Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells

Abstract

G protein-coupled receptors (GPCRs) such as angiotensin II, bradykinin and endothelin-1 (ET-1) are critically involved in the regulation of adrenal function, including aldosterone production from zona glomerulosa cells. Whereas, substantial data are available on the signaling mechanisms of ET-1 in cardiovascular tissues, such information in adrenal glomerulosa cells is lacking. Bovine adrenal glomerulosa (BAG) cells express receptors for endothelin-1 (ET-1) and their stimulation caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), extracellularly regulated signal kinases (ERK1/2), and their dependent proteins, p90 ribosomal S6 kinase (RSK-1) and CREB. ET-1 elicited these responses predominantly through activation of a G(i)-linked cascade with a minor contribution from the G(q)/PKC pathway. Whereas, selective inhibition of EGF-R kinase with AG1478 caused complete inhibition of EGF-induced ERK/RSK-1/CREB activation, it caused only partial reduction (30-40%) of such ET-1-induced responses. Consistent with this, inhibition of matrix metalloproteinases (MMPs) with GM6001 reduced ERK1/2 activation by ET-1, consistent with partial involvement of the MMP-dependent EGF-R activation in this cascade. Activation of ERK/RSK-1/CREB by both ET-1 and EGF was abolished by inhibition of Src, indicating its central role in ET-1 signaling in BAG cells. Moreover, the signaling characteristics of ET-1 in cultured BAG cells closely resembled those observed in clonal adrenocortical H295R cells. The ET-1-induced proliferation of BAG and H295 R cells was much smaller than that induced by Ang II or FGF. These data demonstrate that ET-1 causes ERK/RSK-1/CREB phosphorylation predominantly through activation of G(i) and Src, with a minor contribution from MMP-dependent EGF-R transactivation.

Fig. 1. ET-1 and EGF cause transient activation of ERK1/2

A, Time-course of the effects of ET-1 and EGF on phosphorylation of ERK1/2. Serum-starved BAG cells were treated with ET-1 (100 nM) and EGF (20 ng/ml) for the time periods indicated, lysed in Laemmli sample buffer and analyzed by SDS-PAGE using phospho-specific antibodies against ERK1/2 (Thr202/Tyr204). The blots were stripped and reprobed with ERK1/2. ERK1/2 phosphorylation in unstimulated cells at time zero min (control) was taken as 1 and agonist-induced increases in phosphorylation were compared to control as arbitrary units (a.u.). The data shown are representative of 3 experiments.

Fig. 2. ET-1-induced ERK1/2 phosphorylation in BAG cells is partly dependent on transactivation of the EGF-R

A,B, Concentration-dependent inhibitory effects of EGF-R kinase inhibition on ERK1/2 phosphorylation by ET-1 and EGF. Cells were treated with increasing concentrations of AG1478 for 20 min before stimulation with ET-1 (100 nM) and EGF (20 ng/ml) for 5 min.

Fig. 3. Effects of metalloproteinase inhibitor, GM6001, on agonist-induced ERK1/2 activation in BAG cells

A,B, Cells were treated with increasing concentrations of GM6001 for 20 min and stimulated with ET-1 (100 nM) and EGF (20 ng/ml) for 5 min. C, Effects of HB-EGF antagonist, CRM (10 μg/ml), on agonist-induced ERK1/2 activation.

Fig. 4. Roles of G_i and PKC in agonist-induced phosphorylation of ERK1/2

A,B, Cells were treated with G_i inhibitor, pertussis toxin (PTX ; 100 ng/ml) for 16 h, and PKC inhibitor, Go6983 (1 μM), for 20 min followed by stimulation with ET-1 (100 nM) and PMA (100 nM) for 5 min. C, Effects of ET_A and ET_B antagonists on ET-1-induced ERK1/2 activation. BAG cells were treated with ETA antagonist, BQ610 (---) and ETB antagonist, BQ788 (---) for 20 min followed by stimulation with ET-1 for 5 minutes. Quantitation of data is shown (n=3-4).

Fig. 5. A central role of Src in agonist-induced ERK1/2 activation in BAG cells

A, Concentration-dependent inhibitory effects of Src inhibitor, PP2, on ET-1-induced phosphorylation of ERK1/2 and Src (Y416) and Pyk2 (Y402). BAG cells were treated with increasing concentrations of PP2 for 20 min and stimulated with ET-1 (100 nM for 5 min). Cells were lysed in Laemmli sample buffer and analyzed by SDS-PAGE. C, The quantitation of data is shown (n=3).

Fig. 6. Role of PI3K in agonist-induced phosphorylation of Akt and ERK1/2

A, Concentration-dependent effects of Src inhibition on phosphorylation of Akt at Ser473. B, Effects of PI3K inhibition by wortmannin on phosphorylation of Akt at Ser473 and ERK1/2 by ET-1. Cells were treated with varying concentrations of wortmannin and stimulated with ET-1 (100 nM) for 5 min, then lysed in Laemmli sample buffer and analyzed by SDS-PAGE. C, The quantitation of data is shown (n=3).

Fig. 7. ET-1 signaling occurs through Ras activation in BAG cells

A, Overexpression of dominant negative Ras (dnRas; S17N) plasmid cDNA (1 μg) impairs agonist-induced ERK1/2 activation. Cells were transfected with cDNA as described in Methods. B, Mechanisms involved in Ras activation by ET-1. BAG cells were treated with PTX (100 ng/ml) for 16 h, and GM6001 (20 μM), PP2 (10 μM), and AG1478 (100 nM) for 20 min followed by stimulation with ET-1 (100 nM) for 5 min. Agonist-stimulated Ras activation was measured as described in Methods. C, Mechanisms involved in ET-1-induced phosphorylation of CREB at Ser133 in BAG cells. BAG cells were treated with PTX (100 ng/ml) for 16 h, and PP2 (10 μM), AG1478 (100 nM) and U0126 (1 μM) for 20 min followed by stimulation with ET-1 (100 nM) for 5 min.