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Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes the vascular endothelial growth factor-stimulated MAPKs and downstream effectors AP-1 and CREB in mouse mesangial cells

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Abstract

Along with its natriuretic, diuretic, and vasodilatory properties, atrial natriuretic peptide (ANP), and its guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) exhibit an inhibitory effect on cell growth and proliferation. However, the signaling pathways mediating this inhibition are not well understood. The objective of this study was to determine the effect of ANP–NPRA system on mitogen-activated protein kinases (MAPKs) and the downstream proliferative transcription factors involving activating protein-1 (AP-1) and cAMP-response element binding protein (CREB) in agonist-stimulated mouse mesangial cells (MMCs). We found that ANP inhibited vascular endothelial growth factor (VEGF)-stimulated phosphorylation of MAPKs (Erk1, Erk2, JNK, and p38), to a greater extent in NPRA-transfected cells (50–60 %) relative to vector-transfected cells (25–30 %). The analyses of the phosphorylated transcription factors revealed that ANP inhibited VEGF-stimulated activation of CREB, and the AP-1 subunits (c-jun and c-fos). Gel shift assays demonstrated that ANP inhibited VEGF-stimulated AP-1 and CREB DNA-binding ability by 67 and 62 %, respectively. The addition of the protein kinase G (PKG) inhibitor, KT-5823, restored the VEGF-stimulated activation of MAPKs, AP-1, and CREB, demonstrating the integral role of cGMP/PKG signaling in NPRA-mediated effects. Our results delineate the underlying mechanisms through which ANP-NPRA system exerts an inhibitory effect on MAPKs and down-stream effector molecules, AP-1, and CREB, critical for cell growth and proliferation.

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Abbreviations

ANP:

Atrial natriuretic peptide

GC-A/NPRA:

Guanylyl cyclase/natriuretic peptide receptor-A

VEGF:

Vascular endothelial growth factor, AP-1, activating protein-1

CREB:

cAMP response element-binding protein

MAPKs:

Mitogen-activated protein kinase

MMCs:

Mouse mesangial cells

DMEM:

Dulbecco’s modified Eagle’s medium

PBS:

Phosphate-buffered saline

HEPES:

N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)

IBMX:

3-Isobutyl-1-methyl xanthin

EMSA:

Electrophoretic mobility shift assays

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene fluoride

TBST:

Tris-buffered saline-tween-20

DTT:

Dithiothreitol

PMSF:

Phenylmethylsulfonyl fluoride

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Acknowledgments

Authors wish to thank Huong Nguyen and Gevoni Bolden for technical assistance and Mrs. Kamala Pandey for assistance in typing of this manuscript. Our special thanks are due to Dr. Bharat B. Aggarwal, Department of Experimental Therapeutics and Cytokine Research Laboratory at MD Anderson Cancer Center and Dr. Susan L. Hamilton, Department of Molecular Physiology and Biophysics at Baylor College of Medicine for providing their facilities during our displacement period due to Hurricane Katrina. This work was supported from the National Institutes of Health grant (HL57531).

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  1. Department of Physiology, Tulane University Health Sciences Center, School of Medicine, SL-39, 1430 Tulane Ave, New Orleans, LA, 70112, USA

    Satyabha Tripathi & Kailash N. Pandey

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  1. Satyabha Tripathi
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  2. Kailash N. Pandey
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Correspondence to Kailash N. Pandey.

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Tripathi, S., Pandey, K.N. Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes the vascular endothelial growth factor-stimulated MAPKs and downstream effectors AP-1 and CREB in mouse mesangial cells. Mol Cell Biochem 368, 47–59 (2012). https://doi.org/10.1007/s11010-012-1341-8

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