ABSTRACT
Cross-talk between G protein-coupled receptor (GPCR) signaling pathways serves to fine tune cellular responsiveness by neurohumoral factors. Accumulating evidence has implicated nitric oxide (NO)-based signaling downstream of GPCRs, but the molecular details are unknown. Here, we show that adenosine triphosphate (ATP) decreases angiotensin type 1 receptor (AT(1)R) density through NO-mediated S-nitrosylation of nuclear factor κB (NF-κB) in rat cardiac fibroblasts. Stimulation of purinergic P2Y(2) receptor by ATP increased expression of inducible NO synthase (iNOS) through activation of nuclear factor of activated T cells, NFATc1 and NFATc3. The ATP-induced iNOS interacted with p65 subunit of NF-κB in the cytosol through flavin-binding domain, which was indispensable for the locally generated NO-mediated S-nitrosylation of p65 at Cys38. ß-Arrestins anchored the formation of p65/IκBα/ß-arrestins/iNOS quaternary complex. The S-nitrosylated p65 resulted in decreases in NF-κB transcriptional activity and AT(1)R density. In pressure-overloaded mouse hearts, ATP released from cardiomyocytes led to decrease in AT(1)R density through iNOS-mediated S-nitrosylation of p65. These results show a unique regulatory mechanism of heterologous regulation of GPCRs in which cysteine modification of transcriptional factor rather than protein phosphorylation plays essential roles.
Subject(s)
Down-Regulation , Myocardium/metabolism , Nitric Oxide/metabolism , Receptor, Angiotensin, Type 1/biosynthesis , Receptors, Purinergic P2Y2/metabolism , Transcription Factor RelA/metabolism , Adenosine Triphosphate/pharmacology , Animals , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/genetics , HEK293 Cells , Humans , Male , Mice , Mice, Knockout , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Nitric Oxide/genetics , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Phosphorylation/drug effects , Phosphorylation/genetics , Rats , Receptor, Angiotensin, Type 1/genetics , Receptors, Purinergic P2Y2/genetics , Transcription Factor RelA/geneticsABSTRACT
Sustained elevation of [Ca(2+)](i) has been implicated in many cellular events. We previously reported that alpha subunits of G(12) family G proteins (Galpha(12/13)) participate in sustained Ca(2+) influx required for the activation of nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor, in rat neonatal cardiac fibroblasts. Here, we demonstrate that Galpha(12/13)-mediated up-regulation of canonical transient receptor potential 6 (TRPC6) channels participates in sustained Ca(2+) influx and NFAT activation by endothelin (ET)-1 treatment. Expression of constitutively active Galpha(12) or Galpha(13) increased the expression of TRPC6 proteins and basal Ca(2+) influx activity. The treatment with ET-1 increased TRPC6 protein levels through Galpha(12/13), reactive oxygen species, and c-Jun N-terminal kinase (JNK)-dependent pathways. NFAT is activated by sustained increase in [Ca(2+)](i) through up-regulated TRPC6. A Galpha(12/13)-inhibitory polypeptide derived from the regulator of the G-protein signaling domain of p115-Rho guanine nucleotide exchange factor and a JNK inhibitor, SP600125, suppressed the ET-1-induced increase in expression of marker proteins of myofibroblast formation through a Galpha(12/13)-reactive oxygen species-JNK pathway. The ET-1-induced myofibroblast formation was suppressed by overexpression of TRPC6 and CA NFAT, whereas it was enhanced by TRPC6 small interfering RNAs and cyclosporine A. These results suggest two opposite roles of Galpha(12/13) in cardiac fibroblasts. First, Galpha(12/13) mediate ET-1-induced myofibroblast formation. Second, Galpha(12/13) mediate TRPC6 up-regulation and NFAT activation that negatively regulates ET-1-induced myofibroblast formation. Furthermore, TRPC6 mediates hypertrophic responses in cardiac myocytes but suppresses fibrotic responses in cardiac fibroblasts. Thus, TRPC6 mediates opposite responses in cardiac myocytes and fibroblasts.
