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.
Highlights
Signal transduction is a key process of converting one signal to another, leading to a series of signaling reactions
We investigated the role of MEKK1-MKK4-Jun N-terminal kinase (JNK)-AP1 pathway in regulating Rgs4 expression in rabbit colonic smooth muscle cells (SMC) and showed that JNK inhibition increased while MEKK1/MKK4 overexpression attenuated both constitutive and IL-1b-induced expression of Rgs4
We have shown that the NFkB pathway, as well as the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK pathways enhance while the PI3K/ Akt/GSK3b pathway inhibits the upregulation of Rgs4 expression by IL-1b in colonic SMC [6,39]
Summary
Signal transduction is a key process of converting one signal to another, leading to a series of signaling reactions. One critical class of signal-transduction pathways is the signaling controlled by the guanine–nucleotide-binding heterotrimeric proteins (G proteins). The ligand binding to GPCRs, such as the acetylcholine (ACh) receptor, catalyzes GDP-GTP exchange on the a-subunit of a heterotrimeric G-protein complex. GPCR signaling is terminated by the intrinsic GTPase activity of the Ga-subunit, which is accelerated by the regulator of G-protein signaling (RGS) proteins as GTPaseactivating proteins. Each RGS protein regulates the function of multiple GPCRs, while some RGS proteins have a clear preference for particular receptor-G protein complexes. RGS4 plays an important role in regulating smooth muscle contraction, cardiomyocyte development, neural plasticity and psychiatric disorders [4,5,6,7]. Regulator of G-protein Signaling 4 (RGS4) plays an important role in regulating smooth muscle contraction, cardiac development, neural plasticity and psychiatric disorder. We investigate the effect of the c-Jun N-terminal kinase (JNK) pathway on Rgs expression in rabbit colonic smooth muscle cells
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call