Stretch-induced regulation of angiotensinogen gene expression in cardiac myocytes and fibroblasts: opposing roles of JNK1/2 and p38alpha MAP kinases

Abstract

The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy, remodeling, and fibroblast proliferation in the hemodynamically overloaded heart. However, the intracellular signaling mechanisms responsible for regulation of angiotensinogen (Ao), a substrate of the RAS system, are largely unknown. Here we report the identification of JNK1/2 as a negative, and p38alpha as a major positive regulator of Ao gene expression. Isolated neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRFB) plated on deformable membranes coated with collagen IV, were exposed to 20% equiaxial static-stretch (0-24 h). Mechanical stretch initially depressed Ao gene expression (4 h), whereas after 8 h, Ao gene expression increased in a time-dependent manner. Blockade of JNK1/2 with SP600125 increased basal Ao gene expression in NRVM (10.52+/-1.98 fold, P<0.001) and NRFB (13.32+/-2.07 fold, P<0.001). Adenovirus-mediated expression of wild-type JNK1 significantly inhibited, whereas expression of dominant-negative JNK1 and JNK2 increased basal and stretch-mediated (24 h) Ao gene expression, showing both JNK1 and JNK2 to be negative regulators of Ao gene expression in NRVM and NRFB. Blockade of p38alpha/beta by SB202190 or p38alpha by SB203580 significantly inhibited stretch-induced (24 h) Ao gene expression, whereas expression of wild-type p38alpha increased stretch-induced Ao gene expression in both NRVM (8.41+/-1.50 fold, P<0.001) and NRFB (3.39+/-0.74 fold, P<0.001). Conversely, expression of dominant-negative p38alpha significantly inhibited stretch response. Moreover, expression of constitutively active MKK6b (E) significantly stimulated Ao gene expression in the absence of stretch, indicating that p38 activation alone is sufficient to induce Ao gene expression. Taken together p38alpha was demonstrated to be a positive regulator, whereas JNK1/2 was found to be a negative regulator of Ao gene expression. Prolonged stretch diminished JNK1/2 activation, which was accompanied by a reciprocal increase in p38 activation and Ao gene expression. This suggests that a balance in JNK1/2 and p38alpha activation determines the level of Ao gene expression in myocardial cells.

Figure 1. Time-dependent Ao gene expression in stretched myocytes and cardiac fibroblasts

NRVM (A) and NRFB (B) were stretched (20%) for different time (2 – 24 h), as indicated. After stretch, cells were harvested and RNA was isolated, expression of Ao was determined by Taqman real time PCR in a multiplex system with GAPDH (for normalization). Results are expressed as means ± SEM. N=5 experiments. NS, No stretch; ST, Stretch; NRVM, Neonatal rat ventricular myocytes; NRFB, Neonatal rat ventricular fibroblasts.

Figure 2. Time-course of stretch-induced JNK and p38 activation in cardiac myocytes and fibroblasts

NRVM (A) and NRFB (B) were stretched (20%) for different time (2 min-24 h), as indicated. After stretch, cells were harvested and lysates were analyzed by immunoblotting with phospho-JNK and phospho-p38 antibodies, as indicated (A&B). C&D, Bar graphs show fold changes in JNK (C) and p38 (D) phosphorylation after stretch, when compared to no-stretch (NS). Results are expressed as means ± SEM. N=5 experiments. NRVM, Neonatal rat ventricular myocytes; NRFB, Neonatal rat ventricular fibroblasts.

Figure 3. JNK negatively regulates Ao gene expression

NRVM and NRFB were stretched in presence and absence of specific JNK1/2 inhibitors 20 μM, SP600125 for 24 h. After stretch, cells were harvested and RNA was isolated, and expression of Ao was determined. SP600125 treatment significantly increased basal Ao gene expression in myocytes (A) and NRFB (B). (C&D) Cells were transfected with adenoviruses expressing wild-type (JNK1-WT) or dominant-negative (JNK1-DN) forms of JNK1 or JNK2 (JNK2-DN). After 24 h of transfection, viral medium was replaced with serum free medium and allowed for overnight starvation before starting the stretch experiment. After stretching, cells were lysed, RNA was isolated and Ao expression was determined. Results are expressed as means ± SEM. N=5 experiments. NS, No-stretch; ST, Stretch; NRVM, Neonatal rat ventricular myocytes; NRFB, Neonatal rat ventricular fibroblasts; n.s., Not signficant.

Figure 4. p38 mediates stretch-induced Ao gene expression

NRVM and NRFB were stretched in presence and absence of specific p38 inhibitors (10 μM, SB202190 and SB203580) for 24 h. Inhibition of p38 significantly inhibited stretch-mediated Ao gene expression both in myocytes (A) and NRFB (B). (C&D) Cells were transfected with adenoviruses expressing wild-type (p38-WT) and dominant-negative (p38-DN) forms of p38α. After 24 h of transfection, viral medium was replaced with serum free medium and allowed for overnight starvation before starting the stretch experiment. After stretch, cells were harvested and RNA was isolated, expression of Ao was determined (C&D) Results are expressed as means ± SEM. N=5 experiments. NS, No stretch; ST, Stretch; NRVM, Neonatal rat ventricular myocytes; NRFB, Neonatal rat ventricular fibroblasts; SB190, SB20190; SB580, SB203580.

Figure 5. Activation of p38 alone is sufficient to induce Ao gene expression

NRVM and NRFB were transfected with adenovirus expressing MKK6b (E), in the presence and absence of the specific p38 inhibitor SB202190 (10 μM). A&B, p38 activity was induced by MKK6b (E) expression in NRVM and NRFB as measured from immunoblot to detect levels of phospho-p38 in cell lysates. C&D, real-time PCR analysis of Ao gene expression reveals that MKK6b (E) expression alone significantly induced Ao gene expression and treatment with p38 inhibitor significantly inhibited this response in both cell types. Results are expressed as means ± SEM. N=5 experiments. NRVM, Neonatal rat ventricular myocytes; NRFB, Neonatal rat ventricular fibroblasts; SB190, SB202190.

Figure 6. Increased JNK activation is responsible for downregulation of Ao gene expression in response to acute mechanical stretch

NRVM and NRFB were pretreated (1 h) with specific JNK1/2 inhibitor SP600125 (20 μM), then stretched for 4 h. After stretch, cells were harvested and RNA was isolated, and expression of Ao was determined. SP600125 treatment significantly increased stretch mediated Ao gene expression in NRVM (A) and NRFB (B). Results are expressed as means ± SEM. N=5 experiments. NS, No stretch; ST, Stretch; NRVM, Neonatal rat ventricular myocytes; NRFB, Neonatal rat ventricular fibroblasts.