No abstract

We examined the links between fibrotic and proliferative pathways for the 5-HT2A receptor in rat mesangial cells. Serotonin (5-hydroxytryptamine, 5-HT) induced transforming growth factor-β1 (TGF-β1) mRNA in a concentration-dependent (peak at 30 nM 5-HT) and time-dependent fashion. For 10 nM 5-HT, the effect was noticeable at 1 h and maximal by 6 h. Inhibition of 1) protein kinase C (PKC), 2) mitogen- and extracellular signal-regulated kinase kinase (MEK1) with 2′-amino-3′-methoxyflavone (PD-90859), and 3) extracellular signal-regulated kinase (ERK) with apigenin attenuated this effect. The effect was blocked by antioxidants, N-acetyl-l-cysteine (NAC) and α-lipoic acid, and mimicked by direct application of H2O2. TGF-β1 mRNA induction was also blocked by diphenyleneiodonium and 4-(2-aminoethyl)-benzenesulfonyl fluoride, which inhibit NAD(P)H oxidase, a source of oxidants. 5-HT increased the amount of TGF-β1 protein, validating the mRNA studies and demonstrating that 5-HT potently activates ERK and induces TGF-β1 mRNA and protein in mesangial cells. Mapping studies strongly supported relative positions of the components of the signaling cascade as follow: 5-HT2A receptor → PKC → NAD(P)H oxidase/reactive oxygen species → MEK → ERK → TGF-β1 mRNA. These studies demonstrate that mitogenic signaling components (PKC, MEK, and oxidants) are directly linked to the regulation of TGF-β1, a key mediator of fibrosis. Thus a single stimulus can direct both proliferative and fibrotic signals in renal mesangial cells.

Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells through a G protein-coupled 5-HT(2A) receptor. We tested the hypothesis that oxidants might be involved in the signal transduction pathway linking the receptor to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly increased the activity and phosphorylation of ERK. These effects were blocked by the 5-HT(2A) receptor antagonist ketanserin. The peak effect was noted at 5-10 min, and half-maximal stimulation was achieved at 10-30 nM 5-HT. Chemical inhibitor and activator studies supported the involvement of phospholipase C, protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H(2)O(2) and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK activation cascade. Mapping studies supported a location for the NAD(P)H oxidase enzyme and the ROS downstream from PKC. Our studies are most consistent with an ERK activation pathway as follows: 5-HT(2A) receptor --> G(q) protein --> phospholipase C --> diacylglycerol --> classical PKC --> NAD(P)H oxidase --> superoxide --> superoxide dismutase --> H(2)O(2) --> mitogen-activated extracellular signal-regulated kinase --> ERK. These studies demonstrate a role for the 5-HT(2A) receptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in conveying the stimulatory signal from 5-HT, because 1) chemical antioxidants attenuate the 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar degree, 3) 5-HT produces superoxide and H(2)O(2) in these cells, and 4) a specific enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, which react selectively downstream of classical PKC.