Z Orthop Unfall 2023; 161(02): 201-210
DOI: 10.1055/a-1527-7900
Original Article

Osteoblasts Regulate the Expression of ADAMTS and MMPs in Chondrocytes through ERK Signaling Pathway

Osteoblasten regulieren die Expression von ADAMTS und MMPs in Chondrozyten durch den ERK-Signalweg
Xiao Ding
Department of Orthopaedics, The First Affiliated Hospital of the Medical Colleges, Shihezi University, China
,
Wei Xiang
Department of Orthopaedics, The First Affiliated Hospital of the Medical Colleges, Shihezi University, China
,
Defeng Meng
Department of Orthopaedics, The First Affiliated Hospital of the Medical Colleges, Shihezi University, China
,
Wang Chao
Department of Orthopaedics, The First Affiliated Hospital of the Medical Colleges, Shihezi University, China
,
Han Fei
Department of Orthopaedics, The First Affiliated Hospital of the Medical Colleges, Shihezi University, China
,
Weishan Wang
Department of Orthopaedics, The First Affiliated Hospital of the Medical Colleges, Shihezi University, China
› Author Affiliations

Abstract

Objective Degradative enzymes such as matrix metalloproteinase (MMP) and disintegrin metalloproteinase with platelet thrombin-sensitive protein-like motifs (ADAMTS) play a key role in the development of osteoarthritis (OA). We aimed to investigate the effects of OA subchondral osteoblasts on the expression of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 in chondrocytes and the regulation of mitogen-activated protein kinase (MAPK) signaling pathway.

Methods A rat knee OA model was constructed by cutting the anterior cruciate ligament of the knee joints, and normal rat articular cartilage chondrocytes (N-ACC), OA rat articular cartilage chondrocytes (O-ACC), normal subchondral bone osteoblasts (N-SBO), and OA subchondral bone osteoblasts (O-SBO) were isolated and extracted. The expressions of O-ACC and O-SBO COL1 and COL2 were detected respectively. Chondrocytes were identified by immunofluorescence of COL2 and toluidine blue staining, and osteoblasts were identified by COL1 immunofluorescence, alkaline phosphatase (ALP), and Alizarin Red staining. Gene expression of COL1, COL2, and aggrecan in normal chondrocytes and OA chondrocytes, and gene expression of osteoblast ALP and osteocalcin (OCN) were detected by RT-PCR to identify the two chondrocytes and the two osteoblast phenotypes. The constructing N-ACC group, O-ACC group, N-ACC + N-SBO group, N-ACC + O-SBO group, O-ACC + N-SBO group, O-ACC + O-SBO group, I + N-ACC + O-SBO group, and I + O-ACC + O-SBO group cell cultures, and the expression of ERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes in chondrocytes cultured for 0, 24, 48, and 72 h were detected by RT-PCR. The protein expressions of pERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 were detected by Western blot.

Results

  1. The X-ray showed that the knee joint space of the affected limb became narrow.

  2. The results of RT-PCR of COL2 and aggrecan gene in OA and normal chondrocytes suggest that the relative expression of COL2 in OA articular chondrocytes (0.24 ± 0.07) is significantly lower than that in normal cartilage (0.61 ± 0.07) (p < 0.05). The relative expression of AGG (0.37 ± 0.16) in OA chondrocytes was significantly lower than that of normal chondrocytes AGG (1.30 ± 0.25) (p < 0.05). The expression of COL1 was very low, and was not statistically significant.

  3. The results of RT-PCR of the osteoblast ALP and OCN gene indicated that gene expression of ALP (12.30 ± 1.17) and OCN (20.47 ± 4.19)was upregulated when compared with the relative expression of ALP (4.66 ± 0.71) (p < 0.05) and OCN (12.17 ± 2.76) (p < 0.05) in normal osteoblasts, indicating that osteoblasts of OA have greater osteogenic potential than normal osteoblasts.

  4. The expressions of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes and proteins in OA chondrocytes or normal chondrocytes were basically unchanged when they were cocultured with normal osteoblasts. Indirect coculture of OA osteoblasts and chondrocytes could promote the expression of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes and proteins in chondrocytes. Overexpression of ADAMTS and MMP in coculture systems can be reversed by MAPK-ERK inhibitors.

Conclusions

  1. OA subchondral bone osteoblasts can promote the overexpression of ADAMTS and MMPs in chondrocytes.

  2. The ERK signaling pathway may be involved in the regulation of the effect of subchondral bone osteoblasts on chondrocytes.

Zusammenfassung

Zielsetzung: Enzyme wie Matrix-Metalloproteinase (MMP) und ADAMTS spielen eine Schlüsselrolle bei der Entstehung der Osteoarthritis (OA). Das Ziel der Studie war es, die Auswirkungen subchondraler Osteoblasten auf die Expression von ADAMTS4, ADAMTS5, MMP-3, MMP-9 und MMP-13 in Chondrozyten und die Regulation des Mitogen-aktivierten Proteinkinase-(MAPK-)Signalwegs in der OA zu untersuchen.

Methode: Ein OA-Rattenknie-Modell wurde konstruiert, indem das vordere Kreuzband der Kniegelenke und normale Ratten-Chondrozyten (N-ACC), OA-Ratten-Chondrozyten (O-ACC), normale subchondrale Osteoblasten (N-SBO) und OA subchondrale Osteoblasten (O-SBO) isoliert und extrahiert wurden. Die Expressionen von O-ACC und O-SBO COL1 bzw. COL2 wurden nachgewiesen. Chondrozyten wurden durch Immunfluoreszenz von COL2 und Toluidinblau-Färbung identifiziert, Osteoblasten wurden durch COL1-Immunfluoreszenz, alkalische Phosphatase (ALP) und Alizarin-Rot-Färbung identifiziert. Die Genexpression von COL1, COL2 und Aggrecan in normalen Chondrozyten und OA-Chondrozyten sowie die Genexpression von Osteoblasten-ALP und Osteocalcin (OCN) wurden durch RT-PCR nachgewiesen, um die beiden Chondrozyten und die beiden Osteoblasten-Phänotypen zu identifizieren. Die aufbauende N-CC-Gruppe, O-ACC-Gruppe, N-ACC + NSBO-Gruppe, N-ACC + O-SBO-Gruppe, O-ACC + N-SBO-Gruppe, O-ACC + O-SBO-Gruppe, I + NACC + O-SBO-Gruppe und I + O-ACC + O-SBO-Gruppe-Zellkulturen und die Expression von ERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9- und MMP-13- Gene in 0, 24, 48 und 72 h kultivierten Chondrozyten wurden durch RT-PCR nachgewiesen. Die Proteinexpressionen von pERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9 und MMP-13 wurden durch Western Blot nachgewiesen.

Ergebnisse:

  1. Im Röntgenbild zeigte sich eine Kniegelenkspaltverengung.

  2. Die Ergebnisse der RT-PCR von COL2 und dem Aggrecan Gen in OA und normalen Chondrozyten legen nahe, dass die relative Expression von COL2 in artikulären Chondrozyten von OA (0,24 ± 0,07) signifikant niedriger ist als die in normalem Knorpel (0,61 ± 0,07) (p < 0,05). Die relative Expression von AGG (0,37 ± 0,16) in OA-Chondrozyten war signifikant niedriger als die von normalen Chondrozyten-AGG (1,30 ± 0,25) (p < 0,05). Die Expression von COL1 war sehr gering und statistisch nicht signifikant.

  3. Die Ergebnisse der RT-PCR des Osteoblasten-ALP- und OCN-Gens zeigten, dass die Genexpression von ALP (12,30 ± 1,17) und OCN (20,47 ± 4,19) im Vergleich zur relativen Expression von ALP (4,66 ± 0,71) (p < 0,05) und OCN (12,17 ± 2,76) (p < 0,05) bei normalen Osteoblasten hochreguliert war, was darauf hindeutet, dass Osteoblasten von OA ein größeres osteogenes Potenzial haben als normale Osteoblasten signifikant.

  4. Die Expressionen der ADAMTS4-, ADAMTS5-, MMP-3-, MMP-9- und MMP-13-Gene und -Proteine in OA-Chondrozyten oder normalen Chondrozyten waren im Wesentlichen unverändert, wenn sie mit normalen Osteoblasten kokultiviert wurden. Indirekte Kokultur von OA-Osteoblasten und Chondrozyten könnte die Expression der Gene und Proteine ADAMTS4, ADAMTS5, MMP-3, MMP-9 und MMP-13 in Chondrozyten fördern. Die Überexpression von ADAMTS und MMP in Kokultursystemen kann durch MAPK-ERK-Inhibitoren rückgängig gemacht werden.

Schlussfolgerungen:

  1. Subchondrale Osteoblasten in OA Gelenken können die Überexpression von ADAMTS und MMPs in Chondrozyten fördern.

  2. Der ERK-Signalweg kann an der Regulation der Wirkung subchondraler Knochenosteoblasten auf Chondrozyten beteiligt sein.



Publication History

Article published online:
09 September 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 
  • References

  • 1 Findlay DM, Kuliwaba JS. Bone-cartilage crosstalk: a conversation for understanding osteoarthritis. Bone Res 2016; 4: 16028
  • 2 Clark JM, Huber JD. The structure of the human subchondral plate. J Bone Joint Surg Br 1990; 72: 866-873
  • 3 Amin AK, Huntley JS, Simpson AH. et al. Chondrocyte survival in articular cartilage: the influence of subchondral bone in a bovine model. J Bone Joint Surg Br 2009; 91: 691-699
  • 4 Sanchez C, Horcajada MN, Membrez Scalfo F. et al. Carnosol Inhibits Pro-Inflammatory and Catabolic Mediators of Cartilage Breakdown in Human Osteoarthritic Chondrocytes and Mediates Cross-Talk between Subchondral Bone Osteoblasts and Chondrocytes. PLoS One 2015; 10: e0136118
  • 5 Mobasheri A, Bay-Jensen AC, van Spil WE. et al. Osteoarthritis Year in Review 2016: biomarkers (biochemical markers). Osteoarthritis Cartilage 2017; 25: 199-208
  • 6 Rodríguez-Manzaneque JC, Westling J, Thai SN. et al. ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors. Biochem Biophys Res Commun 2002; 293: 501-508
  • 7 Verma P, Dalal K. ADAMTS-4 and ADAMTS-5: key enzymes in osteoarthritis. J Cell Biochem 2011; 112: 3507-3514
  • 8 Deng S, Zhou JL, Peng H. et al. Local intra-articular injection of vascular endothelial growth factor accelerates articular cartilage degeneration in rat osteoarthritis model. Mol Med Rep 2018; 17: 6311-6318
  • 9 Malemud CJ. Matrix Metalloproteinases and Synovial Joint Pathology. Prog Mol Biol Transl Sci 2017; 148: 305-325
  • 10 Gao SC, Yin HB, Liu HX. et al. [Research progress on MAPK signal pathway in the pathogenesis of osteoarthritis]. Zhongguo Gu Shang 2014; 27: 441-444
  • 11 Rogart JN, Barrach HJ, Chichester CO. Articular collagen degradation in the Hulth-Telhag model of osteoarthritis. Osteoarthritis Cartilage 1999; 7: 539-547
  • 12 Flemming DJ, Gustas-French CN. Rapidly Progressive Osteoarthritis: a Review of the Clinical and Radiologic Presentation. Curr Rheumatol Rep 2017; 19: 42
  • 13 Nagase H, Kashiwagi M. Aggrecanases and cartilage matrix degradation. Arthritis Res Ther 2003; 5: 94-103
  • 14 Verma P, Dalal K, Chopra M. Pharmacophore development and screening for discovery of potential inhibitors of ADAMTS-4 for osteoarthritis therapy. J Mol Model 2016; 22: 178
  • 15 Glasson SS, Askew R, Sheppard B. et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature 2005; 434: 644-648
  • 16 Majumdar MK, Askew R, Schelling S. et al. Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis. Arthritis Rheum 2007; 56: 3670-3674
  • 17 Leeman MF, Curran S, Murray GI. The structure, regulation, and function of human matrix metalloproteinase-13. Crit Rev Biochem Mol Biol 2002; 37: 149-166
  • 18 Varghese S. Matrix metalloproteinases and their inhibitors in bone: an overview of regulation and functions. Front Biosci 2006; 11: 2949-2966
  • 19 Chakraborti S, Mandal M, Das S. et al. Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem 2003; 253: 269-285
  • 20 Ströbel S, Loparic M, Wendt D. et al. Anabolic and catabolic responses of human articular chondrocytes to varying oxygen percentages. Arthritis Res Ther 2010; 12: R34
  • 21 Guilak F, Fermor B, Keefe FJ. et al. The role of biomechanics and inflammation in cartilage injury and repair. Clin Orthop Relat Res 2004; (423) 17-26
  • 22 Pallu S, Francin PJ, Guillaume C. et al. Obesity affects the chondrocyte responsiveness to leptin in patients with osteoarthritis. Arthritis Res Ther 2010; 12: R112
  • 23 Sanchez C, Deberg MA, Piccardi N. et al. Subchondral bone osteoblasts induce phenotypic changes in human osteoarthritic chondrocytes. Osteoarthritis Cartilage 2005; 13: 988-997
  • 24 Sanchez C, Deberg MA, Piccardi N. et al. Osteoblasts from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases expression by chondrocytes. This effect is mimicked by interleukin-6, − 1beta and oncostatin M pre-treated non-sclerotic osteoblasts. Osteoarthritis Cartilage 2005; 13: 979-987
  • 25 Murphy LO, Blenis J. MAPK signal specificity: the right place at the right time. Trends Biochem Sci 2006; 31: 268-275
  • 26 Sokoloff L. Microcracks in the calcified layer of articular cartilage. Arch Pathol Lab Med 1993; 117: 191-195
  • 27 Liacini A, Sylvester J, Li WQ. et al. Inhibition of interleukin-1-stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes. Matrix Biol 2002; 21: 251-262
  • 28 Pelletier JP, Fernandes JC, Brunet J. et al. In vivo selective inhibition of mitogen-activated protein kinase kinase 1/2 in rabbit experimental osteoarthritis is associated with a reduction in the development of structural changes. Arthritis Rheum 2003; 48: 1582-1593
  • 29 Fan Z, Söder S, Oehler S. et al. Activation of interleukin-1 signaling cascades in normal and osteoarthritic articular cartilage. Am J Pathol 2007; 171: 938-946
  • 30 Loeser RF, Erickson EA, Long DL. Mitogen-activated protein kinases as therapeutic targets in osteoarthritis. Curr Opin Rheumatol 2008; 20: 581-586
  • 31 Lories RJ, Luyten FP. The bone-cartilage unit in osteoarthritis. Nat Rev Rheumatol 2011; 7: 43-49
  • 32 Sharif M, George E, Dieppe PA. Correlation between synovial fluid markers of cartilage and bone turnover and scintigraphic scan abnormalities in osteoarthritis of the knee. Arthritis Rheum 1995; 38: 78-81
  • 33 Lajeunesse D, Reboul P. Subchondral bone in osteoarthritis: a biologic link with articular cartilage leading to abnormal remodeling. Curr Opin Rheumatol 2003; 15: 628-633
  • 34 Westacott CI, Webb GR, Warnock MG. et al. Alteration of cartilage metabolism by cells from osteoarthritic bone. Arthritis Rheum 1997; 40: 1282-1291