Interleukin-1β up-regulates RGS4 through the canonical IKK2/IκBα/NF-κB pathway in rabbit colonic smooth muscle

Initial Ca2+-dependent contraction of the intestinal smooth muscle mediated by G(q)-coupled receptors is attenuated by RGS4 (regulator of G-protein signalling 4). Treatment of colonic muscle cells with IL-1 beta (interleukin-1 beta) inhibits acetylcholine-stimulated initial contraction through increasing the expression of RGS4. NF-kappa B (nuclear factor kappa B) signalling is the dominant pathway activated by IL-1 beta. In the present study we show that RGS4 is a new target gene regulated by IL-1 beta/NF-kappa B signalling. Exposure of cultured rabbit colonic muscle cells to IL-1 beta induced a rapid increase in RGS4 mRNA expression, which was abolished by pretreatment with a transcription inhibitor, actinomycin D, implying a transcription-dependent mechanism. Existence of the canonical IKK2 [I kappa B (inhibitor of NF-kappa B) kinase 2]/I kappa B alpha pathway of NF-kappa B activation induced by IL-1 beta in rabbit colonic muscle cells was validated with multiple approaches, including the induction of reporter luciferase activity and endogenous NF kappa B-target gene expression, NF-kappa B-DNA binding activity, p65 nuclear translocation, I kappa B alpha degradation and the phosphorylation of IKK2 at Ser(177/181) and p65 at Ser(536). RGS4 up-regulation by IL-1 beta was blocked by selective inhibitors of IKK2, I kappa B alpha or NF-kappa B activation, by effective siRNA (small interfering RNA) of IKK2, and in cells expressing either the kinase-inactive IKK2 mutant (K44A) or the phosphorylation-deficient I kappa B alpha mutant (S32A/S36A). An IKK2-specific inhibitor or effective siRNA prevented IL-1 beta-induced inhibition of acetylcholine-stimulated PLC-beta (phopsholipase C-P) activation. These results suggest that the canonical IKK2/I kappa B alpha pathway of NF-kappa B activation mediates the up-regulation of RGS4 expression in response to IL-1 beta and contributes to the inhibitory effect of IL-1 beta on acetylcholinestimulated PLC-beta-dependent initial contraction in rabbit colonic smooth muscle.