2026 Volume 49 Issue 2 Pages 281-290
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.
The liver has an extraordinary capacity for regeneration, efficiently restoring lost tissue following injury or surgical resection.1) Hepatocyte proliferation is minimal under typical physiological conditions; however, in the event of substantial hepatic tissue loss, such as after partial hepatectomy, the remaining liver tissue undergoes rapid proliferation, re-establishing its original mass within approximately 2 weeks.1,2) This regenerative process is facilitated by various growth factors, including epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), which together stimulate hepatocyte proliferation.2,3) The mitogen-activated protein (MAP) kinase pathway, activated by receptor tyrosine kinases (RTKs), is a critical signaling pathway implicated in liver regeneration. This signaling cascade culminates in the activation of extracellular signal-regulated kinase (ERK), a serine/threonine kinase that promotes cell proliferation and differentiation.4)
Erythropoietin (EPO) functions as a hematopoietic hormone, promoting both the maturation and expansion of precursor cells in the erythroid lineage, thereby regulating the levels of red blood cells in the peripheral blood.5) Its molecular structure comprises a 193-amino acid polypeptide, with approximately 80% homology between human and mouse EPO. In human EPO, 3 N-linked glycans and a single O-linked glycan are present, and disulfide bridges are formed between Cys7–Cys161 and Cys29–Cys33, respectively.5,6) EPO is synthesized primarily in the liver during fetal development and in the kidneys in adults, where oxygen sensors regulate its expression in response to hypoxic conditions.7) The EPO receptor (EPOR) is composed of an extracellular region of approximately 230 amino acids, a single transmembrane helix, and a cytoplasmic portion of about 230 amino acids that lacks intrinsic enzymatic function.8) In erythroid progenitors, EPO binds to EPOR homodimers, inducing the activation of Janus kinase 2 (JAK2).9) JAK2 activation results in the phosphorylation of tyrosine residues in the EPOR cytoplasmic domain, which then act as anchoring sites for SH2 domain-containing downstream signaling molecules.10) This activation triggers pathways such as signal transducer and activator of transcription 5 (STAT5), MAPK, and phosphatidylinositol-3 kinase (PI3K)/AKT, thereby promoting hematopoiesis.11,12)
EPOR is expressed not only in erythroid progenitor cells but also in tissues such as the liver, kidney, and brain, indicating its involvement in biological activities beyond erythropoiesis.13–15) EPO has been demonstrated to regulate lipid and carbohydrate metabolism, as well as cell differentiation and proliferation in the liver.16) Notably, studies using partial hepatectomy models have demonstrated that EPO facilitates liver regeneration.17,18) Gene expression profiling suggests the involvement of the JAK/STAT and MAPK pathways in EPO-induced hepatocyte proliferation in vitro.19) However, the specific intracellular signaling pathways remain incompletely understood, necessitating further investigation. In a previous study, we showed that many factors (i.e., physiological and non-physiological substances) promote the proliferation of hepatocytes in primary cultures.20) Recent research examined the mechanism of hepatocyte proliferation induced by the growth hormone receptor (GHR), which, like EPOR, is part of the class I cytokine receptor superfamily.21) The findings indicated that GHR stimulation enhances hepatocyte proliferation by inducing autocrine secretion of IGF-I through the JAK2/phospholipase C (PLC)/Ca2+ pathway.22,23) Whether EPO promotes hepatocyte proliferation through a pathway analogous to that of GH or via a mechanism unique to EPO remains uncertain.
This work aimed to dissect the signaling pathways contributing to hepatocyte proliferation triggered by EPO in primary cultures derived from adult rats. To this end, the mechanisms by which EPO promotes hepatocyte proliferation were investigated using specific inhibitors of signal transduction. Cell proliferation was assessed by measuring the hepatocyte nuclear counts and S-phase cell cycle progression. Furthermore, the phosphorylation of MAPK (ERK1/2) and PLC, both involved in hepatocyte proliferation, was analyzed to clarify the specific mechanisms underlying EPO-induced hepatocyte growth.
Male Wistar rats (7–10 weeks old) were purchased from Sankyo Labo Service Corporation (Tokyo, Japan). They were acclimated for 5 d before the start of the experiments, with unrestricted access to food and water throughout this period. All animal handling procedures in this study complied with the Guidelines for the Care and Use of Laboratory Animals of Josai University (No. JU 24027).
Isolation and Primary Culture of HepatocytesHepatocytes were isolated from rats via a modified 2-step collagenase perfusion procedure performed in situ.24) After induction of anesthesia with sodium pentobarbital (45 mg/kg, intraperitoneally [i.p.]), the portal vein was cannulated, and the liver was first perfused with Ca2+-free Hanks’ buffer at 37°C for 10 min, followed by a digestion step using 0.058 U/L collagenase Type II (Worthington Biochemical Corp., Freehold, NJ, U.S.A.) supplemented with 0.75 mg/mL CaCl2 for an additional 11 min. Hepatocyte viability was assessed using the trypan blue exclusion assay, and only preparations with >96% viability were cultured.
Cells were plated at a density of 3.3 × 104 cells/cm2 in Williams’ medium E containing 0.1 nM dexamethasone and 5% newborn bovine serum, and cultured for 3 h to permit attachment. Subsequently, the culture medium was replaced with serum-free medium supplemented with the appropriate test compounds. Hepatocytes were exposed to EPO (Sigma-Aldrich Co., LLC., St. Louis, MO, U.S.A.) alone or in combination with pharmacological inhibitors and activators targeting signaling pathways associated with cell proliferation. The signaling modulators used included TG101209 (JAK2 inhibitor),25) SH-4-54 (STAT3/STAT5 inhibitor),26) U-73122 (PLC inhibitor),27) GF109203X (protein kinase C [PKC] inhibitor),28) BAPTA/AM (intracellular Ca2+ chelator),29) PD98059 (MAPK/ERK kinase [MEK] inhibitor),30) rapamycin (mammalian target of rapamycin [mTOR] inhibitor),31) and somatostatin (granule secretion inhibitor).32) All reagents were obtained from Sigma-Aldrich. The antibodies used were monoclonal antibodies against IGF-I (anti-IGF-I mAb; Santa Cruz Biotechnology, Dallas, TX, U.S.A.). The doses of all signal transduction inhibitors and neutralizing antibodies used in this study were determined based on preliminary dose–response experiments, as well as reference literature and the manufacturer’s specification sheets. These doses were also consistent with those used in previous studies to examine the effects of growth factors and related drugs.
Hepatocyte Proliferation AssayHepatocyte proliferation in response to EPO was assessed by quantifying both the number of hepatocyte nuclei and the number of viable cells. Cell counts were performed using light microscopy.
Hepatocyte nuclear counts were obtained using a modified procedure based on the method reported by Nakamura et al.33) Briefly, hepatocyte nuclei were isolated by treating cells with a solution containing 0.1% Triton X-100 and 0.1 M citric acid. After isolation, the nuclei were stained with 0.3% trypan blue and quantified using a hemocytometer.
In addition, the total number of hepatocytes per 0.01 cm2 (×102 cells/cm2) was determined from phase-contrast microscopic images. Cell numbers were counted in 3 randomly selected fields of view, and the mean of these values was used as a single data point for each experimental condition.34,35)
Detection of Cell Cycle Phases (S Phase and G0/G1 Phase)DNA synthesis and cell cycle progression were evaluated by determining the distribution of hepatocytes in the S phase and G0/G1 phase using a Muse cell analyzer (Merck Millipore, Darmstadt, Germany).36) Hepatocyte nuclei obtained as described above were treated with 5% propidium iodide and incubated in darkness for 15 min. The Muse cell analyzer, a flow cytometry-based instrument, was then used to analyze the stained nuclei and quantify the percentage of cells in each phase of the cell cycle. This system allows for the precise determination of hepatocyte distribution within the S phase and G0/G1 phase based on DNA content indicated by propidium iodide fluorescence intensity.
Measurement of Phosphorylated PLCγ, C-Rapidly Accelerated Fibrosarcoma (C-Raf), and ERK1/2Phosphorylation levels of PLCγ (p155), C-Raf (p74), and ERK1/2 (p44/42) were assessed by Western blot analysis, as previously described.23,37) Hepatocytes were lysed in lysis buffer, and the protein extracts were mixed with Laemmli sample buffer, followed by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed using antibodies specific to both the phosphorylated and total forms of each target protein. The antibody against phosphorylated ERK1/2 was used at a dilution of 1 : 2000, whereas all other primary antibodies were diluted 1 : 1000. Following incubation with primary antibodies, membranes were exposed to horseradish peroxidase (HRP)-linked secondary antibodies. Protein signals were detected by enhanced chemiluminescence (ECL), and band intensities were quantified using imaging software (Bio-Rad, Hercules, CA, U.S.A.). Phosphorylation levels were normalized to the corresponding total protein levels and expressed as phosphorylation ratios. All reagents, including antibodies, lysis buffer, and ECL kit, were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). PVDF membranes, Laemmli sample buffer, and molecular weight markers were obtained from Bio-Rad Laboratories.
Measurement of Ras ActivityRas activity, represented by the GTP-bound active form, was quantified using a 96-well Ras Activation ELISA Kit (Cell Biolabs, Inc., San Diego, CA, U.S.A.) following the manufacturer’s instructions. Briefly, cultured hepatocytes were lysed by adding 200 µL of lysis buffer to each well, followed by incubation on ice for 10 min. The lysates were then centrifuged at 14000 × g for 10 min at 4°C, and 50 µL of the resulting supernatant was used for the ELISA assay. Total protein concentrations in each sample were determined and normalized using the SDS-Lowry method.38)
Statistical AnalysisAll data are presented as mean ± standard error of the mean (S.E.M.) values from 3 independent experiments (biologically distinct samples [experimental units] to which the treatment was applied). Statistical comparisons between control and treatment groups were performed using Dunnett’s multiple comparison test. In addition, specific pairwise comparisons (control vs. EPO and EPO vs. GF109203X-treated groups) were analyzed using Student’s t-test. A p-value of less than 0.05 was considered statistically significant.
The proliferative effects of EPO on hepatocytes were investigated using cultured cells. Initially, isolated hepatocytes were allowed to adhere to culture plates, followed by incubation in serum-free medium containing EPO, either alone or in conjunction with TG101209 or PD98059. Figure 1 shows the phase-contrast microscopic images of hepatocytes after 5 h of culture. The number of hepatocytes was significantly higher in the EPO-treated cells than in the control group (medium alone) (Figs. 1A and 1B). As shown in Fig. 1E, the total number of hepatocytes per field of view (0.01 cm2 [×102 cells/cm2]) was 318 ± 14 in the control cultures, whereas treatment with EPO (10 ng/mL) markedly increased this number to 421 ± 21, representing a 1.32-fold increase. Moreover, EPO-induced hepatocyte proliferation was significantly reduced to control levels by the addition of TG101209 and PD98059 (Figs. 1C and 1D).
Hepatocytes were seeded at a density of 3.3 × 104 cells/cm2 and allowed to attach for 3 h, followed by incubation in serum-free medium with or without EPO for 5 h, as detailed in Materials and Methods. (A) Control (medium alone); (B) EPO alone (10 ng/mL); (C) EPO + TG101209 (10–6 M); (D) EPO + PD98059 (10–6 M). Scale bar = 200 µm. (E) Quantitative analysis of hepatocyte numbers per field of view (0.01 cm2). Data are presented as mean ± S.E.M. values (n = 3). *p = 0.0120 vs. control (Student’s t-test); (a) p = 0.0134, (b) p = 0.0148 vs. EPO alone (Dunnett’s multiple comparison test). EPO: erythropoietin.
To further elucidate the proliferative effects of EPO, hepatocyte nuclei were quantified under time- and dose-dependent conditions (Figs. 2A and 2B). As shown in Fig. 2A, the dose–response relationship of EPO was assessed by measuring the number of hepatocyte nuclei after 5 h of culture in serum-free conditions, with EPO concentrations ranging from 0.01 to 100 ng/mL. EPO elicited a dose-dependent increase in hepatocyte proliferation, reaching a plateau at 10 ng/mL with an EC50 value of 1.05 ng/mL (Fig. 2A). Subsequently, the final EPO concentration was fixed at 10 ng/mL, and cultures were maintained for 0–21 h. As shown in Fig. 2B, EPO-induced proliferation increased over time, becoming significant at approximately 4 h, peaking at 5 h, and persisting for up to 21 h (Fig. 2B).
After seeding hepatocytes at 3.3 × 104 cells/cm2 and allowing attachment, the medium was changed to serum-free medium containing EPO, as outlined in Materials and Methods. (A) Dose-dependent proliferative effects of EPO (0.01–100 ng/mL) after 5 h of incubation. (B) Time course of hepatocyte proliferation in response to EPO (closed circle; 10 ng/mL). The arrow indicates the time point at which drugs were added. Data are expressed as mean ± S.E.M. values (n = 3). Significant differences vs. corresponding controls (Dunnett’s multiple comparison test) are observed as follows (p-values): aa (0.0224), ba (0.0071), ca (0.0230), ab (0.0384), bb (0.0081), cb (0.0070), db (0.0071), and eb (0.0085).
To assess the capacity of EPO to promote DNA synthesis in cultured hepatocytes, a detailed analysis of cell cycle progression, focusing on the S-phase population following EPO exposure, was conducted. As shown in Fig. 3A, the proportion of hepatocytes in S phase was significantly elevated between 3 and 5 h after stimulation with EPO (10 ng/mL) compared with that in the control group. In addition, as shown in Fig. 3B, the percentage of hepatocyte nuclei in the G0/G1 phase exhibited a decreasing trend in the EPO-treated group relative to controls at 3 and 4 h, suggesting a shift of cells into the DNA synthesis phase in response to EPO (Fig. 3B).
Hepatocytes were cultured and treated with EPO (closed circle; 10 ng/mL) as described in Materials and Methods. (A) Time course of the percentage of cells in the S phase following treatment with each compound. (B) Time course of the percentage of cells in the G0/G1 phase under the same conditions. The arrow indicates the time point at which drugs were added. Data are expressed as mean ± S.E.M. values (n = 3). Significant differences vs. corresponding controls (Dunnett’s multiple comparison test) are observed, as follows (p-values): a (0.0194), b (0.0458), and c (0.0478).
To clarify which intracellular signaling cascades participate in EPO-driven hepatocyte proliferation and DNA synthesis, nuclear counts and the advancement of cells into S phase were assessed in cultures exposed to EPO together with selective inhibitors of proliferation-associated signaling pathways. As shown in Figs. 4A and 4B, the proliferative effects of EPO were completely suppressed to control levels by TG101209, U-73122, GF109203X, BAPTA/AM, PD98059, and rapamycin. In contrast, SH-4-54, somatostatin, and anti-IGF-I mAb demonstrated no inhibitory effects on EPO-induced hepatocyte proliferation (Figs. 4A, 4B).
Hepatocytes were seeded, allowed to attach, and then cultured in serum-free medium as described in Materials and Methods. Cells were treated with EPO (10 ng/mL) alone or in combination with various signal transduction inhibitors for 5 h. (A) Percentage of cells in the S phase (indicative of DNA synthesis). (B) Number of nuclei (reflecting cell proliferation). The inhibitors used were as follows: TG101209 (10−6 M), SH-4-54 (10−6 M), U-73122 (10−6 M), GF109203X (10−7 M), BAPTA/AM (10−7 M), PD98059 (10−6 M), rapamycin (10 ng/mL), somatostatin (10−6 M), and anti-insulin-like growth factor I (IGF-I) mAb (100 ng/mL). Data are presented as mean ± S.E.M. values (n = 3). *p = 0.0194, #p = 0.0071, vs. corresponding controls (Student’s t-test). Significant differences vs. EPO alone (Dunnett’s multiple comparison test) are observed as follows (p-values): aa (0.0145), ba (0.0147), ca (0.0151), da (0.0111), ea (0.0112), fa (0.0104), ab (0.0012), bb (0.0006), cb (0.0025), db (0.0019), eb (0.0004), and fb (0.0008). IGF-I: insulin-like growth factor I.
Western blotting was performed to assess ERK1/2 phosphorylation in hepatocytes following EPO stimulation. As shown in Fig. 5, the phosphorylation of the 42-kDa form of ERK2 showed a rapid increase following EPO treatment, achieving peak levels within 5–10 min. At this peak, the phosphorylated ERK2 levels were approximately 2.1 times greater than that observed in the untreated controls. Subsequently, phosphorylation decreased gradually and returned to baseline levels after 30 min of incubation (Fig. 5). Conversely, EPO treatment did not induce any significant increase in the phosphorylation of the 44-kDa form of ERK1 throughout the experimental period (Fig. 5).
After seeding hepatocytes at 3.3 × 104 cells/cm2 and allowing attachment, the medium was changed to serum-free medium containing EPO, as outlined in Materials and Methods. Cells were treated with EPO (10 ng/mL), and the phosphorylation levels of p44/42 ERK1/2 were analyzed by Western blotting. For each graph, representative Western blot bands corresponding to the respective phosphorylation events are shown above the graph, and the quantitative data for phosphorylation levels (expressed as percentages of total protein) are shown below. The arrow indicates the time point at which EPO (10 ng/mL) was added. Data are presented as mean ± S.E.M. values (n = 3). (M) Molecular weight marker. Significant differences vs. corresponding controls (Dunnett’s multiple comparison test) are observed, as follows (p-values): a (0.0227), b (0.0003), and c (0.0006).
To determine whether EPO enhances the phosphorylation of proteins other than ERK2, the phosphorylation levels of PLC and C-Raf were assessed. As shown in Figs. 6A and 6B, EPO led to an increase in the phosphorylation of both PLC and C-Raf. How specific signaling factor inhibitors affect the EPO-induced phosphorylation of PLC, C-Raf, and ERK2 was further investigated. EPO-induced phosphorylation of PLC was inhibited by TG101209 and U-73122 (Fig. 6A). In contrast, EPO-induced phosphorylation of C-Raf was inhibited not only by TG101209 and U-73122 but also by GF109203X and BAPTA/AM (Fig. 6B). Furthermore, EPO-induced phosphorylation of ERK2 was reduced to the control level by all of these inhibitors (Fig. 6C).
Hepatocytes were seeded, allowed to attach, and then cultured in serum-free medium as described in Materials and Methods. Cells were treated with EPO (10 ng/mL), with or without various growth-related signal transduction inhibitors. Phosphorylation levels of p155 PLCγ, p74 C-Raf, and p42 ERK2 were evaluated 5 min after SAC stimulation by Western blot analysis. (A) Phosphorylation of p155 PLCγ. (B) Phosphorylation of p74 C-Raf. (C) Phosphorylation of p42 ERK2. For each graph, representative Western blot bands corresponding to the respective phosphorylation events are shown above the graph, and the quantitative data for phosphorylation levels (expressed as percentages of total protein) are shown below. The inhibitors used were TG101209 (10−6 M), GF109203X (10−7 M), U-73122 (10−6 M), and PD98059 (10−6 M), and BAPTA/AM (10−7 M). Data are presented as mean ± S.E.M. values (n = 3). (M) Molecular weight marker * p = 0.0153, #p = 0.0034, ♭p = 0.0061 vs. corresponding controls (Student’s t-test). Significant differences vs. EPO alone (Dunnett’s multiple comparison test) are observed, as follows (p-values): aa (0.0142), ba (0.0118), ab (0.0019), bb (0.0021), cb (0.0028), db (0.0014), ac (0.0024), bc (0.0029), cc (0.0028), dc (0.0029), and ec (0.0039). PLCγ: phospholipase Cγ; C-Raf: C-rapidly accelerated fibrosarcoma.
To investigate the effect of EPO on intracellular Ca2+ levels in cultured hepatocytes, changes in intracellular Ca2+ concentration were monitored using the Ca2+-sensitive fluorescent probe Fluo-4 AM under fluorescence microscopy. As shown in Fig. 7, treatment with 10 ng/mL EPO resulted in a time-dependent increase in intracellular Ca2+ levels, which peaked within 5 min and subsequently decreased over the following 15 min (Figs. 7A–7E). Quantitative analysis showed that fluorescence intensity at 5 min post-EPO stimulation was approximately 3.4 times higher than the baseline level at 0 min, indicating a significant elevation in intracellular Ca2+ concentration (Fig. 7G).
Hepatocytes were seeded at a density of 3.3 × 104 cells/cm2, allowed to attach, and then incubated with 3 µM Fluo 4-AM in recording buffer for 40 min at 37°C, as described in Materials and Methods. After washing with PBS, cells were treated with EPO (10 ng/mL) and observed over a 15-min period. (A–E) Fluorescence images showing intracellular Ca2+ levels at 0, 1, 3, 5, 10, and 15 min after EPO stimulation, respectively. Scale bar = 20 µm. (F) Phase-contrast image at 0 min. Scale bar = 20 µm. (G) Quantitative analysis of fluorescence intensity per cell. Data are expressed as mean ± S.E.M. values (n = 3). Significant differences vs. control (0 min) (Dunnett’s multiple comparison test) are observed, as follows (p-values): a (0.0058), b (0.0008), c (0.0014), and d (0.0146).
Since GF109203X suppressed EPO-induced C-Raf phosphorylation (Fig. 6), we hypothesized that EPO-activated PKC interacts with C-Raf or its upstream protein Ras. As shown in Fig. 8, a transient increase in the optical density (OD) value at A450 nm, indicative of active Ras (Ras-GTP), was observed in cultured hepatocytes 5–10 min after stimulation with 10 ng/mL EPO. Conversely, a significant inhibition of EPO-induced Ras activity was observed in the group treated with both GF109203X and EPO.
Hepatocytes were seeded at a density of 3.3 × 104 cells/cm2, allowed to attach, and then incubated in fresh medium containing EPO (closed circle: 10 ng/mL), GF109203X alone (open triangle: 10−7 M), or EPO [10 ng/mL] + TG101209 [10−7 M] (closed triangle) for 0–30 min, as detailed in Materials and Methods. After treatment, Ras activation was assessed by ELISA. Data are presented as mean ± S.E.M. values (n = 3). *p = 0.0382, #p = 0.0280 vs. corresponding controls (Dunnett’s multiple comparison test). Significant differences vs. EPO alone (Student’s t-test) are observed, as follows (p-values): a (0.0195) and b (0.0148).
In this study, the proliferative efficacy of EPO was evaluated, and the underlying molecular mechanisms were examined in primary cultured hepatocytes. As presented in Figs. 1 and 2, EPO significantly enhanced hepatocyte proliferation in a time- and concentration-dependent manner. Quantitative analysis demonstrated that EPO treatment led to a marked increase in cell number relative to control cultures, with the maximal proliferative effects observed at a concentration of 10 ng/mL following 5 h of incubation (Figs. 1 and 2B). These results indicate that EPO functions as a potent mitogenic factor in hepatocytes under serum-free conditions. Dose–response analysis showed that EPO-induced proliferation followed a typical sigmoidal pattern, with an EC50 value of 1.05 ng/mL, and reached a plateau at 10 ng/mL (Fig. 2A). Notably, this effective concentration range is consistent with previously reported EPO actions in non-hematopoietic tissues, including endothelial and neuronal cells.39–42) Although the concentration used in the present study (10 ng/mL) was higher than the physiological serum levels in vivo, previous in vivo studies using partial hepatectomy models showed that administration of EPO (1000 IU/kg, i.p.) increased serum EPO levels approximately 150-fold compared with control animals.43) These studies also demonstrated enhanced hepatocyte proliferation, suggesting that relatively high EPO concentrations may be required to promote liver regeneration.
Consistent with the proliferative activity shown in Fig. 2, cell cycle analysis showed a significant increase in the proportion of hepatocytes in the S phase 3 and 5 h after EPO treatment, accompanied by a corresponding decrease in the G0/G1-phase population (Fig. 3). These results indicate that EPO promotes cell cycle progression from G0/G1 to S phase, thereby accelerating DNA synthesis in hepatocytes. Interestingly, similar rapid proliferative responses have been reported for other well-characterized growth factors and hormones, such as EGF and growth hormone (GH). For example, EGF with insulin is known to induce S-phase entry within 5 h after stimulation in primary hepatocyte cultures,33) and we have also previously demonstrated that GH-induced hepatocyte proliferation promotes DNA synthesis and reaches a peak approximately 2–3 h after administration.22) These growth factors, including EPO, appear to act via distinct receptor systems, but they share the ability to trigger early cell cycle progression.
To define the intracellular mechanisms responsible for EPO-induced hepatocyte proliferation, a series of selective inhibitors targeting key molecules implicated in proliferation-related signaling pathways was used. As shown in Fig. 4, EPO-induced increases in both cell number and S-phase entry were entirely suppressed by inhibitors of JAK2 (TG101209), PLC (U-73122), PKC (GF109203X), intracellular Ca2+ chelation (BAPTA/AM), MEK (PD98059), and mTOR (rapamycin). In contrast, inhibition of STAT3/5 (SH-4-54), granule secretion (somatostatin), and anti-IGF-I mAb did not significantly affect EPO-induced proliferation. These findings suggest that EPO stimulates hepatocyte proliferation through a signaling cascade involving JAK2, PLC, PKC, Ca2+ mobilization, MEK/ERK, and mTOR pathways, but is independent of STAT3/5 activation. These results contrast with the classical hematopoietic action of EPO, which relies predominantly on JAK2/STAT5 signaling,44) highlighting potential tissue-specific differences in the EPO receptor signaling pathway.
Moreover, Western blot analysis demonstrated that EPO rapidly increased 42-kDa ERK2 phosphorylation, peaking within 5–10 min and returning to basal levels by 30 min. In contrast, 44-kDa ERK1 phosphorylation remained unchanged (Fig. 5). These results suggest that ERK2, rather than ERK1, plays a pivotal role in EPO-induced hepatocyte proliferation. This finding is consistent with previous reports of isoform-specific functions of ERK1/2 in various cellular processes, including proliferation and regeneration.45)
In addition to the activation of ERK2, this study demonstrated that EPO stimulation triggers the phosphorylation of other key intracellular signaling molecules in hepatocytes, including PLC and C-Raf. As shown in Fig. 6, Western blot analysis showed that EPO-induced PLC phosphorylation was dependent on JAK2 activation, as it was abolished by TG101209 and direct PLC inhibition with U-73122 (Fig. 6A). Furthermore, EPO-induced C-Raf phosphorylation was suppressed not only by JAK2 and PLC inhibitors but also by the PKC inhibitor GF109203X and BAPTA/AM, suggesting that PKC and intracellular Ca2+ act downstream of PLC and upstream of C-Raf in the EPO-initiated signaling cascade (Fig. 6B). Importantly, ERK2 phosphorylation was blocked by all of these inhibitors, confirming that this signaling axis converges on ERK2 activation (Fig. 6C). These observations are consistent with previous findings, suggesting that JAK2 activation regulates the Ras-Raf-MEK-ERK signaling cascade.46)
A rapid increase in intracellular Ca2+ concentration was also observed following EPO stimulation, peaking within 5 min and subsequently decreasing (Fig. 7). This transient Ca2+ influx was likely mediated by PLC activation, leading to inositol 1,4,5-trisphosphate production and the release of Ca2+ from intracellular stores. As shown in Figs. 4 and 6, BAPTA/AM and GF109203X suppressed EPO-induced hepatocyte proliferation, as well as C-Raf and ERK2 phosphorylation, to the control levels, suggesting that EPO-induced intracellular Ca2+ binding to PKC regulates the Ras–MAPK pathway. In other words, conventional PKC (e.g., PKCα, PKCβ, PKCγ) that responds to Ca2+ stimulation may be involved in EPO-induced hepatocyte proliferation.47)
Furthermore, Fig. 8 demonstrates that EPO stimulation induced transient Ras activation in hepatocytes, peaking at 5–10 min. This activation was significantly attenuated by GF109203X, indicating that PKC activity is required for EPO-induced Ras activation. These findings suggest that EPO activates Ras via a PKC-dependent mechanism that is distinct from the classical RTK-mediated Ras activation pathway. PKCβII has been reported to facilitate cell invasion in rat intestinal epithelial cells through a Ras-dependent signaling pathway.48) In the context of the present study, these observations position PKC as a critical mediator linking PLC activation to the Ras–MAPK pathway in hepatocytes exposed to EPO treatment.
We hypothesized that EPO-induced hepatocyte proliferation is mediated by IGF-I secretion, similar to the mechanism observed with GH. However, somatostatin and anti-IGF-I mAb did not suppress EPO-induced hepatocyte proliferation. Although the intracellular signaling pathways of the GH receptor and EPOR share several common components, the differences between these pathways remain incompletely understood. The authors consider that both GH and EPO activate STAT5 phosphorylation, although the activation is generally stronger following GH stimulation.49,50) Since IGF-I secretion depends on robust STAT5 phosphorylation, EPO, which induces relatively weak STAT5 activation may be insufficient to stimulate IGF-I secretion.
The present study focused on the proliferative effect of EPO in primary cultured rat hepatocytes. Preliminary experiments also showed that EPO (1.0–10 ng/mL)-induced hepatocyte proliferation could be observed in the human hepatoma cell line HepG2 (data not shown). Although these findings suggest that the proliferative effect of EPO is not species-specific, further studies using other human liver cell lines, such as Huh7, are required to confirm whether similar signaling pathways are involved.
Knowledge of liver regeneration and related techniques has been applied in living donor liver transplantation (LDLT) for the treatment of end-stage liver disease. Based on the present findings that EPO promotes hepatocyte proliferation via the EPOR JAK2/PLC/PKC-Ca2+ signaling cascade, together with previous reports using liver regeneration models, EPO may potentially accelerate liver regeneration in LDLT donors, thereby reducing their physical burden. Moreover, members of the class I cytokine receptor superfamily, including GH and EPO, could serve as novel targets for the development of drugs that promote hepatic regeneration.
Taken together, these results provide a comprehensive view of the intracellular signaling events triggered by EPO-induced hepatocyte proliferation in hepatocytes. The present findings indicate that EPO engages the EPOR JAK2/PLC/PKC-Ca2+ signaling cascade, leading to the sequential activation of Ras, C-Raf, and ERK2, ultimately promoting hepatocyte proliferation (Fig. 9).
EPO: erythropoietin; JAK2: Janus kinase 2; PLC: phospholipase C; PIP2: phosphatidylinositol 4,5-bisphosphate; DG: diacylglycerol; IP3: inositol trisphosphate; ERK2: extracellular signal-regulated kinase; mTOR: mammalian target of rapamycin; P: phosphorylation C-Raf.
The authors declare no conflict of interest.
