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Protein kinase C- mediates bradykinin-induced cyclooxygenase-2 expression in human airway smooth muscle cells¹

LINHUA PANG^*2, MEI NIE^*, LISA CORBETT^*, RICHARD DONNELLY, SAMUEL GRAY and ALAN J. KNOX^*

^* Division of Respiratory Medicine, City Hospital, University of Nottingham, Nottingham NG5 1PB, UK; and
Division of Vascular Medicine, Derbyshire Royal Infirmary, University of Nottingham, Derby DE1 2QY, UK

²Correspondence: Division of Respiratory Medicine, Clinical Sciences Building, City Hospital, University of Nottingham, Hucknall Road, Nottingham NG5 1PB, UK. E-mail: linhua.pang{at}nottingham.ac.uk

SPECIFIC AIMS

In this study we aimed to identify the specific protein kinase C (PKC) isozyme(s) that mediated the expression of cyclooxygenase-2 (COX-2) by bradykinin (BK) in human airway smooth muscle (HASM) cells.

PRINCIPAL FINDINGS

1. BK induces PKC isozyme activation
Since PKC comprises a family of at least 11 isozymes with differences in regulation and subcellular distribution, to determine which PKC isozymes were expressed in HASM cells we performed Western blot with isozyme-specific antibodies on the total protein samples. Under resting conditions, PKC-, -ßI, -ßII, -, -, and -µ were strongly expressed in these cells. PKC- and PKC- were weakly expressed. No immunoreactivity was detected for PKC-, -, or -. To examine whether the individual PKC isozymes expressed in HASM cells translocate on BK stimulation, the intracellular localization of the PKC isoforms was determined by centrifugal fractionation, SDS-PAGE separation, and Western blot. Western analysis showed that in the unstimulated state, PKC-, -ßI, -, -, -µ, -, and - were mainly distributed in the cytosolic fraction whereas PKC-ßII was distributed mainly in the membrane fraction. After stimulation with BK for 10 min, PKC-, and -ßI of the classic or Ca²⁺-dependent group and PKC-, and - of the novel or Ca²⁺-independent group were translocated from the cytosol to the membrane fraction in a BK concentration-dependent (0.1–10 µM) manner (Fig. 1 ), but little difference was observed in the distribution of other PKC isozymes. Densitometric analysis of the bands revealed a loss of 40%, 50%, 50%, and 30% for cytosolic PKC-, -ßI, -, and -, respectively, and an increase of 390%, 270%, 830%, and 300% for corresponding membrane PKC isozyme at the highest concentration (10 µM) of BK tested (Fig. 1) .

View larger version (53K): [in this window] [in a new window]   Figure 1. Effect of BK on the translocation of PKC-, -ßI, -, and -. HASM cells were incubated with BK (0.1–10 µM) for 10 min. Cytosolic and membrane proteins were prepared and PKC isozyme expression was analyzed by Western blot using isozyme specific antibodies. Densitometric analysis of the bands was performed and data were expressed as fold change. These blots are representative of similar results obtained three times.

2. PKC is involved in BK-induced COX-2 expression and PGE₂ release
We previously reported that BK concentration- and time-dependently induces COX-2 protein expression and PGE₂ accumulation in HASM cells. To assess the role of PKC activation in these processes, we first investigated whether phorbol 12-myristate 13-acetate (PMA), a known direct PKC activator, could exert a similar effect as BK in these cells. PMA (10 µM) mimicked the kinetics of the COX-2 expression by BK (10 µM) with a time-dependent expression pattern almost identical to that of BK (appeared at 1 h, peaked at 4 and 8 h, declined at 16 h, and disappeared at 24 h after treatment); in contrast, COX-1 expression remained unchanged. PMA also stimulated PGE₂ accumulation in a time-dependent manner, with the peak achieved after 16 h of stimulation. The results indicate that PKC activation can lead to COX-2 protein expression and PGE₂ formation.

When HASM cells were pretreated with PKC inhibitors before stimulation with BK, BK-induced COX-2 expression (4 h) was inhibited by the broad spectrum PKC inhibitor bisindolylmaleimide I (Bis I) concentration dependently (0.1–10 µM) but was not affected by the selective Ca²⁺-dependent PKC isozyme inhibitor Go 6976 (0.1–10 µM), the selective PKC- inhibitor rottlerin (0.1–2.5 µM), or the membrane-permeable calcium chelator BAPTA-AM (1–10 µM). In line with the inhibition on COX-2 expression, Bis I also significantly inhibited BK-induced PGE₂ accumulation (4 h) in a dose-dependent manner (P<0.01 for 0.1 µM, P<0.001 for 1 and 10 µM) whereas Go 6976 and rottlerin had no effect. BAPTA-AM markedly inhibited BK-induced PGE₂ accumulation at 15 min (P<0.05 for 1 µM, P<0.01 for 10 µM), was effective only at 10 µM for PGE₂ accumulation at 4 h (P<0.05), and had no effect on BK-induced COX activity, suggesting the involvement of Ca²⁺ in the early release of COX substrate arachidonic acid (AA) but not in COX-2 expression. Since only PKC- and - of the novel group PKC are activated by BK and the involvement of PKC- is unlikely in view of the ineffectiveness of the selective PKC- inhibitor rottlerin, the pharmacological evidence suggests that PKC- is the most likely candidate PKC isozyme that mediates BK-induced COX-2 expression. We performed molecular studies to confirm this.

3. Overexpression of wild-type and dominant negative PKC- modulates BK-induced transcriptional activation of COX-2
When HASM cells cotransfected with the COX-2 promoter firefly luciferase reporter construct phPES2(-1432/+59) and the internal control Renilla luciferase vector pRL-SV40 were stimulated with BK (10 µM), COX-2 promoter activity was increased time dependently, which was significant at 2 h (P<0.05) and peaked at 4 h (P<0.05) compared to controls, and declined at 8 h, suggesting that BK regulates COX-2 expression transcriptionally.

To further establish the role of PKC- and to eliminate the involvement of other PKC isozymes, particularly PKC-, cells were cotransfected with phPES2(-1432/+59), pRL-SV40, and pSRD vectors expressing either wild-type (wt) or dominant negative (dn) PKC-, -, and -, then stimulated with BK (10 µM, 4 h). BK markedly increased COX-2 promoter activity compared to pSRD empty vector controls, overexpression of wtPKC- further enhanced (P<0.05), and overexpression of dnPKC- abolished the effect of BK (P<0.05, Fig. 2 ). In contrast, overexpression of wtPKC- and - and of dnPKC-, and - had no effect. The results confirm that PKC- is the only PKC isozyme required for BK-induced COX-2 expression.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect PKC isozyme overexpression on BK-induced transcriptional activation of COX-2. 50–60% confluent HASM cells were cotransfected with 0.4 µg/well phPES2(-1432/+59) construct, 4 ng/well pRL-SV40 internal control vector, and 0.5 µg/well pSRD vectors expressing wild-type or dominant negative PKC-, -, or - using FuGene 6 transfection reagent and incubated with or without BK (10 µM) for 4 h. Luciferase activities were determined on cell extracts and relative luciferase activity was calculated. Each point represents the mean ± SE of 8 determinations from two independent experiments. *P < 0.05 vs. BK in cells cotransfected with pSRD empty vectors.

CONCLUSIONS

It is well documented that PKC isozymes translocate from the cytosolic to the membrane fraction on activation, and this is widely accepted as a measure of enzyme activation. The present study revealed that among the PKC isozymes present in HASM cells, only PKC-, -ßI, -, and - were activated after BK treatment. Furthermore, the kinetics of BK-induced COX-2 expression was mimicked by the direct PKC activator PMA, suggesting the involvement of phorbol ester-responsive PKC isozymes (classic and novel groups) in COX-2 expression by BK. To identify the specific PKC isozyme(s) responsible for COX-2 expression, we used three inhibitors with varying specificity for PKCs. Bis I inhibits PKC isozymes of all three groups with a ranked order of potency (PKC->-ßI>->->-); Go 6976 is a relatively selective inhibitor for the Ca²⁺-dependent PKC isozymes, with IC₅₀ values of 2.3 nM and 6.2 nM for PKC- and -ßI, respectively, but has no effect on the activity of the Ca²⁺-independent PKC-, -, and - even in micromolar concentrations; Rottlerin is relatively specific for PKC- and is unlikely to affect other PKC isozymes at concentrations that are effective for PKC-. The prevention of BK-induced COX-2 expression by Bis I and the ineffectiveness of Go 6976, up to 10 µM, on BK-induced COX-2 protein expression and PGE₂ release suggest that the Ca²⁺-independent, rather than the Ca²⁺-dependent, PKC isozymes are involved. This was confirmed by the lack of effect of the membrane permeable calcium chelator BAPTA-AM on BK-induced COX-2 protein expression and COX activity despite its inhibition on the early (15 min) and late (4 h) PGE₂ accumulation. The lack of effect of rottlerin suggests that PKC- is not involved. Collectively, the translocation and pharmacological manipulation results suggest that the novel Ca²⁺-independent PKC- is responsible for BK-induced COX-2 expression.

To directly investigate the role of PKC isozymes in regulating BK-induced COX-2 transcription, transient transfections of COX-2 promoter reporter construct and PKC expression vectors were performed. We observed that BK-induced COX-2 promoter activity was further enhanced by overexpression of wild-type PKC- (but not PKC- and -) and was abolished by a dominant negative mutant form of PKC-, but not PKC- and -. The results rule out the involvement of PKC- and demonstrate that PKC- acts as an important intermediary molecule in BK-induced COX-2 transcription.

Two forms of phospholipase A₂ (PLA₂) are involved in AA release from membrane phospholipids and prostaglandin (PG) biosynthesis. An 85 kDa cytosolic form (cPLA₂) is rapidly activated in response to stimulation of G-protein-coupled receptors such as the BK B₂ receptor, involving its translocation by a rise in the concentration of intracellular Ca²⁺ ([Ca²⁺i]) and its phosphorylation by protein kinases such as mitogen-activated protein kinase, and is responsible for the immediate PG biosynthesis. A 14 kDa secreted form (sPLA₂) can be induced by cytokines and other mediators and is responsible for the delayed PG biosynthesis. As we have previously demonstrated that BK induces PGE₂ accumulation in two phases and that BAPTA-AM has different effects on BK-induced PGE₂ release, COX-2 expression, and activity in this study, it is plausible to suggest that the immediate PG biosynthesis is largely due to the ([Ca²⁺i]/cPLA₂-induced AA release and its conversion to PGs by the constitutive COX-1, and that the delayed PG biosynthesis involves cPLA₂ phosphorylation, sPLA₂ expression, PKC--mediated COX-2 expression, and possibly COX-1 (Fig. 3 ). The slight inhibition of BK-induced 4 h PGE₂ accumulation by BAPTA-AM is likely due to inhibition of the immediate PGE₂ biosynthesis that forms part of the 4 h PGE₂ accumulation. How PKC- activation leads to the transcription of the human COX-2 gene remains to be clarified.

View larger version (19K): [in this window] [in a new window]   Figure 3. Schematic diagram of possible mechanisms of BK-induced COX-2 expression and PG biosynthesis in HASM cells. PLC, phospholipase C.

We demonstrate in this study that BK induces the activation of the Ca²⁺-dependent PKC-, -ßI, and the Ca²⁺-independent PKC-, and -, and that PKC- is a vital component of the postreceptor signal transduction pathway of BK that leads to the expression of COX-2 in HASM cells. This is, to the best of our knowledge, the first study to link a specific PKC isozyme to BK-induced COX-2 expression in any cell system.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0169fje; to cite this article, use FASEB J. (July 18, 2002) 10.1096/fj.02-0169fje

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