Skip to main content

Expression profiles uncover the relationship between erythropoietin and cell proliferation in rat hepatocytes after a partial hepatectomy

Abstract

Erythropoietin (EPO) has a beneficial effect on hepatic cell proliferation during liver regeneration. However, the underlying mechanism has not yet been elucidated. To uncover the proliferation response of EPO in rat liver regeneration after partial hepatectomy (PH) at the cellular level, hepatocytes (HCs) were isolated using Percoll density gradient centrifugation. The genes of the EPO-mediated signaling pathway and the target genes of the transcription factor (TF) in the pathway were identified in a pathway and TF database search. Their expression profiles were then detected using Rat Genome 230 2.0 Microarray. The results indicated that the EPO-mediated signaling pathway is involved in 19 paths and that 124 genes participate, of which 32 showed significant changes and could be identified as liver regeneration-related genes. In addition, 443 targets regulated by the TFs of the pathway and 60 genes associated with cell proliferation were contained in the array. Subsequently, the synergetic effect of these genes in liver regeneration was analyzed using the E(t) mathematical model based on their expression profiles. The results demonstrated that the E(t) values of paths 3, 8, 12 and 14–17 were significantly strengthened in the progressing phase of liver regeneration through the RAS/MEK/ERK or PI3K/AκT pathways. The synergetic effect of the target genes, in parallel with target-related cell proliferation, was also enhanced 12–72 h after PH, suggesting a potential positive effect of EPO on HC proliferation during rat liver regeneration. These data imply that the EPO receptor may allow EPO to promote HC proliferation through paths 3, 8, 12 and 14–17, mediating the RAS/MEK/ERK and PI3K/AκT pathways in rat liver regeneration after PH.

Abbreviations

Afp:

alpha-fetoprotein

ALB:

albumin

AκT:

v-Akt murine thymoma viral oncogene

Ctgf:

connective tissue growth factor

EPO:

erythropoietin

ERK:

extracellular regulated protein kinases

Fos:

FBJ osteosarcoma oncogene

G6P:

glucose-6-phosphatase

HCs:

hepatocytes

Hgf:

hepatocyte growth factor

Hif-2:

hypoxia-inducible factor-2

Il1rn:

interleukin 1 receptor antagonist

Jun:

jun protooncogen

MAPK:

mitogen-activated protein kinase

Med1:

mediator complex subunit 1

PH:

partial hepatectomy

PI3K:

phosphoinositide 3-kinase

Sirpa:

signal-regulatory protein alpha

Sox15:

SRY-box 15

SRY:

sex-determining region Y

TF:

transcription factor

TNF:

tumor necrosis factor

Vhlh:

von Hippel-Lindau syndrome homolog

References

  1. Taub, R. Liver regeneration: from myth to mechanism. Nat. Rev. Mol. Cell. Biol. 5 (2004) 836–847.

    PubMed  Article  CAS  Google Scholar 

  2. Michalopoulos, G.K. Liver regenetation. J. Cell Physiol. 213 (2007) 286–300.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  3. Staudinger, J.L. and LIchti, K. Cell signaling and nuclear receptors: new opportunities for molecular pharmaceuticals in liver disease. Mol. Pharm. 5 (2008) 17–34.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  4. Sun, Y., Deng, X., Li, W., Yan, Y., Wei, H., Jiang, Y. and He, F. Liver proteome analysis of adaptive response in rat immediately after partial hepatectomy. Proteomics 7 (2007) 4398–4407.

    PubMed  Article  CAS  Google Scholar 

  5. Jarnagin, W.R., Gonen, M., Fong, Y., DeMatteo, R.P., Ben-Porat, L., Little, S., Corvera, C., Weber, S. and Blumgart, L.H. Improvement in perioperative outcome after hepatic resection: analysis of 1803 consecutive cases over the past decade. Ann. Surg. 236 (2002) 397–406.

    PubMed  Article  PubMed Central  Google Scholar 

  6. Belghiti, J., Hiramatsu, K., Benoist, S., Massault, P., Sauvanet, A. and Farges, O. Seven hundred forty-seven hepatectomies in the 1990s: an update to evaluate the actual risk of liver resection. J. Am. Coll. Surg. 191 (2000) 38–46.

    PubMed  Article  CAS  Google Scholar 

  7. Garcea, G. and Maddern, G.J. Liver failure after major hepatic resection. J. Hepatobiliary Pancreat. Surg. 16 (2009) 145–155.

    PubMed  Article  Google Scholar 

  8. Coleman, T. and Brines, M. Science review: recombinant human erythropoietin in critical illness: a role beyond anemia? Crit. Care 8 (2004) 337–341.

    PubMed  Article  PubMed Central  Google Scholar 

  9. Farrell, F. and Lee, A. The erythropoietin receptor and its expression in tumor cells and other tissues. Oncologist S5 (2004) 18–30.

    Article  Google Scholar 

  10. Drüeke, T.B., Locatelli, F., Clyne, N., Eckardt, K.U., Macdougall, I.C., Tsakiris, D., Burger, H.U. and Scherhag, A. Normalization of hemoglobin level in patients with chronic kidney disease and anemia. N. Engl. J. Med. 355 (2006) 2071–2084.

    PubMed  Article  Google Scholar 

  11. Siebert, N., Xu, W., Grambow, E., Zechner, D. and Vollmar, B. Erythropoietin improves skin wound healing and activates the TGF-β signaling pathway. Lab. Invest. 91 (2011) 1753–1765.

    PubMed  Article  CAS  Google Scholar 

  12. Liu, N., Tian, J., Wang, W., Cheng, J., Hu, D. and Zhang, J. Effect and mechanism of erythropoietin on mesenchymal stem cell proliferation in vitro under the acute kidney injury microenvironment. Exp. Biol. Med. 236 (2011) 1093–1099.

    Article  CAS  Google Scholar 

  13. Sakanaka, M., Wen, T.C., Matsuda, S., Masuda, S., Morishita, E., Nagao, M. and Sasaki, R. In vivo evidence that erythropoetin protects neurons from ischemic damage. Proc. Natl. Acad. Sci. USA 95 (1998) 4635–4640.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  14. Xi, J.Q., Meng, X.K., Zhang, J.J. and Zhang, M. Erythropoletin promotes hepatic regeneration after partial hepatectomy in rats. Chin. J. Clinicians 5 (2011) 967–970.

    Google Scholar 

  15. Schmeding, M., Neumann, U.P., Boas-Knoop, S., Spinelli, A. and Neuhaus, P. Erythropoietin reduces ischemia-reperfusion injury in the rat liver. Eur. Surg. Res. 39 (2007) 189–197.

    PubMed  Article  CAS  Google Scholar 

  16. Schmeding, M., Hunold, G., Ariyakhagorn, V., Rademacher, S., Boas-Knoop, S., Lippert, S., Neuhaus, P. and Neumann, U.P. Erythropoietin reduces ischemia-reperfusion injury after liver transplantation in rats. Transpl. Int. 22 (2009) 738–746.

    PubMed  Article  CAS  Google Scholar 

  17. Bockhorn, M., Fingas, C.D., Rauen, U., Canbay, A., Sotiropoulos, G.C., Frey, U., Sheu, S.Y., Wohlschläger, J., Broelsch, C.E. and Schlaak, J.F. Erythropoietin treatment improves liver regeneration and survival in rat models of extended liver resection and living donor liver transplantation. Transplantation 86 (2008) 1578–1585.

    PubMed  Article  CAS  Google Scholar 

  18. Bader, A., Pavlica, S., Deiwick, A., Lotkova, H., Kucera, O., Darsow, K., Bartel, S., Schulze, M., Lange, H.A. and Cervinkova, Z. Proteomic analysis to display the effect of low doses of erythropoietin on rat liver regeneration. Life Sci. 89 (2011) 827–833.

    PubMed  Article  CAS  Google Scholar 

  19. Greif, F., Ben-Ari, Z., Taya, R., Pappo, O., Kurtzwald, E., Cheporko, Y., Ravid, A. and Hochhauser, E. Dual effect of erythropoietin on liver protection and regeneration after subtotal hepatectomy in rats. Liver Transpl. 16 (2010) 631–638.

    PubMed  Google Scholar 

  20. Vassiliou, I., Lolis, E., Nastos, C., Tympa, A., Theodosopoulos, T., Dafnios, N., Fragulidis, G., Frangou, M., Kondi-Pafiti, A. and Smyrniotis, V. The combined effect of erythropoietin and granulocyte macrophage colony stimulating factor on liver regeneration after major hepatectomy in rats. World J. Surg. Oncol. 8 (2010) 57.

    PubMed  Article  PubMed Central  Google Scholar 

  21. Naughton, B.A., Kaplan, S.M., Roy, M., Burdowski, A.J., Gordon, A.S. and Piliero, S.J. Hepatic regeneration and erythropoietin production in the rat. Science 196 (1977) 301–302.

    PubMed  Article  CAS  Google Scholar 

  22. Klemm, K., Eipel, C., Cantré, D., Abshagen, K., Menger, M.D. and Vollmar, B. Multiple doses of erythropoietin impair liver regeneration by increasing TNF-alpha, the Bax to Bcl-xL ratio and apoptotic cell death. PLoS One 3 (2008) e3924.

    PubMed  Article  PubMed Central  Google Scholar 

  23. Higgins, G.M. and Anderson, R.M. Experimental pathology of the liver: restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 12 (1931) 186–202.

    Google Scholar 

  24. Xu, C., Yang, Y., Yang, J., Chen, X. and Wang, G. Analysis of the role of the integrin signaling pathway in hepatocytes during rat liver regeneration. Cell. Mol. Biol. Lett. 17 (2012) 274–288.

    PubMed  Article  CAS  Google Scholar 

  25. Xu, C.S., Chang, C.F., Yuan, J.Y., Li, W.Q., Han, H.P., Yang, K.J., Zhao, L.F., Li, Y.C., Zhang, H.Y., Rahman, S. and Zhang, J.B. Expressed genes in regenerating rat liver after partial hepatectomy. World J. Gastroenterol. 11 (2005) 2932–2940.

    PubMed  CAS  Google Scholar 

  26. Knepp, J.H., Geahr, M.A., Forman, M.S. and Valsamakis, A. Comparison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA. J. Clin. Microbiol. 41 (2003) 3532–3536.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  27. Mulrane, L., Rexhepaj, E., Smart, V., Callanan, J.J., Orhan, D., Eldem, T., Mally, A., Schroeder, S., Meyer, K., Wendt, M., O’Shea, D. and Gallagher, W.M. Creation of a digital slide and tissue microarray resource from a multiinstitutional predictive toxicology study in the rat: an initial report from the PredTox group. Exp. Toxicol. Pathol. 60 (2008) 235–245.

    PubMed  Article  Google Scholar 

  28. Nikitin, A., Egorov, S., Daraselia, N. and Mazo, I. Pathway studio-the analysis and navigation of molecular networks. Bioinformatics 19 (2003) 2155–2157.

    PubMed  Article  CAS  Google Scholar 

  29. Wang, G.P. and Xu, C.S. Reference gene selection for real-time RT-PCR in eight kinds of rat regenerating hepatic cells. Mol. Biotechnol. 46 (2010) 49–57.

    PubMed  Article  Google Scholar 

  30. Chalmers, J.J., Zborowski, M., Sun, L. and Moore, L. Flow through, immunomagnetic cell separation. Biotechnol. Prog. 14 (1998) 141–148.

    PubMed  Article  CAS  Google Scholar 

  31. Doniger, S.W., Salomonis, N., Dahlquist, K.D., Vranizan, K., Lawlor, S.C. and Conklin, B.R. MAPPFinder: using Gene Ontology and GenMAPP to create a global gene-expression profile from microarray data. Genome Biol. 4 (2003) R7.

    PubMed  Article  PubMed Central  Google Scholar 

  32. Ogata, H., Goto, S., Sato, K., Fujibuchi, W., Bono, H. and Kanehisa, M. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 27 (1999) 29–34.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  33. Zhang, J., Ma, C., Liu, Y., Yang, G., Jiang, Y., Xu, C. Interleukin 18 accelerates the hepatic cell proliferation in rat liver regeneration after partial hepatectomy. Gene 537 (2014) 230–237.

    PubMed  Article  CAS  Google Scholar 

  34. Xu, C.S., Jiang, Y., Zhang, L.X., Chang, C.F., Wang, G.P., Shi, R.J. and Yang, Y.J. The role of Kupffer cells in rat liver regeneration revealed by cell-specific microarray analysis. J. Cell Biochem. 113 (2012) 229–237.

    PubMed  Article  CAS  Google Scholar 

  35. Xu, C., Chen, X., Chang, C., Wang, G., Wang, W., Zhang, L., Zhu, Q. and Wang, L. Characterization of transcriptional profiling of Kupffer cells during liver regeneration in rats. Cell Biol. Int. 36 (2012) 721–732.

    PubMed  Article  CAS  Google Scholar 

  36. Jiang, Y., Zhang, L.X., Chang, C.F., Wang, G.P., Shi, R.J., Yang, Y.J. and Xu, C.S. The number of the genes in a functional category matters during rat liver regeneration after partial hepatectomy. J. Cell Biochem. 112 (2011) 3194–3205.

    PubMed  Article  CAS  Google Scholar 

  37. Xu, C.S., Chen, X.G., Chang, C.F., Wang, G.P., Wang, W.B., Zhang, L.X., Zhu, Q.S. and Wang, L. Analysis of time-course gene expression profiles of sinusoidal endothelial cells during liver regeneration in rats. Mol. Cell Biochem. 350 (2011) 215–227.

    PubMed  Article  CAS  Google Scholar 

  38. Xu, C., Chen, X., Chang, C., Wang, G., Wang, W., Zhang, L., Zhu, Q. and Wang, L. Genome-wide analysis of gene expression in dendritic cells from rat regenerating liver after partial hepatectomy. Cell Biochem. Funct. 29 (2011) 255–264.

    PubMed  Article  CAS  Google Scholar 

  39. Xu, C., Chen, X., Chang, C., Wang, G., Wang, W., Zhang, L., Zhu, Q., Wang, L. and Zhang, F. Transcriptome analysis of hepatocytes after partial hepatectomy in rats. Dev. Genes Evol. 220 (2010) 263–274.

    PubMed  Article  CAS  Google Scholar 

  40. Christ, B. and Pelz, S. (2013) Implication of hepatic stem cells in functional liver repopulation. Cytometry A 83 (1999) 90–102.

    Google Scholar 

  41. Chen, X., Xu, C., Zhang, F. and Ma, J. Microarray approach reveals the relevance of interferon signaling pathways with rat liver restoration post 2/3 hepatectomy at cellular level. J. Interferon Cytokine Res. 30 (2010) 525–539.

    PubMed  Article  CAS  Google Scholar 

  42. Ogiso, T., Nagaki, M., Takai, S., Tsukada, Y., Mukai, T., Kimura, K. and Moriwaki, H. Granulocyte colony-stimulating factor impairs liver regeneration in mice through the up-regulation of interleukin-1beta. J. Hepatol. 47 (2007) 816–825.

    PubMed  Article  CAS  Google Scholar 

  43. Koury, S.T., Bondurant, M.C., Koury, M.J., Semenza, G.L. Localization of cells producing erythropoietin in murine liver by in situ hybridization. Blood 77 (1991) 2497–2503.

    PubMed  CAS  Google Scholar 

  44. Rankin, E.B., Biju, M.P., Liu, Q., Unger, T.L., Rha, J., Johnson, R.S., Simon, M.C., Keith, B. and Haase, V.H. Hypoxia-inducible factor-2 (HIF-2) regulates hepatic erythropoietin in vivo. J. Clin. Invest. 117 (2007) 1068–1077.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  45. Rychtrmoc, D., Hubálková, L., Víšková, A., Libra, A., Bunček, M. and Červinková, Z. Transcriptome temporal and functional analysis of liver regeneration termination. Physiol. Res. 61 (2012) S77–92.

    PubMed  CAS  Google Scholar 

  46. Dransfeld, O., Gehrmann, T., Köhrer, K., Kircheis, G., Holneicher, C., Häussinger, D. and Wettstein, M. Oligonucleotide microarray analysis of differential transporter regulation in the regenerating rat liver. Liver Int. 25 (2005) 1243–1258.

    PubMed  Article  CAS  Google Scholar 

  47. Fukuhara, Y., Hirasawa, A., Li, X.K., Kawasaki, M., Fujino, M., Funeshima, N., Katsuma, S., Shiojima, S., Yamada, M., Okuyama, T., Suzuki, S. and Tsujimoto, G. Gene expression profile in the regenerating rat liver after partial hepatectomy. J. Hepatol. 38 (2003) 784–792.

    PubMed  Article  CAS  Google Scholar 

  48. Li M.H., Zhou X.C., Mei J.X., Geng X.F., Zhou Y., Zhang W.M., Xu C.S. Study on the activity of the signaling pathways regulating hepatocytes from G0 phase into G1 phase during rat liver regeneration. Cell. Mol. Biol. Lett. DOI: 10.2478/s11658-014-0188-2.

  49. Kesselring, F., Spicher, K. and Porzig, H. Changes in G protein pattern and in G protein-dependent signaling during erythropoietin- and dimethylsulfoxide-induced differentiation of murine erythroleukemia cells. Blood 84 (1994) 4088–4098.

    PubMed  CAS  Google Scholar 

  50. Guillard, C., Chrétien, S., Pelus, A.S., Porteu, F., Muller, O., Mayeux, P. and Duprez, V. Activation of the mitogen-activated protein kinases Erk1/2 by erythropoietin receptor via a G(i)protein beta gamma-subunit-initiated pathway. J. Biol. Chem. 278 (2003) 11050–11056.

    PubMed  Article  CAS  Google Scholar 

  51. Zhao, W., Kitidis, C., Fleming, M.D., Lodish, H.F. and Ghaffari, S. Erythropoietin stimulates phosphorylation and activation of GATA-1 via the PI3-kinase/AKT signaling pathway. Blood 107 (2006) 907–915.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Cunshuan Xu.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, J., Yang, Y., He, T. et al. Expression profiles uncover the relationship between erythropoietin and cell proliferation in rat hepatocytes after a partial hepatectomy. Cell Mol Biol Lett 19, 331–346 (2014). https://doi.org/10.2478/s11658-014-0198-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-014-0198-0

Keywords

  • Rat liver regeneration
  • Erythropoietin
  • Rat Genome 230 2.0 Microarray
  • Gene expression profiles
  • Gene synergetic effect