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Regulatory Effects and Mechanism of Adenovirus-Mediated PTEN Gene on Hepatic Stellate Cells

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Abstract

Background

Tension homology deleted on chromosome ten (PTEN) is important in liver fibrosis.

Aims

The purpose of this study was to evaluate the PTEN gene effects and mechanism of action on hepatic stellate cells (HSCs).

Methods

The rat primary HSCs and human LX-2 cells were transfected by an adenovirus containing cDNA constructs encoding the wild-type PTEN (Ad-PTEN), the PTEN mutant G129E gene (Ad-G129E) and RNA interference targeting the PTEN sequence PTEN short hairpin RNA (PTEN shRNA), to up-regulate and down-regulate PTEN expression, respectively. The HSCs were assayed with a fluorescent microscope, real time PCR, Western blot, MTT, flow cytometry and Terminal-deoxynucleoitidyl transferase mediated nick end labeling. In addition, the CCl4 induced rat hepatic fibrosis model was also established to check the in vivo effects of the recombinant adenovirus with various levels of PTEN expression.

Results

The data have shown that the over-expressed PTEN gene led to reduced HSCs activation and viability, caspase-3 activity and cell cycle arrest in the G0/G1 and G2/M phases, as well as negative regulation of the PI3K/Akt and FAK/ERK signaling pathways in vitro. The over-expressed PTEN gene improved liver function, inhibited proliferation and promoted apoptosis of HSCs both in vitro and in vivo.

Conclusions

These data have shown that gene therapy using the recombinant adenovirus encoding wild-type PTEN inhibits proliferation and induces apoptosis of HSCs, which is a potential treatment option for hepatic fibrosis.

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Abbreviations

HSCs:

Hepatic stellate cells

ECM:

Extracellular matrix

α-SMA:

Alpha-smooth muscle actin

PTEN:

Tension homology deleted on chromosome ten

BDL:

Bile duct ligation

shRNA:

Short hairpin RNA

GFP:

Green fluorescent protein

EGFP:

Enhanced green fluorescent protein

H&E:

Hematoxylin and eosin

MT:

Masson’s trichrome

TUNEL:

Terminal-deoxynucleoitidyl transferase mediated nick end labeling

FCM:

Flow cytometry

FAK:

Focal adhesion kinase

ERK:

Extracellular signal-regulated kinase

PI3K:

Phosphoinositol-3-kinase

Akt:

Serine–threonine protein kinase B

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

References

  1. Hernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol. 2011;6:425–456.

    Article  CAS  PubMed  Google Scholar 

  2. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology. 2008;134:1655–1669.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Brenner DA, Kisseleva T, Scholten D, et al. Origin of myofibroblasts in liver fibrosis. Fibrogenesis Tissue Repair. 2012;5:S17.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Asahina K, Zhou B, Pu WT, Tsukamoto H. Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver. Hepatology. 2011;53:983–995.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Scholten D, Reichart D, Paik YH, et al. Migration of fibrocytes in fibrogenic liver injury. Am J Pathol. 2011;179:189–198.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wasmuth HE, Weiskirchen R. Pathogenesis of liver fibrosis: modulation of stellate cells by chemokines. Z Gastroenterol. 2010;48:38–45.

    Article  CAS  PubMed  Google Scholar 

  7. Kisseleva T, Cong M, Paik Y, et al. Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc Natl Acad Sci USA. 2012;109:9448–9453.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu X, Xu J, Brenner DA, Kisseleva T. Reversibility of liver fibrosis and inactivation of fibrogenic myofibroblasts. Curr Pathobiol Rep. 2013;1:209–214.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Kim HA, Kim KJ, Seo KH, Lee HK, Im SY. PTEN/MAPK pathways play a key role in platelet-activating factor-induced experimental pulmonary tumor metastasis. FEBS Lett. 2012;586:4296–4302.

    Article  CAS  PubMed  Google Scholar 

  10. Shi Y, Paluch BE, Wang X, Jiang X. PTEN at a glance. J Cell Sci. 2012;125:4687–4692.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Garcia-Junco-Clemente P, Golshani P. PTEN: a master regulator of neuronal structure, function, and plasticity. Commun Integr Biol. 2014;7:e28358.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Muniyan S, Ingersoll MA, Batra SK, Lin MF. Cellular prostatic acid phosphatase, a PTEN-functional homologue in prostate epithelia, functions as a prostate-specific tumor suppressor. Biochim Biophys Acta. 2014;1846:88–98.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. White ES, Thannickal VJ, Carskadon SL, et al. Integrin α4β1 regulates migration across basement membranes by lung fibroblasts: a role for phosphatase and tensin homologue deleted on chromosome 10. Am J Respir Crit Care Med. 2003;168:436–442.

    Article  PubMed  PubMed Central  Google Scholar 

  14. White ES, Atrasz RG, Hu B, et al. Negative regulation of myofibroblast differentiation by PTEN (phosphatase and tensin homolog deleted on chromosome 10). Am J Respir Crit Care Med. 2006;173:112–121.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lan R, Geng H, Polichnowski AJ, et al. PTEN loss defines a TGF-β-induced tubule phenotype of failed differentiation and JNK signaling during renal fibrosis. Am J Physiol Renal Physiol. 2012;302:F1210–F1223.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vinciguerra M, Veyrat-Durebex C, Moukil MA, Rubbia-Brandt L, Rohner-Jeanrenaud F, Foti M. PTEN down-regulation by unsaturated fatty acids triggers hepatic steatosis via an NF-κBp65/mTOR-dependent mechanism. Gastroenterology. 2008;134:268–280.

    Article  CAS  PubMed  Google Scholar 

  17. Hao LS, Zhang XL, An JY, et al. PTEN expression is down-regulated in liver tissues of rats with hepatic fibrosis induced by biliary stenosis. APMIS. 2009;117:681–691.

    Article  CAS  PubMed  Google Scholar 

  18. Zheng L, Chen X, Guo J, et al. Differential expression of PTEN in hepatic tissue and hepatic stellate cells during rat liver fibrosis and its reversal. Int J Mol Med. 2012;30:1424–1430.

    PubMed  Google Scholar 

  19. Ma J, Li F, Liu L, et al. Raf kinase inhibitor protein inhibits cell proliferation but promotes cell migration in rat hepatic stellate cells. Liver Int. 2009;29:567–574.

    Article  CAS  PubMed  Google Scholar 

  20. Hao LS, Zhang XL, An JY, et al. Adenoviral transduction of PTEN induces apoptosis of cultured hepatic stellate cells. Chin Med J (Engl). 2009;122:2907–2911.

    CAS  Google Scholar 

  21. An J, Zheng L, Xie S, et al. Down-regulation of focal adhesion kinase by short hairpin RNA increased apoptosis of rat hepatic stellate cells. APMIS. 2011;119:319–329.

    Article  CAS  PubMed  Google Scholar 

  22. Knodell RG, Ishak KG, Black WC, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic hepatitis. Hepatology. 1981;1:431–435.

    Article  CAS  PubMed  Google Scholar 

  23. Wang Y, Gao J, Zhang D, et al. New insights into the antifibrotic effects of sorafenib on hepatic stellate cells and liver fibrosis. J Hepatol. 2010;53:132–144.

    Article  CAS  PubMed  Google Scholar 

  24. Singla DK. Akt-mTOR pathway inhibits apoptosis and fibrosis in doxorubicin-induced cardiotoxicity following embryonic stem cell transplantation. Cell Transplant. 2015;24:1031–1042.

    PubMed  Google Scholar 

  25. Takashima M, Parsons CJ, Ikejima K, Watanabe S, White ES, Rippe RA. The tumor suppressor protein PTEN inhibits rat hepatic stellate cell activation. J Gastroenterol. 2009;44:847–855.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Povero D, Busletta C, Novo E, et al. Liver fibrosis: a dynamic and potentially reversible process. Histol Histopathol. 2010;25:1075–1091.

    PubMed  Google Scholar 

  27. Che XH, Jiang WY, Parajuli DR, Zhao YZ, Lee SH, Sohn DH. Apoptotic effect of propyl gallate in activated rat hepatic stellate cells. Arch Pharm Res. 2012;35:2205–2210.

    Article  CAS  PubMed  Google Scholar 

  28. Li J, Li X, Xu W, et al. Antifibrotic effects of luteolin on hepatic stellate cells and liver fibrosis by targeting AKT/mTOR/p70S6 K and TGFβ/Smad signalling pathways. Liver Int. 2015;35:1222–1233.

    Article  PubMed  Google Scholar 

  29. Marin JJ, Hernandez A, Revuelta IE, et al. Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: role of the Akt/mTOR survival pathway and Bcl-2 family proteins. Free Radic Biol Med. 2013;61:218–228.

    Article  CAS  PubMed  Google Scholar 

  30. Van Duijn PW, Ziel-van der Made AC, van der Korput JA, Trapman J. PTEN-mediated G1 cell-cycle arrest in LNCaP prostate cancer cells is associated with altered expression of cell-cycle regulators. Prostate. 2010;70:135–146.

    PubMed  Google Scholar 

  31. Paul-Samojedny M, Suchanek R, Borkowska P, et al. Knockdown of AKT3 (PKBγ) and PI3KCA suppresses cell viability and proliferation and induces the apoptosis of glioblastoma multiforme T98G cells. Biomed Res Int. 2014;2014:768181.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Aarts M, Linardopoulos S, Turner NC. Tumour selective targeting of cell cycle kinases for cancer treatment. Curr Opin Pharmacol. 2013;13:1–7.

    Article  Google Scholar 

  33. Chung JH, Ostrowski MC, Romigh T, Minaguchi T, Waite KA, Eng C. The ERK1/2 pathway modulates nuclear PTEN-mediated cell cycle arrest by cyclin D1 transcriptional regulation. Hum Mol Genet. 2006;15:2553–2559.

    Article  CAS  PubMed  Google Scholar 

  34. Mitrea DM, Yoon MK, Ou L, Kriwacki RW. Disorder-function relationships for the cell cycle regulatory proteins p21 and p27. Biol Chem. 2012;393:259–274.

    Article  CAS  PubMed  Google Scholar 

  35. Brenner DA. Molecular pathogenesis of liver fibrosis. Trans Am Clin Climatol Assoc. 2009;120:361–368.

    PubMed  PubMed Central  Google Scholar 

  36. Wang J, Xu F, Zhu D, et al. Schistosoma japonicum soluble egg antigens facilitate hepatic stellate cell apoptosis by downregulating Akt expression and upregulating p53 and DR5 Expression. PLoS Negl Trop Dis. 2014;8:e3106.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Zhang S, Yu D. PI(3)king apart PTEN’s role in cancer. Clin Cancer Res. 2010;16:4325–4330.

    Article  CAS  PubMed  Google Scholar 

  38. Ming M, Han W, Maddox J, et al. UVB-induced ERK/AKT-dependent PTEN suppression promotes survival of epidermal keratinocytes. Oncogene. 2010;29:492–502.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bunney TD, Katan M. Phosphoinositide signalling in cancer: beyond PI3K and PTEN. Nat Rev Cancer. 2010;10:342–352.

    Article  CAS  PubMed  Google Scholar 

  40. Dubrovska A, Kim S, Salamone RJ, et al. The role of PTEN/Akt/PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations. Proc Natl Acad Sci USA. 2009;106:268–273.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lagares D, Kapoor M. Targeting focal adhesion kinase in fibrotic diseases. BioDrugs. 2013;27:15–23.

    Article  CAS  PubMed  Google Scholar 

  42. Podolska K, Stachurska A, Hajdukiewicz K, Malecki M. Gene therapy prospects-intranasal delivery of therapeutic genes. Adv Clin Exp Med. 2012;21:525–534.

    PubMed  Google Scholar 

  43. Sakashita M, Mochizuki S, Sakurai K. Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry. Bioorg Med Chem. 2014;22:5212–5219.

    Article  CAS  PubMed  Google Scholar 

  44. Baertsch MA, Leber MF, Bossow S, et al. MicroRNA-mediated multi-tissue detargeting of oncolytic measles virus. Cancer Gene Ther. 2014;21:373–380.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant 30872513), Natural Science Foundation of Hebei Province (Grant C2010000565), and Hebei Provincial Science and Technology Department (Grant 09966108D). The authors would like to thank the foundations for their support. We appreciate Gregory X Shen for his valuable revision of written English and owe many thanks to Hong Zhang and Jinbo Guo for their photo contributions.

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    Authors and Affiliations

    1. Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, 215 West Heping Road, Shijiazhuang, 050000, Hebei, China

      Junyan An, Libo Zheng, Shurui Xie, Fengrong Yin, Xiaoxia Huo, Jian Guo & Xiaolan Zhang

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    1. Junyan An
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    Correspondence to Xiaolan Zhang.

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    Junyan An and Libo Zheng have contributed equally to this work.

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    An, J., Zheng, L., Xie, S. et al. Regulatory Effects and Mechanism of Adenovirus-Mediated PTEN Gene on Hepatic Stellate Cells. Dig Dis Sci 61, 1107–1120 (2016). https://doi.org/10.1007/s10620-015-3976-2

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