Serotonin-induced DNA synthesis and proliferation are mediated by autocrine secretion of transforming growth factor-α in primary cultures of adult rat hepatocytes

Abstract

Previously, we demonstrated that serotonin (5-hydroxytryotamine; 5-HT)-induced hepatocyte DNA synthesis and proliferation are mediated through 5-HT 2B receptor. Moreover, 5-HT 2B receptor-mediated hepatocyte mitogenesis involves activation of the Gq/phospholipase C (PLC) pathway and the epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (RTK)/phosphatidylinositol 3-kinase (PI3K)/extracellular signalregulated kinase (ERK) 2/mammalian target of rapamycin (mTOR) pathway. However, how these two signaling pathways are associated with each other remains unknown. Thus we hypothesized that 5-HT 2B receptor stimulated by 5-HT induces secretion of a putative primary mitogen (e.g., TGF-α, insulin-like growth factor (IGF)-I) via the Gq/PLC pathway in primary cultured hepatocytes, followed by induction of DNA synthesis and proliferation that is mediated by the EGF/TGF-α RTK/PI3K/ERK2/mTOR pathway. Hepatocytes were isolated from normal livers by the two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Isolated hepatocytes were plated onto collagen-coated plastic culture dishes and incubated for 3-h in Williams' medium E supplemented with 5% newborn calf serum, 0.1 nM dexamethasone in 5% CO 2 in air at 37ºC to allow attachment. The medium was changed to Williams' medium E without serum and dexamethasone, and 5-HT with or without test substances was added. We investigated effects of monoclonal antibody against TGF-α (1-100 ng/ml) or IGF-I (1-100 ng/ml) on 5-HTinduced hepatocytes DNA synthesis and proliferation. The TGF-α monoclonal antibody, but not IGF-I monoclonal antibody inhibited hepatocytes DNA synthesis and proliferation induced by 5-HT in a dose-dependent manner. Next, we tested to determine autocrine secretion of TGF-α from cultured hepatocytes. 5-HT (10-6 M) significantly increased TGF-α levels in the culture medium. The maximum TGF-α concentration (30 pg/ml) peaked 10 min after addition of 5-HT. The TGF-α secretion induced by 5-HT was completely blocked by a 5-HT 2B receptor antagonist (LY272015). These results suggest that the proliferative mechanism of action of 5-HT is mediated by 5-HT 2B receptor, and stimulation of 5-HT 2B receptor increases autocrine TGF-α secretion from primary cultured hepatocytes. In conclusion, our data indicate that 5-HT stimulates DNA synthesis and proliferation in primary cultures of adult rat hepatocytes by acting via autocrine secretion of TGF-α induced by the serotonin 5-HT 2B receptor/Gq/PLC pathway.

<i>S</i>-Allyl-L-cysteine Promotes Cell Proliferation by Stimulating Growth Hormone Receptor/Janus Kinase 2/Phospholipase C Pathways and Promoting Insulin-Like Growth Factor Type-I Secretion in Primary Cultures of Adult Rat Hepatocytes

“…TGF-α is also an autocrine factor stored in hepatocytes, whose secretion is promoted by stimulation with serotonin or interleukin 1β. 23,34) As shown in Fig. 2, the SAC-induced hepatocyte proliferation effect was suppressed depending on the dose of the monoclonal antibody against IGF-I, but not TGF-α.…”

Autocrine secretion of insulin-like growth factor-I mediates growth hormone-stimulated DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

“…After attachment of hepatocytes for 3 h, cells were rinsed three times with 1.0 ml phosphate-buffered saline (PBS) and then preincubated for 5 min in 1.0 ml PBS supplemented with 1.0 mM CaCl2, 5.5 mM glucose, and 0.10 U/ml aprotinin (pH 7.4). GH (100 ng/ml) was then added in the presence or absence of test molecules (Grosheva et al, 2016, Naito et al, 2016. Conditioned medium (50 µl) was sampled after different incubation times, and IGF-I levels were measured with an ELISA kit (Biosensis, Thebarton, South Australia, Australia).…”

Gene Structure, Expression, and 5-HT2B Receptor Signaling

“…In hepatocyte parenchymal cells grown in serumfree defined medium, the proliferative mechanism of serotonin is mediated mainly through 5-HT 2B receptor-stimulated Gq/phospholipase C (PLC) and epidermal growth factor (EGF)/ transforming growth factor (TGF)-α-receptor/ PI3K/ ERK1/2/ mammalian Target of Rapamycin (mTOR) signaling pathways in primary cultured hepatocytes (108). On the other hand, serotonin-induced phosphorylation of p70S6K, which was blocked by a selective 5-HT 2B receptor antagonist LY272015, a specific PLC inhibitor U-73122, a membrane-permeable Ca 2+ chelator BAPTA/AM, an L-type Ca 2+ channel blocker verapamil, somatostatin, or a specific p70S6K inhibitor LY2584702 (109).…”