Adenosine-5'-triphosphate up-regulates proliferation of human cardiac fibroblasts

Abstract

Background and purpose: ATP is a potent signalling molecule that regulates biological activities including increasing or decreasing proliferation in different types of cells. The aim of the present study was to investigate how ATP regulates the proliferation of human cardiac fibroblasts.

Experimental approach: Reverse transcription (RT)-PCR, Western blot analysis, cell proliferation and migration assays were employed to investigate the effects of ATP on human adult ventricular fibroblasts.

Key results: ATP increased cell proliferation in a concentration-dependent manner. Similarly, the P2X receptor agonist α,β-methylene ATP and P2Y receptor agonist ATP-γS also up-regulated cell proliferation. The P2 receptor antagonists suramin and reactive blue-2 prevented the ATP-induced increase in proliferation and RT-PCR and Western blot analysis revealed that mRNAs of P2X(4/7) and P2Y(2) are abundant in cardiac fibroblasts. ATP increased phosphorylated PKB (Akt) and ERK1/2 levels; an effect antagonized by suramin, reactive blue-2, the PI3-kinase inhibitor, wortmannin, PKB inhibitor, API-2, and MAPK inhibitor, PD98059. These kinase inhibitors also prevented the ATP-induced increase in proliferation. In addition, ATP enhanced the progression of cells from the G0/G1 phase to the S phase by increasing the expression of proteins for cyclin D1 and cyclin E. Silencing the P2X(4/7) and P2Y(2) receptors with siRNA targeting the corresponding receptor diminished ATP-stimulated proliferation and migration of the cardiac fibroblasts.

Conclusion and implication: ATP activates P2X(4/7) and P2Y(2) receptors and up-regulates the proliferation of human cardiac fibroblasts by promoting cell cycling progression. It also increases the migration of these cells. These effects of ATP may be involved in cardiac remodelling of injured hearts.

Figure 1

Stimulation of the proliferation of human cardiac fibroblasts by ATP. (A) Cell proliferation was assayed by MTT in cells incubated with 0.1–300 µM ATP for 24 h (n= 5, *P < 0.05, **P < 0.01 vs. control, 0 µM ATP). (B) [³H]-thymidine incorporation was determined in cells incubated with 0.1–300 µM ATP for 24 h (n= 5, *P < 0.05, **P < 0.01 vs. control, 0 µM ATP).

Figure 2

P2 receptors gene expression and effects of P2 receptor antagonists on [³H]-thymidine incorporation. (A, B) Images of RT-PCR and Western blots for detecting mRNAs and proteins of P2 receptors in human cardiac fibroblasts. (C) [³H]-thymidine incorporation was determined in cells incubated with ATP (100 µM), AMP-CPP (α,β-methylene ATP, 100 µM) or ATP-γS (100 µM) for 24 h. (D) Pre-incubation (for 30 min) with reactive blue-2 (RB-2, 1 µM) or suramin (10 µM) attenuated or abolished the increase in [³H]-thymidine incorporation rate induced by ATP. n= 4, *P < 0.05, **P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. ATP alone.

Figure 3

Effect of ATP on the phosphorylation of PKB and ERK1/2. (A, B) Images of total PKB (Akt) and phosphorylated PKB (P-Akt308, P-Akt407, upper panels) and relative mean values of phosphorylated PKB in cells treated with ATP (100 µM for 60 min incubation), alone or after pretreatment with either suramin (10 µM for 30 min) or reactive blue-2 (RB-2, 1 µM for 30 min) (n= 3, **P < 0.01 vs. vehicle control; ^##P < 0.01 vs. ATP alone). (C) Time-dependent effect of ATP (100 µM) on P-ERK1/2 (n = 3, P < 0.05 or P < 0.01 vs. 0 min). (D) Images of total ERK1/2 and phosphorylated ERK1/2 (P-ERK1/2) and relative mean values of phosphorylated ERK1/2 in cells treated with ATP (100 µM for 60 min incubation), alone or after pretreatment with either suramin (10 µM for 30 min) or reactive blue-2 (RB-2, 1 µM for 30 min) (n= 3, **P < 0.01 vs. vehicle control; ^##P < 0.01 vs. ATP alone).

Figure 4

Effects of kinase inhibitors on the ATP-stimulated ERK1/2 phosphorylation or proliferation of human cardiac fibroblasts. (A) Images of total and phosphorylated ERK1/2 and mean values of phosphorylated ERK1/2 levels (relative to total ERK1/2) in cells treated with 100 µM ATP (60 min), alone or pretreated with the PKB inhibitor API2 (10 µM for 30 min), the PI3K inhibitor wortmannin (1 µM for 30 min) or the MAPK inhibitor PD98059 (1 µM for 30 min) (n= 3, *P < 0.05, **P < 0.01 vs. vehicle control; ^#P < 0.01 vs. ATP alone). (B, C) API2 (10 µM), wortmannin (1 µM) or PD98059 (1 µM) abolished the increase of cell proliferation and [³H]-thymidine incorporation induced by ATP (n= 4, *P < 0.05 **P < 0.01 vs. vehicle control; ^##P < 0.01 vs. ATP alone.

Figure 5

Effect of ATP on cell cycle progression. (A) Representative flow cytometry graphs in cells without (control) and with ATP (100 µM for 16 h) treatment. (B) Mean values of cell cycle distribution in control cells and in cells treated with ATP (100 µM) for 16 h (left panel) and 24 h (right panel) (n= 5, *P < 0.05 vs. control).

Figure 6

Effect of ATP on the expression of cyclin D1 and cyclin E. (A) Images of cyclin D1 protein and GAPDH protein and mean values of cyclin D1 protein level (relative to GAPDH) in cells incubated with 100 µM ATP, alone or in the presence of the P2 receptor antagonists suramin (10 µM) and RB2 (1 µM), the PI3K inhibitor wortmannin (1 µM) or the MAP inhibitor PD98059 (1 µM) (n= 3, **P < 0.01 vs. vehicle control; ^#P < 0.05, ^##P < 0.01 vs. ATP alone). (B) Images of cyclin E protein and GAPDH protein and mean values of cyclin E level (relative to GAPDH) in cells incubated with 100 µM ATP, alone or in the presence of suramin (10 µM), RB2 (1 µM), wortmannin (1 µM), or PD98059 (1 µM) (n= 3, **P < 0.01 vs. vehicle control; ^#P < 0.05, ^##P < 0.01 vs. ATP alone).

Figure 7

Effect of silencing the P2 receptors on ATP-stimulated proliferation of human cardiac fibroblasts. (A) Western blots and mean values of protein expression of P2X₄, P2X₇ and P2Y₂ in cells treated with corresponding siRNA molecules (n= 4, *P < 0.05, **P < 0.01 vs. Lipofectamine or control siRNA). (B) Cell proliferation in cells treated with siRNA molecules (40 nM) targeting P2X₄, P2X₇ and P2Y₂ (n= 3). (C) [³H]-thymidine incorporation in cells treated with siRNA molecules (40 nM) targeting P2X₄, P2X₇ and P2Y₂, respectively (n= 3). **P < 0.01 vs. control siRNA without ATP; ^#P < 0.05, ^##P < 0.01 vs. control siRNA with ATP.

Figure 8

Effect of ATP on the migration of human cardiac fibroblasts. (A) Images of human cardiac fibroblasts in the wound-healing migration assay. Confluent cardiac fibroblasts were scraped off with a pipette tip to induce acellular areas, then treated 10 µM ATP in cells transfected with Lipofectamine, control siRNA, P2X₄, P2X₇ and P2Y₂ siRNAs, respectively (40 nM each). Images were taken after 20 h incubation with 10 µM ATP. Broken white lines indicate the initial acellular wound regions. (B) Mean values for number of migrated human cardiac fibroblasts counted in areas as marked in (A) (n= 4, **P < 0.01 vs. control, ^#P < 0.05, ^##P < 0.01 vs. control siRNA). (C) Mean values for number of migrated human cardiac fibroblasts counted on lower surface of the Transwell membrane (microchemotaxis assay with 10 µM ATP incubation for 6 h, n= 3, **P < 0.01 vs. control, ^#P < 0.05, ^##P < 0.01 vs. control siRNA).