The gene expression and protein profiles of ADAMTS and TIMP in human chondrosarcoma cell lines induced by insulin: The potential mechanisms for skeletal and articular abnormalities in diabetes
More details
Hide details
Beaumont Health System-Research Institute, Royal Oak, MI, USA
Department of Medical Biology, Faculty of Medicine, Turgut Ozal University, Ankara, Turkey
Ankara University Biotechnology Institute, Ankara, Turkey
Biochemist, 7675 Oaks Lane, Beaumont, TX, USA
Department of Medical Biochemistry, Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey
Department of Medical Biochemistry, Faculty of Medicine, Turgut Ozal University, Ankara, Turkey
Michigan Math and Science Academy, Warren, MI, USA
Department of Medical Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey
Sumeyya Akyol   

Beaumont Health System-Research Institute, Royal Oak, MI, USA
Submission date: 2019-07-17
Final revision date: 2019-09-29
Acceptance date: 2019-10-03
Online publication date: 2020-01-16
Publication date: 2020-01-16
Electron J Gen Med 2020;17(1):em186
The delay in wound healing, decrease in the long bones resilience to fracture, and delay in fracture healing are among common complications DM patients, and they still remain as challenging issues to be solved. The mechanism has not been fully understood yet, but high sugar and/or insulin deficiency or unresponsiveness to insulin in blood are potential causes to blame. Extracellular matrix degradation/remodeling is one of the important mechanisms whereby cell differentiation, bone remodeling and wound repair can be regulated. ADAMTS proteins play important roles in cartilage/bone metabolism. This study aimed to determine whether ADAMTS/TIMP proteins were affected by insulin application in OUMS-27 cells.

Material and methods:
OUMS-27 cells were induced by 10μg/mL insulin for 1,3,7,and 11days. Cells were harvested, mRNA and protein extractions were performed. Total mRNA and cDNA levels were measured by qRT-PCR and protein levels were detected by WB.

ADAMTS1,5, and 7 levels were significantly decreased, while TIMP-3 levels were detected increased (mRNA/protein concentrations).

Pathologies and disturbances of cartilage/bone metabolism, delayed fracture healing in particular, in patients with DM may result from insulin deficiency. ADAMTS genes that play a role in healing process are increased during insulin deficiency, which consequently interrupts healing process by causing cartilage ECM degradation.

Mobasheri A, Richardson S, Mobasheri R, Shakibaei M, Hoyland JA. Hypoxia inducible factor-1 and facilitative glucose transporters GLUT1 and GLUT3: putative molecular components of the oxygen and glucose sensing apparatus in articular chondrocytes. Histol Histopathol. 2005;20(4):1327-38.
Mangiavini L, Merceron C, Araldi E, Khatri R, Gerard-O’Riley R, Wilson TL, et al. Loss of VHL in mesenchymal progenitors of the limb bud alters multiple steps of endochondral bone development. Dev Biol. 2014;393(1):124-36. https://doi.org/10.1016/j.ydbi... PMid:24972088 PMCid:PMC4335807.
Riddle RC, Clemens TL. Insulin, osteoblasts, and energy metabolism: why bone counts calories. J Clin Invest. 2014;124(4):1465-7. https://doi.org/10.1172/JCI755... PMid:24642463 PMCid:PMC3973079.
Sicca CM, Corotti MV, Sgarbosa SH, Cestari TM, Cavalcanti MG, Ferreira PM, et al. Comparative histomorphometric and tomographic analysis of maxillary sinus floor augmentation in rabbits using autografts and xenografts. J Biomed Mater Res B Appl Biomater. 2008;86(1):188-96. https://doi.org/10.1002/jbm.b.... PMid:18161780.
Xu H, Shimizu Y, Asai S, Ooya K. Grafting of deproteinized bone particles inhibits bone resorption after maxillary sinus floor elevation. Clin Oral Implants Res. 2004;15(1):126-33. https://doi.org/10.1111/j.1600... PMid:14731186.
Xu H, Shimizu Y, Onodera K, Ooya K. Long-term outcome of augmentation of the maxillary sinus using deproteinised bone particles experimental study in rabbits. Br J Oral Maxillofac Surg. 2005;43(1):40-5. https://doi.org/10.1016/j.bjom... PMid:15620773.
Nevins ML, Karimbux NY, Weber HP, Giannobile WV, Fiorellini JP. Wound healing around endosseous implants in experimental diabetes. Int J Oral Maxillofac Implants. 1998;13(5):620-9.
Lu H, Kraut D, Gerstenfeld LC, Graves DT. Diabetes interferes with the bone formation by affecting the expression of transcription factors that regulate osteoblast differentiation. Endocrinology. 2003;144(1):346-52. https://doi.org/10.1210/en.200... PMid:12488363.
Beam HA, Parsons JR, Lin SS. The effects of blood glucose control upon fracture healing in the BB Wistar rat with diabetes mellitus. J Orthop Res. 2002;20(6):1210-6. https://doi.org/10.1016/S0736-....
Hou CJ, Liu JL, Li X, Bi LJ. Insulin promotes bone formation in augmented maxillary sinus in diabetic rabbits. Int J Oral Maxillofac Surg. 2012;41(3):400-7. https://doi.org/10.1016/j.ijom... PMid:22099315.
Sellmeyer DE, Civitelli R, Hofbauer LC, Khosla S, Lecka-Czernik B, Schwartz AV. Skeletal Metabolism, Fracture Risk, and Fracture Outcomes in Type 1 and Type 2 Diabetes. Diabetes. 2016;65(7):1757-66. https://doi.org/10.2337/db16-0... PMid:27329951 PMCid:PMC4915586.
Jehle PM, Jehle DR, Mohan S, Bohm BO. Serum levels of insulin-like growth factor system components and relationship to bone metabolism in Type 1 and Type 2 diabetes mellitus patients. J Endocrinol. 1998;159(2):297-306. https://doi.org/10.1677/joe.0.... PMid:9795371.
Gerstenfeld LC, Wronski TJ, Hollinger JO, Einhorn TA. Application of histomorphometric methods to the study of bone repair. J Bone Miner Res. 2005;20(10):1715-22. https://doi.org/10.1359/JBMR.0... PMid:16160729.
Loder RT. The influence of diabetes mellitus on the healing of closed fractures. Clin Orthop Relat Res. 1988(232):210-6. https://doi.org/10.1097/000030....
Chaudhary SB, Liporace FA, Gandhi A, Donley BG, Pinzur MS, Lin SS. Complications of ankle fracture in patients with diabetes. J Am Acad Orthop Surg. 2008;16(3):159-70. https://doi.org/10.5435/001246... PMid:18316714.
Gooch HL, Hale JE, Fujioka H, Balian G, Hurwitz SR. Alterations of cartilage and collagen expression during fracture healing in experimental diabetes. Connect Tissue Res. 2000;41(2):81-91. https://doi.org/10.3109/030082... PMid:10992154.
Gandhi A, Beam HA, O’Connor JP, Parsons JR, Lin SS. The effects of local insulin delivery on diabetic fracture healing. Bone. 2005;37(4):482-90. https://doi.org/10.1016/j.bone... PMid:16027060.
Caley MP, Martins VL, O’Toole EA. Metalloproteinases and Wound Healing. Adv Wound Care (New Rochelle). 2015;4(4):225-34. https://doi.org/10.1089/wound.... PMid:25945285 PMCid:PMC4397992.
Chockalingam PS, Zeng W, Morris EA, Flannery CR. Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS-4 (aggrecanase 1) or ADAMTS-5 (aggrecanase 2). Arthritis Rheum. 2004;50(9):2839-48. https://doi.org/10.1002/art.20... PMid:15457452.
Yaykasli KO, Oohashi T, Hirohata S, Hatipoglu OF, Inagawa K, Demircan K, et al. ADAMTS9 activation by interleukin 1 beta via NFATc1 in OUMS-27 chondrosarcoma cells and in human chondrocytes. Mol Cell Biochem. 2009;323(1-2):69-79. https://doi.org/10.1007/s11010... PMid:19052845.
Kumagishi K, Nishida K, Yamaai T, Momota R, Miyaki S, Hirohata S, et al. A disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9) expression by chondrocytes during endochondral ossification. Arch Histol Cytol. 2009;72(3):175-85. https://doi.org/10.1679/aohc.7... PMid:20513980.
Aiken A, Khokha R. Unraveling metalloproteinase function in skeletal biology and disease using genetically altered mice. Biochim Biophys Acta. 2010;1803(1):121-32. https://doi.org/10.1016/j.bbam... PMid:19616584.
Uysal S, Unal ZN, Erdogan S, Akyol S, Ramazan Yigitoglu M, Hirohata S, et al. Augmentation of ADAMTS9 gene expression by IL-1beta is reversed by NFkappaB and MAPK inhibitors, but not PI3 kinase inhibitors. Cell Biochem Funct. 2013;31(7):539-44. https://doi.org/10.1002/cbf.29... PMid:23175174.
Gerstenfeld LC, Thiede M, Seibert K, Mielke C, Phippard D, Svagr B, et al. Differential inhibition of fracture healing by non-selective and cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs. J Orthop Res. 2003;21(4):670-5. https://doi.org/10.1016/S0736-....
Follak N, Kloting L, Wolf E, Merk H. Delayed remodeling in the early period of fracture healing in spontaneously diabetic BB/OK rats depending on the diabetic metabolic state. Histol Histopathol. 2004;19(2):473-86.
White CB, Turner NS, Lee GC, Haidukewych GJ. Open ankle fractures in patients with diabetes mellitus. Clin Orthop Relat Res. 2003(414):37-44. https://doi.org/10.1097/01.blo... PMid:12966274.
Kayal RA, Tsatsas D, Bauer MA, Allen B, Al-Sebaei MO, Kakar S, et al. Diminished bone formation during diabetic fracture healing is related to the premature resorption of cartilage associated with increased osteoclast activity. J Bone Miner Res. 2007;22(4):560-8. https://doi.org/10.1359/jbmr.0... PMid:17243865 PMCid:PMC3109431.
Otsu K, Geary ES, Stevens RL. Aberrant regulation of the metabolism of the insulin receptor in Swarm rat chondrosarcoma chondrocytes. Biochem J. 1988;254(1):203-9. https://doi.org/10.1042/bj2540... PMid:3052429 PMCid:PMC1135057.
Stevens RL, Hascall VC. Characterization of proteoglycans synthesized by rat chondrosarcoma chondrocytes treated with multiplication-stimulating activity and insulin. J Biol Chem. 1981;256(4):2053-8.
Bembenek ME, Willis DH, Jr., Liberti JP. The effect of insulin on collagen production in isolated chondrosarcoma chondrocytes. Biochem Biophys Res Commun. 1982;106(2):338-45. https://doi.org/10.1016/0006-2....
Mason RM, Kimura JH, Hascall VC. Biosynthesis of hyaluronic acid in cultures of chondrocytes from the Swarm rat chondrosarcoma. J Biol Chem. 1982;257(5):2236-45.
Stevens RL, Nissley SP, Kimura JH, Rechler MM, Caplan AI, Hascall VC. Effects of insulin and multiplication-stimulating activity on proteoglycan biosynthesis in chondrocytes from the Swarm rat chondrosarcoma. J Biol Chem. 1981;256(4):2045-52.
Salomon DS, Paglia LM, Verbruggen L. Hormone-dependent growth of a rat chondrosarcoma in vivo. Cancer Res. 1979;39(11):4387-95.
Kevorkian L, Young DA, Darrah C, Donell ST, Shepstone L, Porter S, et al. Expression profiling of metalloproteinases and their inhibitors in cartilage. Arthritis Rheum. 2004;50(1):131-41. https://doi.org/10.1002/art.11... PMid:14730609.
Kayal RA, Alblowi J, McKenzie E, Krothapalli N, Silkman L, Gerstenfeld L, et al. Diabetes causes the accelerated loss of cartilage during fracture repair which is reversed by insulin treatment. Bone. 2009;44(2):357-63. https://doi.org/10.1016/j.bone... PMid:19010456 PMCid:PMC2700945.
Cawston TE, Wilson AJ. Understanding the role of tissue degrading enzymes and their inhibitors in development and disease. Best Pract Res Clin Rheumatol. 2006;20(5):983-1002. https://doi.org/10.1016/j.berh... PMid:16980219.
Stickens D, Behonick DJ, Ortega N, Heyer B, Hartenstein B, Yu Y, et al. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development. 2004;131(23):5883-95. https://doi.org/10.1242/dev.01... PMid:15539485 PMCid:PMC2771178.
Kennedy AM, Inada M, Krane SM, Christie PT, Harding B, Lopez-Otin C, et al. MMP13 mutation causes spondyloepimetaphyseal dysplasia, Missouri type (SEMD(MO). J Clin Invest. 2005;115(10):2832-42. https://doi.org/10.1172/JCI229... PMid:16167086 PMCid:PMC1201660.
Little CB, Mittaz L, Belluoccio D, Rogerson FM, Campbell IK, Meeker CT, et al. ADAMTS-1-knockout mice do not exhibit abnormalities in aggrecan turnover in vitro or in vivo. Arthritis Rheum. 2005;52(5):1461-72. https://doi.org/10.1002/art.21... PMid:15880348.
Stanton H, Rogerson FM, East CJ, Golub SB, Lawlor KE, Meeker CT, et al. ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature. 2005;434(7033):648-52. https://doi.org/10.1038/nature... PMid:15800625.
Kuno K, Kanada N, Nakashima E, Fujiki F, Ichimura F, Matsushima K. Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene. J Biol Chem. 1997;272(1):556-62. https://doi.org/10.1074/jbc.27... PMid:8995297.
Sandy JD, Westling J, Kenagy RD, Iruela-Arispe ML, Verscharen C, Rodriguez-Mazaneque JC, et al. Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4. J Biol Chem. 2001;276(16):13372-8. https://doi.org/10.1074/jbc.M0... PMid:11278559.
Vazquez F, Hastings G, Ortega MA, Lane TF, Oikemus S, Lombardo M, et al. METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity. J Biol Chem. 1999;274(33):23349-57. https://doi.org/10.1074/jbc.27... PMid:10438512.
Twining SS. Regulation of proteolytic activity in tissues. Crit Rev Biochem Mol Biol. 1994;29(5):315-83. https://doi.org/10.3109/104092... PMid:7828434.
Demircan K, Comertoglu I, Akyol S, Yigitoglu BN, Sarikaya E. A new biological marker candidate in female reproductive system diseases: Matrix metalloproteinase with thrombospondin motifs (ADAMTS). J Turk Ger Gynecol Assoc. 2014;15(4):250-5. https://doi.org/10.5152/jtgga.... PMid:25584036 PMCid:PMC4285216.
Akyol S, Ugurcu V, Cakmak O, Altuntas A, Yukselten Y, Akyol O, et al. Evidence for the control of aggrecanases by insulin and glucose in Alzheimer’s disease. Bull Clin Psychopharmacol. 2014;24(1):323-32. https://doi.org/10.5455/bcp.20....
Hardingham TE, Fosang AJ. Proteoglycans: many forms and many functions. FASEB J. 1992;6(3):861-70. https://doi.org/10.1096/fasebj....
Zhang E, Yan X, Zhang M, Chang X, Bai Z, He Y, et al. Aggrecanases in the human synovial fluid at different stages of osteoarthritis. Clin Rheumatol. 2013;32(6):797-803. https://doi.org/10.1007/s10067... PMid:23370724.
Zeng W, Corcoran C, Collins-Racie LA, Lavallie ER, Morris EA, Flannery CR. Glycosaminoglycan-binding properties and aggrecanase activities of truncated ADAMTSs: comparative analyses with ADAMTS-5, -9, -16 and -18. Biochim Biophys Acta. 2006;1760(3):517-24. https://doi.org/10.1016/j.bbag... PMid:16507336.
Yamanishi Y, Boyle DL, Clark M, Maki RA, Tortorella MD, Arner EC, et al. Expression and regulation of aggrecanase in arthritis: the role of TGF-beta. J Immunol. 2002;168(3):1405-12. https://doi.org/10.4049/jimmun... PMid:11801682.
Malfait AM, Liu RQ, Ijiri K, Komiya S, Tortorella MD. Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage. J Biol Chem. 2002;277(25):22201-8. https://doi.org/10.1074/jbc.M2... PMid:11956193.
Tortorella MD, Pratta M, Liu RQ, Austin J, Ross OH, Abbaszade I, et al. Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J Biol Chem. 2000;275(24):18566-73. https://doi.org/10.1074/jbc.M9... PMid:10751421.
Tortorella MD, Liu RQ, Burn T, Newton RC, Arner E. Characterization of human aggrecanase 2 (ADAM-TS5): substrate specificity studies and comparison with aggrecanase 1 (ADAM-TS4). Matrix Biol. 2002;21(6):499-511. https://doi.org/10.1016/S0945-....