TGF-β1, in association with the increased expression of connective tissue growth factor, induce the hypertrophy of the ligamentum flavum through the p38 MAPK pathway

Cao,Yan-Lin; Duan,Yang; Zhu,Li-Xin; Zhan,Ye-Nan; Min,Shao-Xiong; Jin,An-Min

doi:10.3892/ijmm.2016.2631

TGF-β1, in association with the increased expression of connective tissue growth factor, induce the hypertrophy of the ligamentum flavum through the p38 MAPK pathway

Authors:
- Yan-Lin Cao
- Yang Duan
- Li-Xin Zhu
- Ye-Nan Zhan
- Shao-Xiong Min
- An-Min Jin
View Affiliations

Affiliations: Department of Orthopaedic Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, P.R. China, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, P.R. China
Published online on: June 8, 2016 https://doi.org/10.3892/ijmm.2016.2631
Pages: 391-398
Copyright: © Cao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Hypertrophy of the ligamentum flavum (LF) is one of the key pathomechanisms of lumbar spinal stenosis (LSS). Transforming growth factor (TGF)-β1 is abundantly expressed in hypertrophied degenerative LF tissues from LSS. However, the molecular mechanisms underling the association between TGF-β1 and LF hypertrophy have not yet been fully elucidated. In this study, we investigated the important role of the mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of LSS by analyzing the expression of connective tissue growth factor (CTGF) and extracellular matrix (ECM) components (collagen I and collagen III) in TGF-β1-treated LF cells. Cell growth assay revealed that TGF-β1, in association with CTGF, enhanced the the proliferation of LF cells, and we found that TGF-β1 also elevated CTGF expression and subsequently enhanced the mRNA expression of collagen I and collagen III. The increased mRNA expression levels of CTGF, collagen I and collagen III were abolished by p38 inhibitors. Both immunofluorescence imaging and western blot analysis of p38 and p-p38 revealed the increased expression and phosphorylation of p38. Silencing the expression of p38 by siRNA in LF cells decreased the protein expression of p38, p-p38 and CTGF, as well as the mRNA expression of CTGF, collagen I and collagen III. Taken together, our findings indicate that TGF-β1, in association with the increased expression of CTGF, contribute to the homeostasis of the ECM and to the hypertrophy of LF through the p38 MAPK pathway.

View Figures

View References

1	Siebert E, Prüss H, Klingebiel R, Failli V, Einhäupl KM and Schwab JM: Lumbar spinal stenosis: syndrome, diagnostics and treatment. Nat Rev Neurol. 5:392–403. 2009. View Article : Google Scholar : PubMed/NCBI
2	Botwin KP and Gruber RD: Lumbar spinal stenosis: anatomy and pathogenesis. Phys Med Rehabil Clin N Am. 14:1–15. 2003. View Article : Google Scholar : PubMed/NCBI
3	Fukuyama S, Nakamura T, Ikeda T and Takagi K: The effect of mechanical stress on hypertrophy of the lumbar ligamentum flavum. J Spinal Disord. 8:126–130. 1995. View Article : Google Scholar : PubMed/NCBI
4	Behm B, Babilas P, Landthaler M and Schreml S: Cytokines, chemokines and growth factors in wound healing. J Eur Acad Dermatol Venereol. 26:812–820. 2012. View Article : Google Scholar : PubMed/NCBI
5	Matsumoto Y, Fujiwara T, Imamura R, Okada Y, Harimaya K, Doi T, Kawaguchi K, Okada S, Yamada Y, Oda Y and Iwamoto Y: Hematoma of the ligamentum flavum in the thoracic spine: report of two cases and possible role of the transforming growth factor beta-vascular endothelial growth factor signaling axis in its pathogenesis. J Orthop Sci. 18:347–354. 2013. View Article : Google Scholar
6	Maezawa Y, Baba H, Uchida K, Kokubo Y, Kubota C and Noriki S: Ligamentum flavum hematoma in the thoracic spine. Clin Imaging. 25:265–267. 2001. View Article : Google Scholar : PubMed/NCBI
7	Sairyo K, Biyani A, Goel VK, Leaman DW, Booth R Jr, Thomas J, Ebraheim NA, Cowgill IA and Mohan SE: Lumbar ligamentum flavum hypertrophy is due to accumulation of inflammation-related scar tissue. Spine. 32:E340–E347. 2007. View Article : Google Scholar : PubMed/NCBI
8	Kosaka H, Sairyo K, Biyani A, Leaman D, Yeasting R, Higashino K, Sakai T, Katoh S, Sano T, Goel VK and Yasui N: Pathomechanism of loss of elasticity and hypertrophy of lumbar ligamentum flavum in elderly patients with lumbar spinal canal stenosis. Spine. 32:2805–2811. 2007. View Article : Google Scholar
9	Nakamura T, Okada T, Endo M, Kadomatsu T, Taniwaki T, Sei A, Odagiri H, Masuda T, Fujimoto T, Nakamura T, et al: Angiopoietin-like protein 2 induced by mechanical stress accelerates degeneration and hypertrophy of the ligamentum flavum in lumbar spinal canal stenosis. PLoS One. 9:e855422014. View Article : Google Scholar : PubMed/NCBI
10	Nakatani T, Marui T, Hitora T, Doita M, Nishida K and Kurosaka M: Mechanical stretching force promotes collagen synthesis by cultured cells from human ligamentum flavum via transforming growth factor-beta1. J Orthop Res. 20:1380–1386. 2002. View Article : Google Scholar : PubMed/NCBI
11	Ariel A and Timor O: Hanging in the balance: endogenous anti-inflammatory mechanisms in tissue repair and fibrosis. J Pathol. 229:250–263. 2013. View Article : Google Scholar
12	Seko Y, Seko Y, Takahashi N, Shibuya M and Yazaki Y: Pulsatile stretch stimulates vascular endothelial growth factor (VEGF) secretion by cultured rat cardiac myocytes. Biochem Biophys Res Commun. 254:462–465. 1999. View Article : Google Scholar : PubMed/NCBI
13	Löhr M, Hampl JA, Lee JY, Ernestus RI, Deckert M and Stenzel W: Hypertrophy of the lumbar ligamentum flavum is associated with inflammation-related TGF-β expression. Acta Neurochir (Wien). 153:134–141. 2011. View Article : Google Scholar
14	Park JB, Chang H and Lee JK: Quantitative analysis of transforming growth factor-beta 1 in ligamentum flavum of lumbar spinal stenosis and disc herniation. Spine. 26:E492–E495. 2001. View Article : Google Scholar : PubMed/NCBI
15	Chen YT, Wei JD, Wang JP, Lee HH, Chiang ER, Lai HC, Chen LL, Lee YT, Tsai CC, Liu CL, et al: Isolation of mesenchymal stem cells from human ligamentum flavum: implicating etiology of ligamentum flavum hypertrophy. Spine. 36:E1193–E1200. 2011. View Article : Google Scholar : PubMed/NCBI
16	Zhong ZM, Zha DS, Xiao WD, Wu SH, Wu Q, Zhang Y, Liu FQ and Chen JT: Hypertrophy of ligamentum flavum in lumbar spine stenosis associated with the increased expression of connective tissue growth factor. J Orthop Res. 29:1592–1597. 2011. View Article : Google Scholar : PubMed/NCBI
17	Brigstock DR: The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev. 20:189–206. 1999.PubMed/NCBI
18	Taipale J, Miyazono K, Heldin CH and Keski-Oja J: Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein. J Cell Biol. 124:171–181. 1994. View Article : Google Scholar : PubMed/NCBI
19	Igarashi A, Okochi H, Bradham DM and Grotendorst GR: Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell. 4:637–645. 1993. View Article : Google Scholar : PubMed/NCBI
20	Abreu JG, Ketpura NI, Reversade B and De Robertis EM: Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol. 4:599–604. 2002.PubMed/NCBI
21	Chen Z, Gibson TB, Robinson F, Silvestro L, Pearson G, Xu B, Wright A, Vanderbilt C and Cobb MH: MAP kinases. Chem Rev. 101:2449–2476. 2001. View Article : Google Scholar : PubMed/NCBI
22	Gu J, Liu X, Wang QX, Tan HW, Guo M, Jiang WF and Zhou L: Angiotensin II increases CTGF expression via MAPKs/TGF-β1/TRAF6 pathway in atrial fibroblasts. Exp Cell Res. 318:2105–2115. 2012. View Article : Google Scholar : PubMed/NCBI
23	Specchia N, Pagnotta A, Gigante A, Logroscino G and Toesca A: Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis. J Orthop Res. 19:294–300. 2001. View Article : Google Scholar : PubMed/NCBI
24	Zhong ZM and Chen JT: Phenotypic characterization of ligamentum flavum cells from patients with ossification of ligamentum flavum. Yonsei Med J. 50:375–379. 2009. View Article : Google Scholar : PubMed/NCBI
25	Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 65:55–63. 1983. View Article : Google Scholar : PubMed/NCBI
26	Zeng Y, Adamson RH, Curry FR and Tarbell JM: Sphingosine-1-phosphate protects endothelial glycocalyx by inhibiting syndecan-1 shedding. Am J Physiol Heart Circ Physiol. 306:H363–H372. 2014. View Article : Google Scholar :
27	Leask A and Abraham DJ: TGF-beta signaling and the fibrotic response. FASEB J. 18:816–827. 2004. View Article : Google Scholar : PubMed/NCBI
28	Yoshida M, Shima K, Taniguchi Y, Tamaki T and Tanaka T: Hypertrophied ligamentum flavum in lumbar spinal canal stenosis. Pathogenesis and morphologic and immunohistochemical observation. Spine. 17:1353–1360. 1992. View Article : Google Scholar : PubMed/NCBI
29	Feng XH and Derynck R: Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol. 21:659–693. 2005. View Article : Google Scholar : PubMed/NCBI
30	Massagué J: How cells read TGF-beta signals. Nat Rev Mol Cell Biol. 1:169–178. 2000. View Article : Google Scholar
31	Strand DW, Liang YY, Yang F, Barron DA, Ressler SJ, Schauer IG, Feng XH and Rowley DR: TGF-β induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways. Am J Clin Exp Urol. 2:239–248. 2014.
32	Demagny H, Araki T and De Robertis EM: The tumor suppressor Smad4/DPC4 is regulated by phosphorylations that integrate FGF, Wnt, and TGF-β signaling. Cell Reports. 9:688–700. 2014. View Article : Google Scholar
33	Derynck R and Zhang YE: Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 425:577–584. 2003. View Article : Google Scholar : PubMed/NCBI
34	Javelaud D and Mauviel A: Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-beta: implications for carcinogenesis. Oncogene. 24:5742–5750. 2005. View Article : Google Scholar : PubMed/NCBI
35	Binder DK, Schmidt MH and Weinstein PR: Lumbar spinal stenosis. Semin Neurol. 22:157–166. 2002. View Article : Google Scholar
36	Omidi-Kashani F, Hasankhani EG and Ashjazadeh A: Lumbar spinal stenosis: who should be fused? an updated review. Asian Spine J. 8:521–530. 2014. View Article : Google Scholar : PubMed/NCBI