Phosphodiesterase 1C integrates store-operated calcium entry and cAMP signaling in leading-edge protrusions of migrating human arterial myocytes

Abstract

In addition to maintaining cellular ER Ca²⁺ stores, store-operated Ca²⁺ entry (SOCE) regulates several Ca²⁺-sensitive cellular enzymes, including certain adenylyl cyclases (ADCYs), enzymes that synthesize the secondary messenger cyclic AMP (cAMP). Ca²⁺, acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca²⁺-sensitive enzymes. Here, we report that depletion of ER Ca²⁺ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca²⁺/calmodulin-mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca²⁺-insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca²⁺ release activated Ca²⁺ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca²⁺/cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers.

Keywords: adenylate cyclase; calcium; cyclic AMP; human arterial smooth muscle cells; phosphodiesterase 1C; phosphodiesterases; store-operated calcium entry.

Figure 1

SOCE-dependent effects on HASMC cAMP.A, model of the FRET cAMP sensor, mTurq2ΔEPACcp173Ven_Ven, showing the EPAC1 cAMP-binding domain (CD, ΔDEP, Q270E) positioned between a mTurquoise2 (donor domain, blue) and two Venus (acceptor, yellow) domains (top), and representing how cAMP binding increases the distance between the donor and acceptor domains, thus reducing FRET (bottom). B, representative trace of normalized FRET emission ratio measured in H134-expressing HASMCs under initial experimental conditions (Ca²⁺-free Krebs buffer, “A”) during ER(Ca²⁺) store depletion (Ca²⁺-free Krebs buffer supplemented with CPA [10 μM], “B”), during the early (blue, “C”) and late (yellow, “D”) phases of SOCE [Krebs buffer supplemented with CPA (10 μM)] and, lastly, during sensor saturation [Forskolin (10 μM) + IBMX (100 μM), Fsk/IBMX, “E”]. C, representative single cell traces of normalized FRET emission ratios measured in H134-expressing HASMCs treated as in B in the presence of either saline (black) or of SQ 22536 (1 mM). D–G, changes in normalized FRET emission ratios measured in control (n = 20) or SQ22536-treated (n = 10) HASMCs during ER(Ca²⁺) store depletions (D), the early phase of SOCE (E), or the late phase (F and G) (Student’s unpaired t test, ∗p = 0.0420 ∗∗p = 0.0083). H, representative single-cell traces of normalized FRET emission ratios measured in control (siCtrl) or ADCY6-silenced (siADCY6) HASMCs treated as in B. I–L, differences in normalized FRET emission ratios measured in control (n = 26) or ADCY6-silenced (n = 26) HASMCs during ER(Ca²⁺) store depletion (I), the early phase of SOCE (J), or the late phase (K and L) of SOCE, respectively (Student’s unpaired t test, ∗∗p = 0.0076 comparing cAMP increase in Ca²⁺ free Krebs with CPA, ∗∗p = 0.004 comparing cAMP decrease during initial phase of SOCE, ∗p = 0.01 comparing cAMP rate of increase during SOCE). M, representative single-cell traces of normalized FRET emission ratios measured in control (siCtrl) or ADCY8-silenced (siADCY8) HASMCs treated as in B. N–Q, differences in normalized FRET emission ratios measured in control (n = 32) or ADCY8-silenced (n = 22) HASMCs during ER(Ca²⁺) store depletions (N), during the early phase of SOCE (O) or the late phase (P and Q) of SOCE, respectively (Student’s unpaired t test, ∗p = 0.04, ∗∗p = 0.007). R, levels of ADCY6 mRNA in siCtrl and siADCY6 transfected HASMCs (Student’s unpaired t test, ∗∗∗∗p < 0.0001). S, levels of ADCY8 mRNA in siCtrl and siADCY8 transfected HASMCs (Student’s unpaired t test, ∗∗p = 0.0012). ADCY, adenylyl cyclase; cAMP, cyclic AMP; CPA, cyclopiazonic acid; EPAC1, exchange protein activated by cAMP-1; FRET, fluorescence resonance energy transfer; HASMC, human arterial smooth muscle cell; SOCE, store-operated calcium entry.

Figure 2

HASMC PDE1C activation in response to ER (Ca²⁺) store depletion or SOCE activation.A and B, cAMP PDE activity in homogenates generated from control siRNA (A) or PDE1C siRNA (B), transfected HASMCs under control conditions (Krebs), following ER(Ca²⁺) store depletion [Ca²⁺ free + CPA (10 μM)] or following stepwise ER(Ca²⁺) depletion and SOCE activation [Krebs + CPA (10 μM)]. n = 4 experiments where each experiment was carried out in triplicate. One-way ANOVA, F = 3.76, p = 0.0343, Dunnett’s multiple comparison’s test was conducted, ∗p = 0.0485 control cells (Krebs) versus either ER(Ca²⁺) depletion and ∗p = 0.0415 control cells (Krebs) versus SOCE activation. C, HASMC cAMP PDE activity in homogenates without (black) or with Ca²⁺/CaM (red) in the presence of a combination of cilostamide (5 μM) + Ro 20 to 1724 (10 μM), a combination of C33 (1 μM) and cilostamide (5 μM) + Ro 20 to 1724 (10 μM), or a combination of PF-04827736 (1 μM) and cilostamide (5 μM) + Ro-20 to 1724 (10 μM). n = 3 experiments where each experiment was carried out in triplicate. Tukey’s multiple comparisons test was conducted, ∗p = 0.011 compared with control, ∗∗∗∗p < 0.0001 compared with control, ####p < 0.0001 compared with Ca²⁺/CaM. Two-way ANOVA, F (3, 16) = 8.207, p = 0.0016 interaction between control versus Ca²⁺/CaM conditions, F (3, 16) = 157.9 within each individual drug condition (control versus Ca²⁺/CaM treatment), p < 0.0001, F (1, 16) = 19.96, p = 0.0004 between the different drug treatments. Tukey’s multiple comparisons test was conducted, ∗p = 0.011 compared with control, ∗∗∗∗p < 0.0001 compared with control, ####p < 0.0001 compared with Ca²⁺/CaM. D, representative anti-PDE1C immunoblot of lysates generated from control siRNA-transfected HASMCs (siCtrl) or PDE1C-silenced (siPDE1C) HASMCs and quantitation of the efficiency of PDE1C-silencing in these cells (plot). ∗∗∗p < 0.01 versus siCtrl, n = 3, ∗∗∗∗p < 0.001 versus siCtrl, n = 3, Student’s unpaired t test. CaM, calmodulin; cAMP, cyclic AMP; CPA, cyclopiazonic acid; HASMC, human arterial smooth muscle cell; PDE, phosphodiesterase; PDE1C, phosphodiesterase 1C; SOCE, store-operated calcium entry.

Figure 3

PDE1C inhibition or PDE1C silencing reduces SOCE in HASMCs.A, representative single-cell traces of changes in the fluorescence ratio (F/Fo) of fura-2 in control [vehicle, (DMSO, 0.01% v/v), black line] or C33 (1 μM)-treated (red line) HASMCs during ER(Ca²⁺) store depletion [0 Ca²⁺ + CPA (10 μM)] or during subsequent SOCE [2 mM Ca²⁺ + CPA (10 μM)]. B–E, impact of C33 on maximal [Ca²⁺] increases (B) and rate of increase (C) during ER(Ca²⁺) store depletions or maximal [Ca²⁺] increases (D) and rate of increase (E) during SOCE. Control n = 33, C33 n = 32 individual cells in which each treatment was analyzed. Student’s unpaired t test, ∗∗∗∗p < 0.0001 control versus C33 treated cells. F, representative single-cell traces of changes in the fluorescence ratio (F/Fo) of fura-2 in control siRNA transfected (black line) or PDE1C siRNA-transfected (red line) HASMCs during ER(Ca²⁺) store depletion [0 Ca²⁺ + CPA (10 μM)] or during subsequent SOCE [2 mM Ca²⁺ +CPA (10 μM)]. G–J, Impact of PDE1C-silencing on maximal [Ca²⁺] increases (G) and rate of increase (H) during ER(Ca²⁺) store depletions or maximal [Ca²⁺] increases (I) and rate of increase (J) during SOCE. siCtrl n = 35, siPDE1C n = 49 individual cells in which treatment was analyzed. Student’s unpaired t test, ∗p = 0.0340 siCtrl versus siPDE1C transfected cells. CPA, cyclopiazonic acid; HASMC, human arterial smooth muscle cell; PDE, phosphodiesterase; PDE1C, phosphodiesterase 1C; SOCE, store-operated calcium entry.

Figure 4

PDE1C inhibition or PDE1C silencing, inhibit, rather than promote, SOCE-associated increases in HASMCs cAMP.A, representative single cell traces of normalized FRET emission ratios measured in H134-expressing HASMCs treated as in Figure 1B in the absence (black line) or the presence (red line) of C33 (1 μM). B–D, changes in normalized FRET emission ratios measured in control (n = 25) or C33-treated (n = 32) HASMCs during (B) ER(Ca²⁺) store depletions, (C) the early phase of SOCE or (D) the later phase of SOCE. E, rate of change in cAMP during the later phase of SOCE in HASMC; ∗p < 0.05, Student’s unpaired t test. F, representative single cell traces of normalized FRET emission ratios measured in control siRNA-transfected H134-expressing HASMCs (black line) or PDE1C-silenced H134-expressing HASMCs treated as in Figure 1B. G–I, changes in normalized FRET emission ratios measured in control (n = 23) or PDE1C-silenced (n = 32) HASMCs during (G) ER(Ca²⁺) store depletion, (H) the early phase of SOCE or (I) the later phase of SOCE. J, rate of change in cAMP during the later phase of SOCE in HASMCs. ∗p < 0.05, ∗∗∗p < 0.001, Student’s unpaired t test. cAMP, cyclic AMP; FRET, fluorescence resonance energy transfer; HASMC, human arterial smooth muscle cell; PDE, phosphodiesterase; PDE1C, phosphodiesterase 1C; SOCE, store-operated calcium entry.

Figure 5

PDE1C regulates formation of leading-edge protrusions (LEPs) in polarized HASMCs.A, model of the assay used to identify and quantify LEPs which accumulate on the underside of FluoroBlok membranes. B, representative images showing increased numbers of LEPs (tetramethylrhodamine-isothiocyanate-conjugated phalloidin-stained actin, red) formed by PDE1C-silenced HASMCs, compared with controls and inhibition of this process by addition of Forskolin (0.5 μM), scale bars 50 μm. C, LEP quantitation. LEP abundance in multiple individual membranes was determined by averaging phalloidin fluorescence in 4 to 5 separate nonoverlapping areas on individual membranes. n = 4 experiments in which individual experiments were carried out with four separate FluoroBlok membranes. A two-way ANOVA followed by Tukey’s multiple comparisons was conducted, ∗p < 0.05, ∗∗p < 0.01 between experimental conditions. D and E, concentration-dependent inhibition of LEP formation in HASMCs incubated with forskolin (Fsk) or isoproterenol (Iso). n = 3 experiments. A two-way ANOVA followed by Tukey’s multiple comparisons was conducted; 1-way ANOVA: Fsk dose response: F = 21.77, p < 0.0001. Dunnett’s multiple comparisons: ∗p = 0.0222 control versus Fsk 0.1 μM, ∗∗∗∗p < 0.0001 control versus Fsk (0.5 μM, 1 μM, 10 μM). 1-way ANOVA: Iso dose response: F = 14.85, p < 0.0001. Dunnett’s multiple comparisons: ∗p = 0.0080 control versus Iso 0.1 μM, ∗∗∗∗p < 0.0001 control versus Iso 1 μM. F–I, inhibition of LEP formation in HASMCs incubated with (F, G,I and L) the PDE1-family inhibitor C33 (1 μM) or in which cells were transfected with siPDE1C (H) in the absence or presence of 0.5 μM Fsk (F), 1 μM Iso (G), 40 μM myrPKI (H), or 10 μM HT31, or an inactive peptide (40 μM pHT31) (I). n = 3 to 5 experiments. Two-way ANOVA: C33 + siPDE1C: F (1, 90) = 7.486, p = 0.0075 interaction, F (1, 90) = 6.120, p = 0.0152 (control versus C33), F (1, 90) = 14.78, p = 0.0002 (siCtrl versus siPDE1C transfected cells). Two-way ANOVA: PDE1C Fsk: F (1, 76) = 0.6591, p = 0.4194 interaction, F (1, 76) = 17.32, p < 0.0001 siCtrl versus siPDE1C, F (1, 76) = 20.71, p < 0.0001 control versus forskolin treated cells. Tukey’s multiple comparisons: ∗p = 0.0479, ∗∗p = 0.0040 (siCtrl versus siPDE1C control), ∗∗p = 0.0017 (siPDE1C control versus siPDE1C + forskolin). Two-way ANOVA: C33 HT31: F (1, 56) = 3.201, p = 0.0790 interaction, F (1, 56) = 20.21, p < 0.0001 (control versus C33 treated cells), F (1, 56) = 23.97, p < 0.0001 (st-HT31(p) versus st-HT31 treated cells). Tukey’s multiple comparisons: ∗∗∗p = 0.0002 (st-HT31(p) control versus st-HT31(p) + C33), ∗∗∗∗p < 0.0001 (st-HT31(p) + C33 versus st-HT31 + C33). Two-way ANOVA: C33 Iso. F (1, 76) = 12.13, p = 0.0008 interaction, F (1, 76) = 16.60, p = 0.0001 (control versus C33), F (1, 76) = 81.78, p < 0.0001 (control versus isoproterenol treatment). Tukey’s multiple comparisons: ∗∗p = 0.0010 (control versus Iso), ∗∗∗∗p < 0.0001 (control versus C33, and C33 versus C33 + Iso). Two-way ANOVA: C33 Fsk: F (1, 155) = 30.00, p < 0.0001 interaction, F (1, 155) = 12.18, p = 0.0006 (control versus C33 treated cells), F (1, 155) = 129.5, p < 0.0001 (control versus Fsk treated cells). Tukey’s multiple comparisons: ∗∗∗p = 0.0003 (control versus Fsk), ∗∗∗∗p < 0.0001 (control versus C33 and C33 versus C33 + Fsk). Two-way ANOVA: PKI: F (1, 143) = 1.466, p = 0.2280 interaction, F (1, 143) = 8.173, p = 0.0049 (sictrl versus siPDE1C transfected cells), F (1, 143) = 25.46, p < 0.0001 (control versus forskolin treated cells). Tukey’s multiple comparisons: ∗p = 0.0367 (siCtrl control versus PKI), ∗∗p = 0.0080 (siCtrl versus siPDE1C), ∗∗∗p = 0.0001 (siPDE1C control versus siPDE1C + PKI). J and K, silencing HASMC PKA(Cα) (I) inhibited basal HASMC LEP formation and antagonized C33 (1 μM) or PF-04827736 (1 μM)-induced increases in LEP formation; data normalized to siRNA control, n = 3 experiments where each used four FluoroBlok membranes. Two-way ANOVA: siPKA C33 and PF: F (2, 155) = 10.84, p < 0.0001 interaction, F (1, 155) = 128.1, p < 0.0001 (siCtrl versus siPKA transfected cells), F (2, 155) = 10.42, p < 0.0001 [comparison versus drug treatment (control, C33, and PF)]. Tukey’s multiple comparisons: ∗∗p < 0.01 (siCtrl versus siPKA), ∗∗∗∗p < 0.0001 (siCtrl control versus each of C33 and PF), (siCtrl + C33 versus siPKA + C33) and (siCtrl + PF versus siPKA + PF). Two-way ANOVA: siAC8 and siAC6 Fsk: F (2, 154) = 14.65, p < 0.0001 interaction, F (1, 154) = 13.63 p = 0.0003 (control versus C33), F (2, 154) = 40.63, p < 0.0001 [comparison of transfection conditions (siCtrl versus siADCY6 versus siADCY8)]. Tukey’s multiple comparisons test: ∗p = 0.0289 (control versus C33 treated cells), ∗∗∗∗p < 0.0001 (siCtrl versus siADCY6 transfected cells and control versus C33 in siADCY6 transfected cells). ADCY, adenylyl cyclase; HASMC, human arterial smooth muscle cell; PDE, phosphodiesterase; PDE1C, phosphodiesterase 1C.

Figure 6

PDE1C interaction and co-localization with PKA, AKAP79, Orai1, and ADCY8 in HASMC and HASMC LEPs.A, representative immunoblots of anti-PDE1C, or anti-IgG, generated immune complexes analyzed for PDE1C, gravin, AKAP79, PKA-RIIβ, PKA-RIα, Orai1, or STIM1. Proteins isolated along with PDE1C in anti-PDE1C immunoprecipitation experiments, but not in identical anti-IgG immunoprecipitations are indicated (arrows, right side). n = 3 experiments in which distinct HASMC cell lysates were treated identically. B and C, representative high-resolution confocal images of HASMCs, transfected with either myc-Orai1, GFP-STIM1, HA-ADCY8, or FLAG-PDE1C plated on coated coverslips (B) or Fluoroblok membranes (C). Transfected HASMCs plated on coverslips were incubated with primary antibodies for individual expression tags (i.e., myc, GFP, HA, FLAG) or with an anti-AKAP79 antibody. Staining of the expressed proteins, or of endogenous AKAP79, are shown in green while actin tetramethylrhodamine-isothiocyanate-conjugated phalloidin is shown in red. C, HASMCs, transfected with either myc-Orai1, GFP-STIM1, HA-ADCY8, or FLAG-PDE1C were plated on Fluoroblok membranes and allowed to accumulate LEPs on the underside of the membranes. LEP on the underside of Fluoroblok membranes were stained with anti-myc, anti-GFP, anti-HA, anti-FLAG, or anti-AKAP79; visualized by staining with 488-conjugated secondary for anti-myc, anti- GFP, anti-HA, anti-FLAG and anti-AKAP79 and tetramethylrhodamine-isothiocyanate-conjugated phalloidin to visualize F-actin and DAPI for nuclei; scale bars, 20 μm, n = 5 to 7 transfections were analyzed per condition. Arrowheads indicate staining of each protein and their accumulation within LEPs. ADCY, adenylyl cyclase; HASMC, human arterial smooth muscle cell; LEP, leading-edge protrusion; PDE, phosphodiesterase; PDE1C, phosphodiesterase 1C; STIM1, stromal interaction molecule 1.