The PDE1A-PKCalpha signaling pathway is involved in the upregulation of alpha-smooth muscle actin by TGF-beta1 in adventitial fibroblasts

Abstract

Background: Increasing evidence has suggested that differentiation of adventitial fibroblasts (AFs) to myofibroblasts plays an important role in arterial remodeling. The molecular mechanisms by which myofibroblast formation is regulated still remain largely unknown. This study aimed to evaluate the role of cyclic nucleotide phosphodiesterase 1A (PDE1A) in the formation of adventitial myofibroblasts induced by transforming growth factor (TGF)-beta(1).

Methods and results: AFs were cultured by the explant method. Western blot and immunocytochemistry were applied for alpha-smooth muscle actin (SMA) or protein kinase C (PKC) alpha protein analysis. Results showed that TGF-beta(1) upregulated PDE1A protein expression in rat aortic AFs and pharmacological inhibition of PDE1A blocked TGF-beta(1)-induced alpha-SMA expression, a marker of myofibroblast formation, suggesting that the upregulation of PDE1A may mediate TGF-beta(1)-induced AF transformation. Moreover, calphostin C (a PKC inhibitor) inhibited TGF-beta(1)-induced alpha-SMA expression, whereas phorbol-12-myristate-13-acetate (a PKC activator) induced it. Finally, the upregulation of PKCalpha expression by TGF-beta(1) was also inhibited by PDE1A inhibition.

Conclusions: Taken together, our data suggest that TGFbeta(1) induces alpha-SMA expression and myofibroblast formation via a PDE1A-PKCalpha-dependent mechanism. Our study thus unveils a novel signaling mechanism underlying TGF-beta(1)-induced adventitial myofibroblast formation.

Fig. 1

TGF-β₁ upregulated PDE1A expression in rat aortic AFs. Growth-arrested rat aortic AFs were stimulated with 10 ng/ml TGF-β₁ for the indicated time points (a) or various doses of TGF-β₁ for 24 h (b). Protein levels of PDE1A were measured by Western blot analysis. β-Actin was used as loading control. Values are expressed as means ± SEM of triplicates from a representative experiment. Mean values for the percent change in PDE1A were calculated by comparing TGF-β₁-stimulated samples versus samples collected at time 0, or without TGF-β₁, which was arbitrarily set at 100% for each experiment. ^a p < 0.05, ^b p < 0.01, vs. samples at time 0 or without TGF-β₁. Similar results were obtained from at least three independent experiments.

Fig. 2

IBMX and IC86340 inhibited α-SMA expression induced by TGF-β₁. Growth-arrested rat aortic AFs were pretreated with IC86340 (a) or IBMX (b) for 30 min in the presence or absence of 10 ng/ml TGF-β₁ for 24 h. Protein levels of α-SMA were measured by Western blot analysis (a, b) and by immunofluorescent staining using an anti-α-SMA antibody (c). Nuclei were stained with Hoechst 33342. ×200. a, b Mean values for the percent change in α-SMA were calculated in comparison to samples without TGF-β₁, which was arbitrarily set at 100% for each experiment. ^a p < 0.05, ^b p < 0.01, vs. TGF-β₁ at zero; ^c p < 0.05, ^d p < 0.01 vs. TGF-β₁ alone. d Representative microscopic images showing PDE1A localization in cells treated with vehicle (d) or 10 ng/ml TGF-β (e). PDE1A is localized in cytoplasmic regions in non-stimulated cells (d). PDE1A is primarily localized in the nucleus after TGF-β stimulation for 30 min (e).

Fig. 3

PKCα was involved PDE1A-mediated myofibroblast formation induced by TGF-β₁. a TGF-β₁ upregulated PKCα protein expression in a time-dependent manner in rat aortic AF. Growth-arrested rat aortic AFs were stimulated with 10 ng/ml TGF-β₁ for 0, 6, 12 and 24 h. Protein levels of PKCα were measured by Western blot analysis. β-Actin was used as loading control. Values are expressed as means ± SEM of triplicates from a representative experiment. b PMA upregulated α-SMA in AFs. Growth-arrested rat aortic AFs were treated with different doses of PMA for 24 h. Protein levels of α-SMA were measured by Western blot analysis. Data are normalized under serum-free conditions. Values represent means ± SEM of at least three experiments and were compared using one-way ANOVA. c IC86340 and IBMX inhibited PKCα expression induced by TGF-β₁. Growth-arrested rat aortic AFs were pretreated with IC86340 or IBMX for 30 min in the presence or absence of 10 ng/ml TGF-β₁ for 12 h. Protein levels of PKCα were measured by Western blot analysis. d Effects of the PKC inhibitor calphostin C and the PDE1A inhibitor IC86340 on α-SMA expression. AFs were pretreated with 100 μM calphostin C or 15 μM IC86340 or both of them for 30 min and then treated with 10 ng/ml TGF-β₁ for 24 h. Calphostin C or IC86340 alone inhibited α-SMA protein expression. When used together, no difference was observed on the inhibitory effect compared with calphostin C or IC86340 alone. Mean values for the percent change in PKCα or α-SMA were calculated in comparison to samples without TGF-β₁ or PMA, which was arbitrarily set at 100% for each experiment. ^a p < 0.05, ^b p < 0.01, vs. TGF-β₁ or PMA at zero; ^c p < 0.05, ^d p < 0.01, vs. TGF-β₁ alone.