Abstract
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.
Highlights
AUGUST 10, 2007 VOLUME 282 NUMBER 32 tion and nuclear factor of activated T cells (NFAT) activation that negatively regulates ET-1-induced myofibroblast formation
We demonstrated that TRPC6 proteins are up-regulated by ET-1 treatment in cardiac fibroblasts
We demonstrated that the ET-1-induced increase in TRPC6 expression is critical for NFAT activation, which negatively regulates myofibroblast formation
Summary
Materials and Plasmid Construction—JNK inhibitor II (SP600125), cyclosporine A, and PP2 were purchased from Calbiochem. F, effects of TRPC6 siRNAs on CA G␣13-induced increase in NFAT activity. For the measurement of collagen synthesis, cells were harvested with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, and protease inhibitor mixture (Nacalai, Japan). Since the extent of NFAT activation induced by CA G␣13 was larger than that by CA G␣12, CA G␣13-expressing cells were used to examine the involvement of TRPC6 in G␣12/13mediated Ca2ϩ responses. Treatment with TRPC6 siRNAs completely suppressed CA G␣13-induced up-regulation of TRPC6 protein expression. The basal Ca2ϩ influx activity was determined by the increase in [Ca2ϩ]i induced by the addition of 2 mM extracellular Ca2ϩ to cells in Ca2ϩ-free Tyrode’s solution [18] This basal Ca2ϩ influx activity was enhanced by the expression of CA G␣13 and CA G␣12 (Fig. 1D). These results suggest that up-regulation of TRPC6 by G␣13 activation enhances Ca2ϩ influx and NFAT activation in car-
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