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. 2021 Mar 28;296:100606. doi: 10.1016/j.jbc.2021.100606

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© 2021 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

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.