Hydrolysis of extracellular ATP by ectonucleoside triphosphate diphosphohydrolase (ENTPD) establishes the set point for fibrotic activity of cardiac fibroblasts

Abstract

The establishment of set points for cellular activities is essential in regulating homeostasis. Here, we demonstrate key determinants of the fibrogenic set point of cardiac fibroblasts (CFs) by focusing on the pro-fibrotic activity of ATP, which is released by CFs. We tested the hypothesis that the hydrolysis of extracellular ATP by ectonucleoside triphosphate diphosphohydrolases (ENTPDs) regulates pro-fibrotic nucleotide signaling. We detected two ENTPD isoforms, ENTPD-1 and -2, in adult rat ventricular CFs. Partial knockdown of ENTPD-1 and -2 with siRNA increased basal extracellular ATP concentration and enhanced the pro-fibrotic effect of ATP stimulation. Sodium polyoxotungstate-1, an ENTPD inhibitor, not only enhanced the pro-fibrotic effects of exogenously added ATP but also increased basal expression of α-smooth muscle actin, plasminogen activator inhibitor-1 and transforming growth factor (TGF)-β, collagen synthesis, and gel contraction. Furthermore, we found that adenosine, a product of ATP hydrolysis by ENTPD, acts via A2B receptors to counterbalance the pro-fibrotic response to ATP. Removal of extracellular adenosine or inhibition of A2B receptors enhanced pro-fibrotic ATP signaling. Together, these results demonstrate the contribution of basally released ATP in establishing the set point for fibrotic activity in adult rat CFs and identify a key role for the modulation of this activity by hydrolysis of released ATP by ENTPDs. These findings also imply that cellular homeostasis and fibrotic response involve the integration of signaling that is pro-fibrotic by ATP and anti-fibrotic by adenosine and that is regulated by ENTPDs.

Keywords: ATPases; Adenosine Receptor; ENTPD; Fibroblast; Myofibroblast; P2Y; Purinergic Receptor.

FIGURE 1.

Basal ATP signaling stimulates α-SMA expression and CF contraction. A, CFs seeded in 2.5 mg/ml rat tail collagen spontaneously contracted the collagen gels by 24 h. Subsequent 24 h of treatment with apyrase (apy) (1 unit/ml) and P2 inhibition with suramin (sur) (50 μm) reversed spontaneous collagen gel contraction and increased collagen gel surface area compared with the 48-h untreated controls (cont) by 65 and 67%, respectively. B, apyrase (1 unit/ml for 24 h) decreased α-SMA protein expression by 42%. **, p < 0.01; ***, p < 0.001 versus untreated controls; quantitative data are presented as mean ± S.E. of three independent experiments.

FIGURE 2.

Simultaneous siRNA knockdown of ENTPD1 (E1) and ENTPD2 (E2) increases extracellular ATP concentration and enhances the pro-fibrotic effect of ATP. A, ENTPD-1 and -2 were detected in similar abundance in rat ventricular CFs. Co-transfection with siRNA for ENTPD-1 and -2 (E1/E2) decreased expression by 57 and 54%, respectively. B, ENTPD-1/-2 knockdown increased basal extracellular ATP concentration by 2.5-fold. C, knockdown of ENTPD-1/-2 significantly enhanced the pro-fibrotic effect of 10 μm ATP on CFs. CFs transfected with siRNA targeting both ENTPD-1/-2 up-regulated α-SMA, PAI-1, and TGF-β expression by 2.7-, 4.0-, and 1.7-fold, respectively, in response to a 4-h incubation with ATP as compared with control (cont) siRNA-transfected CFs incubated with ATP. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus identically transfected, untreated samples; +, p < 0.05; ++, p < 0.01; +++, p < 0.001 between groups indicated. Gene expression data are presented as mean ± S.E. of three independent experiments; ATP assay data are presented as mean ± S.E. of six independent experiments.

FIGURE 3.

Inhibition of endogenous NTPase activity by POM-1 is pro-fibrotic. A, ATP hydrolysis was measured using a malachite green assay. Addition of 30 μm POM-1 inhibited ENTPD-mediated hydrolysis of exogenously added ATP (30 μm) after 1 h. POM-1 treatment for 24 h increased CF α-SMA and PAI-1 protein expression (B) and collagen synthesis (C) in a concentration-dependent manner. D, POM-1 pretreatment (1 h) increased basal ERK phosphorylation and prolonged 10 μm ATP-stimulated ERK phosphorylation. E, stimulatory effects of 30 μm POM-1 on collagen synthesis and expression of α-SMA and PAI-1 protein were blocked by 50 μm suramin (sur). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus untreated controls; +++, p < 0.001 between groups indicated. Quantitative data are presented as mean ± S.E. of at least three independent experiments. cont, control; WB, Western blot.

FIGURE 4.

POM-1 enhances the effects of ATP on pro-fibrotic marker expression of CFs. A, incubation of CFs with POM-1 together with ATP for 4 h increased expression of α-SMA, PAI-1, and TGF-β mRNA by 1.9-, 3.0-, and 1.7-fold, respectively, compared with CFs incubated only with ATP. B, incubation of CFs with POM-1 for 24 h increased α-SMA and PAI-1 protein expression by 30 and 89%, respectively, as compared with CFs incubated only with ATP. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus untreated controls (cont); +, p < 0.05; ++, p < 0.01 between groups indicated. Data are presented as mean ± S.E. of at least three independent experiments. veh, vehicle.

FIGURE 5.

POM-1 enhances the stimulatory effect of ATP on collagen synthesis and gel contraction. A, incubation of CFs with 30 μm POM-1 increased the effect of 10 μm ATP on collagen synthesis 73% after 24 h as compared with CFs incubated only with ATP. B, POM-1 increased the 12% reduction in collagen gel surface area in response to a 24-h incubation with 10 μm ATP to 32%. *, p < 0.05; **, p < 0.01, ***, p < 0.001 versus untreated controls (cont); +++, p < 0.001 between groups indicated. Quantitative data are presented as mean ± S.E. of three independent experiments.

FIGURE 6.

Adenosine signaling counteracts the pro-fibrotic effects of ATP. Adenosine (ado) decreased POM-1-stimulated α-SMA mRNA (A) and protein (B) expression in a concentration-dependent manner. C, incubation with ATP (10 μm) and adenosine (ado, 30 μm) for 4 h increased CREB phosphorylation, an effect that was blunted with the PKA inhibitors, H89 (10 μm) and (R_p)-cAMPS (50 μm). D, inhibition of A2_B adenosine receptors with PSB 603 (300 nm) blocked ATP-stimulated CREB phosphorylation; A2_A receptor inhibition (300 nm SCH 442416) had no significant effect. *, p < 0.05; **, p < 0.01 versus untreated controls (cont); +, p < 0.05; ++, p < 0.01; +++, p < 0.001 between groups indicated or as compared with control CFs not treated with inhibitors (C). Data are presented as mean ± S.E. of at least three independent experiments. WB, Western blot; veh, vehicle.

FIGURE 7.

Extracellular adenosine deamination and A2_B receptor inhibition enhances pro-fibrotic ATP signaling. Incubation with adenosine deaminase (ADA) increased collagen synthesis by 38% (after 24 h) (A) and the expression of α-SMA and PAI-1 protein 2- and 3-fold, respectively (after 4 h) (B). Incubation of CFs with ADA enhanced the stimulation produced by 10 μm ATP in the expression of α-SMA (42%) and PAI-1 (182%). C, incubation with 300 nm PSB 603 (PSB) enhanced the stimulation by ATP (10 μm, 4 h treatment) of α-SMA and PAI-1 gene expression by 62 and 69%, respectively. D, incubation for 24 h with PSB produced a 33% increase in ATP-promoted enhancement in α-SMA protein levels. PSB did not enhance the UTP-promoted increases in α-SMA or PAI-1 expression. E, effects of 30 μm POM-1 were not enhanced by pretreatment with PSB. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus untreated controls (cont); +, p < 0.05; ++, p < 0.01; +++, p < 0.001 between groups indicated. Data are presented as mean ± S.E. of three independent experiments. WB, Western blot.

FIGURE 8.

Model of the counterbalancing pro-fibrotic ATP-P2Y (P2Y₂) and anti-fibrotic adenosine-P1 signaling pathways initiated by cellular ATP release and regulated by ENTPD and nucleotidase activity.