The mixed-lineage kinase 1-3 signalling pathway regulates stress response in cardiac myocytes via GATA-4 and AP-1 transcription factors

Abstract

Background and purpose: The mixed-lineage kinases (MLKs) act upstream of mitogen-activated protein kinases, but their role in cardiac biology and pathology is largely unknown.

Experimental approach: We investigated the effect of a MLK1-3 inhibitor CEP-11004 on G protein-coupled receptor agonist-induced stress response in neonatal rat cardiac myocytes in culture.

Key results: CEP-11004 administration dose-dependently attenuated phenylephrine and endothelin-1 (ET-1)-induced c-Jun N-terminal kinase activation. MLK inhibition also reduced ET-1- and phenylephrine-induced phosphorylation of p38 mitogen-activated protein kinase. In contrast, phenylephrine-induced extracellular signal-regulated kinase phosphorylation was further up-regulated by CEP-11004. ET-1 increased activator protein-1 binding activity 3.5-fold and GATA-binding protein 4 (GATA-4) binding activity 1.8-fold, both of which were attenuated with CEP-11004 administration by 59% and 63% respectively. Phenylephrine induced activator protein-1 binding activity by 2.6-fold, which was decreased by 81% with CEP-11004 administration. Phenylephrine also induced a 3.7-fold increase in the transcriptional activity of B-type natriuretic peptide (BNP), which was attenuated by 41% with CEP-11004 administration. In agreement, MLK inhibition also reduced hypertrophic agonist-induced secretion of immunoreactive atrial natriuretic peptide and BNP.

Conclusions and implications: These results showed that inhibition of the MLK1-3 signalling pathway was sufficient for suppressing the activity of key nuclear effectors (GATA-4 and activator protein-1 transcription factors) in cardiac hypertrophy, and attenuated the agonist-induced atrial natriuretic peptide secretion and activation of BNP gene transcription.

Figure 3

The effect of mixed-lineage kinase inhibition on extracellular signal-regulated kinase (ERK) phosphorylation. Phosphorylation of ERK was measured by Western blot analysis to study the effect of mixed-lineage kinase inhibition on ERK activity. Myocytes were pretreated with 100 nM CEP-11004 or vehicle for 4 h before treatment with endothelin-1 (ET-1) or phenylephrine (PE) alone or in combination with CEP-11004 for 15 min. (A) ET-1 induces an increase in ERK phosphorylation, which is further enhanced by administration of CEP-11004. (B) PE exposure induces ERK phosphorylation, which is further increased by treatment of cell with CEP-11004. Representative Western blots are shown below. Results are the mean ± SE, n= 7–11. **P < 0.01; ***P < 0.001 versus non-treated control; ^#P < 0.05; ^###P < 0.001 versus ET-1 or PE treatment.

Figure 2

The effect of CEP-11004 administration on p38 mitogen-activated protein kinase (MAPK) phosphorylation. The effect of mixed-lineage kinase (MLK) inhibition on p38 MAPK activity was studied by measuring the phosphorylation of p38 MAPK by Western blot analysis. Myocytes were pretreated with 100 nM CEP-11004 or vehicle for 4 h before treatment with endothelin-1 (ET-1) or phenylephrine (PE) alone or in combination with CEP-11004 for 15 min. (A) Activation of p38 MAPK by ET-1 is completely blocked by MLK1–3 inhibition with CEP-11004. (B) Administration of CEP-11004 abolishes PE-induced activation of p38 MAPK. Representative Western blots are shown below. Results are the mean ± SE, n= 7–11. *P < 0.05; **P < 0.01 versus non-treated control; ^#P < 0.05 versus ET-1 or PE treatment.

Figure 1

Effect of CEP-11004 administration on c-Jun phosphorylation. Effect of CEP-11004 on c-Jun N-terminal kinase (JNK) activity was studied by measuring the phosphorylation of JNK's main target, c-Jun as described in Methods. Myocytes were treated with hypertrophic agonists; either endothelin-1 (ET-1) or phenylephrine (PE) alone or in combination with CEP-11004, for 15 min and phospho-c-Jun (p-c-Jun) formation was measured by Western blot analysis. (A) CEP-11004 dose-dependently decreases ET-1-induced phosphorylation of c-Jun. Sub-maximal inhibition is seen with 100 nM of CEP-11004. CEP-11004 treatment alone has no effect on basal c-Jun phosphorylation. (B) CEP-11004 dose-dependently attenuates PE-induced increase in c-Jun phosphorylation. (A,B) Panels are representative of three separate experiments.

Figure 4

The effect of mixed-lineage kinase inhibition on transcription factor GATA-4 or activator protein-1 (AP-1) DNA binding activity. Myocytes were pretreated with 100 nM CEP-11004 or vehicle for 4 h prior to treatment with endothelin-1 (ET-1) or phenylephrine (PE) alone, or in combination with CEP-11004 for 1 h. The nuclear proteins were extracted and binding reactions containing 3 µg of extracts were probed with radiolabelled rBNP-90 GATA or rBNP-373 AP-1 probe and subjected to electrophoretic mobility shift assay (EMSA) as described in Methods. (A) ET-1-induced GATA-4 transcription factor DNA binding activity is significantly inhibited by CEP-11004. (B) CEP treatment inhibits ET-1-induced increase in AP-1 transcription factor DNA binding activity. (C) PE-induced AP-1 binding activity is significantly attenuated by CEP-11004 treatment. Representative EMSAs are shown below. Results are the mean ± SE, n= 7–16. *P < 0.05; ***P < 0.001 versus non-treated control; ^#P < 0.05; ^##P < 0.01 versus ET-1 or PE treatment.

Figure 5

The effect of mixed-lineage kinase inhibition on atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) secretion. Myocytes were pretreated with 100 nM CEP-11004 or vehicle for 4 h before exposure to the hypertrophic agonists endothelin-1 (ET-1) or phenylephrine (PE) for 24 h. The natriuretic peptide levels in the incubation medium were measured by radioimmunoassay. (A) CEP-11004 treatment reduces the ET-1-induced secretion of BNP. (B) ET-1-induced ANP secretion is attenuated by treatment with CEP-11004. (C) The increase in BNP secretion stimulated by PE is attenuated by treatment with CEP-11004. (D) CEP-11004 treatment reduces the PE-induced increase in ANP secretion. Results are the mean ± SE, n= 11–22. ***P < 0.001 versus non-treated control; ^#P < 0.05; ^###P < 0.001 versus ET-1 or PE treatment.

Figure 6

The effect of mixed-lineage kinases 1–3 inhibition on rat B-type natriuretic peptide (BNP) promoter transcription. Luciferase reporter construct containing (Δ-534 bp/+8 bp) of the rat BNP promoter was cotransfected with RSV-β-Gal-plasmid into neonatal rat ventricular myocytes. Myocytes were incubated for 4 h with 100 nM CEP-11004 or vehicle before 24 h treatment with 50 µM phenylephrine (PE) alone or in combination with CEP-11004. CEP-11004 significantly inhibits the PE-induced activation of the BNP promoter (rBNP), but has no effect on the basal transcriptional activity. Results are the mean ± SE, n= 8–10. *P < 0.001 versus non-treated control; ^#P < 0.01 versus PE treatment.