Abstract
Activation of the 5-hydroxytryptamine receptor 2B (5-HT(2B)), a G(q/11) protein-coupled receptor, results in proliferation of various cell types. The 5-HT(2B) receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT(2B) receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT(2B) receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by LY294002 decreased base-line proliferation but had no effect on 5-HT(2B) receptor-mediated proliferation. Proliferation of ICC through the 5-HT(2B) receptor was inhibited by the phospholipase C inhibitor U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the alpha, beta, gamma, and micro protein kinase C (PKC) inhibitor Gö6976, and the alpha, beta, gamma, delta, and zeta PKC inhibitor Gö6983 inhibited 5-HT(2B) receptor-mediated proliferation, indicating the involvement of PKC alpha, beta, or gamma. Of all the PKC isoforms blocked by Gö6976, PKCgamma and micro mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT(2B) receptor activation in primary cell cultures obtained from PKCgamma(-/-) mice did not result in a proliferative response, further indicating the requirement for PKCgamma in the proliferative response to 5-HT(2B) receptor activation. The data demonstrate that the 5-HT(2B) receptor-induced proliferative response of ICC is through phospholipase C, [Ca(2+)](i), and PKCgamma, implicating this PKC isoform in the regulation of cellular proliferation.
Highlights
Tight control of cell proliferation is essential to maintain organ size and function
This study demonstrates that proliferation mediated by the 5-hydroxytryptamine receptor 2B (5-HT2B) receptor requires phospholipase C (PLC), intracellular calcium release, and the ERK/mitogen-activated protein kinase (MAPK) signaling pathway and identifies the protein kinase C (PKC) isoform activated by the 5-HT2B receptor in interstitial cells of Cajal (ICC) as PKC␥
The proliferative response of ICC to 5-HT2B receptor activation was completely blocked by the U73122 (Fig. 2B), indicating that proliferation mediated by activation of the 5-HT2B receptor requires PLC
Summary
Tight control of cell proliferation is essential to maintain organ size and function. There is substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT2B [3,4,5,6,7,8,9]). Activation of the 5-HT2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves [10], but they conduct and amplify neuronal signals [11, 12], release carbon monoxide to set the intestinal smooth muscle membrane potential gradient [13], and act as mechanosensors [14, 15]. The other hand, the 5-HT2 subfamily of receptors are known to couple to phospholipase C (PLC) (26 –28)
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