The effects of insulin on the expression levels of ADAMTS6 & 19 in OUMS-27 cell

Öz

Objective: A Disintegrin-like Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteins are kind of matrix metalloproteinase enzymes that primarily founds in the extracellular matrix (ECM). Insulin is an important anabolic hormone, which acts on many tissues. The aim of this study is to evaluate the time-dependent effects of insulin on the two functionally unknown enzyme expressions (ADAMTS6 & 19) in OUMS-27 human chondrosarcoma cell line. Methods: OUMS-27 cells were cultured in Dulbecco\'s modified Eagle\' medium (DMEM) alone and DMEM containing 10 μg/mL insulin. The medium was changed every other day up to 11th day. Cells were harvested at 1, 3, 7, and 11th days and RNA isolation was performed at appropriate times according to study setup. The levels of RNA expression of ADAMTS6 and 19 were estimated by qRT-PCR using appropriate primers. Results: According to qRT-PCR analysis, ADAMTS6 mRNA expression was found to be decreased as early as one day after insulin application and continued up to day 11, the last day of insulin induction (p=0.008). The ratio of ADAMTS6 in insulin-applied groups was changed between 1/2 and 1/4 of control values. The changes in ADAMTS19 mRNA levels in insulin-applied cells were not statistically significant compared to control cell group. Conclusion: Our results demonstrated that insulin has a potential effect on alleviation of loss of extracellular matrix compounds by diminishing ADAMTS6 levels. To test this hypothesis and finding, more investigations are needed to recognize the real functions of orphan ADAMTS proteins.

Anahtar Kelimeler

• Insulin, ADAMTS6, ADAMTS19, chondrosarcoma, OUMS-27, RNA

İnsülinin OUMS-27 hücrelerinde ADAMTS6 ve 19 ekspresyon düzeylerine etkileri

Öz

Amaç: A Disintegrin-like Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteinleri ekstraselüler matrikste (ECM) bulunan bir çeşit matriks metalloproteinaz enzimidir. İnsulin birçok dokuda etki gösteren önemli bir anabolik hormondur. Bu çalışmanın amacı OUMS-27 insan kondrosarkom hücre kültüründe insülinin, fonksiyonları tam olarak bilinmeyen ADAMTS enzimlerinden ADAMTS6 ve 19 ekspresyonu üzerine zamana bağımlı olarak etkilerini değerlendirmek ve insülinin anabolik etkilerinden dolayı ADAMTS ekspresyonunu azaltma hipotezini test etmektir. Yöntemler: OUMS-27 hücrelerinin, 10 μg/mL insulin içeren ve içermeyen Dulbecco\'s modified Eagle besiyerinde (DMEM) kültürü yapıldı. 11. güne kadar iki günde bir besiyeri ile birlikte insülin takviyesi yapıldı. Hücreler 1, 3, 7 ve 11. günlerde harvest edilerek uygun zamanlarda RNA izolasyonu yapıldı. ADAMTS6 ve 19 RNA ekspresyonları uygun primerler kullanılarak qRT-PCR yöntemi ile ölçüldü. Bulgular: qRT-PCR cihazının verilerine göre, insülin uygulanmasından 1 gün sonra başlamak üzere deneyin son günü olan 11. güne kadar ADAMTS6\'nın mRNA düzeyinde ciddi bir azalma meydana geldi (p=0.008). İnsülin uygulanan gruplarda ADAMTS6 oranı kontrol grubuna kıyasla 1/2 ve 1/4 aralığında değişti. İnsülin verilen hücre grubunda ADAMTS19 mRNA düzeyindeki değişiklikler ise kontrol grubuna kıyasla istatistiksel olarak anlamsızdı. Sonuç: Sonuçlarımız, insülinin ADAMTS6 düzeylerini azaltarak ekstrasellüler matriks bileşenlerinin kaybının telafi edilmesinde potansiyel bir etkisinin olabileceğini gösterdi. Bu hipotez ve bulgunun anlaşılması için yetim ADAMTS proteinlerinin gerçek fonksiyonlarının inceleneceği çok sayıda araştırma yapılmasına ihtiyaç vardır.

Anahtar Kelimeler

• İnsulin, ADAMTS6, ADAMTS19, kondrosarkom, OUMS-27, RNA

Kaynakça

1. Demircan K, Hirohata S, Nishida K, et al. ADAMTS-9 is synergistically induced by interleukin-1beta and tumor ne- crosis factor alpha in OUMS-27 chondrosarcoma cells and in human chondrocytes. Arthritis Rheum 2005;52:1451- 1460.
2. Koshy PJ, Lundy CJ, Rowan AD, et al. The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin-1 and oncostatin M: a time-course study using real-time quantitative reverse transcription-polymerase chain reaction. Arthritis Rheum 2002;46:961-967.
3. Apte SS. A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family. Int J Biochem Cell Biol 2004;36:981-985.
4. Kumar S, Rao N, Ge R. Emerging Roles of ADAMTSs in Angiogenesis and Cancer. Cancers (Basel) 2012;4:1252- 1299.
5. Porter S, Clark IM, Kevorkian L, Edwards DR. The AD- AMTS metalloproteinases. Biochem J 2005;386:15-27.
6. WHO: Cartilage tumours. In: World health organization clas- sification of tumours. Pathology and genetics - Tumours of soft tissue and bone, Fletcher CDM, Unni KK, Mertens F (Eds), 2002. p.234.
7. Dorfman HD, Czerniak B. Bone cancers. Cancer 1995; 75: 203-210.
8. Murray RK, Granner DK, Mayes PA, Rodwell VW, Harper’s Illustrated Biochemistry, 26th Edition. USA: McGraw-Hill Companies, 2003:466.

9. Schiller S, Dorfman A. The metabolism of mucopolysaccha- rides in animals. IV. The influence of insulin. J Biol Chem 1957;227:625-632.
10. Kayal RA, Alblowi J, McKenzie E, et al. Diabetes causes the accelerated loss of cartilage during fracture repair which is reversed by insulin treatment. Bone 2009;44:357-363.
11. Hou CJ, Liu JL, Li X, Bi LJ. Insulin promotes bone forma- tion in augmented maxillary sinus in diabetic rabbits. Int J Oral Maxillofac Surg 2012;41:400-407.
12. Mandal M, Mandal A, Das S, et al. Clinical implica- tions of matrix metalloproteinases. Mol Cell Biochem 2003;252:305-329.
13. Kevorkian L, Young DA, Darrah C, et al. Expression profil- ing of metalloproteinases and their inhibitors in cartilage. Arthritis Rheum 2004;50:131-141.
14. Maor G, Vasiliver-Shamis G, Hazan-Brill R, et al. GLUT4 in murine bone growth: from uptake and translocation to proliferation and differentiation. Am J Physiol Endocrinol Metab 2011;300:E613-623.
15. Stevens RL, Nissley SP, Kimura JH, et al. Effects of insu- lin and multiplication-stimulating activity on proteoglycan biosynthesis in chondrocytes from the Swarm rat chondro- sarcoma. J Biol Chem 1981;256:2045-2052.
16. McCumbee WD, Lebovitz HE. Hormone responsiveness of a transplantable rat chondrosarcoma. I. In vitro effects of growth hormone-dependent serum factors and insulin. Endocrinology 1980;106:905-910.
17. Bembenek ME, Willis DH, Jr., Liberti JP. The effect of insulin on collagen production in isolated chondrosar- coma chondrocytes. Biochem Biophys Res Commun 1982;106:338-345.
18. Mason RM, Kimura JH, Hascall VC. Biosynthesis of hyal- uronic acid in cultures of chondrocytes from the Swarm rat chondrosarcoma. J Biol Chem 1982;257:2236-2245.
19. Stevens RL, Hascall VC. Characterization of proteoglycans synthesized by rat chondrosarcoma chondrocytes treated with multiplication-stimulating activity and insulin. J Biol Chem 1981;256:2053-2058.
20. Glade MJ, Kanwar YS, Stern PH. Insulin and thyroid hor- mones stimulate matrix metabolism in primary cultures of articular chondrocytes from young rabbits independently and in combination. Connect Tissue Res 1994;31:37-44.
21. Fırat R, Akyol S, Kurşunlu SF, et al. ADAMTS13 expres- sion in human chondrosarcoma cells induced by insulin. J Clin Exp Invest 2014;5:226-232.