EPIDERMAI GROWTH FACTOR SECRETED BY ASTROCYTES STIMULATED WITH TACROLIMUS PROMOTING NEURITE OUTGROWTH_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CAIJun ¹ , SUNYu ¹ , GEYingbin ² ,  CAOXiaojian ¹

1. Department of Orthopedics, the First Affiliated Hospital of Nanjing Medical University, Nanjing Jiangsu, 210029, P. R. China;
2. Department of Physiology, Nanjing Medical University;

Corresponding author：

CAOXiaojian, Email: xiaojiancao@gmail.com

Keywords：

Tacrolimus; Spinal neurons; Spinal astrocytes; Epidermal growth factor; Rat

DOI：

10.7507/1002-1892.20150305

Video：

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Objective To illustrate the role of epidermal growth factor (EGF) secreted by astrocytes in the process of tacrolimus (FK506) in promoting neurite outgrowth. Methods The spinal cord astrocytes and neuronal cells were isolated respectively from 2-day-old Sprague Dawley (SD) rats and 15-day SD pregnant rats, and cultured in vitro and identified by immunofluorescence staining. The spinal cord astrocytes were cultured with 20 μmol/L FK506 medium in the experimental group, and with FK506 free medium in the control group. The supernatant was collected after 24 hours for preparing conditioned medium, and astrocytes were collected. EGF proteins in the conditioned medium were detected with ELISA, and EGF gene expressions of astrocytes were detected with real-time quantitative PCR (RT-qPCR). The spinal cord neurons were cultured respectively with conditioned medium from the experimental group (FK506-CM) and the control group (C-CM) in group A and group B, also with neutralized C-CM and neutralized FK506-CM with anti-EGF neutralizing antibodies in group C and group D. Both the total neurite length and the longest neurite length were measured and compared among groups. Results Both astrocytes and neurons were confirmed by immunofluorescence staining. The EGF content of experimental group (0.241±0.044) was significantly higher than that of the control group (0.166±0.014) (t=3.93, P=0.01); EGF gene expression of the experimental group (1.12±0.25) was significantly higher than that of the control group (0.46±0.11) (t=5.78, P=0.00). The neurite length measurement displayed that the total neurite length and the longest neurite length of groups C and D were significantly shorter than those of groups A and B (P<0.05). Both the total and longest neurite length of group A were significantly longer than those of group B (P<0.05), but no significant difference was shown between groups C and D (P>0.05). Conclusion The EGF secreted by spinal cord astrocytes can promote the neurite outgrowth. So spinal cord astrocytes can be used as an important intermediary target of FK506 to promote the recovery of neurological function.

Citation： CAIJun, SUNYu, GEYingbin, CAOXiaojian. EPIDERMAI GROWTH FACTOR SECRETED BY ASTROCYTES STIMULATED WITH TACROLIMUS PROMOTING NEURITE OUTGROWTH. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(11): 1423-1428. doi: 10.7507/1002-1892.20150305 Copy

1.	Zhang J, Zhang A, Sun Y, et al. Treatment with immunosuppressants FTY720 and tacrolimus promotes functional recovery after spinal cord injury in rats. Tohoku J Exp Med, 2009, 219(4):295-302.
2.	Martinez R, Gomes FC. Neuritogenesis induced by thyroid hormone-treated astrocytes is mediated by epidermal growth factor/mitogen-activated protein kinase-phosphatidylinositol 3-kinase pathways and involves modulation of extracellular matrix proteins. J Biol Chem, 2002, 277(51):49311-49318.
3.	Zawadzka M, Kaminska B. Immunosuppressant FK506 affects multiple signaling pathways and modulates gene expression in astrocytes. Mol Cell Neurosci, 2003, 22(2):202-209.
4.	Volterra A, Meldolesi J. Astrocytes, from brain glue to communication elements:the revolution continues. Nat Rev Neurosci, 2005, 6(8):626-640.
5.	Halassa MM, Fellin T, Haydon PG. The tripartite synapse:roles for gliotransmission in health and disease. Trends Mol Med, 2007, 13(2):54-63.
6.	Rossi DJ, Brady JD, Mohr C. Astrocyte metabolism and signaling during brain ischemia. Nat Neurosci, 2007, 10(11):1377-1386.
7.	Han SS, Kang DY, Mujtaba T. Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord. Exp Neurol, 2002, 177(2):360-375.
8.	Brewer GJ, Torricelli JR. Isolation and culture of adult neurons and neurospheres. Nat Protoc, 2007, 2(6):1490-1498.
9.	Stipursky J, Gomes FC. TGF-beta1/SMAD signaling induces astrocyte fate commitment in vitro:implications for radial glia development. Glia, 2007, 55(10):1023-1033.
10.	E Spohr TC, Dezonne RS, Rehen SK, et al. Astrocytes treated by lysophosphatidic acid induce axonal outgrowth of cortical progenitors through extracellular matrix protein and epidermal growth factor signaling pathway. J Neurochem, 2011, 119(1):113-123.
11.	Madsen JR, MacDonald P, Irwin N, et al. Tacrolimus (FK506) increases neuronal expression of GAP-43 and improves functional recovery after spinal cord injury in rats. Exp Neurol, 1998, 154(2):673-683.
12.	Kaymaz M, Emmez H, Bukan N, et al. Effectiveness of FK506 on lipid peroxidation in the spinal cord following experimental traumatic injury. Spinal Cord, 2005, 43(1):22-26.
13.	Szydlowska K, Gozdz A, Dabrowski M, et al. Prolonged activation of ERK triggers glutamate-induced apoptosis of astrocytes:neuroprotective effect of FK506. J Neurochem, 2010, 113(4):904-918.
14.	Nottingham S, Knapp P, Springer J. FK506 treatment inhibits caspase-3 activation and promotes oligodendroglial survival following traumatic spinal cord injury. Exp Neurol, 2002, 177(1):242-251.
15.	McKeon RJ, Schreiber RC, Rudge JS, et al. Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes. J Neurosci, 1991, 11(11):3398-3411.
16.	Rolls A, Shechter R, Schwartz M. The bright side of the glial scar in CNS repair. Nat Rev Neurosci, 2009, 10(3):235-241.
17.	Norenberg MD. Astrocyte responses to CNS injury. J Neuropathol Exp Neurol, 1994, 53(3):213-220.
18.	Bush TG, Puvanachandra N, Horner CH, et al. Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice. Neuron, 1999, 23(2):297-308.
19.	Wong RW, Guillaud L. The role of epidermal growth factor and its receptors in mammalian CNS. Cytokine Growth Factor Rev, 2004, 15(2-3):147-156.
20.	Fricker-Gates RA, Winkler C, Kirik D, et al. EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum. Exp Neurol, 2000, 165(2):237-247.
21.	Yamada M, Ikeuchi T, Hatanaka H. The neurotrophic action and signalling of epidermal growth factor. Prog Neurobiol, 1997, 51(1):19-37.
22.	Goldshmit Y, Greenhalgh CJ, Turnley AM. Suppressor of cytokine signalling-2 and epidermal growth factor regulate neurite outgrowth of cortical neurons. Eur J Neurosci, 2004, 20(9):2260-2266.
23.	Xiang YY, Dong H, Wan Y, et al. Versican G3 domain regulates neurite growth and synaptic transmission of hippocampal neurons by activation of epidermal growth factor receptor. J Biol Chem, 2006, 281(28):19358-19368.
24.	Choi-Lundberg DL, Lin Q, Chang YN, et al. Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science, 1997, 275(5301):838-841.
25.	Engele J, Bohn MC. The neurotrophic effects of fibroblast growth factors on dopaminergic neurons in vitro are mediated by mesencephalic glia. J Neurosci, 1991, 11(10):3070-3078.
26.	Maxwell GD, Reid K, Elefanty A, et al. Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures. Proc Natl Acad Sci U S A, 1996, 93(23):13274-13279.

1. Zhang J, Zhang A, Sun Y, et al. Treatment with immunosuppressants FTY720 and tacrolimus promotes functional recovery after spinal cord injury in rats. Tohoku J Exp Med, 2009, 219(4):295-302.
2. Martinez R, Gomes FC. Neuritogenesis induced by thyroid hormone-treated astrocytes is mediated by epidermal growth factor/mitogen-activated protein kinase-phosphatidylinositol 3-kinase pathways and involves modulation of extracellular matrix proteins. J Biol Chem, 2002, 277(51):49311-49318.
3. Zawadzka M, Kaminska B. Immunosuppressant FK506 affects multiple signaling pathways and modulates gene expression in astrocytes. Mol Cell Neurosci, 2003, 22(2):202-209.
4. Volterra A, Meldolesi J. Astrocytes, from brain glue to communication elements:the revolution continues. Nat Rev Neurosci, 2005, 6(8):626-640.
5. Halassa MM, Fellin T, Haydon PG. The tripartite synapse:roles for gliotransmission in health and disease. Trends Mol Med, 2007, 13(2):54-63.
6. Rossi DJ, Brady JD, Mohr C. Astrocyte metabolism and signaling during brain ischemia. Nat Neurosci, 2007, 10(11):1377-1386.
7. Han SS, Kang DY, Mujtaba T. Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord. Exp Neurol, 2002, 177(2):360-375.
8. Brewer GJ, Torricelli JR. Isolation and culture of adult neurons and neurospheres. Nat Protoc, 2007, 2(6):1490-1498.
9. Stipursky J, Gomes FC. TGF-beta1/SMAD signaling induces astrocyte fate commitment in vitro:implications for radial glia development. Glia, 2007, 55(10):1023-1033.
10. E Spohr TC, Dezonne RS, Rehen SK, et al. Astrocytes treated by lysophosphatidic acid induce axonal outgrowth of cortical progenitors through extracellular matrix protein and epidermal growth factor signaling pathway. J Neurochem, 2011, 119(1):113-123.
11. Madsen JR, MacDonald P, Irwin N, et al. Tacrolimus (FK506) increases neuronal expression of GAP-43 and improves functional recovery after spinal cord injury in rats. Exp Neurol, 1998, 154(2):673-683.
12. Kaymaz M, Emmez H, Bukan N, et al. Effectiveness of FK506 on lipid peroxidation in the spinal cord following experimental traumatic injury. Spinal Cord, 2005, 43(1):22-26.
13. Szydlowska K, Gozdz A, Dabrowski M, et al. Prolonged activation of ERK triggers glutamate-induced apoptosis of astrocytes:neuroprotective effect of FK506. J Neurochem, 2010, 113(4):904-918.
14. Nottingham S, Knapp P, Springer J. FK506 treatment inhibits caspase-3 activation and promotes oligodendroglial survival following traumatic spinal cord injury. Exp Neurol, 2002, 177(1):242-251.
15. McKeon RJ, Schreiber RC, Rudge JS, et al. Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes. J Neurosci, 1991, 11(11):3398-3411.
16. Rolls A, Shechter R, Schwartz M. The bright side of the glial scar in CNS repair. Nat Rev Neurosci, 2009, 10(3):235-241.
17. Norenberg MD. Astrocyte responses to CNS injury. J Neuropathol Exp Neurol, 1994, 53(3):213-220.
18. Bush TG, Puvanachandra N, Horner CH, et al. Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice. Neuron, 1999, 23(2):297-308.
19. Wong RW, Guillaud L. The role of epidermal growth factor and its receptors in mammalian CNS. Cytokine Growth Factor Rev, 2004, 15(2-3):147-156.
20. Fricker-Gates RA, Winkler C, Kirik D, et al. EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum. Exp Neurol, 2000, 165(2):237-247.
21. Yamada M, Ikeuchi T, Hatanaka H. The neurotrophic action and signalling of epidermal growth factor. Prog Neurobiol, 1997, 51(1):19-37.
22. Goldshmit Y, Greenhalgh CJ, Turnley AM. Suppressor of cytokine signalling-2 and epidermal growth factor regulate neurite outgrowth of cortical neurons. Eur J Neurosci, 2004, 20(9):2260-2266.
23. Xiang YY, Dong H, Wan Y, et al. Versican G3 domain regulates neurite growth and synaptic transmission of hippocampal neurons by activation of epidermal growth factor receptor. J Biol Chem, 2006, 281(28):19358-19368.
24. Choi-Lundberg DL, Lin Q, Chang YN, et al. Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science, 1997, 275(5301):838-841.
25. Engele J, Bohn MC. The neurotrophic effects of fibroblast growth factors on dopaminergic neurons in vitro are mediated by mesencephalic glia. J Neurosci, 1991, 11(10):3070-3078.
26. Maxwell GD, Reid K, Elefanty A, et al. Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures. Proc Natl Acad Sci U S A, 1996, 93(23):13274-13279.

Chinese Journal of Reparative and Reconstructive Surgery

EPIDERMAI GROWTH FACTOR SECRETED BY ASTROCYTES STIMULATED WITH TACROLIMUS PROMOTING NEURITE OUTGROWTH

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