IP Receptor Agonist–Induced DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes: the Involvement of Endogenous Transforming Growth Factor-α

Abstract

Abstract. To elucidate the mechanism of action of prostaglandin I 2 (PGI 2 ) and carbaprostacyclin, we studied their effect on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Hepatocyte parenchymal cells, maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of PGI 2 or carbaprostacyclin in a time-and dose-dependent manner. PGI 2 was less potent than carbaprostacyclin in stimulating hepatocyte mitogenesis. These effects of PGI 2 and carbaprostacyclin were abolished by treatment with a specific IP-receptor antagonist, CAY10441 (10 −9 -10 −7 M). Hepatocyte mitogenesis induced by the IP-receptor agonists was almost completely blocked by specific inhibitors of growth-related signal transducers such as AG1478 (5 × 10 −7 M), LY294002 (10 −7 M), PD98059 (10 −6 M), and rapamycin (10 ng/ ml). In addition, PGI 2 or carbaprostacyclin significantly increased the kinase activity of a (p175 kDa) receptor tyrosine kinase and the phosphorylation of extracellular signalregulated kinase (ERK) 2. Addition of a monoclonal antibody against transforming growth factor (TGF)-α, but not insulin-like growth factor-I, to the culture dose-dependently inhibited the PGI 2 -or carbaprostacyclin-induced hepatocyte mitogenesis. Furthermore, treatment with the IPreceptor agonists significantly increased the secretion of TGF-α to the culture medium. These results indicate that the IP receptor agonist-induced hepatocyte mitogenesis is mediated by autocrine secretion of TGF-α followed by activation of a receptor tyrosine kinase / ERK pathway.

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

“…1) In support of this hypothesis, we previously showed in hepatocytes that DNA synthesis and proliferation are stimulated by prostaglandin EP 1 and a prostacyclin IP receptor agonist via autocrine secretion of TGF-α, which then stimulates hepatocyte mitogenesis via the EGF/ TGF-α RTK/ERK2/mTOR pathway. 17,34) Consistent with our hypothesis, DNA synthesis and proliferation in hepatocytes induced by 5-HT or BW723C86 were blocked by monoclonal antibodies recognizing TGF-α, but not antibodies recognizing IGF-I (Fig. 4).…”

Suppression of acute hepatic injury by a synthetic prostacyclin agonist through hepatocyte growth factor expression

“…Some reports showed that prostaglandins alone did not stimulate basal DNA synthesis, whereas prostaglandins augmented the mitogenic action of EGF (6). Others indicated that PGI 2 enhanced DNA synthesis of hepatocytes, whereas it was abolished by neutralization of TGF-␣ (14). Perhaps the discrepancy may be related to differences in experimental conditions; however, these results indicate that prostaglandins have no mitogenic activity for hepatocytes, at least directly.…”

“…Total RNA was extracted using Sepasol-RNA I Super (Nacalai Tesque). First-strand cDNAs were synthesized using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) with oligo(dT) [12][13][14][15][16][17][18] primers. The primer sequences for mRNA quantification are listed in Fig.…”

Signal Transduction Mechanism for Serotonin 5-HT_2B Receptor-Mediated DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes

“…38,39) This in turn stimulates hepatocyte mitogenesis through a EGF/TGF-α receptor tyrosine Hepatocytes were plated at a cell density of 3.3×10 4 cells/cm 2 and cultured for 3 h as described in the legend to Fig. 1.…”