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.

Key words serotonin (5-hydroxytryptamine); signal transduction; DNA synthesis; proliferation (cultured hepatocyte); transforming growth factor-α Although adult rat hepatocytes rarely undergo cell division under normal physiological conditions, due to the ensuing regenerative response to recruit mature hepatocytes into the cell cycle in vivo after 70% partial hepatectomy, a phenomenon known as liver regeneration, 1,2) they do remain capable of proliferation. Among the numerous growth-promoting factors that pertain to liver regeneration, platelet-derived serotonin (5-hydroxytryptamine; 5-HT) is reportedly directly involved. 3-7)5-HT is not only a neurotransmitter, but also a kind of hormone with various extraneural functions. 5-HT mediates a diverse array of responses-including platelet aggregation, vascular constriction, and cell proliferation-by interacting with 5-HT receptor subtypes in mammals.8) Seven receptor classes including 14 subtypes of 5-HT receptors have been identified to date, 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 cyclic AMP production and are further linked to a variety of downstream pathways. [9][10][11] In the presence of epidermal growth factor (EGF) and insulin, 5-HT has been shown to cause a dose-dependent increase in DNA synthesis in primary cultures of rat hepatocytes, suggesting that it is also a co-mitogen.12) In addition, interaction has also been seen between 5-HT and various hematological factors such as hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF). Although 5-HT has been shown to play significant roles in the stimulation and acceleration of hepatocyte proliferation, 6) few studies have been conducted on the possible involvement of 5-HT 2 receptor subtypes and their intracellular signal transduction pathways in 5-HT-induced hepatocyte DNA synthesis and proliferation in primary cultures of adult rat hepatocytes.Some recently identified selective 5-HT receptor agonists and antagonists have shown promise as useful tools for differentiating between closely related 5-HT 2 receptor subtypes. 8,[13][14][15][16] Therefore, in this study, we used these agents to investigate the involvement of 5-HT 2 receptor subtypes and their intracellular signal transduction pathways in 5-HT-induced hepatocyte DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. MATERIALS AND METHODSAnimals Male Wistar rats (weight range, 200-220 g) were purchased from Tokyo Experimental Animal Co. (Tokyo, Japan) and adapted to a light-, humidity-and temperature-controlled room over a minimum 3-d period prior to the start of the experiments. All rats were fed with a standard laboratory diet and given tap water ad libitum. All rats used in this study were given human care in compliance with Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanes...

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.

Serotonin (5-hydroxytryptamine; 5-HT) can induce hepatocyte DNA synthesis and proliferation by autocrine secretion of transforming growth factor (TGF)-α through 5-HT 2B receptor/phospholipase C (PLC)/Ca 2 and a signaling pathway involving epidermal growth factor (EGF)/TGF-α receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase 2 (ERK2)/mammalian target of rapamycin (mTOR). In the present study, we investigated whether 5-HT or a selective 5-HT 2B receptor agonist BW723C86, would stimulate phosphorylation of TGF-α RTK and ribosomal p70 S6 kinase (p70S6K) in primary cultures of adult rat hepatocytes. Western blotting analysis was used to detect 5-HT-or BW723C86 (10 6 M)-induced phosphorylation of EGF/TGF-α RTK and p70S6K. Our results showed that 5-HT-or BW723C86 (10 6 M)-induced phosphorylation of EGF/TGF-α RTK peaked at between 5 and 10 min. On the other hand, 5-HT-or BW723C86 (10 6 M)induced phosphorylation of p70S6K peaked at about 30 min. Furthermore, 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, and a specific p70S6K inhibitor LY2584702 completely abolished the phosphorylation of p70S6K induced by both 5-HT and BW723C86. These results indicate that phosphorylation of p70S6K is dependent on the 5-HT 2B-receptor-mediated autocrine secretion of TGF-α. In addition, these results demonstrate that the hepatocyte proliferating action of 5-HT and BW723C86 are mediated by phosphorylation of p70S6K, a downstream element of the EGF/TGF-α RTK signaling pathway.

Previously, we reported that serotonin (5-hydoxytryptamine; 5-HT) induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. The 5-HT effect was due to transforming growth factor (TGF)-α secreted by activation of 5-HT 2B receptor/phospholipase C (PLC)/Ca 2+ pathway. In this study, we investigated whether 5-HT would stimulate phosphorylation of ribosomal p70 S6 kinase (p70S6K) in the cultured cells. Phosphorylated p70S6K was identified by Western blotting analysis using anti-phospho-p70S6K monoclonal antibody. The phosphorylated p70S6K was increased at 5 min, and reached a peak at 30 min after 5-HT addition. On the other hand, the phosphorylation of p70S6K induced by 5-HT was completely abolished the 5-HT 2B receptor antagonist, LY272015, U -73122, a PLC inhibitor, BAPTA/AM, a membrane-permeable Ca 2+ chelator, verapamil, L-type Ca 2+ channel blocker, and somatostatin. Moreover, specific inhibitors of growth-related signal transducers (e.g., LY294002, PD98059, and rapamycin) blocked phosphorylation of p70S6K induced by 5-HT. These results suggest that secretion of TGF-α accelerates hepatocyte proliferation through the mitogen-activated protein kinase (MAPK)/p70S6K pathway.