Mechanism of p38 mitogen activated protein kinase signaling pathway on promoting the hypertrophy of human lumbar ligamentum flavum via transforming growth factor β<sub>1</sub>/connective tissue growth factor_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LU Changhuai ¹ , LIU Zhijun ¹ , ZHANG Hongbo ¹ ,  DUAN Yang ² , CAO Yanlin ²

1. Department of Spine Disease Area of Orthopedics and Traumatology, No.1 Traditional Chinese Medicine Hospital of Changde, Changde Hospital Affiliated to Hunan University of Traditional Chinese Medicine, Changde Hunan, 415000, P.R.China;
2. Department of Spinal Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou Guangdong, 510282, P.R.China;

Corresponding author：

DUAN Yang, Email: duanyang2011@163.com

Keywords：

Mitogen activated protein kinase; transforming growth factor β₁; connective tissue growth factor; ligamentum flavum

DOI：

10.7507/1002-1892.201811140

Video：

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Objective To investigate the mechanism of p38 mitogen activated protein kinase (MAPK) signaling pathway in regulating the hyperplasia and hypertrophy of human lumbar ligamentum flavum via transforming growth factor β₁ (TGF-β₁)/connective tissue growth factor (CTGF). Methods The lumbar ligamentum flavum tissue taken from patient with lumbar intervertebral disc herniation was isolated by collagenase-predigested explant cultures. The ligamentum flavum cells were treated with the extracellular regulated protein kinase pathway blocker PD98059, c-Jun N-terminal kinase pathway blocker SP600125, and p38 pathway blocker SB203580, and then the mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR (qRT-PCR). The ligamentum flavum cells were divided into 4 groups, and transfected with small interfering RNA (siRNA), p38 siRNA, siRNA+3 ng/mL TGF-β₁, and p38 siRNA+3 ng/mL TGF-β₁ in groups A, B, C, and D, respectively. After 24 hours of transfection, immunofluorescence staining was performed to observe the expressions of p38 and phosphorylation p38 (p-p38); the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRT-PCR; the protein expression of CTGF in each group was detected by Western blot. Results p38 pathway blocker SB203580 could significantly reduce the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ (P<0.05). After 24 hours of transfection, immunofluorescence staining showed positive staining with p38 and p-p38 expressions in groups A, C, and D and negative staining in group B. Compared with group A, the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ and relative protein expression of CTGF in group B decreased significantly (P<0.05), while those in groups C and D increased significantly (P<0.05); and those indicators significantly increased in group C than in group D (P<0.05). Conclusion TGF-β₁/CTGF based on the p38 MAPK signaling pathway play an important role in the occurance and development of hypertrophy of human lumbar ligamentum flavum.

Citation： LU Changhuai, LIU Zhijun, ZHANG Hongbo, DUAN Yang, CAO Yanlin. Mechanism of p38 mitogen activated protein kinase signaling pathway on promoting the hypertrophy of human lumbar ligamentum flavum via transforming growth factor β₁/connective tissue growth factor. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(6): 730-735. doi: 10.7507/1002-1892.201811140 Copy

1.	Binder DK, Schmidt MH, Weinstein PR. Lumbar spinal stenosis. Semin Neurol, 2002, 22(2): 157-166.
2.	Gu J, Liu X, Wang QX, et al. Angiotensin Ⅱ increases CTGF expression via MAPKs/TGF-β₁/TRAF6 pathway in atrial fibroblasts. Exp Cell Res, 2012, 318(16): 2105-2115.
3.	Maezawa Y, Baba H, Uchida K, et al. Ligamentum flavum hematoma in the thoracic spine. Clin Imaging, 2001, 25(4): 265-267.
4.	Behm B, Babilas P, Landthaler M, et al. Cytokines, chemokines and growth factors in wound healing. J Eur Acad Dermatol Venereol, 2012, 26(7): 812-820.
5.	Zhong ZM, Zha DS, Xiao WD, et al. Hypertrophy of ligamentum flavum in lumbar spine stenosis associated with the increased expression of connective tissue growth factor. J Orthop Res, 2011, 29(10): 1592-1597.
6.	Amudong A, Muheremu A, Abudourexiti T. Hypertrophy of the ligamentum flavum and expression of transforming growth factor beta. J Int Med Res, 2017, 45(6): 2036-2041.
7.	方清清, 李志忠, 周建, 等. 信号分子 p38 参与低频脉冲电磁场促进成骨细胞矿化成熟的实验研究. 中国修复重建外科杂志, 2016, 30(10): 1238-1243.
8.	New L, Han J. The p38 MAP kinase pathway and its biological function. Trends Cardiovasc Med, 1998, 8(5): 220-228.
9.	Hale KK, Trollinger D, Rihanek M, et al. Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma, and delta in inflammatory cell lineages. J Immunol, 1999, 162(7): 4246-4252.
10.	Specchia N, Pagnotta A, Gigante A, et al. Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis. J Orthop Res, 2001, 19(2): 294-300.
11.	Zhong ZM, Chen JT. Phenotypic characterization of ligamentum flavum cells from patients with ossification of ligamentum flavum. Yonsei Med J, 2009, 50(3): 375-379.
12.	Zhang M, Fraser D, Phillips A. ERK, p38, and Smad signaling pathways differentially regulate transforming growth factor-beta1 autoinduction in proximal tubular epithelial cells. Am J Pathol, 2006, 169(4): 1282-1293.
13.	Holmes A, Abraham DJ, Sa S, et al. CTGF and SMADs, maintenance of scleroderma phenotype is independent of SMAD signaling. J Biol Chem, 2001, 276(14): 10594-10601.
14.	Du QC, Zhang DZ, Chen XJ, et al. The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblast differentiation. PLoS One, 2013, 8(10): e75635.
15.	Morales MG, Vazquez Y, Acuña MJ, et al. Angiotensin Ⅱ-induced pro-fibrotic effects require p38MAPK activity and transforming growth factor beta 1 expression in skeletal muscle cells. Int J Biochem Cell Biol, 2012, 44(11): 1993-2002.
16.	Park JB, Chang H, Lee JK. Quantitative analysis of transforming growth factor-beta 1 in ligamentum flavum of lumbar spinal stenosis and disc herniation. Spine (Phila Pa 1976), 2001, 26(21): E492-E495.
17.	Han J, Lee JD, Bibbs L, et al. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science, 1994, 265(5173): 808-811.
18.	Jiang Y, Gram H, Zhao M, et al. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta. J Biol Chem, 1997, 272(48): 30122-30128.
19.	Mavropoulos A, Orfanidou T, Liaskos C, et al. p38 MAPK signaling in pemphigus: implications for skin autoimmunity. Autoimmune Dis, 2013, 2013: 728529.
20.	Whitmarsh AJ. A central role for p38 MAPK in the early transcriptional response to stress. BMC Biol, 2010, 8: 47.

1. Binder DK, Schmidt MH, Weinstein PR. Lumbar spinal stenosis. Semin Neurol, 2002, 22(2): 157-166.
2. Gu J, Liu X, Wang QX, et al. Angiotensin Ⅱ increases CTGF expression via MAPKs/TGF-β₁/TRAF6 pathway in atrial fibroblasts. Exp Cell Res, 2012, 318(16): 2105-2115.
3. Maezawa Y, Baba H, Uchida K, et al. Ligamentum flavum hematoma in the thoracic spine. Clin Imaging, 2001, 25(4): 265-267.
4. Behm B, Babilas P, Landthaler M, et al. Cytokines, chemokines and growth factors in wound healing. J Eur Acad Dermatol Venereol, 2012, 26(7): 812-820.
5. Zhong ZM, Zha DS, Xiao WD, et al. Hypertrophy of ligamentum flavum in lumbar spine stenosis associated with the increased expression of connective tissue growth factor. J Orthop Res, 2011, 29(10): 1592-1597.
6. Amudong A, Muheremu A, Abudourexiti T. Hypertrophy of the ligamentum flavum and expression of transforming growth factor beta. J Int Med Res, 2017, 45(6): 2036-2041.
7. 方清清, 李志忠, 周建, 等. 信号分子 p38 参与低频脉冲电磁场促进成骨细胞矿化成熟的实验研究. 中国修复重建外科杂志, 2016, 30(10): 1238-1243.
8. New L, Han J. The p38 MAP kinase pathway and its biological function. Trends Cardiovasc Med, 1998, 8(5): 220-228.
9. Hale KK, Trollinger D, Rihanek M, et al. Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma, and delta in inflammatory cell lineages. J Immunol, 1999, 162(7): 4246-4252.
10. Specchia N, Pagnotta A, Gigante A, et al. Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis. J Orthop Res, 2001, 19(2): 294-300.
11. Zhong ZM, Chen JT. Phenotypic characterization of ligamentum flavum cells from patients with ossification of ligamentum flavum. Yonsei Med J, 2009, 50(3): 375-379.
12. Zhang M, Fraser D, Phillips A. ERK, p38, and Smad signaling pathways differentially regulate transforming growth factor-beta1 autoinduction in proximal tubular epithelial cells. Am J Pathol, 2006, 169(4): 1282-1293.
13. Holmes A, Abraham DJ, Sa S, et al. CTGF and SMADs, maintenance of scleroderma phenotype is independent of SMAD signaling. J Biol Chem, 2001, 276(14): 10594-10601.
14. Du QC, Zhang DZ, Chen XJ, et al. The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblast differentiation. PLoS One, 2013, 8(10): e75635.
15. Morales MG, Vazquez Y, Acuña MJ, et al. Angiotensin Ⅱ-induced pro-fibrotic effects require p38MAPK activity and transforming growth factor beta 1 expression in skeletal muscle cells. Int J Biochem Cell Biol, 2012, 44(11): 1993-2002.
16. Park JB, Chang H, Lee JK. Quantitative analysis of transforming growth factor-beta 1 in ligamentum flavum of lumbar spinal stenosis and disc herniation. Spine (Phila Pa 1976), 2001, 26(21): E492-E495.
17. Han J, Lee JD, Bibbs L, et al. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science, 1994, 265(5173): 808-811.
18. Jiang Y, Gram H, Zhao M, et al. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta. J Biol Chem, 1997, 272(48): 30122-30128.
19. Mavropoulos A, Orfanidou T, Liaskos C, et al. p38 MAPK signaling in pemphigus: implications for skin autoimmunity. Autoimmune Dis, 2013, 2013: 728529.
20. Whitmarsh AJ. A central role for p38 MAPK in the early transcriptional response to stress. BMC Biol, 2010, 8: 47.

Chinese Journal of Reparative and Reconstructive Surgery

Mechanism of p38 mitogen activated protein kinase signaling pathway on promoting the hypertrophy of human lumbar ligamentum flavum via transforming growth factor β₁/connective tissue growth factor

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Chinese Journal of Reparative and Reconstructive Surgery

Mechanism of p38 mitogen activated protein kinase signaling pathway on promoting the hypertrophy of human lumbar ligamentum flavum via transforming growth factor β1/connective tissue growth factor

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Mechanism of p38 mitogen activated protein kinase signaling pathway on promoting the hypertrophy of human lumbar ligamentum flavum via transforming growth factor β₁/connective tissue growth factor