Upregulation of RGS4 expression by IL-1β in colonic smooth muscle is enhanced by ERK1/2 and p38 MAPK and inhibited by the PI3K/Akt/GSK3β pathway

Abstract

Initial Ca2+-dependent contraction of intestinal smooth muscle is inhibited upon IL-1β treatment. The decrease in contraction reflects the upregulation of regulator of G protein signaling-4 (RGS4) via the canonical inhibitor of NF-κB kinase-2 (IKK2)/IκB-α/NF-κB pathway. Here, we show that the activation of various protein kinases, including ERK1/2, p38 MAPK, and phosphoinositide 3-kinase (PI3K), differentially modulates IL-1β-induced upregulation of RGS4 in rabbit colonic muscle cells. IL-1β treatment caused a transient phosphorylation of ERK1/2 and p38 MAPK. It also caused the phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β), sequential downstream effectors of PI3K. Pretreatment with PD-98059 (an ERK inhibitor) and SB-203580 (a p38 MAPK inhibitor) significantly inhibited IL-1β-induced RGS4 expression. In contrast, LY-294002 (a PI3K inhibitor) augmented, whereas GSK3β inhibitors inhibited, IL-1β-induced RGS4 expression. PD-98059 blocked IL-1β-induced phosphorylation of IKK2, degradation of IκB-α, and phosphorylation and nuclear translocation of NF-κB subunit p65, whereas SB-203580 had a marginal effect, implying that the effect of ERK1/2 is exerted on the canonical IKK2/IκB-α/p65 pathway of NF-κB activation but that the effect of p38 MAPK may not predominantly involve NF-κB signaling. The increase in RGS4 expression enhanced by LY-294002 was accompanied by an increase in the phosphorylation of IKK2/IκB-α/p65 and blocked by pretreatment with inhibitors of IKK2 (IKK2-IV) and IκB-α (MG-132). Inhibition of GSK3β abolished IL-1β-induced phosphorylation of IKK2/p65. These findings suggest that ERK1/2 and p38 MAPK enhance IL-1β-induced upregulation of RGS4; the effect of ERK1/2 reflects its ability to promote IKK2 phosphorylation and increase NF-κB activity. GSK3β acts normally to augment the activation of the canonical NF-κB signaling. The PI3K/Akt/GSK3β pathway attenuates IL-1β-induced upregulation of RGS4 expression by inhibiting NF-κB activation.