The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP - PubMed (original) (raw)

The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP

Tomomi E Kimura et al. J Mol Cell Cardiol. 2016 Jan.

Abstract

Aims: Inhibition of vascular smooth muscle cell (VSMC) proliferation by intracellular cAMP prevents excessive neointima formation and hence angioplasty restenosis and vein-graft failure. These protective effects are mediated via actin-cytoskeleton remodelling and subsequent regulation of gene expression by mechanisms that are incompletely understood. Here we investigated the role of components of the growth-regulatory Hippo pathway, specifically the transcription factor TEAD and its co-factors YAP and TAZ in VSMC.

Methods and results: Elevation of cAMP using forskolin, dibutyryl-cAMP or the physiological agonists, Cicaprost or adenosine, significantly increased phosphorylation and nuclear export YAP and TAZ and inhibited TEAD-luciferase report gene activity. Similar effects were obtained by inhibiting RhoA activity with C3-transferase, its downstream kinase, ROCK, with Y27632, or actin-polymerisation with Latrunculin-B. Conversely, expression of constitutively-active RhoA reversed the inhibitory effects of forskolin on TEAD-luciferase. Forskolin significantly inhibited the mRNA expression of the pro-mitogenic genes, CCN1, CTGF, c-MYC and TGFB2 and this was reversed by expression of constitutively-active YAP or TAZ phospho-mutants. Inhibition of YAP and TAZ function with RNAi or Verteporfin significantly reduced VSMC proliferation. Furthermore, the anti-mitogenic effects of forskolin were reversed by overexpression of constitutively-active YAP or TAZ.

Conclusion: Taken together, these data demonstrate that cAMP-induced actin-cytoskeleton remodelling inhibits YAP/TAZ-TEAD dependent expression of pro-mitogenic genes in VSMC. This mechanism contributes novel insight into the anti-mitogenic effects of cAMP in VSMC and suggests a new target for intervention.

Keywords: 3′-5′-Cyclic adenosine monophosphate; TAZ; TEAD; VSMC; YAP; cAMP.

Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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Graphical abstract

Fig. 1

Fig. 1

Elevated cAMP induces phosphorylation of YAP and TAZ in VSMC. VSMC were stimulated with 25 μM forskolin (A, C, E) or 500 μM db-cAMP (B, D, F) for the indicated times and total cell lysates analysed by Western blotting for phospho-YAP (S127 and S397; A, B, E, F) and total-TAZ phosphorylation (C–F). Western blots were quantified using densitometry (A–D). All experiments are at least n = 4. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Fig. 2

Fig. 2

Elevated cAMP induces YAP/TAZ nuclear export and TAZ degradation. VSMC were stimulated with 25 μM forskolin for the indicated times. (A) Total cell lysates were analysed fot YAP and TAZ (n = 3). (B) Cytoplasmic and nuclear fractions were analysed for YAP, TAZ, Lamin A/C and GAPDH. *** indicates p < 0.001.

Fig. 3

Fig. 3

Elevated cAMP inhibited TEAD-dependent transcription. VSMCs were transfected with either TEAD-luciferase plasmid (8xTEAD) or TnT-minimal promoter-luciferase plasmid (TnT-minP) that lacks TEAD elements. Cells were stimulated with 25 μM forskolin (A and C) or 500 μM db-cAMP (B) for the indicated times. Cell lysates were assayed for luciferase activity. (C) VSMCs were transfected with TEAD-luciferase plasmid together with active YAP (YAPS127A), active TAZ (TAZ4SA), or empty control vector. The cells ere stimulated with 25 μM forskolin for 8 h. All data is n = 3 * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Fig. 4

Fig. 4

Inhibition of RhoA/ROCK-mediated actin polymerisation underlies cAMP-mediated repression of YAP/TAZ and TEAD. VSMCs were treated with RhoA inhibitor (C3 Transferase; 2 μg/ml), ROCK inhibitor (Y-27,632; 10 μM) or actin-polymerisation inhibitor (Latrunculin B; 5 μg/ml) for 30 min and phosphorylation of YAP/TAZ was quantified Western blotting (A–C). VSMCs were transfected with TEAD-luciferase plasmid (8xTEAD-LUC) or TnT-minimal promoter-luciferase plasmid (TnT-minP) and treated with indicated RhoA/ROCK pathway inhibitors for 8 h (D). Cells infected with either active RhoA (RhoAG14V) or control adenovirus were stimulated with 25 μM forskolin for 8 h (E). All data is n = 3. * indicates p < 0.05, ** indicates p < 0.01. ## indicates p < 0.01 and ### indicates p < 0.001 on log transformed data.

Fig. 5

Fig. 5

cAMP inhibits the expression YAP/TAZ-dependent pro-mitogenic genes. VSMCs were stimulated with 25 μM forskolin for the indicated times (A–F) and analysed by qRT-PCR for CCN1 (A; n = 4), CTGF (B; n = 4), cMYC (C; n = 8), TGFB2 (D; n = 8), 36B4 (E; n = 4). Cells were infected with control adenovirus, YAPS127A adenovirus or TAZ4SA adenovirus and stimulated with 25 μM forskolin (F–J). Expression of CCN1 (F; n = 4), CTGF (G; n = 4), cMYC (H; n = 4), TGFB2 (I; n = 4), 36B4 (J; n = 4) was analysed by qRT-PCR. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Fig. 6

Fig. 6

YAP and TAZ cooperate to regulate VSMC proliferation. VSMC were transfected with siRNA targeting YAP, TAZ or YAP plus TAZ. Total cell lysates were prepared 24 h post transfection and analysed by Western blotting for YAP, TAZ, phospho-Rb, GAPDH and ACTIN (A). Phospho-Rb levels were quantified by densitometry (B; n = 3). siRNA-transfected cells were labelled with BrdU between 24 and 36 h post transfection (C; n = 3). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Fig. 7

Fig. 7

The YAP–TEAD inhibitor Veteporfin inhibits VSMC proliferation. VSMC were transfected with TEAD-luciferase reporter plasmid (8xTEAD) or a TnT-minimal promoter-luciferase plasmid (TnT-minP) and treated with the indicated concentrations of Verteporfin for 8 h (A; n = 3). VSMCs were treated with the indicated concentrations of Verteporfin for 18 h and total cell lysates analysed for phospho-Rb, GAPDH and ACTIN levels by Western blotting (B). Phospho-Rb Western blots were quantified by densitometry (C; n = 3). VSMCs were treated with Verteporfin for 18 h followed by BrdU labelling for 6 h (D; n = 3). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Fig. 8

Fig. 8

Constitutively active YAP and TAZ reverse the anti-mitogenic effect of forskolin in VSMC. VSMCs were infected with 3 × 107 pfu/ml of control adenovirus or adenovirus expressing constitutively active YAPS127A or TAZ4SA. Cells were stimulated with 25 μM forskolin for 18 h (A and B). Total cell lysates were analysed for phospho-Rb levels by Western blotting and densitometry (A; n = 3). Cells were labelled with BrDU for 6 h immediately after forskolin stimulations (B; n = 3). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

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