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First published online 11 December 2007
doi: 10.1242/jcs.011692

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Spectrin-anchored phosphodiesterase 4D4 restricts cAMP from disrupting microtubules and inducing endothelial cell gap formation

¹ Center for Lung Biology, The University of South Alabama College of Medicine, Mobile, AL 36688, USA
² Department of Pharmacology, The University of South Alabama College of Medicine, Mobile, AL 36688, USA
³ Department of Cell Biology and Neuroscience, The University of South Alabama College of Medicine, Mobile, AL 36688, USA

View larger version (25K): [in this window] [in a new window]   Fig. 1. Ca2+-sensitive adenylyl cyclase and PDE4 activities dominate membrane cAMP synthesis and hydrolysis in PMVECs. (A) Using membranes obtained from a 30-40% sucrose gradient, increasing buffer Ca2+ concentration from 100 nM Ca2+ to 10 µM Ca2+ inhibited 97% of adenylyl cyclase activity in PMVECs compared with 22% in PAECs. (B) In buffer containing 100 nM Ca2+, rolipram (10 µM, EC100) inhibited sufficient PDE4 cAMP hydrolysis to increase cAMP accumulation by 330% in PMVEC membranes, but had no effect on cAMP accumulation in PAEC membranes. Whole cell (C) and membrane-specific (D) cAMP hydrolysis is similar in PMVECs and PAECs. However, rolipram (10 µM, EC100) inhibited 75% of cAMP PDE activity in PMVECs compared with 20% inhibition in PAECs (D). Data are means ± s.e.m.*P<0.05, n=3.

View larger version (21K): [in this window] [in a new window]   Fig. 2. PDE4D4 is expressed in PMVECs. (A) Western blot analysis of PDE4 protein expression in caveolin-containing membrane fractions revealed a 115 kDa band similar to the expected molecular mass of PDE4D4 in PMVECs. (B) Schematic diagram depicts the nine known splice variants encoded by the PDE4D gene. (C) Western blot using antibody specific to PDE4D4 identified a 115 kDa protein (center lane) consistent with results using pan PDE4 antibody. Overexpression of PDE4D4 in PMVECs was used as a positive control (right lane). (D) Semi-quantitative RT-PCR confirmed PDE4D4 expression in PMVECs (β actin, loading control).

View larger version (16K): [in this window] [in a new window]   Fig. 3. PDE4D4 interacts with spectrin in pulmonary endothelial cells. (A) Non-erythroid spectrin is comprised of II and βII subunits, which form a heterotetramer. PDE4D4 dimers bind II subunit SH3 domains. (B) Co-immunoprecipitation studies using II spectrin antibody followed by immunoblotting with antibody to PDE4D reveal a 115 kDa band in PMVECs consistent with the PDE4D splice variant PDE4D4. Overexpression of PDE4D4 in PAECs resolved a 115 kDa band that co-immunoprecipitated with II spectrin.

View larger version (41K): [in this window] [in a new window]   Fig. 4. PDE4D4 localizes with spectrin at cell-cell borders in PMVECs. (A) Confocal microscopy using antibodies to PDE4 and βII spectrin indicates both proteins localize to cell-cell borders in PMVECs (top panel), but not in PAECs (bottom panel). (B) Schematic depicts comparison of full-length PDE4D4 to the catalytically inactive construct comprised of the N-terminal (1-166 residues) of PDE4D4 fused to GFP, which was expressed in PMVECs. (C) Confocal microscopy of PMVECs expressing PDE4D4-GFP fusion protein (top panel) indicates the peptide localized to cell-cell borders with βII spectrin, similarly to endogenous PDE4D4. GFP vector control, bottom panel. (D) Control and PDE4D41-166-expressing cells were grown to confluence and separated into pellet, cytosolic and membrane fractions. Western analysis revealed that most PDE4D4 enzyme was present in caveolin-containing membrane fractions. The PDE4D41-166 fragment did not shift the location of the endogenous PDE4D4 enzyme. (E) In PMVEC membranes expressing the catalytically inactive PDE4D4-GFP fusion protein, cAMP PDE activity was reduced by 30% compared with that in control cell membranes. Rolipram (10 µM for 10 minutes) inhibited 85% of the PDE4 activity in control PMVECs and 65% of the PDE4 activity in PDE4D41-166 expressing cells (*P<0.05, n=3). (F) Radioimmunoassay revealed that PDE4D41-166-expressing cells possessed higher basal cAMP concentrations, and greater forskolin-stimulated (1 µM, 3 minutes) cAMP responses (P<0.05, n=3). (G) Expression of PDE4D4 in PAECs resulted in co-immunoprecipitation of II spectrin with PDE4 (right panel), which was not observed in control cells (left panel). (H) The PDE4D41-166 construct was expressed in PAECs, which express little PDE4D4. Cells treated with rolipram (10 µM for 10 minutes) show similarly reduced phosphodiesterase activity in control and PDE4D41-166-expressing cells (P<0.05, n=3).

View larger version (15K): [in this window] [in a new window]   Fig. 5. PDE4D41-166 expressing PMVECs possess greater membrane and cytosolic PKA activity. Forskolin (1 µM, 30 minutes) stimulated more PKA activity in PDE4D41-166-expressing cells than in control cells, in both membrane and cytosolic fractions. Total PKA activity represents maximal kinase activity in the presence of 2 µM cAMP in the reaction buffer (*P<0.05, n=4).

View larger version (43K): [in this window] [in a new window]   Fig. 6. PKA-mediated phosphorylation of tau at Ser214 reorganizes microtubules. (A) Forskolin (1 µM, 30 minutes) increased co-immunoprecipitation of phosphorylated tau-Ser214 with β-tubulin in the cytosol of PDE4D41-166-expressing PMVECs, which was not seen in vector controls. Such enhanced co-immunoprecipitation of phosphorylated tau with β-tubulin was not observed using the phosphorylated tau-Ser262 antibody under the same conditions. (B) The forskolin-stimulated increase in tau-Ser214 phosphorylation was prominent in the depolymerized microtubule-enriched fractions, and was not observed in polymerized fractions. (C) H89 pretreatment (10 µM, 10 minutes before forskolin stimulation) blocked the forskolin-induced phosphorylation of tau-Ser214 binding to β-tubulin in cytosol. (D) Phosphorylated tau-Ser214 (red) and microtubules (green) are shown in control and PDE4D41-166-expressing PMVECs. Whereas PDE4D41-166 did not influence resting tau-Ser214 or microtubule distribution (top panel), the application of forskolin (bottom panel) abruptly reorganized microtubules into bundles. (White boxes denote area enlarged in right panel of each image set.)

View larger version (86K): [in this window] [in a new window]   Fig. 7. PDE4D4 activity is necessary to maintain PMVEC barrier integrity in the presence of increased cAMP synthesis. Direct stimulation of adenylyl cyclase using 1 µM forskolin does not alter cell-cell interaction in confluent control PMVEC monolayers (top row) or in cells expressing the GFP vector (bottom row). However, forskolin induces gap formation in PMVECs expressing the catalytically inactive PDE4D4-GFP fusion peptide (second row). Regions of the images in the second panel (white boxes) were expanded to highlight areas of gap formation (third row). Arrows indicate initial gap formation. Time-lapse videos corresponding to these images are included as Movies 1-3 in supplementary material.