Suppression of Cyclic GMP-dependent Protein Kinase Is Essential to the Wnt/cGMP/Ca2+ Pathway

Li Ma,Hsien-Yu Wang

doi:10.1074/jbc.m603603200

Abstract

Novel downstream effectors sensing changes in intracellular concentrations of Ca2+ and cyclic GMP in response to activation of the Wnt/Frizzled-2 pathway were sought. Activation of Frizzled-2 suppressed protein kinase G activity while activating NF-AT-dependent transcription. Each of these responses was abolished by pertussis toxin and by knock-down of the expression of either Galphat2 or Galphao. Activation of NF-AT-dependent transcription in response to Wnt5a stimulation was suppressed by activation of protein kinase G and by buffering intracellular Ca2+. Elevation of intracellular cyclic GMP either by inhibition of cyclic GMP phosphodiesterase or by addition of 8-bromocyclic GMP was shown to activate protein kinase G, to block Ca2+ mobilization, as well as to markedly attenuate activation of NF-AT-dependent transcription in response to Wnt5a stimulation. Chemical inhibition of protein kinase G by Rp-8-pCPT-cGMP, conversely, was shown to provoke increased NF-AT gene transcription and Ca2+ mobilization in the absence of Wnt stimulation. Protein kinase G is shown to be a critical downstream effector of the noncanonical Wnt-Frizzled-2/cGMP/Ca2+ pathway.

Highlights

Unlike the situation for downstream effectors sensitive to Ca2ϩ mobilization, elucidation of the downstream effector(s) for Wnt-regulated changes in the intracellular concentration of cyclic GMP has remained elusive
Stimulating zebrafish embryos expressing ␤2AR/rat Frizzled-2 (Rfz2) chimera with isoproterenol leads to Ca2ϩ transients (13), identical in character to those stimulated by Wnt5a alone (12)
The increase in intracellular Ca2ϩ stimulated by activation of the Frizzled-2 chimera with agonist could be abolished by simultaneous treatment with the ␤-adrenergic antagonist propranolol (Fig. 1B). Expression of this Frizzled-2 chimera in zebrafish embryos by-passed the need for purified Wnt5a, allowing the use of isoproterenol to activate the noncanonical pathway, a response that was sensitive to antagonism by propranolol (13)

Summary

Introduction

Unlike the situation for downstream effectors sensitive to Ca2ϩ mobilization, elucidation of the downstream effector(s) for Wnt-regulated changes in the intracellular concentration of cyclic GMP has remained elusive. It is not known, for example, whether the signals from Wnt5a mediated via Frizzled-2 to Ca2ϩ mobilization and to cyclic GMP PDE operate in concert or act independently of each other in the Wnt/Ca2ϩ/cGMP pathway. Probed in zebrafish embryos and mouse F9 cells, Ca2ϩ mobilization in response to Wnt signaling has not been investigated in the context of immediate upstream regulatory element. The Wnt/Ca2ϩ/cyclic GMP pathway constitutes a noncanonical pathway that is mediated by Frizzled-2 (6 – 8). Ca2ϩ imaging with Fura-2 dye in whole embryos of zebrafish has revealed Wnt5a-stimu-

Methods

Results

Conclusion