Intracellular Signaling Pathways for Erythropoietin-Induced Cell Proliferation in Primary Cultured Hepatocytes

The mechanisms by which erythropoietin (EPO) promotes hepatocyte proliferation in primary cultures of adult rat hepatocytes were studied. EPO stimulated cell proliferation in a time- and dose-dependent manner, significantly increasing the number of hepatocyte nuclei and DNA synthesis. EPO-induced hepatocyte proliferation was completely suppressed by specific inhibitors targeting Janus kinase 2 (JAK 2), phospholipase C (PLC), protein kinase C (PKC), intracellular Ca2+ mobilization, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), and mammalian target of rapamycin (mTOR). In contrast, inhibition of signal transducer and activator of transcription 3/5 (STAT 3/5) or granule secretion had no effect, indicating that EPO acts through a pathway distinct from the classical JAK2–STAT signaling pathway. Western blot analysis showed rapid phosphorylation of ERK 2, but not ERK 1, following EPO stimulation. In addition, EPO induced phosphorylation of PLC and C-rapidly accelerated fibrosarcoma (C-Raf), with PKC acting downstream of PLC and upstream of C-Raf. In contrast, intracellular Ca2+ concentration and activated Ras were transiently increased in hepatocytes after EPO stimulation, and EPO-induced activated Ras was significantly suppressed by the specific PKC inhibitor GF109203X. These results indicate that EPO engages the JAK2/PLC/PKC-Ca2+ signaling cascade, leading to the sequential activation of Ras, C-Raf, and ERK2, ultimately promoting hepatocyte proliferation in vitro.