Serotonin 5-HT_2B Receptor-Stimulated DNA Synthesis and Proliferation Are Mediated by Autocrine Secretion of Transforming Growth Factor-α in Primary Cultures of Adult Rat Hepatocytes

Abstract

The mechanism of serotonin 5-HT 2 receptor subtype-stimulated DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction pathways. DNA synthesis and proliferation were detected in hepatocyte parenchymal cells grown in serumfree, defined medium containing 5-HT ( Under normal physiological conditions, the mature rat liver is quiescent and does not proliferate. However, the liver has a remarkable capacity to regenerate. For example, after 70% partial hepatectomy, the remaining hepatocytes proliferate to restore the liver to its original mass within 2 weeks. 1) Among the many growth-promoting factors important for liver regeneration, platelet-derived serotonin (5-hydroxytryptamine; 5-HT) is directly involved in liver regeneration. [2][3][4][5] In addition to its role as a neurotransmitter, 5-HT also functions as a hormone with various peripheral effects. 5-HT mediates many types of biological responses such as platelet aggregation, vascular constriction, and cell proliferation and mediates a diverse array of responses by interacting with 5-HT receptor subtypes in mammals.6) Seven receptor classes including 14 subtypes of 5-HT receptors have been identified, reflecting the diversity of 5-HT actions. With the exception of the 5-HT 3 receptors, which are ligand-gated ion channels, all known 5-HT receptors are G-protein-coupled receptors that are positively linked to phosphatidylinositol turnover or cAMP production and further linked to a variety of downstream pathways. 7,8) In a previous report, we demonstrated that 5-HT-induced hepatocyte DNA synthesis and proliferation are mediated through 5-HT 2B receptors. 9) 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 signal-regulated kinase (ERK)2/mammalian target of rapamycin (mTOR) pathway. However, how these two signaling pathways are associated with each other remains unknown. We hypothesized that 5-HT 2B receptor stimulation induces secretion of a putative primary mitogen via the Gq/ PLC pathway in hepatocytes, followed by induction of DNA synthesis and proliferation that is mediated by the EGF/TGF-α RTK/PI3K/ERK2/mTOR pathway. 9)To test this model, demonstrating that treatment of primary cultured hepatocytes with 5-HT or the selective 5-HT 2B receptor agonist BW723C86 10) leads to an increase in secretion of a putative primary mitogen is important. If so, determining how autocrine secretion of this mitogen via 5-HT 2B receptor activation is regulated is also important. Here we show that in primary cultured hepatocytes, 5-HT or BW723C86 induces autocrine secretion of TGF-α, which then activates EGF/ TGF-α RTK/PI3K/ERK2/mTOR signaling to induce DNA synthesis and proliferation.

Effect of Selective Serotonin (5-HT)_2B Receptor Agonist BW723C86 on Epidermal Growth Factor/Transforming Growth Factor-α Receptor Tyrosine Kinase and Ribosomal p70 S6 Kinase Activities in Primary Cultures of Adult Rat Hepatocytes

“…Animals Male Wistar rats obtained from Tokyo Experi-mental Animal Co. (Tokyo, Japan) were used in the present study as previously descried. 13) All rats were treated in compliance with the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society and Josai University.…”

“…Isolated hepatocytes were plated at a density of 3.3 × 10 4 cells/cm 2 onto plastic and collagen-coated 33-mm culture dishes (Iwaki Glass Co., Tokyo, Japan) and were cultured, as previously study. 13) The medium was then removed by aspiration and the cells were cultured in dexamethasone-and serum-free Williams' medium E supplemented with 5-HT or BW723C86. Finally, the following were added as appropriate: 5-HT or BW723C86 with or without selective 5-HT 2A (ketanserin), 5-HT 2B (LY272015), and 5-HT 2C receptor antagonist (SB242084), U-73122, U-73343, GF109203X, H-89, 2,4-dideoxyadenosine, BAPTA/AM, verapamil, somatostatin, monoclonal antibody against TGF-α and growth-related signal transducer inhibitor (AG1478, LY294002, PD98059, rapamycin, and LY2584702).…”

“…21) Determination of Cell Proliferation The number of nuclei was determined as previously descried. 13) Nuclei rather than cells were counted to assess proliferation. 15) Materials Regarding the selective antagonists for 5-HT 2 receptor subtypes, ketanserin tartrate was obtained from Data Analysis and Statistics Data were expressed as the mean ± standard error of the mean (S.E.M.).…”

“…10,11) Previously, we demonstrated that 5-HT and a selective 5-HT 2B receptor agonist BW723C86, 12) induce DNA synthesis and proliferation and increase EGF/transforming growth factor (TGF)-α receptor tyrosine kinase (RTK) phosphorylation and extracellular signal-regulated kinase 2 (ERK2) phosphorylation in primary cultures of rat adult hepatocytes. 13) In these hepatocytes, the proliferative effect induced by 5-HT or BW723C86 is mediated through the activation of a signaling pathway involving 5-HT 2B receptor/Gq/phospholipase C (PLC)/Ca 2+ via autocrine secretion of TGF-α. 14) TGF-α secreted by cultured hepatocytes accelerates DNA synthesis and proliferation via activation of a signaling pathway involving EGF/TGF-α RTK, phosphatidylinositol 3-kinase (PI3K), ERK2, and mammalian target of rapamycin (mTOR).…”

“…13) In these hepatocytes, the proliferative effect induced by 5-HT or BW723C86 is mediated through the activation of a signaling pathway involving 5-HT 2B receptor/Gq/phospholipase C (PLC)/Ca 2+ via autocrine secretion of TGF-α. 14) TGF-α secreted by cultured hepatocytes accelerates DNA synthesis and proliferation via activation of a signaling pathway involving EGF/TGF-α RTK, phosphatidylinositol 3-kinase (PI3K), ERK2, and mammalian target of rapamycin (mTOR). Proliferating signals by TGF-α may be transmitted to a downstream signaling element, mTOR and ribosomal p70 S6 kinase (p70S6K).…”