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.

Serotonin (5-hydroxytryptamine; 5-HT) can induce hepatocyte DNA synthesis and proliferation by autocrine secretion of transforming growth factor (TGF)-α through 5-HT2B receptor/phospholipase C (PLC)/Ca2+ 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-HT2B 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-HT2B receptor antagonist LY272015, a specific PLC inhibitor U-73122, a membrane-permeable Ca2+ chelator BAPTA/AM, an L-type Ca2+ 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-HT2B-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.

Serotonin 5-HT2B Receptor-Stimulated DNA Synthesis and Proliferation Are Mediated by Autocrine Secretion of Transforming Growth Factor-α in Primary Cultures of Adult Rat Hepatocytes.

The mechanism of serotonin 5-HT2 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 serum-free, defined medium containing 5-HT (10(-6) M) or the selective 5-HT2B receptor agonist BW723C86 (10(-6) M). In addition, exogenous transforming growth factor (TGF)-α (1.0 ng/mL) significantly increased hepatocyte DNA synthesis and proliferation, which reached plateau after 4 h of culture. Use of blocking monoclonal antibodies demonstrated that TGF-α, but not insulin-like growth factor-I, was involved in hepatocyte proliferation mediated by 5-HT or BW723C86. TGF-α levels in the culture medium increased significantly versus baseline within 5 min in response to 5-HT (10(-6) M) or BW723C86 (10(-6) M), and the maximum TGF-α level (30 pg/mL) was reached 10 min after 5-HT or BW723C86 stimulation. Secretion of TGF-α into the culture medium was inhibited by addition of the selective phospholipase C (PLC) inhibitor, U-73122 (10(-6) M), or somatostatin (10(-7) M). These results indicate that the proliferative mechanism of action of 5-HT is mediated mainly through a 5-HT2B receptor/Gq/PLC-stimulated increase in autocrine secretion of TGF-α from primary cultured hepatocytes.

Signal Transduction Mechanism for Serotonin 5-HT2B Receptor-Mediated DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes.

The involvement of serotonin (5-hydroxytryptamine; 5-HT) and the 5-HT2 receptor subtypes in the induction of DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction mechanisms. Hepatocyte parenchymal cells maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of 5-HT or a selective 5-HT2B receptor agonist, BW723C86, but not in the presence of 5-HT2A, or 5-HT2C receptor agonists (TCB-2 and CP809101, respectively), in a time- and dose-dependent manner. A selective 5-HT2B receptor antagonist, LY272015 (10(-7) M), and a specific phospholipase C (PLC) inhibitor, U-73122 (10(-6) M), as well as specific inhibitors of growth-related signal transducers-including AG1478, LY294002, PD98059, and rapamycin-completely inhibited 5-HT (10(-6) M)- or BW723C86 (10(-6) M)-induced hepatocyte DNA synthesis and proliferation. Both 5-HT and BW723C86 were shown to significantly stimulate the phosphorylation of epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (p175 kDa) and extracellular signal-regulated kinase (ERK) 2 on Western blot analysis. These results suggest that the proliferative mechanism of activating 5-HT is mediated mainly through 5-HT2B receptor-stimulated Gq/PLC and EGF/TGF-α-receptor/phosphatidylinositol 3-kinase (PI3K)/ERK2/mammalian target of rapamycin (mTOR) signaling pathways in primary cultured hepatocytes.