MEKK1-MKK4-JNK-AP1 Pathway Negatively Regulates Rgs4 Expression in Colonic Smooth Muscle Cells

Abstract

Background Regulator of G-protein Signaling 4 (RGS4) plays an important role in regulating smooth muscle contraction, cardiac development, neural plasticity and psychiatric disorder. However, the underlying regulatory mechanisms remain elusive. Our recent studies have shown that upregulation of Rgs4 by interleukin (IL)-1β is mediated by the activation of NFκB signaling and modulated by extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, and phosphoinositide-3 kinase. Here we investigate the effect of the c-Jun N-terminal kinase (JNK) pathway on Rgs4 expression in rabbit colonic smooth muscle cells.Methodology/Principal FindingsCultured cells at first passage were treated with or without IL-1β (10 ng/ml) in the presence or absence of the selective JNK inhibitor (SP600125) or JNK small hairpin RNA (shRNA). The expression levels of Rgs4 mRNA and protein were determined by real-time RT-PCR and Western blot respectively. SP600125 or JNK shRNA increased Rgs4 expression in the absence or presence of IL-1β stimulation. Overexpression of MEKK1, the key upstream kinase of JNK, inhibited Rgs4 expression, which was reversed by co-expression of JNK shRNA or dominant-negative mutants for MKK4 or JNK. Both constitutive and inducible upregulation of Rgs4 expression by SP600125 was significantly inhibited by pretreatment with the transcription inhibitor, actinomycin D. Dual reporter assay showed that pretreatment with SP600125 sensitized the promoter activity of Rgs4 in response to IL-1β. Mutation of the AP1-binding site within Rgs4 promoter increased the promoter activity. Western blot analysis confirmed that IL-1β treatment increased the phosphorylation of JNK, ATF-2 and c-Jun. Gel shift and chromatin immunoprecipitation assays validated that IL-1β increased the in vitro and ex vivo binding activities of AP1 within rabbit Rgs4 promoter.Conclusion/SignificanceActivation of MEKK1-MKK4-JNK-AP1 signal pathway plays a tonic inhibitory role in regulating Rgs4 transcription in rabbit colonic smooth muscle cells. This negative regulation may aid in maintaining the transient level of RGS4 expression.

Regulator of G protein signaling 4 is a novel target of GATA-6 transcription factor

“…As shown in Fig. 1C, mutation of NFκB binding site inhibited while mutation of AP1 binding site enhanced the promoter activity of P4, consistent with our previous observation that NFκB signaling stimulates while AP1 signaling represses the transcription of RGS4[17, 19, 20]. Dual deletion of NFκB and AP1 sites induced a mixed effect on the P4 promoter activity.…”

“…The increase in RGS4 expression is responsible for IL-1β-induced inhibition of initial Ca 2+ -dependent smooth muscle contraction[16]. Up-regulation of RGS4 expression by IL-1β is mediated by the canonical IKK2/IκBα/NFκB signaling[17], enhanced by extracellular signal-regulated kinases (ERK1/2) and p38 mitogen-activated protein (MAP) kinase[18], and inhibited by phosphoinositide-3 kinase[18] and JNK pathway[19]. …”

Involvement of Interleukin-17A-Induced Hypercontractility of Intestinal Smooth Muscle Cells in Persistent Gut Motor Dysfunction

“…They reported the activation of ERK1/2 and p38 MAPK enhances, and PI3K/Akt/GSK3β signaling reduces, the IL-1β-induced, NFκB-mediated RGS4 upregulation in rabbit colonic SMCs [60]. Furthermore, they also recently reported that JNK activation decreases RGS4 expression in the presence or absence of IL-1β stimulation [61]. Their results are opposite to those of the present study in terms of the direction of action of P38 MAPK and JNK, and differ in the presence or absence of ERK1/2 activation (we have not observed evident ERK1/2 activation by IL-1β).…”

IRE1α and TRB3 do not contribute to the disruption of proximal insulin signaling caused by palmitate in C2C12 myotubes

“…This apparent discrepancy could be due to different durations of the phosphorylation state of JNK and c-Jun. In smooth muscle cells, the duration of JNK phosphorylation induced by interleukin-1b is shorter (30 min) than that of c-Jun (>2 h) (Zhang et al, 2012). As we analyzed 17 h after palmitate exposure, we may only have observed the long lasting c-Jun phosphorylation.…”

“…In smooth muscle cells, the duration of JNK phosphorylation induced by interleukin-1b is shorter (30 min) than that of c-Jun (>2 h) (Zhang et al, 2012). This apparent discrepancy could be due to different durations of the phosphorylation state of JNK and c-Jun.…”