepared and subjected to western blot analysis by probing with specific antibodies to endogenous levels of PKA regulatory (PKA RI ) and catalytic (PKA CI ) subunits. As shown in Fig. 14, 16HBE14o À cells express equal levels of both PKA RI and PKA CI subunits compared with MCF-7 cells used as a positive control. Expression differences were normalized for loading by probing for total bactin levels. Values are given as fold differences in PKA expression between MCF-7 and 16HBE14o À cell lysates. Values are displayed as mean ± SEM (n ¼ 3). n.s. denotes not significant (P > 0.05) between MCF-7 and 16HBE14o À cells in PKA CI and PKA RI expression. Statistical analysis was performed by the Student's paired t-test for three independent experiments. B: Representative Western blot analysis of PKA CI and PKA RI subunit in cellular extracts of 16HBE14o À and MCF-7 cells. Total protein (50 mg/lane) was transferred to nitrocellulose membranes after fractionating by SDS-PAGE and blotted with anti-PKA RI (48kDa) and anti-PKA CI (40kDa). b-actin (42 kDa) was used as an internal control to estimate protein loading.

Dexamethasone effects on cAMP-dependent protein kinase A activity
The cAMP-adenylyl-PKA signaling pathway is clearly expressed in 16HBE14o À cells. We therefore examined the effects of dexamethasone on PKA activity. To determine the time course of dexamethasone activation of PKA in 16HBE14o À cells, serum starved cells were exposed to dexamethasone (1 nM) or equivalent vehicle (methanol, 0.001% v/v) for the duration of 2, 5 and 10 mins. PKA activity was rapidly and dramatically upregulated after 5 mins in response to dexamethasone (1 nM) treatment and Fig. 15. PKA is rapidly activated in response to dexamethasone in 16HBE14o ¡ cells. A: The graph represents densitometric analysis at specific time points of dexamethasone treatment. Values are given as fold changes in PKA phosphorylation of the F-Kemptide PepTag for 16HBE14o À cell lysates. Values are displayed as mean ± SEM (n ¼ 6). ** Denotes significance (p < 0.001) between control and treated values. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison tests. B: Representative image of PKA activation of an F-Kemptide PepTag in cellular extracts from 16HBE14o À cells. PKA activity phosphorylated the F-Kemptide PepTag peptide changing its net charge from þ1 to À1. This allows the phosphorylated and nonphosphorylated forms of the substrate to be rapidly separated on agarose gel. Lane 1, lysate control, Lane 2, forskolin (20 mM/5 min), Lane 3, dexamethasone (1 nM, 2 min), Lane 4, dexamethasone, (1 nM, 5 min), Lane 5, dexamethasone (1 nM, 10 min), Lane 6, vehicle control (methanol 0.001% v/v/2 min), Lane 7, vehicle control (methanol 0.001% v/v, 5 min), Lane 8, vehicle control (methanol 0.001% v/v, 10 min).

Table 9
Summary of the fold increases in PKA activity following dexamethasone treatment. 1.05 ± 0.08 returned to basal levels after 10 mins (Fig. 15). The effect was comparable to forskolin (20 mM) a wellknown activator of AC. A summary of the fold increases in PKA activity for each treatment is shown in Table 9.

PKA and cAMP effects on cAMP-dependent protein kinase activity
The adenylyl cyclase activator, forskolin, and the PKA antagonist, R p -cAMP[S], were used as positive and negative controls for PKA stimulation, respectively. Dexamethasone increased PKA activity and this effect was inhibited by R p -cAMP[S] (Fig. 16). The effect was comparable to forskolin, a known activator of adenylyl cyclase. Table 10 shows a summary of fold increases in PKA activity.

Dexamethasone induces rapid non-genomic PKA activation
The rapid time course (5 minutes) of the dexamethasone effect on PKA activity suggests that this response does not involve a classical genomic mechanism. In order to verify this hypothesis we investigated the dexamethasone response in the presence of cycloheximide, an inhibitor of mRNA translation. Dexamethasone (1 nM) stimulated a 2.62 ± 0.29 -fold increase in total PKA activity in 16HBE14o À cells after 5min (Fig. 17). This increase was not inhibited by preincubation with cycloheximide (1 mM) for 1 hour. The vehicle control and cycloheximide alone did not stimulate a significant change in PKA activity relative to untreated control. Table 11 shows a summary for the fold changes in   1.77 ± 0.21 Fig. 18. Dexamethasone induces PKA activity independent of the classical GR and MR receptor antagonists. A: The graph represents densitometric analysis at specific time points of dexamethasone treatment. Values are given as fold changes in PKA activity measured as phosphorylation of the F-Kemptide PepTag for 16HBE14o À cell lysates. Values are displayed as mean ± SEM (n ¼ 4). n.s. denotes not significant between dexamethasone and dexamethasone and inhibitor treated values. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison tests. B: UV-illuminated agarose gel of the products of reactions run with F-Kemptide and 16HBE14o À homogenate. Lane 1, lysate control, Lane 2, dexamethasone (1 nM, 5 min), Lane 3, RU486 (prior treatment 1 mM/30 min) and dexamethasone (1 nM, 5 min), Lane 4, vehicle control (methanol, 0.001% v/v, 5 min), Lane 5, RU486 þ vehicle control (methanol 0.001% v/v, 5 min), Lane 6, dexamethasone (1 nM, 5 min), Lane 7, spironolactone (prior treatment 10 mM/30 min) and dexamethasone, (1 nM, 5 min), Lane 8, spironolactone þ vehicle control (methanol 0.001% v/v, 5 min). PKA activity. This result indicates that the dexamethasone induced increase in PKA activity observed after 5 min treatment is not dependent on changes in gene translation.
2.14. Role of MR and GR in dexamethasone PKA response? -Effect of the classic glucocorticoid antagonist, RU486 and the mineralocorticoid antagonist, spironolactone on dexamethasone-induced PKA activity In order to investigate the receptor involved in the rapid response to dexamethasone, RU486 and spironolactone were used as antagonists of the GR and the MR, respectively. In control experiments the receptor antagonists did not affect basal PKA activity. Furthermore, neither RU486 nor spironolactone Fig. 19. Role of pertussis toxin sensitive G Proteinecoupled receptor in the dexamethasone-induced PKA activity. A: The graph represents densitometric analysis at specific time points of dexamethasone treatment. Values are given as fold changes in PKA activity measured as phosphorylation of the F-Kemptide PepTag for 16HBE14o À cell lysates. Values are displayed as mean ± SEM (n ¼ 4). ** Denotes significance (p < 0.001) between dexamethasone and dexamethasone þ pertussis toxin treated values. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison tests. W UV-illuminated agarose gel of the products of reactions run with F-Kemptide and 16HBE14o À cell homogenate. PKA activity phosphorylated the PepTag peptide (F-Kemptide) changing its net charge from þ1 to À1. Lane 1, forskolin (20 mM/5 min); lane 2, dexamethasone (1 nM/5 min); lane 3, PD98059 (prior treatment 50 mM/40 min) and dexamethasone (1 nM/5 min); lane 4, pertussis toxin (prior treatment 2 mg/l, 20 min) and dexamethasone (1 nM/5 min); lane 5, vehicle control (methanol 0.001% v/v), (Verriere et al., 2005).

Table 13
Summary of the fold increases in PKA activity.

Role of pertussis toxin sensitive G protein-coupled receptors in the PKA response to dexamethasone
The role of G protein-coupled receptors (GPCRs) in the rapid response to dexamethasone was investigated using pertussis toxin (PTX). As shown in Fig. 19 and Table 13, PKA activity was upregulated in less than 5 min by 40% over control (p < 0.01) following 1 nM dexamethasone treatment. The G i protein inhibitor, PTX (2 mg/l) significantly inhibited the activation of PKA by dexamethasone. The cells pretreated with PTX before dexamethasone did not show a significantly increased phosphorylation compared with untreated control cells (p > 0.1). In contrast, the MEK 1 inhibitor, PD98059 (50 mM), had no significant effect on PKA activation by the steroid (p > 0.1). In these experiments, the AC activator, forskolin, and the PKA antagonist, (R p )-cAMP, were used as positive and negative controls for PKA stimulation, respectively. The vehicle control (methanol 0.001% v/v) did not stimulate PKA activity. These data suggest a role for a PTX sensitive G protein-coupled receptor in the rapid PKA response to dexamethasone.

Activation of the ERK1/2 MAPK pathway by dexamethasone
PKA is known to activate downstream ERK1/2 MAPK signaling to induce non-genomic responses to steroid hormones. The basal expression level of ERK1/2 MAPK in 16HBE14o À cells was examined. Total untreated cellular lysates of 16HBE14o À cells were prepared, subjected to western blot analysis and probed using a specific antibody recognising ERK1/2 MAPK. Expression differences were normalized for loading by probing for total b-actin levels. MCF-7 cells were used as a positive control as they are known to express ERK1/2 MAPK. This result clearly showed that ERK1/2 MAPK was expressed in 16HBE14o À cells (Fig. 20).

Dexamethasone activation of ERK1/2 MAPK
The effect of dexamethasone on the activation of ERK1/2 MAPK was next examined. Phosphorylation of ERK1/2 MAPK on key threonine 202 and tyrosine 204 residues is strongly correlated with an increased activation of these kinases.
An antibody specific for phospho-ERK1/2 MAPK was used to determine phosphorylation in response to dexamethasone by western blot analysis. As shown in Fig. 21, dexamethasone (1 nM) produced a biphasic activation of ERK1/2 MAPK. ERK1/2 activation was observed as early as 2 minutes reaching a maximal activation at 10 minutes, thereafter returning to basal levels by 15 minutes. Dexamethasone treatment increased ERK1/2 MAPK phosphorylation levels compared to vehicle controls (Table 14). In 16HBE14o À cells the rapid activation of ERK1/2 MAPK occurs in a time frame consistent with the rapid activation of PKA by dexamethasone.

Non-genomic response -dexamethasone induces ERK1/2 MAPK activation independent of mRNA translation
The time course (5 minutes) of the dexamethasone effect on ERK1/2 MAPK activity suggests that this response does not involve a classical genomic mechanism. In order to verify this hypothesis we Fig. 21. ERK1/2 MAPK is rapidly activated in response to dexamethasone in 16HBE14o ¡ cells. A: The graph represents densitometric analysis at specific time points of dexamethasone treatment. Values are given as fold changes in ERK1/2 MAPK activation in 16HBE14o À cell lysates. Values are displayed as mean ± SEM (n ¼ 4). ** Denotes significance (p < 0.001) between control and treated values. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison tests. B: Representative western blot analysis of phospho-ERK1/2 MAPK in cellular extracts of 16HBE14o À . By employing antibodies specific to the phosphorylated form of ERK1/2 (Thr 202/Tyr 204), its activation by dexamethasone was monitored by Western blot analysis. Dexamethasone (1 nM) produced a biphasic activation of ERK1/2 that peaked at 2 and 10 minutes. tested the dexamethasone (1 nM) response in the presence of cycloheximide, an inhibitor of mRNA translation. As shown in Fig. 22, the increase in ERK1/2 MAPK activity following dexamethasone treatment was not reduced by preincubation with cycloheximide (1 mM) (Table 15). EGF (100 ng/mg) was used as a positive control for MAPK activation. These results indicate a mechanism of dexamethasone-induced activation of ERK1/2 MAPK that is independent of de novo protein synthesis.

Table 14
Summary of the fold increases in ERK1/2 MAPK activity induced by dexamethasone.

Dexamethasone PKA activation is upstream of MAPK activation
The potential role of PKA in dexamethasone-induced activation of ERK 1/2 MAPK was examined. As shown in Fig. 23, dexamethasone (1 nM) stimulated ERK1/2 MAPK phosphorylation in 16HBE14o e cells (p < 0.001) (lane 2). The rapid activation of MAPK by dexamethasone was significantly inhibited (p < 0.001) by the PKA inhibitor H89 (10 mM) (lane 4) demonstrating that PKA is activated upstream of ERK1/2 MAPK. As an internal control the MEK 1 inhibitor PD98059 (25 mM) (lane 5) inhibited the ERK1/ 2 MAPK activity. Table 16 shows a summary of fold increases in ERK1/2 MAPK activity.

PKC activation is upstream of PKA
The data indicate that dexamethasone induced MAP kinase activation is dependent on upstream PKA activity. What dexamethasone modulated kinase, if any, is upstream of PKA ?  In order to determine whether PKC activation was involved in the dexamethasone induced activation of PKA, the effects of the PKCa inhibitor 2,2 0 ,3,3 0 ,4,4 0 -Hexahydroxy-1,1 0 -biphenyl-6,6 0 -dimethanol Dimethyl Ether (HBDDE), a selective inhibitor of PKCa and PKCg (IC 50 concentration: PKCa, 43 mM and PKCg 50 mM) was examined. PKCg was not expressed in the human airway epithelial cells (it is Representative western blot analysis of phospho-PKD1 in cellular extracts of 16HBE14o À . By employing antibodies specific to the phosphorylated form of PKD1 (Ser 916), the activation by dexamethasone was monitored by Western blot analysis. b-actin (42 kDa) was used as an internal control to estimate protein loading. The graph represents densitometric analysis at specific time points of dexamethasone treatment. Bombesin (BOM) was used as a positive control for PKD1 activity. Values are given as fold changes in PKCε activation of 16HBE14o À cell lysates. Values are displayed as mean ± SEM (n ¼ 3); n.s. denotes no significance between control and treated values. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison tests.

Table 18
Summary of the fold increases in PKD activity.
Treatment PKD activation (phosphorylation at Ser 729) exclusively expressed in brain and spinal tissue). Dexamethasone induced PKA activation was calculated as fold increases (arbitrary units, A.U.). Serum starved cells were treated with HBDDE (100 mM) for 30 mins before dexamethasone treatment (1 nM). Vehicle and HBDDE on its own had no effect on basal PKA activity. However, pretreatment with the PKCa inhibitor HBDDE (100 mM) prevented dexamethasone induced PKA activation (Fig. 24, Table 17). This result shows that PKA activated in response to dexamethasone is downstream of PKCa.
2.21. Effect of dexamethasone on PKD1 activation in 16HBE14o À cells Protein kinase D is a potential downstream target of cPKC and nPKCs. To determine whether dexamethasone stimulated activation of PKD1 in 16HBE14o À cells, serum starved cells were treated with dexamethasone (1 nM) and PKD1 activity was assessed by probing with an antibody specific to phosphorylation to Ser916. Residue Ser916 is a site of autophosphorylation in the PKD1 structure that occurs subsequent to phosphorylation at Ser744/748 by PKC. Phosphorylation at Ser916 is therefore indicative of PKD activation. Treatment of 16HBE14o À cells with dexamethasone had no significant effect on PKD1 phosphorylation levels (Fig. 25, Table 18). Bombesin was used as a positive control as it is known to activate PKD1.