Interleukin-1beta up-regulates RGS4 through the canonical IKK2/IkappaBalpha/NF-kappaB 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-1beta (interleukin-1beta) inhibits acetylcholine-stimulated initial contraction through increasing the expression of RGS4. NF-kappaB (nuclear factor kappaB) signalling is the dominant pathway activated by IL-1beta. In the present study we show that RGS4 is a new target gene regulated by IL-1beta/NF-kappaB signalling. Exposure of cultured rabbit colonic muscle cells to IL-1beta 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 [IkappaB (inhibitor of NF-kappaB) kinase 2]/IkappaBalpha pathway of NF-kappaB activation induced by IL-1beta in rabbit colonic muscle cells was validated with multiple approaches, including the induction of reporter luciferase activity and endogenous NF-kappaB-target gene expression, NF-kappaB-DNA binding activity, p65 nuclear translocation, IkappaBalpha degradation and the phosphorylation of IKK2 at Ser(177/181) and p65 at Ser(536). RGS4 up-regulation by IL-1beta was blocked by selective inhibitors of IKK2, IkappaBalpha or NF-kappaB activation, by effective siRNA (small interfering RNA) of IKK2, and in cells expressing either the kinase-inactive IKK2 mutant (K44A) or the phosphorylation-deficient IkappaBalpha mutant (S32A/S36A). An IKK2-specific inhibitor or effective siRNA prevented IL-1beta-induced inhibition of acetylcholine-stimulated PLC-beta (phopsholipase C-beta) activation. These results suggest that the canonical IKK2/IkappaBalpha pathway of NF-kappaB activation mediates the up-regulation of RGS4 expression in response to IL-1beta and contributes to the inhibitory effect of IL-1beta on acetylcholine-stimulated PLC-beta-dependent initial contraction in rabbit colonic smooth muscle.
Hu
Wenhui
W
Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA. whu@vcu.edu
Li
Fang
F
Mahavadi
Sunila
S
Murthy
Karnam S
KS
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003106:Colon / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D053583:Interleukin-1beta / Q000494:pharmacology*; D009119:Muscle Contraction / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D054799:Phospholipase C beta / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D020710:RGS Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D015854:Up-Regulation
D020672:I-kappa B Proteins; D053583:Interleukin-1beta; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D020710:RGS Proteins; D012333:RNA, Messenger; D000072000:NF-KappaB Inhibitor alpha; C098799:RGS4 protein; D051550:I-kappa B Kinase; D054799:Phospholipase C beta; D000109:Acetylcholine