EXPRESSION AND ROLE OF KIP1 UBIQUITYLATION-PROMOTING COMPLEX 2 IN ASTROCYTES AFTER SPINAL CORD INJURY IN RATS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XULiang ^1,2 , LIUFan ¹ ,  ZHUJianwei ¹ , ZHULifan ² , JIANGFugui ²

1. Department of Orthopedics, Affiliated Hospital of Nantong University, Nantong Jiangsu, 226001, P. R. China;
2. Department of Orthopedics, the Wujiang Hospital Affiliated to Nantong University, University, Nantong Jiangsu, 226001, P. R. China;

Corresponding?author：

ZHUJianwei, Email: zhujianweint@126.com

Keywords：

Kip1 ubiquitylation-promoting complex 2; P27kip1; Spinal cord injury; Astrocytes; Proliferation; Rat

DOI：

10.7507/1002-1892.20150211

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Objective To explore the biological functions of Kip1 ubiquitylation-promoting complex 2 (KPC2) in the repair process of spinal cord injury (SCI) by studying the expression and cellular localization of KPC2 in rat SCI models. Methods Fifty-six adult Sprague-Dawley rats were randomly divided into 2 groups: in the control group (n=7), simple T₉ laminectomy was performed;in the experimental group (n=49), the SCI model was established at T₉, 7 rats were used to detect follow indexs at 6 hours, 12 hours, 1 day, 3 days, 5 days, 7 days, and 14 days after SCI. Western blot analysis was used to detect the protein expressions of P27^kip1, KPC2, CyclinA and proliferating cell nuclear antigen (PCNA) after SCI. Immunohistochemistry was used to observed the cellular localization of KPC2 after SCI, double-labeling immunofluorescence staining to observe the co-localization of KPC2 with neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) and PCNA. in vitro astrocytes proliferation model was used to further validate these results, Western blot to detect KPC2, P27^kip1, and PCNA expressions. The interaction of P27^kip1, KPC1, and KPC2 in cell proliferation was analyzed by co-immunoprecipitation. Results The Western blot analysis showed a significant down-regulation of P27^kip1 and a concomitant up-regulation of KPC2, CyclinA, and PCNA after SCI. Immunohistochemistry staining revealed a wide distribution of KPC2 positive signals in the gray matter and white matter of the spinal cord. The number of KPC2 positive cells in the experimental group was significantly higher than that in the control group (t=10.982, P=0.000). Double-labeling immunofluorescence staining revealed the number of KPC2/NeuN co-expression cells in the gray matter of spinal cord was (0.43±0.53)/visual field in the control group and (0.57±0.53)/visual field in the experimental group, showing no significant difference (t=0.548, P=0.604);in the white matter of spinal cord, the number of KPC2/PCNA co-expression cells was (3.86±0.90)/visual field in the control group and (0.71±0.49)/visual field in the experimental group, showing significant difference (t=7.778, P=0.000). And then, the number of KPC2/PCNA co-expression cells were (0.57±0.53)/visual field in the control group and (5.57±1.13)/visual field in the experimental group, showing significant difference (t=8.101, P=0.000). Concomitantly, there was a similar kinetic in proliferating astrocytes in vitro. The Western blot analysis showed a significant down-regulation of P27^kip1 and a concomitant up-regulation of KPC2 and PCNA after serum stimulated. Co-immunoprecipitation demonstrated increased interactions between P27^kip1, KPC1, and KPC2 after stimulation. Conclusion The up-regulated expression of KPC2 after SCI is related to the down-regulation of P27^kip1, this event may be involved in the proliferation of astrocytes after SCI.

Citation： XULiang, LIUFan, ZHUJianwei, ZHULifan, JIANGFugui. EXPRESSION AND ROLE OF KIP1 UBIQUITYLATION-PROMOTING COMPLEX 2 IN ASTROCYTES AFTER SPINAL CORD INJURY IN RATS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(8): 978-985. doi: 10.7507/1002-1892.20150211 Copy

1.	胥少汀.脊髓原發和繼發損傷.中華骨科雜志,2005,25(9):575-576.
2.	黃其林,蔡文琴,張可成.反義膠原纖維酸性蛋白逆轉錄病毒對膠質瘢痕增生的影響.中華創傷雜志,2000,16(2):103-105.
3.	McDonald JW,Sadowsky C.Spinal-cord injury.Lancet,2002,359(9304):417-425.
4.	武亮,李建軍,陳亮,等.抑制脊髓損傷后星形膠質細胞增殖和膠質瘢痕形成的研究進展.中國康復理論與實踐,2010,16(3):201-204.
5.	侯超,武衡.反應性星形膠質細胞對中樞神經系統損傷的保護作用.社區醫學雜志,2012,10(2):49-51.
6.	Becker EB,Bonni A.Cell cycle regulation of neuronal apoptosis in development and disease.Prog Neurobiol,2004,72(1):1-25.
7.	Sgambato A,Cittadini A,Faraglia B,et al.Multiple functions of p27(Kip1) and its alterations in tumor cells:a review.J Cell Physiol,2000,183(1):18-27.
8.	Miyazawa K,Himi T,Garcia V,et al.A role for p27/Kip1 in the control of cerebellar granule cell precursor proliferation.J Neurosci,2000,20(15):5756-5763.
9.	Kotoshiba S,Kamura T,Hara T,et al.Molecular dissection of the interaction between p27 and Kip1 ubiquitylation-promoting complex,the ubiquitin ligase that regulates proteolysis of p27 in G1 phase.J Biol Chem,2005,280(18):17694-17700.
10.	Kamura T,Hara T,Matsumoto M,et al.Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase.Nat Cell Biol,2004,6(12):1229-1235.
11.	Lee JG,Kay EP.Involvement of two distinct ubiquitin E3 ligase systems for p27 degradation in corneal endothelial cells.Invest Ophthalmol Vis Sci,2008,49(1):189-196.
12.	Zhao J,Zhang S,Wu X,et al.KPC1 expression and essential role after acute spinal cord injury in adult rat.Neurochem Res,2011,36(3):549-558.
13.	McCarthy KD,de Vellis J.Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.J Cell Biol,1980,85(3):890-902.
14.	潘勇,周躍.脊髓損傷后再生研究進展.中華創傷雜志,2001,17(11):698-700.
15.	初同偉,焦文倉,周躍.脊髓損傷后的細胞反應和分子變化.中華創傷雜志,2005,21(9):717-720.
16.	Sekhon LH,Fehlings MG.Epidemiology,demographics,and pathophysiology of acute spinal cord injury.Spine (Phila Pa 1976),2001,26(24 Suppl):S2-12.
17.	McGraw J,Hiebert GW,Steeves JD.Modulating astrogliosis after neurotrauma.J Neurosci Res,2001,63(2):109-115.
18.	陳哲宇,黃愛軍,路長林,等.膠質細胞源性神經營養因子對脊髓前角神經元的保護作用.中華創傷雜志,2000,16(12):723-726.
19.	Davies SJ,Field PM,Raisman G.Regeneration of cut adult axons fails even in the presence of continuous aligned glial pathways.Exp Neurol,1996,142(2):203-216.
20.	Silver J,Miller JH.Regeneration beyond the glial scar.Nat Rev Neurosci,2004,5(2):146-156.
21.	Di Giovanni S,Movsesyan V,Ahmed F,et al.Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury.Proc Natl Acad Sci U S A,2005,102(23):8333-8338.
22.	Byrnes KR,Stoica BA,Fricke S,et al.Cell cycle activation contributes to post-mitotic cell death and secondary damage after spinal cord injury.Brain,2007,130(Pt 11):2977-2992.

1. 胥少汀.脊髓原發和繼發損傷.中華骨科雜志,2005,25(9):575-576.
2. 黃其林,蔡文琴,張可成.反義膠原纖維酸性蛋白逆轉錄病毒對膠質瘢痕增生的影響.中華創傷雜志,2000,16(2):103-105.
3. McDonald JW,Sadowsky C.Spinal-cord injury.Lancet,2002,359(9304):417-425.
4. 武亮,李建軍,陳亮,等.抑制脊髓損傷后星形膠質細胞增殖和膠質瘢痕形成的研究進展.中國康復理論與實踐,2010,16(3):201-204.
5. 侯超,武衡.反應性星形膠質細胞對中樞神經系統損傷的保護作用.社區醫學雜志,2012,10(2):49-51.
6. Becker EB,Bonni A.Cell cycle regulation of neuronal apoptosis in development and disease.Prog Neurobiol,2004,72(1):1-25.
7. Sgambato A,Cittadini A,Faraglia B,et al.Multiple functions of p27(Kip1) and its alterations in tumor cells:a review.J Cell Physiol,2000,183(1):18-27.
8. Miyazawa K,Himi T,Garcia V,et al.A role for p27/Kip1 in the control of cerebellar granule cell precursor proliferation.J Neurosci,2000,20(15):5756-5763.
9. Kotoshiba S,Kamura T,Hara T,et al.Molecular dissection of the interaction between p27 and Kip1 ubiquitylation-promoting complex,the ubiquitin ligase that regulates proteolysis of p27 in G1 phase.J Biol Chem,2005,280(18):17694-17700.
10. Kamura T,Hara T,Matsumoto M,et al.Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase.Nat Cell Biol,2004,6(12):1229-1235.
11. Lee JG,Kay EP.Involvement of two distinct ubiquitin E3 ligase systems for p27 degradation in corneal endothelial cells.Invest Ophthalmol Vis Sci,2008,49(1):189-196.
12. Zhao J,Zhang S,Wu X,et al.KPC1 expression and essential role after acute spinal cord injury in adult rat.Neurochem Res,2011,36(3):549-558.
13. McCarthy KD,de Vellis J.Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.J Cell Biol,1980,85(3):890-902.
14. 潘勇,周躍.脊髓損傷后再生研究進展.中華創傷雜志,2001,17(11):698-700.
15. 初同偉,焦文倉,周躍.脊髓損傷后的細胞反應和分子變化.中華創傷雜志,2005,21(9):717-720.
16. Sekhon LH,Fehlings MG.Epidemiology,demographics,and pathophysiology of acute spinal cord injury.Spine (Phila Pa 1976),2001,26(24 Suppl):S2-12.
17. McGraw J,Hiebert GW,Steeves JD.Modulating astrogliosis after neurotrauma.J Neurosci Res,2001,63(2):109-115.
18. 陳哲宇,黃愛軍,路長林,等.膠質細胞源性神經營養因子對脊髓前角神經元的保護作用.中華創傷雜志,2000,16(12):723-726.
19. Davies SJ,Field PM,Raisman G.Regeneration of cut adult axons fails even in the presence of continuous aligned glial pathways.Exp Neurol,1996,142(2):203-216.
20. Silver J,Miller JH.Regeneration beyond the glial scar.Nat Rev Neurosci,2004,5(2):146-156.
21. Di Giovanni S,Movsesyan V,Ahmed F,et al.Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury.Proc Natl Acad Sci U S A,2005,102(23):8333-8338.
22. Byrnes KR,Stoica BA,Fricke S,et al.Cell cycle activation contributes to post-mitotic cell death and secondary damage after spinal cord injury.Brain,2007,130(Pt 11):2977-2992.

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EXPRESSION AND ROLE OF KIP1 UBIQUITYLATION-PROMOTING COMPLEX 2 IN ASTROCYTES AFTER SPINAL CORD INJURY IN RATS

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