What question did this study set out to answer?

This research aims to explore how erythropoietin (EPO) stimulates the proliferation of hepatocytes in culture.

February 14, 2026Biological and Pharmaceutical Bulletin3 citationsOpen Access

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

HMHajime MotekiJosai University MOMasahiko OgiharaJosai University MKMitsutoshi KimuraJosai University

Key Points

This research aims to explore how erythropoietin (EPO) stimulates the proliferation of hepatocytes in culture.
Primary cultures of adult rat hepatocytes were used.
EPO stimulation was varied based on time and dose.
Specific inhibitors targeting various pathways were applied to identify mechanisms.
Western blot analysis assessed protein phosphorylation following EPO treatment.
EPO significantly increased hepatocyte nuclei and DNA synthesis in a dose- and time-dependent manner.
Hepatocyte proliferation was inhibited by targeting JAK2, PLC, PKC, Ca<sup>2+</sup>, MEK, and mTOR.
Phosphorylation of ERK2 occurred rapidly after EPO stimulation, while ERK1 phosphorylation was not observed.
EPO-induced activation of Ras was suppressed by a specific PKC inhibitor.

Abstract

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.

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